Michael R. Stallcup - US grants

virus genetics; genetic transcription; gene expression; glucocorticoids; hormone regulation /control mechanism; mouse mammary tumor virus; provirus; structural genes; virus RNA; virus; genetic manipulation; cell free system; hybridomas; molecular cloning; mouse leukemia; mutant; genetic recombination; cell bank /registry; radiotracer; nucleic acid inhibitor;

Transcription of mouse mammary tumor virus (MMTV) RNA from integrated proviral DNA is stimulated rapidly and specifically by glucocorticoids. Molecular and genetic approaches will be used to identify genes and gene products that are involved in glucocorticoid regulation of MMTV gene expression. A procedure employing ribonucleoside (beta-S)triphosphates, Hg-Sepharose column chromatography, and nucleic acid filter hybridization has been established for measuring initiation of new MMTV RNA chains in preparations of cell nuclei. This cell free system will be used to determine whether glucocorticoids control MMTV RNA synthesis at the level of transcription initiation. The sites of initiation of MMTV RNA chains in vitro and in vivo will be compared by S1 nuclear mapping. Nuclei from MMTV-infected cells or, alternatively, nuclei from cells transfected with SV40/MMTV recombinant vectors, will be used to establish a system in which initiation of MMTV RNA chains is stimulated by the addition of glucocorticoid hormone and receptors. A combination of immunological and pharmacological procedures will be used to select novel glucocorticoid response variants from clones of MMTV-infected mouse lymphomacells with two responses: stimulation of MMTV gene expression and glucocorticoid-induced cytolysis. Specific programs are proposed for obtaining and charcterizing (biochemically and genetically) variants with lesions in MMTV structural genes, MMTV DNA sequences that regulate transcription and hormone inductibility, cellular genes necessry for posttranscriptional processing of MMTV proteins, and other cellular genes (including but not limited to the glucocorticoid receptor) that may be necessary for each hormone response. Programmed regulation of gene expression is essential for the normal development of an organism. Cancer is caused by a breakdown in thenormal control of specific genes. The proposed project will increase understanding of the role of hormones in development, differentiation, and malignancy.

virus genetics; genetic transcription; hormone regulation /control mechanism; glucocorticoids; gene expression; mouse mammary tumor virus; structural genes; cell differentiation; nucleic acid sequence; murine leukemia virus; cell free system; genetic manipulation; genetic recombination; mutant; molecular cloning; monoclonal antibody; simian virus 40;

We will investigate the molecular mechanisms by which peptide hormones regulate the transcription of specific genes in cultured mammalian cell lines. Biochemical studies in many laboratories have determined the first intracellular steps in these hormone response pathways, but the later steps that lead to regulation of transcription remain unknown. We will use a combination of molecular biology and somatic cell genetics to define the steps and components of these pathways. Promoters and regulatory sequences of hormonally responsive genes (prolactin and SV40 early) will be fused to the structural gene for Eco gpt, thus creating hormonally responsive selectable marker genes. These hybrid genes will be introduced into hormonally responsive cultured cell lines (GH3 and HepG2, respectively) where they can be regulated by a variety of hormones and inducers (TRH, EGF, phorbol esters, and calcium for prolactin; phorbol esters for SV40). Selective culture media can be used to select for or against the expression of the gpt gene either in the presence or the absence of inducers. We will select for cell variants that lack the normal hormone responses and then characterize these variants genetically and biochemically. These same response pathways are involved in regulation of cell proliferation; in fact most of the currently known oncogene products are believed to be components of these pathways. Thus, the biochemical details of these pathways are essential for understanding the nature of cancer.

