WO2012051301A1

WO2012051301A1 - Methods for identifying modulators of triglyceride metabolism, for modulating triglyceride metabolism and for identifying subjects at risk for abnormal triglyceride metabolism

Info

Publication number: WO2012051301A1
Application number: PCT/US2011/055967
Authority: WO
Inventors: Laurie H. Glimcher; Ann-Hwee Lee
Original assignee: President And Fellows Of Harvard College
Priority date: 2010-10-12
Filing date: 2011-10-12
Publication date: 2012-04-19
Also published as: WO2012051301A9

Abstract

The present invention is based, at least in part, on the discovery that the transcription factor CREB-H modulates lipid metabolism. Mice bearing a null mutation in CREB-H exhibit a hypertriglyceridemia secondary to inefficient triglyceride clearance. Mutations in CREB3L3 have also been identified which are predictive of the propensity to develop abnormal triglyceride metabolism. Accordingly, methods for identifying agents that increase CREB-H expression and/or activity, methods for increasing CREB-H protein levels in cells, and methods for identifying subjects at risk for abnormal triglyceride metabolism are provided.

Description

METHODS FOR IDENTIFYING MODULATORS OF TRIGLYCERIDE METABOLISM, FOR MODULATING TRIGLYCERIDE METABOLISM AND FOR IDENTIFYING SUBECTS AT RISK FOR ABNORMAL TRIGLYCERIDE

METABOLISM

Background of the Invention

Transcription factors are a group of molecules within the cell that function to connect the pathways from extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes.

Dysregulation of lipid metabolism leading to increased plasma cholesterol and triglyceride (TG) levels is closely associated with coronary artery disease (CAD or atherosclerosis), obesity and type 2 diabetes. Transcription factors play central roles in metabolic regulation in mammals by controlling the synthesis of key metabolic proteins in response to nutritional and hormonal cues. Thus, transcription factors such as nuclear hormone receptors have emerged as potential drug targets for treating metabolic disorders.

Further elucidation of the factors influencing triglyceride metabolism would be of value in identifying agents capable of normalizing triglyceride metabolism. The identification of such agents and methods of using such agents would be of great benefit in the treatment of metabolic disorders.

Summary of the Invention

The present invention is based, at least in part, on the discovery that the transcription factor CREB-H modulates lipid metabolism. Mice bearing a null mutation CREB-H exhibit a hypertriglyceridemia secondary to inefficient triglceride clearance. Mutations in CREB3L3 have also been identified which are predictive of the propensity to develop abnormal triglyceride metabolism.

Accordingly, in one aspect, the invention pertains to a method for identifying compounds useful in maintaining normal triglyceride levels in a subject comprising,

a) providing a cell comprising the regulatory region of the CREB3L3gQUQ genetically fused to an indicator gene encoding a polypeptide;

b) contacting the cell with each member of a library of test compounds; c) selecting from the library of test compounds a compound of interest that increases the transcription of the indicator gene, wherein the ability of the compound to increase transcription of the indicator gene indicates that the compound is useful in maintaining normal triglyceride levels in a subject.

In another aspect, the invention pertains to a method for identifying compounds useful in maintaining normal triglyceride levels in a subject comprising, a) providing a cell comprising CREB-H or the amino terminal portion thereof and the regulatory region of a gene transcriptionally regulated by CREB-H genetically fused to an indicator gene encoding a polypeptide;

b) contacting the cell with each member of a library of test compounds; c) selecting from the library of test compounds a compound of interest that modulates the transcription of the indicator gene, wherein the ability of the compound to modulate CREB-H-mediated transcription of the indicator gene indicates that the compound is useful in maintaining normal triglyceride levels in a subject.

In one embodiment, the regulatory region regulates expression of a gene selected from the group consisting of Fgf21, Apoc2, Apoa4, and Apoa5 and wherein increased expression of the indicator gene indicates that the compound reduces triglyceride levels.

In one embodiment, the method further comprises testing the effect of the compound on triglyceride clearance.

In another embodiment, the triglyceride level in the very low density lipoprotein (VLDL) or low density (LDL) fraction of plasma is measured.

In another aspect, the invention pertains to a method for identifying a subject at risk for having abnormal triglyceride and lipoprotein metabolism comprising, contacting a biological sample derived from the subject with an agent capable of detecting the presence or absence of a CREB-H loss of function mutation, wherein the presence of the CREB-H loss of function mutation indicates that the subject is at risk for developing abnormal triglyceride and lipoprotein metabolism, thereby identifying a subject at risk for having abnormal triglyceride and lipoprotein metabolism.

In still another aspect, the invention pertains to a method for identifying a subject at risk for having abnormal triglyceride and lipoprotein metabolism comprising, contacting a biological sample derived from the subject with an agent capable of detecting the presence or absence of a mutation present in the CREB3L3 gene, wherein the presence of the mutation indicates that the subject is at risk for developing abnormal triglyceride and lipoprotein metabolism, thereby identifying a subject at risk for having abnormal triglyceride and lipoprotein metabolism.

In yet another aspect, the invention pertains to a method for reducing serum triglyceride levels in a subject, comprising administering to the subject an active form of CREB-H to thereby reduce serum triglyceride levels in the subject.

In yet another aspect, the invention pertains to a kit for predicting whether a subject is at risk for having abnormal triglyceride and lipoprotein metabolism, the kit comprising means for determining the presence or absence of a CREB-H loss of function mutation in a biological sample obtained from said subject and instructions for using the kit to predict whether the subject is at risk for having abnormal triglyceride and lipoprotein metabolism.

Figure Legends

Figure 1 panels A-H show that CREB-H^{" "} mice display severe

hypertriglyceridemia. (A) Plasma TG, (B) FFA, and (C) cholesterol levels were measured after a 16 h fast. Each dot represents an individual mouse. (D) Lipoproteins were separated by density gradient ultracentrifugation from pooled plasma (N=3 per each) after a 24 hr fast. Each 1 ml fraction sequentially collected from top to bottom was measured for density, and assayed for TG and cholesterol levels. (E) WT (N=9) and CREB-H^{" "} (N=7) male mice were deprived of food and bled at indicated times to measure plasma TG levels, starting at 8 AM. (F) Hepatic TG levels measured at fed state or after a 24hr fast. N=5 per group. (G) After concentration, fractions from (D) were separated on 4-20% gradient SDS-polyacrylamide gels. The gel was stained by

Coomassie Brilliant Blue G-250. Arrowhead indicates apoC-III identified by mass spectrometry. KO, CREB-H^{" "} (H) ApoC-III western blot of VLDL fractions. VLDL, d <1.006 g/ml; IDL d= 1.006-1.019 g/ml; LDL, d=l .019-1.063 g/ml; HDL, d=l .063-1.21 g/ml. *, p<0.05, pO.0001, compared to WT mice.

Figure 2 panels A-E show that loss of CREB-H impairs LPL-mediated TG clearance, which was reversed by apoC-II transfusion. (A) Two month-old female mice were i.v. injected with tyloxapol (500 mg/kg) after a 4hr fasting, and then plasma TG levels were measured at indicated times. N=4 per group. (B) Seven week-old female mice received an oral gavage of olive oil (10 ml/kg) after overnight fasting, and plasma TG levels were measured at indicated times. N=6 per group. (C) Post-heparin LPL activity (N=6 per group) was measured in the presence of heat inactivated serum pooled from WT or CREB-H^"7" mice (N=3 per group) as ApoC sources. (D) Purified LPL was incubated with triolein substrate in the presence of plasma collected from WT or CREB- H^"7" mice. Data were obtained by measuring FFA levels released from triolein. N=8 per group. (E) CREB-H^"7" mice were i.v. injected with 100 μΐ of freshly prepared plasma from WT or CREB-H^"7" mice after fasting for 4 hrs (N=10 per group). Blood samples were collected at indicated times for TG assays. Data are shown as mean ± SEM. *, p<0.05

Figure 3 panels A-C show that CREB-H is required for expression of genes implicated in hypertriglyceridemia and is regulated by feeding status. (A) Total RNAs were prepared from the liver of WT and CREB-H^"7" mice fasted for 24h. mRNA levels of select genes identified in microarray analysis were determined by real-time RT- PCR analysis. N=3 mice per group. Error bars indicate standard deviation. (B)

Expression of Apoc2, Apoa4, Apoa5 and Fgf21 mRNAs in WT and CREB-H^"7" mice as determined by real-time PCR. N=4 mice per group. Mice were sacrificed at fed state or after a 24hr fast. (C) CREB-H^"7" mice were i.v. injected with 25 μg of recombinant apoC- II proteins or saline (N=4 per group). Plasma TG levels were measured along the indicated time course. *, p<0.05, **, p<0.01, pO.0001, compared to WT mice. Figure 4 panels A-C show that Mutations of CREB3L3 contribute to human hypertriglyceridemia. (A) Locations of the nonsynonymous mutations that were found in patients with hypertriglyceridemia, and predicted amino acid changes. bZIP: basic leucine zipper; TM: transmembrane domain. (B) Pedigrees in which CREB3L3 nonsense mutations segregate. The probands are indicated by arrows. The lipid profiles are shown below each individual. Abbreviations: BMI, body mass index; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; ND, not determined. (C) Hepal .6 cells were co-transfected with Apoa4 reporter and the indicated CREB-H constructs. Values represent fold induction of luciferase activities compared to the reporter only transfection.

Figure 5. Expression of CREB-H mRNA and protein in liver and small intestine.

(A) Total RNAs were isolated from various organs of C57BL/6 male mice and subjected to northern blot analysis to detect CREB-H mRNA. (B) Microsomal fractions and nuclear extracts were isolated from WT and CREB-H^"7" mouse liver, and subjected to a western blot using CREB-H specific antibody. Whole protein samples extracted from intestinal epithelial cells were also tested for CREB-H by western blot. ^Nonspecific bands. CREB-H bands were not detectable in whole liver lysates due to the limited sensitivity of the antibody (not shown).

Figure 6. CREB-H is induced by fasting in mouse liver. (A) Mice were sacrificed at fed state or after a 24hr fast. CREB-H mRNA level was determined by Real-time PCR. N=4 mice per group. (B) Hepatic CREB-H(N) levels were measured by western blot of nuclear extracts of mice fed or fasted for 24hr before sacrifice. SP1 protein served as a loading control. **, p<0.01

Figure 7. Normal expression of LPL mRNA in adipose tissue and skeletal muscle in CREBH^{" "} mice. LPL mRNA levels in adipose tissue and skeletal muscle were determined by real-time RT-PCR. N=4 per group. N.S., not significant (p>0.05).

Figure 8. Microarray analysis of gene expression in CREB-H^{" "} liver. Total RNAs were isolated from liver after fasting the animals for 24h, and subjected to microarray analysis using Illumina WG6 chips. Differentially expressed genes were sorted according to P values from three independent experiments per group. Listed are genes which are downregulated in CREB-H^"7" liver by >1.5 folds with P values of <0.05 for comparison between WT vs. CREB-H^"7". *P value for Apoc2 was 0.086. Genes that are known to be associated with TG metabolism in human or mice are highlighted in bold face.

Figure 9. CREB-H is required for the expression of apoC-II and apoA-IV mRNAs in small intestine but dispensable for apoC-III. (A) Total RNAs were prepared from the duodenum of WT and CREB-H^"7" mice. Expression of apo genes was determined by real-time RT-PCR analysis. N=8 mice per group. (B) ApoC-III mRNA expression in liver and small intestine. N=8 mice per group. *** p<0.0001, N.S., not significant

(p>0.05).

Figure 10. Identification of CREB-H target genes by transgenic overexpression. (A) Transgenic vector contains amino acids 1-318 of CREB-H (CREB-H(N)), encompassing the N-terminal portion of the protein extending to the predicted S2P protease cleavage site. (B-C) Expression of the endogenous and transgenic CREB-H protein and mRNA were revealed by western and northern blots, respectively. Two WT and transgenic mice were examined in these representative experiments. (D) Microarray analyses were performed on total RNAs isolated from WT and CREB-H(N) transgenic mouse liver using the GeneChip® Mouse Genome 43 OA 2.0 Array (Affymetrix). Genes that were induced by more than three fold in two separate experiments were selected and sorted according to the signal intensities in transgenic mouse liver. Genes involved in TG metabolism are highlighted in bold face. (E) Total RNAs were isolated from WT and CREB-H(N) transgenic liver, and mRNA levels of apolipoprotein genes in

Apoe/cl/c4/c2 and Apoal/c3/a4/a5 clusters Fgf21, and Cidec were measured by quantitative real time PCR. (N=4 mice per group). (F) Luciferase reporter constructs containing 0.43 kb Apoa4, 0.5 kb Apoc2 or 1.5 kb Fgf21 promoters were transfected into Hepal .6 cells together with control or CREB-H(N) expression plasmid for luciferase assays.

