Genome-wide analysis of RXR( binding in mouse liver chromatin with ChIP-SEQ Abstract: This proposal will address core items in Challenge Area 08 (Genomics), specifically targeting Challenge Topic 08-DK-107, Nuclear receptor mediated assembly of functional transcription units. In relation to this application, this Challenge Topic states: '... The application of genome-wide analyses of response element occupancy has the potential to rapidly and comprehensively reveal novel mechanisms of gene regulation.'... A substantial proportion of metabolic and responsive gene regulation in the liver occurs via members of the Nuclear Receptor (NR) superfamily, specifically those regulated by RXR(-containing heterodimer pairs, yet we only know a small fraction of RXR( binding sites in the genome. A gene-by-gene assessment of binding sites is not feasible, while recent technological advances strongly indicate that our current bias towards studying binding sites near the transcription start sites (TSS) vastly underestimates the actual binding that occurs in living cells. In most genome-wide analyses performed to date, for both NR family members and other transcription factors, > 50% of in vivo binding takes place > 10 kb from the TSS, and has not yet been explored. The major unmet Challenges that are now surmountable with current technology, and specifically addressed in this proposal, are: 1.) to identify the global array of genomic binding sites for RXR( in both male and female mouse liver; 2.) correlate RXR( binding to chromatin with gene expression; and 3.) identify roles for other functional domains of RXR( apart from DNA-binding, that drive its participation in chromatin binding and gene expression. These goals will be addressed via the following 2 Aims:
Aim 1 ChIP-SEQ of RXR( in mouse livers vivo: We hypothesize that global delineation of ChIP-SEQ in mouse liver will identify all RXR( binding sites in chromatin. With correlation to Pol II binding, we will identify those regions where RXR( binding is linked to liver gene expression, and compare the findings in male and female mice. We will also explore the mechanisms where regions other than the DNA-binding Domain (DBD) of RXR( participate in gene regulation in mouse liver. We will perform ChIP-SEQ using livers from a uniquely-available hepatocyte-selective DBD-deficient RXR( mice (hs-? DBD- RXR(). Preliminary data indicate informative and non-predictable effects of this internally-truncated RXR( on previously reported RXR(-regulated genes, indicating unexpected roles for other domains and interactions that participate in RXR('s effectiveness in gene regulation.
Aim 2 Validation of RXR( binding in vivo and after IL-1 induced inflammation: We hypothesize that the findings of Aim1 will need to be validated in detail for select genes and regions in order to confirm the global findings. A focus upon genes and regions that show discrepancies between male and female livers, and wt and hs-?DBD- RXR(, will provide the rational framework for select validations. Methodologies will include western blotting, ChIP qPCR, RNA quantitation by rtPCR, exploration by ChIP of other members of the transcription complexes, and gel shifts. We will utilize the findings from previous Aims, and our lab's ongoing delineation of alterations in hepatobiliary transporter gene expression in inflammation, to explore and correlate the consequences in chromatin in response to IL-1 signaling. Genome-wide mapping of RXR( binding regions in mouse liver will provide a framework to jumpstart ongoing investigations into identification of all RXR(-regulated genes in liver in both academia and pharma. With current therapeutic emphases on exploring NR ligands as therapeutics for a variety of liver diseases, these data are critical for finding therapeutic targets, avoiding toxicities, and screening likely candidate molecules. In addition, studies of the hs-?DBD- RXR(, provide a unique window into basic NR functioning, and are likely to provide novel insights into which domains of RXR( are utilized in different transcription complexes.
There is a dire need for effective therapeutics for most liver diseases, since liver disease affects over 10% of the adult US population, and is the 9th leading cause of death. Among the more attractive candidates are drugs that modify liver function through changing activities of master gene regulators, and this proposal aims to fully map all the thousands of sites in the DNA where the master liver gene regulator RXR( binds and functions. Such knowledge is expected to rapidly accelerate rationally-designed and validated drug discovery for liver disease.
|Kosters, Astrid; Sun, Deqiang; Wu, Hao et al. (2013) Sexually dimorphic genome-wide binding of retinoid X receptor alpha (RXR?) determines male-female differences in the expression of hepatic lipid processing genes in mice. PLoS One 8:e71538|
|Kosters, Astrid; Tian, Feng; Wan, Yu-Jui Yvonne et al. (2012) Gene-specific alterations of hepatic gene expression by ligand activation or hepatocyte-selective inhibition of retinoid X receptor-ýý signalling during inflammation. Liver Int 32:321-30|