The major functional domains of the glucocorticoid receptor (GR) include a sequence-specific DNA binding domain near the center of the polypeptide chain and a hormone binding domain in the Cterminal region. The aim of this proposal is to establish a detailed genetic map of the 250-amino acid hormone binding domain, by obtaining single amino acid substitutions that affect hormone binding affinity and/or specificity. This map will be essential for a complete understanding of the structure and function of the hormone binding domain, which determines the ligand affinity and specificity of the GR and regulates the activity of the GR protein as a conditional gene-specific transcriptional activator. The analysis will be performed using two complementary genetic approaches. First, a somatic cell genetics approach will make use of the glucocorticoid induced cytolytic response of the mouse lymphoma cell fine W7MG1 to select hormone-resistant cell lines containing mutant forms of the GR with altered hormone binding affinity and/or specificity. GR cDNA clones will be obtained from the mutant cell lines after polymerase chain reaction (PCR) amplification of single-stranded cDNA. Site directed mutagenesis will be performed in subregions dictated by the somatic cell genetics data and by biochemical data indicating that specific residues are involved in hormone binding. The mutations will be introduced by using PCR primed by synthetic oligonucleotides containing specifically designed point mutations. The phenotype of each mutant GR cDNA will be tested by co-transfecting the GR-negative COS-7 cell line with a GR-cDNA expression vector and a hormone responsive reporter gene, consisting of the chloramphenicol acetyl transferase (CAT) gene drive by the mouse mammary tumor virus promoter. The health relatedness of this project lies in the fact that steroid receptors play a pivotal role in steroid hormone action. In addition, a variety of malignant tumors treated clinically with steroid agonists and antagonists. The tumors frequently develop steroid resistance, which may arise from a subpopulation of the tumor cells that have sustained a mutation that alters hormone binding affinity and/or specificity. Understanding of this phenomenon will depend upon a detailed structure-function analysis of the hormone binding pockets of these steroid receptor proteins.

DESCRIPTION (Adapted from the applicant's abstract): This project will investigate how steroid receptors interact with the transcription machinery to activate transcription of specific genes. An 810 amino acid fragment of a recently identified protein called GRIP1 interacts with the hormone binding domain of steroid receptors in an agonist dependent manner. In yeast, GRIP1 serves as a transcriptional coactivator for steroid receptors by interacting with the AF2 transcriptional activation domain of steroid receptors. Characterization of GRIP1 will be continued by first completing the cloning and sequencing of the coding region. The possibility that GRIP1 may interact with other nuclear receptors will be investigated. The functional domains of GRIP1 will be mapped as well as the specific subregions of the steroid receptors that GRIP1 interacts with. Cell and tissue specificity of GRIP1 expression will be determined. The ability of GRIP1 to interact with and serve as a coactivator for steroid receptors in mammalian cells will be examined. Finally, the mechanism of transcriptional coactivation by GRIP1 will be studied by looking for interactions between GRIP1 and known components of the transcription machinery, as well as by initiating studies with an in vitro transcription system.

protein protein interaction; steroid hormone receptor; protein structure function; transcription factor; protein binding; vitamin D receptors; enzyme activity; nuclear matrix; histones; protein sequence; biological signal transduction; cell type; hormone regulation /control mechanism; chromatin; gene expression; genetic transcription; retinoid binding proteins; hormone receptor; acyltransferase; yeast two hybrid system; genetic library; mutant; cell line; western blottings; northern blottings;

The nuclear receptor (NR) family includes the receptors for steroid, thyroid, retinoid, and vitamin D hormones; NRs are hormone-regulated transcription factors which bind enhancer elements and recruit coactivators to activate target genes by modulating local chromatin structure and recruiting RNA polymerase II to the promoter. The current study focuses on the three p160 coactivators (p160 CoAs) (SRC-1, GRIP1, and p/CIP) which bind to AF-2 and (in some cases) AF-1 activation domains of NRs. The p160 CoAs also recruit secondary coactivators such as p300/CBP, p/CAF, and CARM1 which act synergistically with p160 CoAs by acetylating or methylating histones and other proteins in the transcription machinery. Many studies now indicate that the p160 CoAs play central roles in NR function. The p160 CoAs also serve as coactivators for other transcription factors, such as MEF-2C and myogenin which promote muscle cell differentiation. We propose to focus on the role of the N-terminal region of GRIP1, which is the most highly conserved but least understood region of p160 CoAs. By mutational analysis, we will map the functionally important subregions of the N-terminal domain, using both transiently transfected and stably integrated reporter genes and chromatin immunoprecipitation (ChIP) assays to study the coactivator function of GRIP1 with NRs, MEF-2C, and myogenin. The yeast 2-hybrid system will be used to identify p160- interacting proteins (p160-IP) which bind the N-terminal region of GRIP1. We will characterize the functions of p160-IPs as coactivators that may collaborate with GRIP1 or as transcriptional activators which use GRIP1 as a coactivator. We have recently developed transient transfection conditions under which NR function depends on co-expression of three different coactivators (GRIP1 + CARM1 + p/CAF); these conditions will be used to characterize the mechanism of synergy, with a focus on the nature of the interaction between GRIP1 and p/CAF. The role of GRIP1 as a coactivator for MEF-2C and myogenin will be studied in transient transfections; and the role of various GRIP1 domains in muscle cell differentiation will be studied in cultured C2C12 cells which require GRIP1 for differentiation into myotubes in culture.