Figure 11. Identification of CREB-H binding sites in the Apoa4 promoter. (A)

Luciferase reporter constructs containing various fragments of the Apoa4 promoter with site specific mutations were generated and tested for their inducibility by CREB-H(N) cotransfection in Helal .6 and MODE-K cells. (B) Apoa4 promoter contains two E-box like elements in the region responsible for CREB-H transactivation, which are conserved in human and mouse. (C) Double strand oligonucleotides containing the distal CREB-H binding site were used for EMSA with in vitro translated CREB-H(N) protein. Control or CREB-H specific antibodies were added in reactions for lanes 3 and 4. Increasing amount (5x, 25x, 125x) of WT or MUT probe with disrupted E-box motif were used as cold competitors in reactions for lanes 5 and 10. Figure 12. CREB3L3 sequencing electropherograms. The panel shows the heterozygous CREB3L3 mutations found in HTG patients. The trivial name for each mutation is indicated at the top of each panel. The electropherogram sections show the mutant sequence in each patient; the lines of text show normal codons, and the amino acid change predicted by the mutant codon. Mutation sites are indicated by arrows.

Detailed Description of the Invention

The present invention is based, at least in part, on the finding that CREB-

H modulates lipid metabolism. Mice bearing a null mutation in CREB-H exhibit a hypertriglyceridemia secondary to inefficient triglceride clearance. Mutations in CREB3L3 have also been identified which are predictive of the propensity to develop abnormal triglyceride metabolism.

Various aspects of the invention are described in further detail in the following subsections:

I. Definitions

Cyclic AMP responsive element binding protein 3 -like protein 3 (CREB- H, also known as CREBH; HYST1481; MGC126553; MGC126557; and CREB3L3) is an endoplasmic reticulum (ER)-bound transcription factor of the CREB/ATF

transcription factor family that is highly and selectively expressed only in the liver and the small intestine (1, 2). CREB-H activation requires a sequential cleavage of its precursor protein by Golgi proteases that liberate the mature N-terminal portion of the protein, which localizes to the nucleus to act as a transcriptional transactivator (3). It has been recently demonstrated that CREB-H mRNA is induced by fatty acids, the fatty acid oxidation regulator PPARa, and fasting in the liver, suggesting that it might participate in nutrient and energy metabolism (4, 5)).

The nucleotide sequence of CREB3L3 gene which specifies CREB-H is known in the art and can be found, e.g., in Omori et al. Nucleic Acids Res. 29 (10), 2154-2162 (2001) as well as under reference number GI: 14211948 at the NCBI web site. The protein sequence can be found under reference number GI: 14211949 at the NCBI web site. CREB-H contains a b-Zip domain at amino acids 323-339 (inclusive, as numbered by Omori et al.) which comprises the sequence

LPLTKYEERVLKKIRRKIRNKQSAQESRK KKEYIDGLETRMSACTAQNQELQR KVLHLEKQNLSLLEQLKRLQATVVQSTSKSAQTGTCVAVLLLSFALT.

As used herein the term "fibroblast growth factor 21 (Fgf21)" refers to the protein encoded by the nucleic acid molecule having the sequence found at GI reference number GL224589810 or GL224514627. The protein sequence information for Fgf21 can be found at GL9506597.

As used herein the term "apolipoprotein C II (ApoC2)" refers to the protein encoded by the nucleic acid molecule having the sequence found at GI reference number GL224589810 or GL224514627. The protein sequence information for ApoC2 can be found at GI :32130518.

As used herein the term "apolipoprotein A IV (ApoA4)" refers to the protein encoded by the nucleic acid molecule having the sequence found at GI reference number GL224589802 or GL224514928. The protein sequence information for ApoA4 can be found at GL32130518.

As used herein the term "apolipoprotein A V (ApoA5)" refers to the protein encoded by the nucleic acid molecule having the sequence found at GI reference number GL224589802 or GL224514928. The protein sequence information for ApoA5 can be found at GL262231737 or GL63079709, which represent alternative transcripts.

As used herein the term "regulatory region of a gene transcriptionally regulated by CREB-H" or "CREB-H-responsive element" refers to a DNA sequence that is directly or indirectly regulated by the activity of the CREB-H (whereby activity of CREB-H can be monitored, for example, via transcription of a reporter gene). As used herein, the term "triglyceride levels" refers to levels of triglycerides in the serum of a subject. Triglyceride levels are influenced, e.g., by clearance from the plasma as well as by production of triglycerides from the liver.

As used herein the term CREB-H loss of function mutation refers to mutations in the CREB3L3 gene which result in a CREB-H protein that has reduced function or no function as compared to wild-type CREB-H.

"Very low density lipoproteins" are approximately 25-90 nm in size (MW 6-27 million), with a density of -0.98. They contain 5-12% protein, 50-55% triglycerides, 18-20%) phospholipids, 12-15%) cholesteryl esters and 8-10% cholesterol. VLDL also contains several types of apolipoproteins including apo-BlOO, apo-CI, II & III and apo-E. VLDL also obtains apo-CII and apo-E from plasma HDL. VLDL assembly in the liver involves the early association of lipids with apo-BlOO mediated by microsomal triglyceride transfer protein while apo-BlOO is translocated to the lumen of the ER. Lipoprotein lipase also removes triglycerides from VLDL in the same way as from chylomicrons.

"Intermediate density lipoproteins" are smaller than VLDL (40 nm) and more dense (~1.0). They contain the same apolipoproteins as VLDL. They are composed of 10-12%o protein, 24-30%> triglycerides, 25-27%> phospholipids, 32-35%> cholesteryl esters and 8-10% cholesterol. IDLs are derived from triglyceride depletion of VLDL. IDLs can be taken up by the liver for reprocessing, or upon further triglyceride depletion, become LDL.

"Low density lipoproteins" are smaller than IDL (26 nm) (MW approximately 3.5 million) and more dense (-1.04). They contain the apolipoprotein apo-BlOO. LDL contains 20-22%o protein, 10-15%> triglycerides, 20-28%> phospholipids, 37-48%) cholesteryl esters and 8-10% cholesterol.

LDL and HDL transport both dietary and endogenous cholesterol in the plasma. LDL is the main transporter of cholesterol and cholesteryl esters and makes up more than half of the total lipoprotein in plasma. LDL is absorbed by the liver and other tissues via receptor mediated endocytosis. The cytoplasmic domain of the LDL receptor facilitates the formation of coated pits; receptor-rich regions of the membrane. The ligand binding domain of the receptor recognizes apo-BlOO on LDL, resulting in the formation of a clathrin-coated vesicle. ATP-dependent proton pumps lower the pH inside the vesicle resulting dissociation of LDL from its receptor. After loss of the clathrin coat the vesicles fuse with lysozomes, resulting in peptide and cholesteryl ester enzymatic hydrolysis. The LDL receptor can be recycled to the cell membrane. Insulin, tri-iodothyronine and dexamethasome have shown to be involved with the regulation of LDL receptor mediated uptake.

"Lipoprotein(a)" is similar in structure to LDL. However, it contains a additional protein, apolipoprotein(a) (apo-(a)), covalently bound to apo-B. Apo-(a) has been found to have a high sequence homology with plasminogen. It contains variable amounts of repeating kringle regions and more than 40 isoforms with a MW range of 400-700 kD. Its function is thought to be related to triglyceride metabolism and possibly thrombotic and atherogenic pathways.

"High density lipoproteins" are the smallest of the lipoproteins (6-12.5 nm) (MW 175-500KD) and most dense (-1.12). HDL contains several types of apolipoproteins including apo-ΑΙ,ΙΙ & IV, apo-CI, II & III, apo-D and apo-E. HDL contains approximately 55% protein, 3-15% triglycerides, 26-46%) phospholipids, IS30%o cholesteryl esters and 2-10% cholesterol.

HDL is produced as a protein rich particle in the liver and intestine, and serves as a circulating source of Apo-CI & II and Apo-E proteins. The HDL protein particle accumulates cholesteryl esters by the esterification of cholesterol by lecithin- cholesterol acyl-transferase (LCAT). LCAT is activated by apo-AI on HDL. HDL can acquire cholesterol from cell membranes and can transfer cholesteryl esters to VLDL and LDL via transferase activity in apo-D. HDL can return to the liver where cholesterol is removed by reverse cholesterol transport, thus serving as a scavenger to free cholesterol. The liver can then excrete excess cholesterol in the form of bile acids.

As used herein the term "dyslipidemia" refers to a disruption in the amount of lipids, e.g., cholesterols {e.g., total cholesterol, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density liporprotein (LDL), high density liporprotein (HDL)), triglycerides, in the blood. In one embodiment, dyslipidemia is hyperlipidemia, i.e., elevated levels of lipids (cholesterol and/or triglycerides) in the blood, e.g., total cholesterol (TC) >200 mg/dL (>5.17 mmol/L), LDL >100 mg/dL (>3.36mmol/L), HDL >60 mg/dL (>1.55 mmol/L), VLDL >50mg/dL, triglycerides (TG) >150 mg/dL (>1.695 mmol/L). In another embodiment, dyslipidemia is hypolipidemia, i.e., decreased levels of lipids in the blood, e.g., total cholesterol (TC) < 120 mg/dL (< 3.1 mmol/L) or LDL < 50 mg/dL (< 0.13 mmol/L), HDL <40 mg/dL (>1.03 mmol/L), VLDL >8mg/dL, triglycerides (TG) <80 mg/dL. A subject with dyslipidemia may be identified by, for example, measuring serum lipid levels, e.g., fasting serum lipid levels, using methods routine to one of skill in the art. In one embodiment, non-HDL cholesterol is measured, e.g., the amount of total choplesterol minus the amount of HDL cholesterol.

As used herein, "atherosclerosis" is a disease affecting arterial blood vessels. It is a chronic inflammatory response in the walls of arteries, in large part due to the accumulation of macrophage white blood cells and promoted by low density

(especially small particle) lipoproteins (plasma proteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high density lipoproteins (HDL). It is commonly referred to as a "hardening" or "furring" of the arteries (and is also referred to as coronary artery disease or CAD). It is caused by the formation of multiple plaques within the arteries.

A subject with atherosclerosis may be identified by, for example, angiography, stress-testing, coronary calcium scoring by CT, carotid IMT (intimal media thickness) measurement by ultrasound, Intravascular ultrasound (IVUS), lipoprotein subclass analysis, Glycosylated (or glycated) hemoglobin (HbAlc), C-reactive protein (CRP), homocysteine, anatomic (abdominal girth) and physiologic (blood pressure, elevated blood glucose) methods.

As used herein, "obesity" is a condition in which excess body fat has accumulated to such an extent that health may be negatively affected. It is commonly defined as a body mass index (BMI) of 30 kg/m2 or higher which distinguishes it from being overweight as defined by a BMI of 25 kg/m2 or higher (see, e.g., World Health Organization (2000) (PDF). Technical report series 894: Obesity: Preventing and managing the global epidemic. Geneva: World Health Organization). Excessive body weight is associated with various diseases, particularly cardiovascular diseases, diabetes mellitus type 2, obstructive sleep apnea, certain types of cancer, and osteoarthritis..

A subject with obesity may be identified by, for example, by determining BMI (BMI is calculated by dividing the subject's mass by the square of his or her height), waist circumference and waist-hip ratio (the absolute waist circumference

(>102 cm in men and >88 cm in women) and the waist-hip ratio (the circumference of the waist divided by that of the hips of >0.9 for men and >0.85 for women) (see, e.g., Yusuf S, et al, (2004). Lancet 364: 937-52), and/or body fat percentage (total body fat expressed as a percentage of total body weight men with more than 25% body fat and women with more than 33% body fat are obese; body fat percentage can be estimated from a person's BMI by the following formula: Bodyfat% = (1.2 * BMI) + (0.23 * age) - 5.4 - (10.8 * gender), where gender is 0 if female and 1 if male). Body fat percentage measurement techniques include , for example, computed tomography (CT scan), magnetic resonance imaging (MRI), and dual energy X-ray absorptiometry (DEXA).

