The Nuclear Receptor Signaling Atlas: Development of a Functional Atlas of Nuclear Receptors

Ronald N. Margolis, Ronald M. Evans, Bert W. O’Malley and the NURSA Atlas Consortium1

National Institute of Diabetes and Digestive and Kidney Diseases (R.N.M.), National Institutes of Health, Bethesda, Maryland 20892; The Salk Institute (R.M.E.), La Jolla, California 92037; and Baylor College of Medicine (B.W.O.), Houston, Texas 77030

Address all correspondence and requests for reprints to: Ronald N. Margolis, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Endocrinology, 6707 Democracy Boulevard, Bethesda, Maryland 20892-6600. E-mail rm76f{at}nih.gov.


    ABSTRACT
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The Nuclear Receptor Signaling Atlas (NURSA) was developed by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the National Institute on Aging (NIA), and the National Cancer Institute (NCI) of the National Institutes of Health (NIH); the aim of NURSA is to utilize classical approaches to validate existing hypotheses and exploit new and emerging technologies to formulate and test new hypotheses that might elucidate the program of nuclear receptor (NR) structure, function, and role in disease. The means for carrying out this ambitious program required development of interactions among investigators and the combined application of new high-throughput technologies and existing approaches to allow for both mechanistic studies and accrual of large datasets in a discovery-based research effort, all leading to advances with implications for the missions of the NIDDK, NIA, and NCI. A team-based multidisciplinary approach has allowed for both objectives to proceed simultaneously, tied together via a central bioinformatics resource and one web-accessible venue (www.nursa.org). The ultimate goals for the NURSA consortium are to: 1) establish the mechanistic principles of NR function, 2) characterize NR-coregulator complex formation and regulation, 3) map protein-protein interactions for coregulators, 4) identify candidate downstream target genes of NR action, 5) identify target tissue expression of NRs, 6) understand the regulation of NR expression and, 7) integrate existing and emerging information through NURSA bioinformatics tools.

MAJOR PROGRESS OVER the past four decades has revealed nuclear receptors (NRs) as ligand-dependent and -independent transcription factors that play important roles in development, reproduction, and metabolism (1). More recent advances have shown that the keys to understanding the mechanism(s) of action of NRs are both complex and interrelated (2). Studies of the genomic effects of NRs have been a staple of investigation into the mechanism of action of NRs (3), with the emerging understanding that nongenomic effects also represent part of the signaling web of NR action, creating new venues for understanding function (4). Classical steroid hormone ligands have been complemented by classes of compounds drawn from diet, metabolism, and elsewhere, whereas for other NRs, particularly the orphan nuclear receptors (ONR), identification of definitive ligands (if any) remains elusive (1, 2). Superimposed on the receptors themselves are classes of cytoplasmic and nuclear proteins and chromatin remodeling/transcription complexes as well as RNA transcripts that act as chaperones, and/or components in signaling cascades (5). Posttranslational modification involving protein-protein interactions that lead to histone acetylation/deacetylation/methylation, ubiquitination/sumoylation, and/or phosphorylation/dephosphorylation have defined crucial roles for coactivators, corepressors, and mediators in carrying out the cellular program of NR action (6).

The presentation of full drafts of the human, mouse, and rat genomes has resulted in the identification of as many as 49 NRs (1, 7, 8, 9), although in lower organisms many more have been identified, and new algorithms for data mining suggest the presence of additional NRs in areas of the genome that have not yet been characterized for functions that are not yet understood (10). NR action may thus be seen as a complex problem in biomedical science requiring application of systematic, multidisciplinary approaches.

With that in mind, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), together with the National Institute on Aging (NIA) and the National Cancer Institute (NCI) [all of the National Institutes of Health (NIH)], initiated the Nuclear Receptor Signaling Atlas (NURSA) through a request for application issued in 2001 (http://grants1.nih.gov/grants/guide/rfa-files/RFA-DK-01–026.html) that solicited applications for development of a functional atlas of orphan nuclear receptors (ONRs) to coalesce, develop, and advance knowledge in this area. The initial focus on ONRs was based on an emerging understanding at the time of their importance to diseases and conditions central to the missions of NIDDK, NIA, and NCI, including obesity, diabetes and its complications, osteoporosis, hormone-dependent cancers, digestive diseases, processes of aging, and xenobiotic metabolism. The concept of an "atlas" was designed to bring to play a blend of approaches, some classical hypothesis driven and some discovery based and driven by exploitation of novel and emerging technologies, to foster greater understanding of NR structure/function and role in disease. The atlas was funded in August 2002 with Bert O’Malley (Baylor College of Medicine) and Ron Evans (Salk Institute) as principal investigator and co-principal investigator, respectively, with the following overriding objectives: 1) to provide important data to the community in a timely fashion and 2) to serve as a catalyst for progress by leveraging ongoing and newly initiated efforts to elucidate the structure and function of ONRs. Soon after start-up, however, the scope was expanded to include all NRs. With that, the functional atlas of ONRs became the NURSA and the goal became to utilize classical approaches to validate existing hypotheses and exploit new and emerging technologies to pinpoint precisely where to delve further to formulate and test new hypotheses. The means for carrying out this ambitious program required development of team-based interactions among individual investigators.