The nuclear receptor (NR) family includes the receptors for steroid, thyroid, retinoid, and vitamin D hormones; NRs are hormone-regulated transcription factors which bind enhancer elements and recruit coactivators to activate target genes by modulating local chromatin structure and recruiting RNA polymerase II to the promoter. The current study focuses on the three p160 coactivators (p160 CoAs) (SRC-1, GRIP1, and p/CIP) which bind to AF-2 and (in some cases) AF-1 activation domains of NRs. The p160 CoAs also recruit secondary coactivators such as p300/CBP, p/CAF, and CARM1 which act synergistically with p160 CoAs by acetylating or methylating histones and other proteins in the transcription machinery. Many studies now indicate that the p160 CoAs play central roles in NR function. The p160 CoAs also serve as coactivators for other transcription factors, such as MEF-2C and myogenin which promote muscle cell differentiation. We propose to focus on the role of the N-terminal region of GRIP1, which is the most highly conserved but least understood region of p160 CoAs. By mutational analysis, we will map the functionally important subregions of the N-terminal domain, using both transiently transfected and stably integrated reporter genes and chromatin immunoprecipitation (ChIP) assays to study the coactivator function of GRIP1 with NRs, MEF-2C, and myogenin. The yeast 2-hybrid system will be used to identify p160- interacting proteins (p160-IP) which bind the N-terminal region of GRIP1. We will characterize the functions of p160-IPs as coactivators that may collaborate with GRIP1 or as transcriptional activators which use GRIP1 as a coactivator. We have recently developed transient transfection conditions under which NR function depends on co-expression of three different coactivators (GRIP1 + CARM1 + p/CAF); these conditions will be used to characterize the mechanism of synergy, with a focus on the nature of the interaction between GRIP1 and p/CAF. The role of GRIP1 as a coactivator for MEF-2C and myogenin will be studied in transient transfections; and the role of various GRIP1 domains in muscle cell differentiation will be studied in cultured C2C12 cells which require GRIP1 for differentiation into myotubes in culture.

DESCRIPTION (provided by applicant): Nuclear receptors (NR) activate transcription by recruiting complexes of coactivators which locally remodel chromatin structure in the promoter and help to recruit and activate an RNA polymerase II complex. Multiple complexes of coactivators participate in transcription initiation, and some recruited coactivators may also facilitate the subsequent coupled steps of transcription elongation and RNA processing. There is clear evidence for physiological relevance of one coactivator complex, which includes the p160 family (GRIP1, SRC-1, and p/CIP), the protein acetyltransferases p300 and CBP, and the protein arginine methyltransferases CARM1 and PRMT1. The p160 coactivators bind directly to NRs and recruit the acetyltransferases and methyltransferases, which modify histones and other proteins in the transcription complex. Another coactivator, PGC-1, is an example of a tissue specific coactivator whose level of expression in liver, brown fat, and muscle plays a key role in controlling the expression of genes involved in processes such as gluconeogenesis and thermogenesis. The studies of these specific coactivators, in our lab and others, have been on the forefront in advancing our understanding of the complex mechanism of transcriptional regulation, but the specific mechanistic roles of the methyltransferases and their methylation of histones and other proteins is not clear. The proposed studies will focus on the roles of these methyltransferases as coactivators and their mechanism of synergistic cooperation with p300/CBP and with PGC-I. We will define: 1) the roles of arginine-specific methylation of histones H3 and H4 by CARM1 and PRMT1, respectively, in transcriptional activation, by using chromatin immunoprecipitation and by identifying proteins which preferentially bind to the methylated histones; 2) the functional domains of CARM1, their specific roles in coactivator function, and proteins that bind to critical domains of CARM1; 3) the mechanisms of CARM1-p300/CBP and PRMT1-PGC-1 coactivator synergy and the roles which methylation of p300/CBP by CARM1 and of PGC-1 by PRMT1 play in synergy; 4) the sub-nuclear locations and co-localization of SR and their coactivators, the assembly of SR and their coactivators onto stably integrated promoters of steroid responsive genes in vivo, and the role of coactivators in chromatin remodeling by SR on reconstituted chromatin in vitro.