As used herein, the various forms of the term "modulate" include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).

As used herein, the terms "a modulator of CREB-H" includes modulators of CREB-H expression, processing, post-translational modification, stability, and/or activity. The term includes agents, for example a compound or compounds which modulates transcription of a CREB-H gene, translation of CREB-H mRNA, post- translational modification of a CREB-H protein (e.g., proteolysis or glycosylation), or activity of a CREB-H protein. In one embodiment, a modulator modulates one or more of the above. In preferred embodiments, the activity of CREB-H is modulated.

A "modulator of CREB-H activity" includes compounds that directly or indirectly modulate CREB-H activity. For example, an indirect modulator of CREB-H activity can modulate a non- CREB-H molecule which is in a signal transduction pathway that includes CREB-H. Examples of modulators that directly modulate CREB- H expression, processing, post-translational modification, and/or activity include nucleic acid molecules encoding a biologically active portion of CREB-H, expression vectors encoding CREB-H that allow for increased expression of CREB-H activity in a cell, active forms of CREB-H protein, as well as chemical compounds that act to specifically modulate the activity or expression of CREB-H.

As used interchangeably herein, the terms "CREB-H activity," "biological activity of CREB-H" or "functional activity CREB-H," include activities exerted by CREB-H protein on a CREB-H responsive cell or tissue, e.g., a hepatocyte or cell of the small intestine, or on a CREB-H nucleic acid molecule or protein target molecule, as determined in vivo, or in vitro, according to standard techniques. CREB-H activity can be a direct activity, such as an association with a CREB-H -target molecule e.g., binding of CREB-H to a regulatory region of a gene responsive to CREB-H (for example, a gene such as Fgf21, Apoc2, Apoa4, or Apoa5). Exemplary biological activities of CREB-H are described herein and include: e.g., reduction of plasma triglycerides.

As used herein, a "substrate" or "target molecule" or "binding partner" is a molecule with which a protein binds or interacts in nature, such that the protein's function {e.g., modulation of triglyceride clearance) is achieved. For example, a target molecule can be a protein or a nucleic acid molecule.

As used herein, the term "contacting" {e.g., contacting a cell, with a compound) includes incubating the compound and the cell together in vitro {e.g., adding the compound to cells in culture) as well as administering the compound to a subject such that the compound and cells of the subject are contacted in vivo. The term "contacting" does not include exposure of cells to a CREB-H modulator that may occur naturally in a subject {i.e., exposure that may occur as a result of a natural physiological process).

As used herein, the term "test compound" refers to a compound that has not previously been identified as, or recognized to be, a modulator of the activity being tested. The term "library of test compounds" refers to a panel comprising a multiplicity of test compounds.

As used herein, the term "indicator composition" refers to a composition that includes a protein of interest {e.g., CREB-H), for example, a cell that naturally expresses the protein, a cell that has been engineered to express the protein by introducing an expression vector encoding the protein into the cell, or a cell free composition that contains the protein {e.g., purified naturally-occurring protein or recombinantly- engineered protein).

As used herein, the term "cell" includes prokaryotic and eukaryotic cells. In one embodiment, a cell of the invention is a bacterial cell. In another embodiment, a cell of the invention is a fungal cell, such as a yeast cell. In another embodiment, a cell of the invention is a vertebrate cell, e.g., an avian or mammalian cell. In a preferred embodiment, a cell of the invention is a murine or human cell.

As used herein, the term "engineered" (as in an engineered cell) refers to a cell into which a nucleic acid molecule e.g., encoding a CREB-H has been introduced.

As used herein, the term "cell free composition" refers to an isolated

composition, which does not contain intact cells. Examples of cell free compositions include cell extracts and compositions containing isolated proteins. As used herein, the term "reporter gene" refers to any gene that expresses a detectable gene product, e.g., RNA or protein. As used herein the term "reporter protein" refers to a protein encoded by a reporter gene. Preferred reporter genes are those that are readily detectable. The reporter gene can also be included in a construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties. Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1 : 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol. 216:362- 368) and green fluorescent protein (U.S. patent 5,491,084; WO 96/23898).

In one embodiment, small molecules can be used as test compounds. The term "small molecule" is a term of the art and includes molecules that are less than about 7500, less than about 5000, less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules {e.g., Cane et al. 1998. Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic. For example, a small molecule is preferably not itself the product of transcription or translation. In one embodiment, small molecule compounds are present on a microarray, see, e.g., Bradner JE, et al. 2006. Chem Biol. 13(5):493-504.

Various aspects of the present invention are described in further detail in the following subsections.

II. Methods of Treatment and/or Prevention

As is set forth in the instant examples, CREB-H is important in triglyceride clearance. Accordingly, the invention provides for the prevention and/or treatment, and/or amelioration of at least one symptom, and/or normalization of at least one indicator of dyslipidemia, (that can lead to, e.g., atherosclerosis, obesity, type II diabetes, or other metabolic disorders) by increasing CREB-H expression or activity in a subject e.g., by directly or indirectly increasing CREB-H protein levels in cells, e.g., either in vitro or in vivo.

Accordingly, the invention features methods for treating and/or preventing a dyslipidemia by administering to a subject that would benefit from decreased plasma triglyceride levels by contacting a cell from such a subject with a modulator of CREB-H expression, processing, post-translational modification, and/or activity. The claimed methods are not meant to include naturally occurring events. For example, the step of contacting includes administering the modulator in a treatment protocol and, in one embodiment the term "agent" or "modulator" is not meant to embrace endogenous mediators produced by the cells of a subject.

The subject methods employ agents that directly modulate CREB-H expression, processing, post-translational modification, or activity (or the expression, processing, post-translational modification, or that indirectly modulate CREB-H by modulating activity of another molecule in a CREB-H signaling pathway such that a biological activity of CREB-H, e.g., triglyceride clearance is modulated.

In one embodiment, the methods and compositions of the invention can be used to modulate CREB-H expression, processing, post-translational modification, and/or activity in a cell. In one embodiment, the cell is a mammalian cell. In another embodiment, the cell is a human cell. In one embodiment, the cell is a hepatocyte. In one embodiment, the hepatocyte is an adult hepatocyte, i.e., a cell from a postnatal subject. Such modulation can occur in vitro or in vivo.

In one embodiment, cells in which, e.g., CREB-H, is modulated in vitro can be introduced, e.g., into an allogeneic subject, or e.g., reintroduced into a subject. In one embodiment, the invention also allows for modulation of CREB-H in vivo, by administering to the subject an amount of a modulator of CREB-H such that at least one symptom or indicator of triglyceride clearance in a subject is modulated.

In one embodiment, a modulatory agent of the invention directly affects the expression, post-translational modification, and/or activity of CREB-H. In another embodiment, the expression of CREB-H is modulated. In another embodiment, the post-translational modification of CREB-H is modulated. In another embodiment, the activity of CREB-H is modulated, e.g., triglyceride metabolism.

In one embodiment, the agent is an active form of CREB-H protein. In another embodiment, the agent is a nucleic acid molecule encoding an active form of the CREB- H protein. In yet another embodiment, the agent is a compound identified using one of the screening methods described herein.

In another embodiment, a modulatory agent of the invention indirectly affects the expression, post-translational modification, and/or activity of CREB-H.

The term "subject" is intended to include living organisms but preferred subjects are mammals. Examples of subjects include mammals such as, e.g., humans, monkeys, dogs, cats, mice, rats cows, horses, goats, and sheep.

Modulation of CREB-H activity in a subject provides a means to regulate disorders arising from aberrant CREB-H activity in various disease states. Examples of disorders in which such inhibitory methods can be useful include, dyslipidemia (i.e., increased levels of plasma triglycerides) which can lead to e.g., atherosclerosis, obesity, type 2 diabetes, and other metabolic disorders.

Application of the modulatory methods of the invention for the prevention, treatment, and/or amelioration of at least one symptom, or normalization of at least one indicator of a disorder can result in curing the disorder, a decrease in at least one symptom associated with the disorder, either in the long term or short term (i.e., amelioration of the condition) or simply a transient beneficial effect to the subject.

The methods of modulating CREB-H can be practiced either in vitro or in vivo.

For practicing the method in vitro, cells can be obtained from a subject by standard methods and incubated (i.e., cultured) in vitro with a stimulatory or inhibitory compound of the invention to stimulate or inhibit, respectively, the activity of CREB-H. Methods for isolating cells are known in the art.

Cells treated in vitro with either a stimulatory compound can be administered to a subject to influence the biological effects of CREB-H. For example, cells can be isolated from a subject, expanded in number in vitro and the activity of, e.g., CREB-H, activity in the cells using a stimulatory agent, and then the cells can be readministered to the same subject, or another subject tissue compatible with the donor of the cells.

Accordingly, in another embodiment, the modulatory method of the invention comprises culturing cells in vitro with e.g., a CREB-H modulator and further comprises

administering the cells to a subject. For administration of cells to a subject, it may be preferable to first remove residual compounds in the culture from the cells before administering them to the subject. This can be done for example by gradient

centrifugation of the cells or by washing of the tissue. For further discussion of ex vivo genetic modification of cells followed by readministration to a subject, see also U.S. Patent No. 5,399,346 by W.F. Anderson et al.

In other embodiments, a stimulatory or inhibitory compound is administered to a subject in vivo. Such methods can be used to treat disorders, e.g., as detailed above. In one embodiment, a stim or inhib compound is delivered directly to hepatocytes, e.g., adult hepatocytes, using methods known in the art.

For stimulatory or inhibitory agents that comprise nucleic acids {e.g. ,

recombinant expression vectors encoding, e.g., CREB-H), the compounds can be introduced into cells of a subject using methods known in the art for introducing nucleic acid {e.g., DNA) into cells. Examples of such methods include:

Direct Injection: Naked DNA can be introduced into cells in vivo by directly injecting the DNA into the cells (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). For example, a delivery apparatus {e.g., a "gene gun") for injecting DNA into cells in vivo can be used. Such an apparatus is commercially available {e.g., from BioRad).

Receptor-Mediated DNA Uptake: Naked DNA can also be introduced into cells in vivo by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C.H. (1988) J. Biol. Chem. 263: 14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Patent No. 5,166,320). Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).

Retroviruses: Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). A recombinant retrovirus can be constructed having a nucleotide sequences of interest incorporated into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.

Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include

ψ2 and ψΑιη. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014- 3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al.

(1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van

Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA

89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No.

4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell.

Adenoviruses: The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al.

(1992) Cell 68: 143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus {e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482- 6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj- Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral El and E3 genes but retain as much as 80 % of the adenoviral genetic material.

Adeno- Associated Viruses: Adeno-associated virus (AAV) is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and

McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51 :611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

The efficacy of a particular expression vector system and method of introducing nucleic acid into a cell can be assessed by standard approaches routinely used in the art. For example, DNA introduced into a cell can be detected by a filter hybridization technique (e.g. , Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR). The gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product, such as an enzymatic assay.

In one embodiment, a modulatory agent of the mention may be specifically targeted to hepatocytes or cells of the small intestine.

In one embodiment, the stimulatory or inhibitory compounds can be administered to a subject as a pharmaceutical composition. In one embodiment, the invention is directed to an active compound (e.g., a modulator of CREB-H) and a carrier. Such compositions typically comprise the stimulatory or inhibitory compounds, e.g., as described herein or as identified in a screening assay, e.g., as described herein, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and methods of administration to a subject are described herein.

In one embodiment, the active compounds of the invention are administered in combination with other agents. For example, in one embodiment, an active compound of the invention, e.g., a compound that modulates a CREB-H signal transduction pathway (e.g., by directly modulating CREB-H activity) is administered with another compound known in the art to be useful in treatment of a particular condition or disease. For example, in one embodiment, an active compound of the invention (e.g., a compound that directly modulates CREB-H activity) can be administered or in combination with an HMG-CoA reductase inhibitor. HMG-CoA reductase inhibitors, also referred to as statins, are a class of hypolipidemic drugs used to lower cholesterol levels in subjects with or at risk of cardiovascular disease. They lower cholesterol by inhibiting the enzyme HMG-CoA reductase, which is the rate-limiting enzyme of the mevalonate pathway of cholesterol synthesis. Inhibition of this enzyme in the liver stimulates LDL receptors, resulting in an increased clearance of low-density lipoprotein (LDL) from the bloodstream and a decrease in blood cholesterol levels.