In so doing, individual investigators were asked to work together on the following: 1) discovery-based applications (e.g. high throughput) designed for data collection to both add to the knowledge base of NRs and serve as the means for the generation of new hypotheses and 2) the traditional hypothesis-driven reductional research mode designed to elucidate underlying mechanism(s) of action. All efforts were designed to lead to advances with implications for the missions of the NIDDK, NIA, and NCI. The team-based approach has allowed for both objectives to proceed simultaneously, with a central bioinformatics resource core acting to centralize the flow of information and ideas in one web-accessible venue.

The application of discovery-based research brings an unbiased approach to NRs, allowing for informed follow-up, and the development of new lines of research to:

Establish the basis for studies on the mechanistic principles of NR function
Characterize NR-coregulator complex formation and regulation
Map protein-protein interactions for coregulators
Identify candidate downstream target genes of NR action
Identify target tissue expression of NRs
Understand the regulation of NR expression
Integrate existing and emerging information through NURSA bioinformatics tools

The NURSA teams were oriented around three core resources and two parallel research (bridging) tracks. Specifically, resource cores devoted to microarray/quantitative PCR (QPCR), expression profiling, and bioinformatics have been used to tie together two parallel research tracks focused on receptors and coregulators, respectively. The cores have fostered an environment of innovation resulting in rapid development and implementation of technologies and the accrual of large and relevant datasets. The bioinformatics resource core has sought to capture these datasets in formats that permit presentation in useful and interactive ways. Further development of the capabilities of this resource are crucial to the goal of broadening the reach of NURSA by incorporating validated datasets developed by others in the research community both here and abroad. The development of a web portal (www.nursa.org) represents the window on this process and the starting point through which the community of investigators may access the efforts of NURSA investigators.

The interrelated components of NURSA include the resource cores to facilitate support for a series of bridging strands. Cores included the following:

Microarray/QPCR: Designed to develop and use cDNA and oligonucleotide microarrays to interrogate the expression of thousands of genes to explore the roles of specific receptors in regulating complex programs of gene expression and map the integrated function of genes that serve as targets for NRs. In addition, the application of high-throughput robotic QPCR methods permits collection of datasets that map the expression of NRs and coregulators themselves.
Expression profiling: Determine cell-specific expression profiles of NRs and coregulators. Through the generation of transgenic lines expressing selected NRs in different cell types during various stages of development, shed light upon NR function in vivo in transgenic models that mimic the active state of a NR.
Bioinformatics: To act as an atlas- and community-accessible repository for both archived literature and database information on nuclear receptors, as well as data generated by the NURSA components. In addition, this core will develop and adapt bioinformatics tools to facilitate further mining and interrogation of data.

Bridging strands included the following:

Bridging strand A (receptors): Characterize the expression patterns of NRs in 49 tissues of adult mice using a standardized high-throughput methodology. Follow-on studies will focus on gene expression profiling of NR target genes in wild-type and null mice and cultured cell lines by cDNA and oligonucleotide microarrays. One focus will be to profile expression of metabolically relevant nuclear receptors in tissue arrays to understand underlying physiology.
Bridging strand B (coregulators): Development of proteomics approaches using traditional and medium-throughput methods to identify specific NR-coactivator protein complexes in HeLa, and other, cells. Composition of complexes will be analyzed in cell-specific and differentiation status- and cellular signaling-dependent contexts with an emphasis on identification of putative posttranslational modifications relevant to function. Creation and characterization of stable cell lines overexpressing corepressors will help to identify interacting proteins and target genes in living cells. Finally, efforts to identify, characterize, and categorize peptides that interact efficiently and specifically with coactivators and corepressors in complex with specific NRs will be undertaken.