DESCRIPTION (provided by applicant): The University of Southern California Training Program in Genetic, Molecular and Cellular Biology will prepare students for careers in interdisciplinary biomedical research and related occupations. Throughout their doctoral training and research, trainees will interact with students and faculty engaged in research from the entire spectrum of biomedical and biological research. They may select a research mentor from at least 9 different Ph.D. programs at USC spanning the entire universe of biomedical and biological research. In their courses and Training Program activities they will interact with faculty and students from many different programs and thereby form connections which will foster interdisciplinary interactions and approaches to research. Students in the Training Program will take at least one formal course in computational methods to promote their interest in and ability to apply sophisticated computational skills to their research, either as primary or supporting technology. This last opportunity will take advantage of areas of special strength at our University to prepare students for the new emphasis on quantitative treatments of complex data sets which is becoming increasingly important in biomedical and biological research. In addition to its broad strengths in other areas of the biomedical and biological sciences, the University of Southern California has faculty groups who rank among the best in the world in the application of computational methods to the study of complex genomes and in the field of molecular cancer epidemiology. Trainees will also participate in a variety of workshops on teaching and other career opportunities. Most students participating in the Training Program will be supported by the Program during their first three years in graduate school and will continue to participate in program activities until the completion of their Ph.D. degree.

DESCRIPTION (provided by applicant): Hormone-activated nuclear receptors (NR) bind to target gene promoters and recruit complexes of coactivators to help activate transcription. The p160 coactivator complex consists of a p160 protein, which binds directly to activated NRs, and several secondary coactivators. Our global strategy is to characterize the functional domains of coactivators and search for proteins that bind to their activation domains and mediate downstream events in the coactivator signaling pathway. By reiterative application of this process, we will find the ultimate targets of the p160 coactivators in the chromatin or basal transcription machinery. The p160 proteins were previously shown to have two C-terminal activation domains for signal transmission: AD1 binds CBP/p300, while AD2 binds CARM1. We recently identified a new activation domain in the N-terminal region of p160 coactivators (AD3) and identified two proteins which bind to the p160 N-terminus and cooperate synergistically with p160 proteins as coactivators for NRs: Coiled-coil Coactivator (CoCoA) and Flightless I (Flil). Here, we will determine the mechanisms by which each of these proteins cooperates with other NR coactivators and contributes to transcriptional activation by NRs. We will define the functional subdomains of CoCoA and Flil by defining their sites of interaction with NRs, p160 coactivators, and/or other known protein interaction partners. We will also define their activation domains, which are used to transmit the activating signal to the transcription machinery, and identify proteins that bind to the activation domains and are thus downstream in the activation signaling pathway. The involvement of CoCoA and Flil as coactivators with other classes of transcription factors will also be investigated. Finally, we will study the variable requirements for CoCoA and Flil and their various functional domains on multiple steroid hormone-regulated promoters (some hormone inducible and some hormone repressible) in the A549 cell line to investigate the relationship between mechanism of coactivator function and promoter architecture.