Statins are divided into two groups: fermentation-derived and synthetic which uinclude, for example Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, Simvastatin+Ezetimibe,

Lovastatin+Niacin, Atorvastatin+Amlodipine, Simvastatin+Niacin. Compositions that can be used in the methods of the invention is described in further detail below.

III. Pharmaceutical Compositions

CREB-H stimulatory compounds can be used in the prevention and/or treatment of disorders in which CREB-H activity and/or expression is undesirably reduced, inhibited, downregulated, or the like. In one embodiment, the stimulatory methods of the invention, a subject is treated with a stimulatory compound that stimulates expression and/or activity of CREB-H.

Examples of stimulatory compounds include CREB-H polypeptides, proteins, or biologically active fragments thereof, nucleic acid molecules encoding CREB-H proteins or biologically active fragments thereof, and chemical agents that stimulate expression and/or activity of CREB-H.

In one embodiment, stimulatory compound is a nucleic acid molecule encoding CREB-H wherein the nucleic acid molecule is introduced into the subject in a form suitable for expression of the protein in the cells of the subject. For example, a CREB-H cDNA (full length or partial cDNA sequence) is cloned into a recombinant expression vector and the vector is transfected into cells using standard molecular biology techniques. The CREB-H cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library. The nucleotide sequences of CREB-H cDNA are known in the art and can be used for the design of PCR primers that allow for amplification of a cDNA by standard PCR methods or for the design of a hybridization probe that can be used to screen a cDNA library using standard hybridization methods.

Following isolation or amplification of CREB-H cDNA or cDNA encoding a molecule in a signal transduction pathway involving CREB-H, the DNA fragment is introduced into a suitable expression vector, as described above. For example, nucleic acid molecules encoding CREB-H in the form suitable for expression of the CREB-H in a host cell, can be prepared as described above using nucleotide sequences known in the art. The nucleotide sequences can be used for the design of PCR primers that allow for amplification of a cDNA by standard PCR methods or for the design of a hybridization probe that can be used to screen a cDNA library using standard hybridization methods. In one embodiment, a stimulatory agent can be present in an inducible construct. In another embodiment, a stimulatory agent can be present in a construct which leads to constitutive expression.

In one embodiment, a stimulatory agent can be directly targeted to hepatocytes. Another form of a stimulatory compound for stimulating expression of CREB-H or a molecule in a signal transduction pathway involving CREB-H in a cell is a chemical compound that specifically stimulates the expression, processing, post-translational modification, or activity of endogenous CREB-H. Such compounds can be identified using screening assays that select for compounds that stimulate the expression or activity of CREB-H as described herein.

The peptidic compounds of the invention can be made intracellularly in cells by introducing into the cells an expression vector encoding the peptide. Such expression vectors can be made by standard techniques using oligonucleotides that encode the amino acid sequence of the peptidic compound. The peptide can be expressed in intracellularly as a fusion with another protein or peptide (e.g., a GST fusion).

Alternative to recombinant synthesis of the peptides in the cells, the peptides can be made by chemical synthesis using standard peptide synthesis techniques. Synthesized peptides can then be introduced into cells by a variety of means known in the art for introducing peptides into cells (e.g., liposome and the like).

A pharmaceutical composition comprising a compound of the invention, e.g., a stimulatory molecule of the invention or a compound identified in the subject screening assays, is formulated to be compatible with its intended route of administration. For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and compounds for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the

extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^M (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition will preferably be sterile and should be fluid to the extent that easy syringability exists. It will preferably be stable under the conditions of

manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, for example, parabens,

chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic compounds, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an compound which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding compounds, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as

micro crystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring.

In one embodiment, compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from, e.g., Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

In one embodiment, a modulatory agent of the invention is administered in amount sufficient to bring triglyceride levels to within the normal range. Indicators of triglyceride levels may be measured according to methods routine to one of ordinary skill in the art.

IV. Screening Assays

In one embodiment, the invention provides methods (also referred to herein as "screening assays") for identifying agents for preventing and/or treating {e.g., modulating at least one symptom of) dyslipidemia e.g., that can lead to atherosclerosis, hepatic steatosis, steatohepatitis, hpercholesteremia, obesity, and/or type II diabetes, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules, nucleic acid molecules) which modulate CREB-H expression and/or activity. The subject assays involve testing the effect of a candidate agent on CREB-H expression or activity using methods known in ther art or described herein. In one embodiment, the subject assays further comprise a step in which the effect of the agent an activity of

CREB-H is measured. For example, the ability of the agent to have an effect on CREB- H expression and/or activity is measured (e.g., in vitro or in silico), and then the ability of the compound to have an effect on triglyceride levels in vivo is measured.

In another embodiment, the ability of a compound to directly modulate the expression, post-translational modification (e.g., glycosylation or proteolysis)of CREB- H is measured in a screening assay of the invention.

The indicator composition can be a cell that expresses the CREB-H protein, for example, a cell that naturally expresses or, more preferably, a cell that has been engineered to express the protein by introducing into the cell an expression vector encoding the protein. Preferably, the cell is a mammalian cell, e.g., a human cell. In another embodiment, the cell is a hepatocyte. In one embodiment, the hepatocyte is an adult hepatocyte, i.e., a cell from a postnatal subject. Alternatively, the indicator composition can be a cell-free composition that includes the protein (e.g., a cell extract or a composition that includes e.g., either purified natural or recombinant protein).

Compounds identified as upmodulating the expression, activity, and/or stability of CREB-H and/or using, e.g., the assays described herein are useful for preventing and/or treating dyslipidemia, e.g., hyperlipidemia.

For example, in one embodiment, a modulating agent identified using the cell- based or cell-free assays described herein may be assayed in a non-human animal model of obesity and/or insulin resistance, e.g., a genetic model of obesity and/or insulin resistance, e.g., ob, db animals, and/or dietary models of obesity and/or insulin resistance, e.g., a high carbohydrate diet. Such methods generally comprise

administering the test compound to the non-human animal and determining the effect of the agent on for example, body weight, serum triglycerides, total cholesterol, blood glucose, glucose tolerance, insulin tolerance, and glucose-stimulated insulin secretion in the presence and absence of the test compound.

In another embodiment, a modulating agent identified using the cell- based or cell-free assays described herein may be assayed in a non-human animal model of hypercholesterolemia and/or atherosclerosis, e.g. a genetic model of

hypercholesterolemia and/or atherosclerosis, e.g., ApoE, ApoB, LDLR, and/or dietary models of hypercholesterolemia and/or atherosclerosis, e.g., a high fat diet. Such methods generally comprise administering the test compound to the non-human animal and determining the effect of the compound on, for example, serum triglycerides, total cholesterol, distribution of cholesterol among HDL, IDL, VLDL, and LDL, and presence of atherosclerotic lesions as assessed by standard hostologic analysis, as described in, for example, Palinski W, et al. Arterioscler Thromb.. 1994;14:605; Nunnari JJ, et al. Exp Mol Pathol. 1989;51 : 1.

Liver function tests can be performed on serum samples using an automated analyzer and can include, for example, measurement of serum lactate dehydrogenase (LDH), serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvate transaminase (SGPT), and serum bilirubin..

In another embodiment, it will be understood that similar screening assays can be used to identify compounds that indirectly modulate the activity and/or expression of CREB-H, e.g., by performing screening assays such as those described above using molecules with which CREB-H interacts, e.g., molecules that act either upstream or downstream of CREB-H in a signal transduction pathway.

The cell based and cell free assays of the invention are described in more detail below.

A. Cell Based Assays

The indicator compositions of the invention can be a cell that expresses a CREB- H protein, for example, a cell that naturally expresses endogenous CREB-H or, more preferably, a cell that has been engineered to express an exogenous CREB-H protein by introducing into the cell an expression vector encoding the protein. Alternatively, the indicator composition can be a cell-free composition that includes CREB-H or a composition that includes purified CREB-H.

Compounds that modulate expression and/or activity of CREB-H can be identified using various "read-outs." In one embodiment, during the detecting step, one or more components is transformed {e.g., by labeling).

For example, an indicator cell can be transfected with a CREB-H expression vector, incubated in the presence and in the absence of a test compound, and the effect of the compound on the expression of the molecule or on a biological response regulated by CREB-H can be determined. In one embodiment, CREB-H can be expressed in a cell. The biological activities of CREB-H include activities determined in vivo, or in vitro, according to standard techniques. A CREB-H activity can be a direct activity, such as an association with a CREB-H-target molecule {e.g., a nucleic acid molecule to which CREB-H binds such as the transcriptional regulatory region of a gene).

Alternatively, a CREB-H activity is an indirect activity, such as a cellular signaling activity or alteration in gene expression occurring downstream of the interaction of the CREB-H protein with a CREB-H target molecule or a biological effect occurring as a result of the signaling cascade triggered by that interaction. For example, biological activities of CREB-H described herein include: reduction in serum triglyceride levels.

To determine whether a test compound modulates CREB-H, assays to measuring serum triglyceride levels may be used. In one embodiment, LDL, HDL, IDL, and/or VLDL are measured. Additionally, modulation of CREB-H may be determined by assaying the direct expression and/or activity of a CREB-H polypeptide, e.g., by assaying the ability of a CREB-H polypeptide to bind to a binding partner, and/or the promoter of a gene directly regulated by CREB-H, e.g., Fgf21, Apoc2, Apoa4, and/or Apoa5, and/or to activate a reporter gene operably linked to a regulatory element responsive to the CREB-H polypeptide.

To determine whether a test compound modulates CREB-H protein expression, in vitro transcriptional assays can be performed. In one example of such an assay, the full length CREB-H gene or promoter and enhancer of CREB-H operably linked to a reporter gene such as chloramphenicol acetyltransferase (CAT) or luciferase and introduced into host cells. The expression or activity of CREB-H or the reporter gene can be measured using techniques known in the art. The ability of a test compound to regulate the expression or activity of a molecule in a signal transduction pathway involving CREB-H can be similarly tested.

As used interchangeably herein, the terms "operably linked" and "operatively linked" are intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence in a host cell (or by a cell extract).

In another embodiment, modulation of expression of a protein whose expression is regulated by CREB-H is measured. Regulatory sequences are art-recognized and can be selected to direct expression of the desired protein in an appropriate host cell. The term regulatory sequence is intended to include promoters, enhancers, polyadenylation signals and other expression control elements. Such regulatory sequences are known to those skilled in the art and are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transfected and/or the type and/or amount of protein desired to be expressed.

A variety of reporter genes are known in the art and are suitable for use in the screening assays of the invention. Examples of suitable reporter genes include those which encode chloramphenicol acetyltransferase, beta-galactosidase, alkaline

phosphatase or luciferase. Standard methods for measuring the activity of these gene products are known in the art.

A variety of cell types are suitable for use as an indicator cell in the screening assay. Preferably a cell line is used which expresses low levels of endogenous CREB-H, and is then engineered to express recombinant CREB-H. Cells for use in the subject assays include both eukaryotic and prokaryotic cells. For example, in one embodiment, a cell is a bacterial cell. In another embodiment, a cell is a fungal cell, such as a yeast cell. In another embodiment, a cell is a vertebrate cell, e.g., an avian cell or a

mammalian cell {e.g., a murine cell, or a human cell).

In one embodiment, the level of expression of the reporter gene in the indicator cell in the presence of the test compound is higher than the level of expression of the reporter gene in the indicator cell in the absence of the test compound and the test compound is identified as a compound that stimulates the expression of the molecule.

In another embodiment, the level of expression of genes whose expression is regulated by CREB-H {e.g., lipogenic gene, e.g., Fgf21, Apoc2, Apoa4, and/or Apoa5 can be measured using standard techniques. The sequences of such genes are known in the art.

In yet another embodiment, the ability of a compound to modulate translocation of CREB-H to the nucleus can be determined. Translocation of CREB-H to the nucleus can be measured, e.g., by nuclear translocation assays in which the emission of two or more fluorescently-labeled species is detected simultaneously. For example, the cell nucleus can be labeled with a known fluorophore specific for DNA, such as Hoechst 33342. The CREB-H protein can be labeled by a variety of methods, including expression as a fusion with GFP or contacting the sample with a fluorescently-labeled antibody specific CREB-H. The amount CREB-H that translocates to the nucleus can be determined by determining the amount of a first fluorescently-labeled species, i.e., the nucleus, that is distributed in a correlated or anti-correlated manner with respect to a second fluorescently-labeled species, i.e., CREB-H, as described in U.S. Patent No. 6,400,487, the contents of which are hereby incorporated by reference.