The development of a consortium, utilizing a cooperative funding agreement, provides a discrete administrative structure involving governance through a Steering Committee. The latter is chaired by the principal investigator and co-principal investigator to oversee the application of systematic, multidisciplinary approaches. An NIH Scientific Program Officer is a member of the Steering Committee and, through this vehicle, NIH input is guaranteed. Use of a Steering Committee for governance fosters development of consensus among the participants on procedures, objectives, and prioritization. Regular consultation among the members of the consortium results, in turn, in more rapid progress than might be possible for individual investigators working either alone or in small collaborations. Added flexibility is contained in a pilot and feasibility program of small-budget, short-duration supplements made to individuals not initially part of the NURSA and designed to add fresh ideas and new expertise. The combined effect has been to increase and broaden the reach of the NURSA and permit important upgrades to capabilities. Through the use of milestones, the consortium periodically evaluates progress and determines the pace and direction of its movement. Metrics for success include important publications in the literature, new collaborations with investigators outside the NURSA (at least partly the result of web-based interfaces), and transformations in the current state of the art. The utilization of milestones has permitted a timely assessment of progress set against the backdrop of specific tasks and goals. These have been updated periodically and are considered fluid, as the Steering Committee responds to progress and advances in technology.

Integral to defining the success of the NURSA is the concept of translation. "Big science" projects, like the NURSA, designed to answer complex problems in biological science have a reference point that interdigitates with the overriding mission of the NIH to improve the health of the American people. Thus, an important goal of the NURSA is to work toward identification of translational potential(s) and move the agenda forward to pursue outcomes that have a positive effect on public health. Equally important is the need to provide for outreach to bring members of interested research communities to NURSA and vice versa. The primary vehicle for communication will be the NURSA web site through which datasets and results of NURSA activities will be presented. Initial activity on the web site has indicated that investigators both here and abroad have made the NURSA web site a regular source for information about NRs (data not shown). Participation by industry has been forthcoming through the provision of datasets for NR ligands. Development of bioinformatics resources necessary to integrate these datasets is underway and will ultimately allow for data mining for a particular NR, associated coregulator(s), and putative ligand(s).

Although NURSA has been incorporated into current funding plans of the contributing NIH institutes, continued support will depend on overall funding levels for the NIH and the pace of achievement of the goals of www.NURSA.org . Continuation of individual components of NURSA will depend on the course taken by these components and could involve independent funding through the traditional investigator-initiated regular research grant. Clearly, the central role played by the bioinformatics core resource and the datasets collected and presented to the general research community through the web site represent a legacy whose continuation will be of paramount importance. Planning for long-term maintenance of this valuable resource will entail discussions between the investigators, the NIH, scientific organizations (e.g. The Endocrine Society), and potentially industry.

With this discrete series of publications of NURSA work, representing a sampling of ongoing efforts, the full potential of the goal to catalyze progress in NR research has begun to be revealed. A mix of discovery-based, hypothesis-generating, and traditional hypothesis-driven mechanistic work demonstrates the power of a team science approach when applied to a complex problem in biomedical science. It is the intent of the NURSA consortium to continue to provide these results in a manner and fashion that invites participation by all members of the scientific community in further validation and utilization of the data. It is the hope that, by doing so, knowledge in the entire field may be advanced, new hypotheses generated, and movement made toward greater progress in revealing the many facets of NR signaling, structure, function, and role in health and disease.


    ACKNOWLEDGMENTS
 
The NURSA Atlas Consortium consists of the following members:

NIH

Ronald Margolis (NIDDK)
Philip Smith (NIDDK)
Frank Bellino (NIA)
Suresh Mohla (NCI)
Baylor College of Medicine
Bert O’Malley
Ming-Jer Tsai
David Moore
Sophia Tsai
Neil McKenna
Franco DeMayo
Austin Cooney
Michael Mancini
Jun Qin
Rainer Lanz
Zeljko Jericevic
Orla Conneely
Adam Antebi

Salk Institute

Ron Evans
Michael Downes
Ruth Yu
Andrew Dillin

University of Pennsylvania

Mitch Lazar

University of Texas Southwestern Medical School

David Mangelsdorf
Steve Kliewer
Angie Bookout

Duke University

Donald McDonnell

Van Andel Institute

Eric Xu

Beckman Res. Institute

Barry Forman

University of Rochester

Yi-Fen Lee

University of California San Diego

Christopher Glass


    FOOTNOTES
 
Funding of the NURSA is through a grant provided by the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute on Aging, and the National Cancer Institute (U19DK062434).

First Published Online July 28, 2005

1 See Acknowledgments for a list of members of the NURSA Atlas Consortium. Back

Abbreviations: NR, Nuclear receptor; ONR, orphan nuclear receptor; QPCR, quantitative PCR;

Received for publication November 16, 2004. Accepted for publication July 6, 2005.


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