Principal Investigator/Program Director (Last, first, middle): Stallcup, Michael, Robert RESEARCH &RELATED Other Project Information 1. * Are Human Subjects Involved? m Yes l No 1.a. If YES to Human Subjects Is the IRB review Pending? m Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 2. * Are Vertebrate Animals Used? m Yes l No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? m Yes m No IACUC Approval Date: Animal Welfare Assurance Number 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 7762-PROJECT_SUMMARY.pdf Mime Type: application/pdf 7. * Project Narrative 5806-PROJECT_NARRATIVE.pdf Mime Type: application/pdf 8. Bibliography &References Cited 9321-BIBLIOGRAPHY_AND_REFERENMCEimSe_CTyITpEeD: a.pdpflication/pdf 9. Facilities &Other Resources 9312-FACILITIES_AND_OTHER_RESOMURimCeETSy.pdef: application/pdf 10. Equipment 1550-EQUIPMENT.pdf Mime Type: application/pdf Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): Stallcup, Michael, Robert PROJECT SUMMARY Nuclear receptors (NRs) and other DNA-binding transcription factors regulate transcription of their target genes by recruiting coregulator proteins to the promoter of the target genes. Many coregulators can assist NRs as either coactivators or corepressors, depending on the regulatory context of the promoter. However, the mechanisms that govern whether a specific coregulator functions as coactivator or corepressor is unknown and will be a central focus of this application. Transcriptional repression involves recruitment of corepressor complexes which often include enzymes that deacetylate and make repressive methylation marks on histones. In particular, di- and trimethylation of lysine 9 of histone H3 (H3 K9) in gene promoters has been associated with gene repression. Knock-out mouse studies of the euchromatin-associated H3 K9 methyltransferases G9a and GLP indicated that these two enzymes are responsible for the majority of mono- and dimethylation of H3 K9 in cells. The knock-out mouse results plus additional biochemical studies indicate that G9a and GLP function as heterodimer partners for at least some of their functions. G9a is also associated with corepressor complexes that mediate the effects of several repressive transcription factors. G9a and GLP can also function as coactivators for NRs, suggesting that G9a may play a critical role as a regulatory switch between activation and repression of transcription, depending on the regulatory context on a particular promoter. The goal of this project is to understand the mechanisms of coactivator and corepressor function by G9a and GLP. The central hypothesis is that specific protein- protein interactions determine whether G9a and GLP function as coactivators or corepressors on a given promoter. Among other protein-protein interactions, the ability of G9a and GLP to bind preferentially to histone H3 that is dimethylated at lysine 9 (recently discovered in this laboratory) will be investigated for its role in coregulator function. In addition, common, distinct, and complementary aspects of G9a and GLP function will be defined. Toward that end, the domains and specific protein- protein interactions of the domains of G9a and GLP which are important for their functions as coactivators and corepressors will be determined. Analyses will be performed on both transiently transfected reporter genes and endogenous target genes of G9a and GLP. These studies will thus significantly extend our understanding of the specific contributions of coregulators and histone modifications to transcriptional regulation of genes. In addition, since NRs play many critical roles in normal and pathological regulation of endocrine and metabolic physiology, the proposed studies will provide new knowledge that has important implications for human health. Project Description Page 6

[unreadable] DESCRIPTION (provided by applicant): The University of Southern California (USC) Training Program in Cellular, Biochemical, and Molecular Sciences (CBM Training Program) prepares students for careers in interdisciplinary biomedical research and related occupations. Major goals of the Program are to promote interdisciplinary training and to make students aware of computational methods which will be useful for their current and future research and for their careers. Trainees are selected from 14 different Ph.D. programs at USC which span the entire spectrum of biomedical and biological sciences. This ensures that Trainees receive a multidisciplinary experience in the CBM Program and are the "cream of the crop" at USC. CBM Trainees have diverse backgrounds and research interests, come from many parts of the U.S. and foreign countries, and include under-represented minority students. In addition to the requirements of their Ph.D. programs, Trainees benefit from Training Program-specific activities, including monthly Trainee research presentations, workshops with computational or career development themes, interactions with selected speakers from outside USC, participation in recruitment of new graduate students, training in ethical standards of research, and an annual retreat. The computational theme of the CBM Program takes advantage of areas of special strength at USC in computational genomics and cancer epidemiology, both of which emphasize quantitative treatments of complex data sets. Most CBM Trainees are supported during their second and third years of graduate school and continue to participate in the Program until completion of their Ph.D. degree. The CBM Program works closely with USC's outstanding graduate student recruitment mechanism, called Program in Biomedical and Biological Sciences (PIBBS). During their first year at USC, students in PIBBS rotate with several faculty, chosen from any biologically-oriented Ph.D. program at USC. The outstanding PIBBS web site attracts more than 100 U.S. and more than 500 total applicants per year. Faculty and staff attend national and regional recruitment fairs, including those targeted to under-represented minority students. We also have formal partnerships with BRIDGES-to-the-Ph.D. programs at local minority-serving institutions. These measures ensure that we will continue to attract a diverse group of high-quality trainees. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) has been shown to be an important coactivator that regulates the activity of several nuclear receptors (NR) such as estrogen receptor (ER) and glucocorticoid receptor (GR);as well as transcription factors such as nuclear respiratory factor 1 (NRF-1).