In another embodiment, a different (i.e., non-CREB-H) molecule acting in a pathway involving CREB-H can be included in an indicator composition for use in a screening assay.

The cells used in the instant assays can be eukaryotic or prokaryotic in origin.

For example, in one embodiment, the cell is a bacterial cell. In another embodiment, the cell is a fungal cell, e.g., a yeast cell. In another embodiment, the cell is a vertebrate cell, e.g., an avian or a mammalian cell. In a preferred embodiment, the cell is a human cell. In another preferred embodiment the cell is a hepatocyte, e.g., a primary

hepatocyte.

The cells of the invention can express endogenous CREB-H, or can be engineered to do so. For example, a cell that has been engineered to express the CREB- H protein and/or a non CREB-H protein can be produced by introducing into the cell an expression vector encoding the protein.

Recombinant expression vectors that can be used for expression of CREB-H, or a molecule in a signal transduction pathway involving CREB-H (e.g., a protein which acts upstream or downstream of CREB-H) or a molecule in a signal transduction pathway involving CREB-H in the indicator cell are known in the art. For example, the CREB-H cDNA is first introduced into a recombinant expression vector using standard molecular biology techniques. A cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library. The nucleotide sequences of cDNAs for CREB-H or a molecule in a signal transduction pathway involving CREB-H (e.g., human, murine and yeast) are known in the art and can be used for the design of PCR primers that allow for amplification of a cDNA by standard PCR methods or for the design of a hybridization probe that can be used to screen a cDNA library using standard hybridization methods.

Following isolation or amplification of a cDNA molecule encoding, for example, CREB-H, the DNA fragment is introduced into an expression vector. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid molecule in a form suitable for expression of the nucleic acid molecule in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression and the level of expression desired, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology:

Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell, those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) or those which direct expression of the nucleotide sequence only under certain conditions (e.g., inducible regulatory sequences).

When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus, cytomegalovirus and Simian Virus 40. Non-limiting examples of mammalian expression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 5:187-195). A variety of mammalian expression vectors carrying different regulatory sequences are commercially available. For constitutive expression of the nucleic acid in a mammalian host cell, a preferred regulatory element is the cytomegalovirus promoter/enhancer. Moreover, inducible regulatory systems for use in mammalian cells are known in the art, for example systems in which gene expression is regulated by heavy metal ions (see e.g., Mayo et al. (1982) Cell 29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985) Mol. Cell. Biol. 5:1480-1489), heat shock (see e.g., Nouer et al. (1991) in Heat Shock Response, e.d. Nouer, L. , CRC, Boca Raton , FL, pp 167-220), hormones (see e.g., Lee et al. (1981) Nature 294:228-232; Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038-2042; Klock et al. (1987) Nature 329:734-736; Israel & Kaufman (1989) Nucl. Acids Res. 17:2589-2604; and PCT Publication No. WO 93/23431), FK506-related molecules (see e.g., PCT Publication No. WO 94/18317) or tetracyclines (Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al.

(1995) Science 268: 1766-1769; PCT Publication No. WO 94/29442; and PCT

Publication No. WO 96/01313). Still further, many tissue-specific regulatory sequences are known in the art, including the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and

Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916) and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally- regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter

(Campes and Tilghman (1989) Genes Dev. 3:537-546).

Vector DNA can be introduced into mammalian cells via conventional trans fection techniques. As used herein, the various forms of the term "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid {e.g., DNA) into mammalian host cells, including calcium phosphate co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transfecting host cells can be found in Sambrook et al. {Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker {e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on a separate vector from that encoding CREB-H or, more preferably, on the same vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection {e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

In one embodiment, within the expression vector coding sequences are operatively linked to regulatory sequences that allow for constitutive expression of the molecule in the indicator cell {e.g., viral regulatory sequences, such as a

cytomegalovirus promoter/enhancer, can be used). Use of a recombinant expression vector that allows for constitutive expression of, for example, CREB-H in the indicator cell is preferred for identification of compounds that enhance or inhibit the activity of the molecule. In an alternative embodiment, within the expression vector the coding sequences are operatively linked to regulatory sequences of the endogenous gene for CREB-H {i.e., the promoter regulatory region derived from the endogenous gene). Use of a recombinant expression vector in which expression is controlled by the endogenous regulatory sequences is preferred for identification of compounds that enhance or inhibit the transcriptional expression of the molecule. C. Cell-free assays

In another embodiment, the indicator composition is a cell free composition. CREB-H protein expressed by recombinant methods in a host cells or culture medium can be isolated from the host cells, or cell culture medium using standard methods for protein purification. For example, ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies can be used to produce a purified or semi -purified protein that can be used in a cell free composition. Alternatively, a lysate or an extract of cells expressing the protein of interest can be prepared for use as cell-free composition.

In one embodiment, compounds that specifically modulate CREB-H activity are identified based on their ability to modulate the interaction of CREB-H with a target molecule to which CREB-H binds. The target molecule can be a DNA molecule, e.g., a CREB-H-responsive element, such as the regulatory region of a chaperone gene, lipogenic gene) or a protein molecule. Suitable assays are known in the art that allow for the detection of protein-protein interactions {e.g., immunoprecipitations, two-hybrid assays and the like) or that allow for the detection of interactions between a DNA binding protein with a target DNA sequence {e.g., electrophoretic mobility shift assays, DNAse I footprinting assays, chromatin immunoprecipitations assays and the like). By performing such assays in the presence and absence of test compounds, these assays can be used to identify compounds that modulate {e.g., inhibit or enhance) the interaction of CREB-H with a target molecule.

In one embodiment, the amount of binding of CREB-H to the target molecule in the presence of the test compound is greater than the amount of binding of CREB-H to the target molecule in the absence of the test compound, in which case the test compound is identified as a compound that enhances binding of CREB-H to a target.

Binding of the test compound to CREB-H can be determined either directly or indirectly as described above. Determining the ability of CREB-H protein to bind to a test compound can also be accomplished using a technology such as real-time

Biomolecular Interaction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants {e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In the methods of the invention for identifying test compounds that modulate an interaction between CREB-H protein and a target molecule, the complete CREB-H protein can be used in the method, or, alternatively, only portions of the protein can be used. For example, an isolated CREB-H b-ZIP structure (or a larger subregion of CREB-H that includes the b-ZIP structure) can be used. The degree of interaction between the protein and the target molecule can be determined, for example, by labeling one of the proteins with a detectable substance (e.g., a radiolabel), isolating the non- labeled protein and quantitating the amount of detectable substance that has become associated with the non-labeled protein. The assay can be used to identify test compounds that either stimulate or inhibit the interaction between the CREB-H protein and a target molecule. A test compound that stimulates the interaction between the protein and a target molecule is identified based upon its ability to increase the degree of interaction between, e.g., CREB-H and a target molecule as compared to the degree of interaction in the absence of the test compound and such a compound would be expected to increase the activity of CREB-H in the cell.

In one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either CREB-H or a respective target molecule for example, to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, or to accommodate automation of the assay. Binding of a test compound to, for example, a CREB-H protein, or interaction of a CREB-H protein with a target molecule in the presence and absence of a test compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided in which a domain that allows one or both of the proteins to be bound to a matrix is added to one or more of the molecules. For example, glutathione- S -transferase fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or CREB-H protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix is immobilized in the case of beads, and complex formation is determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a CREB-H protein or a molecule in a signal transduction pathway involving CREB-H, or a target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

Alternatively, antibodies which are reactive with protein or target molecules but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and unbound target or CREB-H protein is trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with CREB-H or a molecule in a signal

transduction pathway involving CREB-H or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the CREB-H, protein or target molecule.

In yet another aspect of the invention, the CREB-H protein or fragments thereof can be used as "bait proteins" e.g., in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with CREB-H ("binding proteins" or " bp") and are involved in CREB-H activity. Such CREB-H-binding proteins are also likely to be involved in the propagation of signals by the CREB-H proteins or CREB-H targets such as, for example, downstream elements of a CREB-H-mediated signaling pathway.

Alternatively, such CREB-H-binding proteins can be CREB-H inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a CREB-H protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a CREB-H dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the CREB-H protein or a molecule in a signal transduction pathway involving CREB-H.

D. Test Compounds

A variety of test compounds can be evaluated using the screening assays described herein. The term "test compound" includes any reagent or test agent which is employed in the assays of the invention and assayed for its ability to influence the expression and/or activity of CREB-H More than one compound, e.g., a plurality of compounds, can be tested at the same time for their ability to modulate the expression and/or activity of, e.g., CREB-H, in a screening assay. The term "screening assay" preferably refers to assays which test the ability of a plurality of compounds to influence the readout of choice rather than to tests which test the ability of one compound to influence a readout. Preferably, the subject assays identify compounds not previously known to have the effect that is being screened for. In one embodiment, high throughput screening can be used to assay for the activity of a compound.

In certain embodiments, the compounds to be tested can be derived from libraries (i.e., are members of a library of compounds). While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin et al.

(1992). J. Am. Chem. Soc. 114: 10987; DeWitt et al. (1993). Proc. Natl. Acad. Sci. USA 90:6909) peptoids (Zuckermann. (1994). J. Med. Chem. 37:2678) oligocarbamates (Cho et al. (1993). Science. 261 : 1303), and hydantoins (DeWitt et al. supra). An approach for the synthesis of molecular libraries of small organic molecules with a diversity of 104-105 as been described (Carell et al. (1994). Angew. Chem. Int. Ed. Engl. 33:2059- ; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061).

The compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the One-bead one-compound' library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12: 145). Other exemplary methods for the synthesis of molecular libraries can be found in the art, for example in: Erb et al. (1994). Proc. Natl. Acad. Sci. USA 91 : 11422; Horwell et al. (1996)

Immunopharmacology 33:68- ; and in Gallop et al. (1994); J. Med. Chem. 37: 1233-.

Libraries of compounds can be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP ^*409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310). In still another embodiment, the combinatorial polypeptides are produced from a cDNA library.

Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries.

Candidate/test compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K.S. et al. (1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature 354:84-86) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides {e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang, Z. et al. (1993) Cell 72:767-778); 3) antibodies {e.g., polyclonal, monoclonal, humanized, anti- idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')2, Fab expression library fragments, and epitope-binding fragments of antibodies); 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries); 5) enzymes (e.g., endoribonucleases, hydrolases, nucleases, proteases, synthatases, isomerases, polymerases, kinases, phosphatases, oxido-reductases and ATPases), and 6) mutant forms of CREB-H molecules.

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One -bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12: 145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261 : 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds can be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP ^•409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

Compounds identified in the subject screening assays can be used in methods of modulating one or more of the biological responses regulated by CREB-H. It will be understood that it may be desirable to formulate such compound(s) as pharmaceutical compositions (described supra) prior to contacting them with cells.

Once a test compound is identified that directly or indirectly modulates, e.g.,

CREB-H expression or activity, by one of the variety of methods described

hereinbefore, the selected test compound (or "compound of interest") can then be further evaluated for its effect on cells, for example by contacting the compound of interest with cells either in vivo {e.g., by administering the compound of interest to a subject) or ex vivo {e.g., by isolating cells from the subject and contacting the isolated cells with the compound of interest or, alternatively, by contacting the compound of interest with a cell line) and determining the effect of the compound of interest on the cells, as compared to an appropriate control (such as untreated cells or cells treated with a control compound, or carrier, that does not modulate the biological response).

E. Computer Assisted Design of Modulators of CREB-H

Computer-based analysis of a protein with a known structure can also be used to identify molecules which will bind to the protein. Such methods rank molecules based on their shape complementary to a receptor site. For example, using a 3-D database, a program such as DOCK can be used to identify molecules which will bind to CREB-H or a molecule in a signal transduction pathway involving CREB-H. See DesJarlias et al. (1988) J. Med. Chem. 31 :722; Meng et al. (1992) J. Computer Chem. 13:505; Meng et al. (1993) Proteins 17:266; Shoichet et al. (1993) Science 259: 1445. In addition, the electronic complementarity of a molecule to a targeted protein can also be analyzed to identify molecules which bind to the target. This can be determined using, for example, a molecular mechanics force field as described in Meng et al. (1992) J. Computer Chem. 13:505 and Meng et al. (1993) Proteins 17:266. Other programs which can be used include CLIX which uses a GRID force field in docking of putative ligands. See

Lawrence et al. (1992) Proteins 12:31; Goodford et al. (1985) J. Med. Chem. 28:849; Boobbyer et al. (1989) J. Med. Chem. 32: 1083.

The instant invention also pertains to compounds identified in the subject screening assays. V. DIAGNOSTICS

As is set forth in the Examples, mutations to CREB3L3 that result in CREB-H loss of function result in increased serum triglyceride levels. Accordingly, in one embodiment, the invention pertains to methods for identifying subjects at risk for developing increased serum triglyceride levels (e.g., brought about by decreased clearance). In one embodiment, mutations in the coding region of the CREB3L3 gene are detected. In another embodiment, mutations N-terminal to the b-Zip domain are detected. In still another embodiment, mutations in the b-Zip domain are detected. Practical applications of techniques for identifying and detecting polymorphisms relate to many fields including disease diagnosis.

In one embodiment, a single nucleotide polymorphism (SNP) is detected. DNA polymorphisms can occur, e.g., when one nucleotide sequence comprises at least one of 1) a deletion of one or more nucleotides from a polymorphic sequence; 2) an addition of one or more nucleotides to a polymorphic sequence; 3) a substitution of one or more nucleotides of a polymorphic sequence, or 4) a chromosomal rearrangement of a polymorphic sequence as compared with another sequence. As described herein, there are a large number of assay techniques known in the art which can be used for detecting alterations in a polymorphic sequence (suitable detection methods are disclosed, for example, in US Patent Number 7,306,913, which is hereby incorporated by refernce in its entirety).

In one embodiment, analysis of polymorphisms is amenable to highly sensitive PCR approaches using specific primers flanking the sequence of interest.

Oligonucleotide primers corresponding to CREB-H sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer. In one embodiment, detection of the polymorphism involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 : 1077-1080; and Nakazawa et al. (1994) PNAS 91 :360-364). In one embodiment, genomic DNA of a cell is exposed to two PCR primers and amplification for a number of cycles sufficient to produce the required amount of amplified DNA.

This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid {e.g., genomic, DNA) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically amplify a subject SNP under conditions such that hybridization and amplification of the sequence occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting polymorphisms described herein. In one preferred embodiment, detection of single nucleotide polymorphisms ("SNP") and point mutations in nucleic acid molecules is based on primer extension of PCR products by DNA polymerase. This method is based on the fact that the nucleoside immediately 5' adjacent to any SNP/point mutation site is known, and the neighboring sequence immediately 3' adjacent to the site is also known. A primer complementary to the sequence directly adjacent to the SNP on the 3' side in a target polynucleotide is used for chain elongation. The polymerase reaction mixture contains one chain-terminating nucleotide having a base complementary to the nucleotide directly adjacent to the SNP on the 5' side in the target polynucleotide. An additional dNTP may be added to produce a primer with the maximum of a two-base extension. The resultant

elongation/termination reaction products are analyzed for the length of chain extension of the primer, or for the amount of label incorporation from a labeled form of the terminator nucleotide. (See, e.g., U.S. Patent No. 6,972,174, the contents of which are incorporated by reference).

In one preferred embodiment, a polymorphism is detected by primer extension of PCR products, as described above, followed by chip-based laser deionization time-of- flight (MALDI-TOF) analysis, as described in, for example U.S. Patent No. 6,602,662, the contents of which are incorporated by reference.

Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al, 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, 1989, Proc. Natl. Acad. Sci. USA 86: 1173- 1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In one embodiment, after extraction of genomic DNA, amplification is performed using standard PCR methods, followed by molecular size analysis of the amplified product (Tautz, 1993; Vogel, 1997). In one embodiment, DNA amplification products are labeled by the incorporation of radiolabelled nucleotides or phosphate end groups followed by fractionation on sequencing gels alongside standard dideoxy DNA sequencing ladders. By autoradiography, the size of the repeated sequence can be visualized and detected heterogeneity in alleles recorded. In another embodiment, the incorporation of fluorescently labeled nucleotides in PCR reactions is followed by automated sequencing. (Yanagawa, T., et al., (1995). J Clin Endocrinol Metab 80: 41-5 Huang, D., et al., (1998). J Neuroimmunol 88: 192-8.

In other embodiments, polymorphisms can be identified by hybridizing a sample and control nucleic acids to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, polymorphisms can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of polymorphisms. This step is followed by a second hybridization array that allows the characterization of specific polymorphisms by using smaller, specialized probe arrays complementary to all polymorphisms detected.

In one embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence CREB-H, or a region surrounding CREB-H and detect allelic variants, e.g., mutations, by comparing the sequence of the sample sequence with the corresponding reference (control) sequence. Exemplary sequencing reactions include those based on techniques developed by Maxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays (Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example, U.S. Patent No. 5,547,835 and international patent application Publication Number WO 94/16101, entitled DNA Sequencing by Mass Spectrometry by H. Koster; U.S. Patent No. 5,547,835 and international patent application Publication Number WO 94/21822 entitled "DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation" by H. Koster), and U.S Patent No.5,605,798 and International Patent Application No. PCT/US96/03651 entitled DNA Diagnostics Based on Mass Spectrometry by H. Koster; Cohen et al.

(1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159). It will be evident to one skilled in the art that, for certain embodiments, the occurrence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. For instance, A-track or the like, e.g., where only one nucleotide is detected, can be carried out.

Yet other sequencing methods are disclosed, e.g., in U.S. Patent No. 5,580,732 entitled "Method of DNA sequencing employing a mixed DNA-polymer chain probe" and U.S. Patent No. 5,571,676 entitled "Method for mismatch-directed in vitro DNA sequencing".

In some cases, the presence of a specific polymorphism of CREB-H in DNA from a subject can be shown by restriction enzyme analysis. For example, a specific nucleotide polymorphism can result in a nucleotide sequence comprising a restriction site which is absent from the nucleotide sequence of another allelic variant.

In a further embodiment, protection from cleavage agents (such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine) can be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA heteroduplexes (Myers, et al. (1985) Science 230: 1242). In general, the technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing a control nucleic acid, which is optionally labeled, e.g., RNA or DNA, comprising a nucleotide sequence of an CREB-H allelic variant with a sample nucleic acid, e.g., RNA or DNA, obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single- stranded regions of the duplex such as duplexes formed based on basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S 1 nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine whether the control and sample nucleic acids have an identical nucleotide sequence or in which nucleotides they are different. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol.

217:286-295. In a preferred embodiment, the control or sample nucleic acid is labeled for detection.

In another embodiment, an allelic variant can be identified by denaturing high- performance liquid chromatography (DHPLC) (Oefner and Underhill, (1995) Am. J. Human Gen. 57:Suppl. A266). DHPLC uses reverse-phase ion-pairing chromatography to detect the heteroduplexes that are generated during amplification of PCR fragments from individuals who are heterozygous at a particular nucleotide locus within that fragment (Oefner and Underhill (1995) Am. J. Human Gen. 57:Suppl. A266). In general, PCR products are produced using PCR primers flanking the DNA of interest. DHPLC analysis is carried out and the resulting chromatograms are analyzed to identify base pair alterations or deletions based on specific chromatographic profiles (see O'Donovan et al. (1998) Genomics 52:44-49).

In other embodiments, alterations in electrophoretic mobility is used to identify the type of CREB-H polymorphism. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766; see also Cotton (1993) Mutat Res 285: 125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids are denatured and allowed to renature. The secondary structure of single - stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In another preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the identity of an allelic variant of a polymorphic region is obtained by analyzing the movement of a nucleic acid comprising the polymorphic region in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265: 1275). Examples of techniques for detecting differences of at least one nucleotide between two nucleic acids include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide probes may be prepared in which the known polymorphic nucleotide is placed centrally (allele-specific probes) and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al.

(1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543). Such allele specific oligonucleotide hybridization techniques may be used for the simultaneous detection of several nucleotide changes in different polylmorphic regions of CREB-H. For example, oligonucleotides having nucleotide sequences of specific allelic variants are attached to a hybridizing membrane and this membrane is then hybridized with labeled sample nucleic acid. Analysis of the hybridization signal will then reveal the identity of the nucleotides of the sample nucleic acid.

Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the allelic variant of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238; Newton et al. (1989) Nucl. Acids Res. 17:2503). This technique is also termed "PROBE" for Probe Oligo Base Extension. In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6: 1).

In another embodiment, identification of the allelic variant is carried out using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Patent No. 4,998,617 and in Landegren, U. et al, (1988) Science 241 : 1077-1080. The OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target. One of the oligonucleotides is linked to a separation marker, e.g., biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. Nickerson, D.A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al., (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927. In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.

Several techniques based on this OLA method have been developed and can be used to detect specific allelic variants of a polymorphic region of an CREB-H gene. For example, U.S. Patent No. 5593826 discloses an OLA using an oligonucleotide having 3 '-amino group and a 5'-phosphorylated oligonucleotide to form a conjugate having a phosphoramidate linkage. In another variation of OLA described in Tobe et al. ((1996) Nucleic Acids Res 24: 3728), OLA combined with PCR permits typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e. digoxigenin and fluorescein, each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors.

In another embodiment, the single base polymorphism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Patent No. 4,656,127). According to the method, a primer complementary to the allelic sequence immediately 3' to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular

exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is used for determining the identity of the nucleotide of a polymorphic site (Cohen, D. et al. (French Patent 2,650,840; PCT Application No. W091/02087). As in the Mundy method of U.S. Patent No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3' to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ is described by Goelet, P. et al. (PCT Application No. 92/15712). The method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymorphic site. The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087) the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.

Several primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA have been described (Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A. -C, et al, Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al, Proc. Natl. Acad. Sci. (U.S.A.) 88: 1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1 : 159-164 (1992); Ugozzoli, L. et al, GATA 9: 107-112 (1992); Nyren, P. et αΙ., ΑηαΙ. Biochem.

208:171-175 (1993)). These methods differ from GBA™ in that they all rely on the incorporation of labeled deoxynucleotides to discriminate between bases at a

polymorphic site. In such a format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms that occur in runs of the same nucleotide can result in signals that are proportional to the length of the run (Syvanen, A. -C, et al, AmerJ. Hum. Genet. 52:46-59 (1993)).

Exemplary mutations which may be detected include those localizing to the N- terminal region of the protein preceding the bZIP domain (e.g., as shown in Fig. 4A). One mutation identified in the working examples is the complex mutation designated

245fs, which consisted of a G insertion in the first nucleotide of codon 245 together with a A>T point mutation 7 nucleotides downstream; the frameshift predicted nonsense amino acid sequence between residue 245 onwards and 338, with premature truncation (Fig. 12). Other mutations include the W46X mutation, G105R, P166L, V180M, D182N, and 240K mutations (clinical features summarized in Table SI). Patients may be heterozygous or homozygous for mutations in CREB3L3.

The methods described herein may be performed, for example, by utilizing pre- packaged diagnostic kits comprising at least one probe/primer nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a polymorphic elements. In addition, a readily available commercial service can be used to analyze samples for the polymorphic elements of the invention.

VI. Kits of the Invention

Another aspect of the invention pertains to kits for carrying out the screening assays or modulatory methods of the invention. For example, a kit for carrying out a screening assay of the invention can include an indicator composition comprising

CREB-H, means for measuring a readout {e.g., protein secretion) and instructions for using the kit to identify modulators of biological effects of CREB-H. In another embodiment, a kit for carrying out a screening assay of the invention can include cells deficient in CREB-H or a molecule in a signal transduction pathway involving CREB-H, means for measuring the readout and instructions for using the kit to identify modulators of a biological effect of CREB-H.

In another embodiment, the invention provides a kit for carrying out a

modulatory method of the invention. The kit can include, for example, a modulatory agent of the invention {e.g., CREB-H stimulatory agent) in a suitable carrier and packaged in a suitable container with instructions for use of the modulator to modulate a biological effect of CREB-H.