DESCRIPTION (provided by applicant): Steroid hormone receptors (SR) are hormone-regulated transcription factors belonging to the nuclear receptor family. Upon binding hormone, SR bind to specific enhancer or silencer elements on DNA, recruit many coregulators which remodel chromatin, regulate assembly of the transcription complex, and regulate transcription of the neighboring promoters. Defining the specific molecular functions of the many coregulators and how their recruitment and actions are coordinated are key to understanding how transcription is regulated. Among the many coregulators identified, three complexes are known to have critical roles: the steroid receptor coactivators (SRC) bind directly to SRs and anchor histone modifying enzymes and other coregulators to the promoter;the SWI/SNF complex remodels chromatin in an ATP-dependent manner;and the Mediator complex recruits RNA polymerase II. We have recently discovered two SRC-associated coregulators which are required for recruitment of the SWI/SNF and Mediator complexes to steroid hormone-regulated promoters. With this knowledge we can now progress from coactivator discovery and characterization, which has occupied us and the field for the past 15 years, to the definition of what each coregulator contributes to the process of hormone- stimulated transcription activation and how the recruitment and activities of many of the coregulators are coordinated. This will be done by using RNA interference to systematically deplete the endogenous levels of selected coregulators, followed by chromatin immunoprecipitation assays to examine the effects of coregulator depletion on the stepwise modification of histones and assembly of the transcription complex, which occur on endogenous SR target genes in response to the hormone. This study will go beyond the simple cataloguing of promoter occupancy by defining the stepwise requirements for chromatin modifications and transcription factor assembly and by establishing the functional relationships among the various coregulators, histone modifications, and transcription complex components. We will also define the functions of specific coregulator domains by testing the effect of specific mutations (which disrupt coregulator interactions or functions) on the hormone-dependent regulation of endogenous target genes of SRs. To complement these mechanistic studies, we will also define the physiological gene programs controlled by individual coregulators. We and others have shown that individual coregulators are required for the hormonal regulation of some, but not all, of the target genes of a particular SR in a particular cell line. We will use microarray and bioinformatics analyses to define the subset of SR target genes that require individual coregulators. Then we will examine the specific cellular properties or programs (such as growth and migration) controlled by coregulator-specific subsets of steroid hormone regulated genes. This comprehensive analysis will provide the information necessary to assess whether coregulators are reasonable targets for intervention in breast and prostate cancer and in other steroid hormone-related diseases. PUBLIC HEALTH RELEVANCE: The proposed project will generate a new level of understanding for the molecular mechanism of regulation of genes and cellular activities by steroid hormones. Steroid hormones control normal development and play important roles in diseases such as cancer, diabetes, and autoimmune diseases. The new basic knowledge obtained in this project will identify new potential targets for therapeutic intervention in these diseases.

Project Summary/Abstract The Epigenetics and Regulation Program cultivates research collaborations between its members as well as with researchers from other USC Norris Research Programs and peer institutions. These efforts have resulted in: 1) breakthroughs in understanding the basic mechanisms that regulate growth and behavior of normal and cancer cells; and 2) translation of USC Norris findings into new methods for cancer prevention, detection, prognosis, and treatment. The Program integrates research in genomics and epigenetics, cell signaling, and normal and cancer stem cells in order to achieve the overarching goal of understanding regulation in cancer. Members jointly develop and employ novel genomic and epigenomic technologies and computational approaches in order to analyze genes and their regulation and genetic variation in cancers, at the mechanistic level and in large-scale comparative studies. They also study selected critical signaling pathways that drive cancer development and progression with the ultimate goals of understanding cancer risk loci, developing new diagnostic tools, identifying new therapeutic targets, and translating discoveries through collaborations with clinical researchers. Member interactions are fostered through retreats and weekly meetings focused on regulation, epigenetics, bioinformatics, and stem cells that have resulted in a number of inter- and intra- programmatic collaborations. The Program fosters member interactions and drives novel collaborations through its leadership in cancer-focused PhD training programs and by including predoctoral and postdoctoral trainees in weekly Program meetings. Drs. Michael Stallcup and Peggy Farnham co-lead the Program. Dr. Stallcup is an expert in steroid hormone signaling and the regulation of chromatin and transcription by transcription factors and their coregulators, while Dr. Farnham is a leader in genomics, epigenetics, and genome-wide methods of analysis. The Program has 26 members from 18 departments and five schools. Program members have $10M in peer-reviewed funding (direct costs), of which 14% is from NCI, 50% is from NIH, and 30% from other peer-review funding sources. During the project period, members had 445 cancer- relevant publications, of which 31% were inter-programmatic, 23% were intra-programmatic, and 32% were inter-institutional.