In still another embodiment, the invention provides a kit for carrying out a diagnosis of a subject having a loss of function mutation in CREB-H by detecting a mutation in a CREB3L3 gene. The kit can include, for example, a agent for detecting a mutation packaged in a suitable container with instructions for detecting the mutation. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); MuUis et al. U.S. Patent NO: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the figures and the sequence listing, are hereby incorporated by reference. EXAMPLES

The following materials and methods were used throughout the

Examples:

Materials and Methods

Animal experiments To generate CREB-H(N) transgenic mice, a cDNA fragment encompassing amino acids 1-318 of mouse CREB-H that extended to the predicted S2P protease cleavage site was cloned into the pLiv.7 transgenic vector that contained the apolipoprotein E (ApoE) promoter for liver specific transgene expression (6).

Microinjection of the transgenic DNA into the pronuclei of fertilized ICR embryos was performed as described previously (7). CREB-H ^{~ ~} mice have been described previously (1). WT control and experimental groups (CREB-H^+/~ and CREB-H ^{~ ~}) were generated by intercrossing the heterozygous CREB-H^+/" mice. Age and sex matched littermates were used as controls throughout the study. Mice were housed in a specific pathogen free facility at the Harvard School of Public Health on a 12h light/dark cycles and had free access to standard chow diet (PicoLab Rodent diet 20, #5058, Lab diet).

Blood chemistry and lipid analysis Plasma TG, cholesterol and FFA concentrations were determined using assay kits from Sigma, Invitrogen and Wako Chemicals, respectively. VLDL secretion rate in vivo was measured as previously described (8). Briefly, 4 hr- fasted mice were injected with triton WR1339 (500 mg/kg in saline) via the tail vein. Blood samples were drawn at indicated time points for TG assays. For plasma/ APOC-II transfusion, mice were fasted for 4 hr and injected with 100 ml of plasma or 25 mg of apoC-II protein (Athens Biotechnology Company) through the tail vein. Plasma samples were collected at indicated time points after injection, and measured for TG levels. Liver tissues were homogenized and lipids were extracted with chloroform/methanol mixture (2: 1 v/v), as described previously (9). TG contents were determined using an assay kit from Sigma.

LPL assay LPL activity in post-heparin plasma was determined as described previously (10), with some modifications. Briefly, post-heparin plasma was prepared thirty minutes after i.p injection of 200U sodium heparin into mice. Substrate for LPL was prepared by mixing 1.12 mCi of 3H-triolein (99 μg), 300 mg of unlabeled triolein, and 18 mg of egg phosphatidylcholine. Radiolabeled substrate was mixed with 10 μΐ of post-heparin plasma and 15 μΐ of heat inactivated serum isolated from WT or CREB-H ^{~ ~} mice.

Liberated FA was measured by scintillation counting.

LPL assay using recombinant LPL protein was performed as described elsewhere (11). Briefly, the substrate was prepared by sonicating 83 mg of triolein (Sigma) in 1.785 ml of 0.2 M Tris buffer (pH 8.2) containing 150 mg/ml gum arabic. 150 μΐ of the sonicated substrate was mixed with 140 μΐ of 2x assay buffer containing 0.33 M NaCl, 165 mg/ml fatty acid-free albumin and 10 μΐ of plasma prepared from WT or CREB-H^{" "} mice, and incubated for 80 min at 37°C to allow the transfer of serum ApoCs to the substrate. 0.93 μg of purified LPL (Sigma) was added to the substrate, and incubated for 30 min. The reaction was stopped by adding cold NaCl to a final concentration of 1 M. FFA content in the reaction mixture released from triolein by LPL was measured as described above.

Fractionation of lipoproteins Five hundred μΐ of pooled plasma (3 per group) were fractionated by discontinuous gradient density ultracentrifugation as described previously (12). Briefly, 1 g of potassium bromide (KBr) and 50 mg of sucrose were added to the plasma, which was then brought to a final volume of 3.5 ml with density solution (p=1.006 g/ml KBr). The discontinuous gradient consisted of plasma (3 ml) and density solutions with p=l .21 g/mL KBr (2 ml), p=1.08 g/ml KBr (3 ml), and p=1.00 g/ml (3 ml) layered from bottom to top in a 12 ml ultracentrifuge tube. The samples were then centrifuged at 35,000 rpm for 18hr at 8°C. Fractions (1 ml) were sequentially collected from top to bottom, determined for their densities, and assayed for TG and cholesterol contents. Fractions were desalted and concentrated into 50 μΐ of PBS using Centricon columns. Fifteen μΐ of fractions were separated on a 4-20% gradient SDS- polyacrylamide gel. The gel was stained by Coomassie Brilliant Blue G-250. RNA isolation, microarray, northern blot and real time PCR Total RNAs were isolated using TRIZOL (Invitrogen) according to the manufacturer's recommendation. Northern blot analysis of CREB-H mRNA was performed as described previously (13). Complementary DNAs were generated using the High Capacity cDNA Reverse

Transcription kit (Applied Biosystems), and subjected to SYBR-based real-time PCR using the Mx3005P™ system (Stratagene). Primer sequences are listed in Table S2. Microarray and data analysis were carried out at the Harvard-Partners Center for Genetics and Genomics (Harvard Medical School). Three liver RNA samples per group were further purified using MiniElute Column (Qiagen), and hybridized on mouse WG- 6 2.0 chips (Illumina).

Western blot analysis Liver nuclear extracts and microsomal fractions were prepared as described previously (8, 14). Primary mouse intestinal epithelial cells were isolated using dispase as described previously (15). CREB-H western blot was performed using a rabbit polyclonal antibody that was raised against bacterially produced mouse CREB-H amino acids 1-232. For apoC-III western blotting using a rabbit polyclonal antibody, 5 of VLDL fractions were separated on a 12 % NuPAGE No vex Bis-Tris gel

(Invitrogen) and transferred into nitrocellose membrane.

Plasmid constructs, cell culture and reporter gene assay Mouse CREB-H cDNA was isolated by PCR amplification using a cDNA clone (IMAGE :4211480, BCO 10786) as template, and inserted into HA-Cruz-C mammalian expression vector (Santa Cruz). Cruz-CREB-H(N) which expressed amino aids 1-318 of CREB-H was also similarly generated by PCR with the following primers : forward, 5'- GATATCCTGGAAAGATGGCGTCCC-3 ' ; reverse, 5'-

AGATCTC AGGTGCCTGCATGGGCTG-3 ' . An EST clone containing human CREB- H cDNA (IMAGE: 8069010, BC101504) was obtained from OpenBiosystems and validated by DNA sequencing. Full length CREB-H and the N-terminal amino acids 1- 322 were amplified by PCR and cloned into pCDNA3.1 vector (Invitrogen). W46X, G105R, P166L, V180M, D182N, E240K and 245fs mutations were introduced to the full length CREB-H and CREB-H(N) using QuikChange Site-Directed Mutagenesis Kit (Stratagene). Proximal ApoA4 promoter (-900/+40) was isolated by PCR using RP24- 302M3 BAC plasmid (BACPAC Resources Center, CHORI) containing Apoa4 gene as template, and cloned into pGL3-basic vector (Promega). Sequential deletion and site directed mutagenesis were performed to generate additional shorter or mutated reporter constructs. Apoc2 and Fgf21 promoter-luciferase reporter plasmids were kindly provided by Dr. Peter A. Edwards (UCLA, Dept of Biological Chemistry and Medicine) and Dr. Steven A. Kliewer (UT Southwest Medical Center), respectively.

MODE-K, Hepal .6 and 293T cells were maintained in DMEM medium supplemented with 10% fetal bovine serum. Cells were transfected using Lipofectamine 2000

(Invitrogen) and harvested for luciferase assays using Dual-luciferase reporter assay kit (Promega). Trans fection efficiency was normalized to Renilla activity after

cotransfection with pRL/CMV (Promega).

Electrophoretic mobility shift assay (EMSA) EMSA was carried out using ³²P- labeled oligonucleotides and CREB-H(N) proteins prepared using in vitro TNT^® quick coupled transcription/translation system (Promega), as described previously (16). Double stranded probes were synthesized based on following sequences: WT, 5 '- TTACGCGTCAGCTTCC ACGTGTCTTAGGGCC-3 ' ; Mut, 5 '- TTACGCGTCAGCTTCCtttcTGTCTTAGGGCC-3 ' (mutated nucleotides are in lower cases).

Sequencing of human genomic DNA. Sequencing, using primers and conditions shown in Table S3, was performed on genomic DNA of patients who were part of a tertiary referral clinic screening cohort with hypertriglyceridemia (plasma triglycerides > 1000 mg/dL) and matched controls, as described (17).

Example 1. CREB-H deficiency decreases LPL-mediated TG clearance

Fasting plasma TG concentration reflects a balance between the production of VLDL from the liver and the clearance of TG-rich lipoproteins from the circulation, governed predominantly by LPL present on the surface of vascular endothelium in various organs (18). To determine if CREB-H plays a role in VLDL secretion from the liver, we measured the rate of TG accumulation in plasma after administration of an LPL inhibitor tyloxapol. The VLDL secretion rate was not significantly different between WT and CREB-H^"7" mice, arguing against a role for CREB-H in VLDL secretion (Fig. 2A). We next examined if CREB-H deficiency decreased LPL-mediated TG clearance. Olive oil gavage resulted in a marked hypertriglyceridemia in CREB-H^"7" mice (Fig. 2B), suggesting a defect in TG clearance in the absence of CREB-H. Post-heparin LPL activity was decreased by -25% in CREB-H^"7" mice (Fig. 2C), despite normal LPL mRNA levels in adipose tissue and skeletal muscle (Fig. 7), suggesting that plasma cofactors for LPL might be limiting in CREB-H^"7" mice. Consistent with this hypothesis, the addition of WT serum as a source of Apoc proteins significantly increased the LPL activity retained in post-heparin plasma of CREB-H^"7" mice (Fig 2C). The potency of CREB-H^"7" serum in augmenting LPL-mediated hydrolysis of triolein substrate was also attenuated compared to that of WT serum (Fig. 2D). Further, transfusion of WT but not CREB-H^"7" plasma efficiently reduced TG levels in CREB-H^"7" mice (Fig. 2E).

Collectively, these data establish that the hypertriglyceridemia observed in CREB-H^"7" mice is a consequence primarily of decreased TG clearance from plasma, rather than increased TG output from the liver. Example 2. Identification of CREB-H target genes

To identify CREB-H target genes that might contribute to the hypertriglyceridemia phenotype of CREB-H^"7" mice, we performed microarray analysis of RNAs isolated from WT and CREB-H^"7" mouse liver after a 24h fast. Statistical analysis revealed a subset of genes downregulated in CREB-H deficient liver that are known to be involved in TG metabolism in human or mouse (Fig. S4). Quantitative RT-PCR confirmed the decreased expression in CREB-H^"7" liver of Fadsl , Fads2, Elovl2, Cidec, Apoc2 and Apoa5, which have been associated with human TG metabolism (Fig. 3 A) (19, 20). Fgf21 , Apoa4, and Elov5, and G0s2 mRNAs were also decreased in CREB-H liver (Fig. 3A). Fgf21 is induced by fasting and reduces plasma TG level (21). Fads and Elovl genes encode fatty acid desaturases and elongases, respectively, and play important roles in the synthesis of long chain polyunsaturated fatty acids and the regulation of fatty acid metabolism (22). G02s and Cidec are induced in the liver by fasting, and are known to regulate TG hydrolysis and lipid droplet formation, respectively (23-25). We note that Apoa4 (apoA- IV), Apoa5 (apoA-V), and Apoc2 (apoC-II) genes were significantly downregulated in CREB-H^"7" liver. These apolipoprotein genes together with Fgf21 were induced in the liver by fasting, an effect which was abrogated in CREB-H^"7" mice (Fig. 3B). Apoa4 and Apoc2 are normally highly expressed in mouse small intestine. Intestinal mRNA levels of these genes were also decreased in CREB-H^"7" mice (Fig. S5A). ApoA-IV, apoA-V, and apoC-II activate LPL to facilitate the delivery of hydro lyzed fatty acids to peripheral cells and hence lower plasma TG levels (26, 27). Despite a marked increase of VLDL associated apoC-III protein, its mRNA levels were not significantly changed in CREB- H^"7" liver or intestine (Fig. 9B), suggesting a post-transcriptional control of apoC-III by CREB-H. We postulated that the concomitant reduction of the LPL activators, ApoA- IV, apoA-V, and apoC-II, and induction of LPL inhibitor, apoC-III impaired TG clearance, resulting in increased plasma TG levels in CREB-H deficient mice. Indeed, injection of recombinant apoC-II protein effectively reduced plasma TG levels in CREB-H^"7" mice (Fig. 3C). To further explore the identity of CREB-H target genes involved in TG metabolism, we generated transgenic mice that over-expressed a constitutively active CREB-H(N) in the liver (Fig. 10A). Both the endogenously processed and the transgenic CREB-H(N) were expressed as 42 kDa proteins in the liver (Fig. 10B). CREB-H(N) protein levels were increased by -14 fold in the transgenic mouse liver, despite only a -1.25 fold increase of the transgenic mR A (Fig. IOC), suggesting that -10% of CREB- H protein is basally processed to the mature form in the liver of mice fed standard chow. Gene profiling revealed that CREB-H(N) overexpression strongly induced Apoc2, Apoa4, Fgf21, and Cidec mRNAs (Fig. 10D and E), mirroring the downregulation of these genes in CREB-H ^{~ ~} mice. Other apolipoprotein genes located within the same gene clusters as Apoc2 and Apoa4 on chromosomes 7 and 9, respectively, were unaffected by CREB-H(N) overexpression (Fig. 10E). Transient transfection assays showed that CREB-H strongly transactivated the Apoa4, Apoc2 and Fgf21 promoters (Fig. 10F), indicating direct CREB-H mediated transcription of these genes. Deletional and site directed mutagenesis analysis coupled with mobility shift assays revealed that CREB- H(N) directly binds to two cis-acting elements in the Apoa4 promoter that are highly conserved across the human and murine genome (Fig. 11).

Example 3. Mutations in CREB3L3 Contribute to Hypertriglyceridemia in Humans

Since CREB-H deficiency caused a dramatic increase in plasma TG with no other overt abnormalities in mice, we asked whether mutations in CREB3L3 could similarly contribute to hypertriglyceridemia in humans. Common variants in CREB3L3 were not observed to be associated with TG in recent large genome wide association studies (28-30). Hence, we searched for rare mutations in the CREB3L3 gene in an established clinical cohort with hypertriglyceridemia (HTG) (31). We sequenced CREB3L3 coding regions in 449 unrelated HTG patients of European Caucasian ancestry (TG >3.37 mmol/L) and normal LPL, APOC2, and APOA5 genes, as well as in 238 matched normolipidemic controls (TG<2.3 mmol/L) (31). Remarkably,

heterozygous nonsynonymous or insertional mutations in CREB3L3 were identified in 12 out of 449 HTG patients (9 who had elevated VLDL only, and three of whom had elevated VLDL plus fasting chylomicrons), but were completely absent from 238 controls (P=0.01, 2-sided Fisher's exact test; Table SI). All mutations localize to the N- terminal region of the protein preceding the bZIP domain (Fig. 4A). Three unrelated HTG patients were heterozygous for the same complex mutation (designated 245fs), which consisted of a G insertion in the first nucleotide of codon 245 together with a A>T point mutation 7 nucleotides downstream; the frameshift predicted nonsense amino acid sequence between residue 245 onwards and 338, with premature truncation (Fig. 12). One HTG patient was heterozygous for the nonsense CREB3L3 W46X mutation. Eight HTG probands had heterozygous missense mutations, which included two probands with G105R, two with P166L, one with V180M, one with D182N, and two with 240K mutations (clinical features summarized in Table SI). Lipoprotein profiles of the nuclear families of the 4 patients with CREB3L3 nonsense mutations showed a significantly elevated mean plasma TG level in 11 mutation carriers compared with 5 non-carrier first-degree relatives (9.67±4.70 vs. 1.66±0.55 mmol/L, P=0.021, Wilcoxon test) (Fig. 4B). Together, these human genetic data strongly suggest that rare heterozygous variants in CREB3L3 are significantly associated with the polygenic trait of HTG, similar to other established HTG-associated genes such as LPL and APOA5 (31). However, incomplete penetrance of the disease phenotype in mutation carriers suggests that heterozygous mutations in CREB3L3 alone are not sufficient to cause HTG and likely require factors such as diet and genetic variations in other loci contributing to TG metabolism.

Mutant CREB-H proteins encoded by these human variants were functionally evaluated in transactivation assays using a luciferase reporter driven by the proximal Apoa4 promoter. The W46X and 245 fs mutations precluded translation of the DNA binding and bZIP domains, and as expected, the resulting mutant proteins failed to transactivate the Apoa4 reporter (Fig. 4C). The E240K mutation also severely abrogated the induction of the Apoa4 reporter by CREB-H cotransfection. In contrast, the G105R, P166L, V180M and D182N mutations did not significantly affect CREB-H

transactivation of the Apoa4 promoter (Fig. 4C). It is unlikely that these mutations that failed to alter Apoa4 transactivation are incidentally present in six HTG patients, since they were not detected in a large sample of normotriglyceridemic individuals, or are represented in any current sequence database. In summary, we identified multiple heterozygous nonsynonymous mutations in CREB3L3 that produced a hypomorphic or nonfunctional CREB-H protein, and were associated with markedly elevated plasma TG levels. Table SI. Clinical features of mutation carriers in CREB3L3 gene

HTG or Mutation Polyphen TC TG HDL

GL Mutation Age Sex BMI DM CONTROL Type prediction (mM) (mM) (mM)

HTG 1329 nonsense W46X truncation 43 male 29 .7 4.97 6.59 0 .49 no

HTG 818 missense G105R possibly damaging 58 male 27 .8 9 5.23 ND no

HTG 1042 missense G105R possibly damaging 54 male 22 .7 8 5.99 1 .98 no

HTG 1591 missense P166L benign 60 male 32 .2 6.1 11.8 0 .95 yes

HTG 1040 missense P166L benign 43 female 26 .8 7.1 4.61 1 .26 no

HTG 4901 missense V180M benign 35 female 29 15.1 56.3 0 .66 no

HTG 1020 missense D182N possibly damaging 53 female 25 .8 6.22 6.03 0 .76 no

HTG 4887 missense E240K possibly damaging 58 male 28 .3 11.7 32.5 1 .72 no

HTG 2430 missense E240K possibly damaging 36 male 32 8.3 5.2 1.2 no

HTG 2657 nonsense 245fs truncation 57 male 30 .3 12.3 51 0 .31 yes

HTG 1189 nonsense 245fs truncation 42 male 36 17.6 27.5 0 .23 yes

HTG 4953 nonsense 245fs truncation 33 male 40 .7 6.3 4.92 0.8 no

Abbreviations: HTG, hypertriglyceridemia; GL, identification number; BMI, body mass index; TC, total cholesterol; TG, triglycerides; HDL, high-density lipoprotein cholesterol ; DM, Diabetes Mellitus; ND, not determined.

Table S2. Sequences of real time PCR primers

Gene Forward Reverse Source

CREB-H GGCCATTGACCTGGACATGT TTCACAGTGAGGTTGAAGCGG This study

Fgf21 GGAGCTCTCTATGGATCGCCT TGTAACCGTCCTCCAGCAGC PrimerBank*

Fadsl AGCACATGCCATACAACCATC TTTCCGCTGAACCACAAAATAGA PrimerBank

Fads2 AAGGGAGGTAACCAGGGAGAG CCGCTGGGACCATTTGGTAA PrimerBank

Elovl2 CCTGCTCTCGATATGGCTGG AAGAAGTGTGATTGCGAGGTTAT PrimerBank

Elovl5 ATGGAACATTTCGATGCGTCA GTCCCAGCCATACAATGAGTAAG PrimerBank

Cidec ATGGACTACGCCATGAAGTCT CGGTGCTAACACGACAGGG PrimerBank

G0s2 TAGTGAAGCTATACGTTCTGGGC GTCTCAACTAGGCCGAGCA PrimerBank apoal TCCTGACAGGGAGCCAGG TGTCCCATTGGGACTGGG PrimerBank apoa4 CCAGCTAAGCAACAATGCCA TGGAAGAGGGTACTGAGCTGC PrimerBank

Apoa5 AGGCAGCAGTTGAAACCCTA TGAGCCTTGGTGTCTTCTCC This study

Apocl TCCTGTCCTGATTGTGGTCGT CCAAAGTGTTCCCAAACTCCTT PrimerBank

Apoc2 GCATGGATGAGAAACTCAGGG AAAATGCCTGCGTAAGTGCTC PrimerBank

Apoc3 TACAGGGCTACATGGAACAAGC CAGGGATCTGAAGTGATTGTCC PrimerBank

Apoc4 GAGCTGTCCAGGGCTTTATG GGCTGTGGGTCTTGTTTAGG PrimerBank

Apoe CTGACAGGATGCCTAGCCG CGCAGGTAATCCCAGAAGC PrimerBank

Lpl GGGAGTTTGGCTCCAGAGTTT TGTGTCTTCAGGGGTCCTTAG PrimerBank

* Harvard University PrimerBank

Table S3. Primers for genomic sequencing of CREB3L3

5%

Exon Forward Reverse ^anneal DMSO

(°C)

added

1 ACAGAGGGCTGTGAGCTTG TTCTCTGGGCCTCAGTCTTC 60 no

2 AGCGGCAACTGAACTCTAGC GCTAAAATCAAGCACCGTGA 60 no

3 CATCTTGGAGAAGGGAAGGACACC ACACCTAGCCAAGGGAGACACGTG 63 yes

4 CTTGGGGACTCCAAACTCTG AAATTCCACGCCTTTCTGTG 60 no

5 CCTGGGGTGATAGTGTTTTCG AAGCTGAGATCGTGCCACTG 60 yes

6 CCTGACCTCAGGTGATACGC ATCTTTCCATCCCTGCAATC 60 yes

7 TGGGTTCTCTTGGCTTGTAACGTGAGG TGAGATTACAGGCGTGAGCCACTGCAC 63 yes

8+9 CCTTGAAGAATGGATGGAATTTGG ACAAGGTGGAGGTGGGGTCCTATG 60 no

10 TTGCGCCTGTACGAGGTAG ATCTCTGCTGGGGTCTTGG 60 no

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method for identifying compounds useful in maintaining normal triglyceride levels in a subject comprising,

a) providing a cell comprising the regulatory region of the CREB-H gene genetically fused to an indicator gene encoding a polypeptide;

2. A method for identifying compounds useful in maintaining normal triglyceride levels in a subject comprising,

a) providing a cell comprising CREB-H or the amino terminal portion thereof and the regulatory region of a gene transcriptionally regulated by CREB-H genetically fused to an indicator gene encoding a polypeptide;

3. The method of claim 2, wherein the regulatory region regulates expression of a gene selected from the group consisting of Fgf21, Apoc2, Apoa4, and Apoa5 and wherein increased expression of the indicator gene indicates that the compound reduces triglyceride levels.

4. The method of any one of claims 1-3, further comprising testing the effect of the compound on triglyceride clearance.

5. The method of claim 4, wherein the triglyceride level in the low density lipoprotein (VLDL) fraction of plasma is measured.

6. A method for identifying a subject at risk for having abnormal triglyceride and lipoprotein metabolism comprising, contacting a biological sample derived from the subject with an agent capable of detecting the presence or absence of a CREB-H loss of function mutation, wherein the presence of the CREB-H loss of function mutation indicates that the subject is at risk for developing abnormal triglyceride and lipoprotein metabolism, thereby identifying a subject at risk for having abnormal triglyceride and lipoprotein metabolism.

7. A method for identifying a subject at risk for having abnormal triglyceride and lipoprotein metabolism comprising, contacting a biological sample derived from the subject with an agent capable of detecting the presence or absence of a mutation present in the region of the CREB-H protein, wherein the presence of the mutation indicates that the subject is at risk for developing abnormal triglyceride and lipoprotein metabolism, thereby identifying a subject at risk for having abnormal triglyceride and lipoprotein metabolism.

8. A method for reducing serum triglyceride levels in a subject, comprising administering to the subject an active form of CREB-H to thereby reduce serum triglyceride levels in the subject.

9. A kit for predicting whether a subject is at risk for having abnormal triglyceride and lipoprotein metabolism, the kit comprising means for determining the presence or absence of a CREB-H loss of function mutation in a biological sample obtained from said subject and instructions for using the kit to predict whether the subject is at risk for having abnormal triglyceride and lipoprotein metabolism.