This application addresses the broad Challenge Area (08) Genomics and specific Challenge Topic 08-DK-107: Nuclear Receptor mediated assembly of functional transcriptional units. A major goal of this laboratory is to understand the transcriptional mechanisms by which nuclear receptors (NRs) regulate circadian and metabolic physiology. The heme receptor Rev-erb was identified by the PI in 1989, and has since emerged as an important repressor of key genes controlling circadian rhythm and glucose metabolism. Prior studies of Rev- erb have been based on candidate gene approaches focused on promoters. Thus, it is not surprising that only about ten Rev-erb-target genes are known, given that genome-wide location analyses for other nuclear receptors have revealed that most binding occurs outside of promoters. We hypothesize that Rev-erb functions through binding of hundreds of genes, many tissue-specifically. To address this, we will perform a genome-wide analysis of Rev-erb binding in mouse liver and white adipose tissues.
Specific Aim 1 is to identify Rev-erb gene targets on a genome-wide scale in liver at different circadian times, and understand their modes of regulation. We hypothesize that circadian fluctuation of Rev-erb protein level will affect its ability to access recognition sequences in the genome, which, in turn, will have downstream effects on circadian and metabolic processes. We will test this by genome-wide location analysis using chromatin immunoprecipitation (ChIP) followed by sequencing of the isolated DNA (ChIP-seq).
Specific Aim 2 is to identify Rev-erb gene targets on a genome-wide scale in liver from fed versus fasted mice and from mice under restricted feeding, and understand their modes of regulation. We hypothesize that metabolic regulation of Rev-erb activity will alter Rev-erb binding at many genes, and will test this by ChIP- seq using liver isolated from fed and fasted mice. Preliminary data reveal that Rev-erb protein level is higher in liver from fasted mice than in liver from fed mice, suggesting that Rev-erb binding activity may be modulated by metabolic signals.
Specific Aim 3 is to identify Rev-erb gene targets on a genome-wide scale in white adipose tissue, and understand their modes of regulation. We hypothesize that Rev-erb mediates circadian expression of adipocyte genes and will test this by performing ChIP-seq for adipose Rev- erb at different circadian times and feeding regimens. This will expand our understanding of Rev-erb function in adipose biology and, when combined with the liver data set, it will identify tissue-specific targets and functions for Rev-erb in liver and adipose tissues. These studies can be accomplished in a two-year time frame because we have validated antibodies for ChIP of endogenous Rev-erb in metabolic tissues, and the experimental paradigms involve short-term studies. The genome-wide insights into in vivo tissue-specific regulation of circadian and metabolic genes will be enormous, and contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States.
Metabolic disorders such as diabetes and obesity are affected by sleep patterns and shift work. Thus, it is exciting that the focus of this proposal, the nuclear receptor Rev-erb, has emerged as a critical link between circadian and metabolic physiology. The proposed experiments aim to determine the extent that Rev-erb contributes to, or even controls, circadian and metabolic processes and the crosstalk that occurs between them. Ultimately, greater insight into the regulation of circadian rhythm and metabolism by Rev-erb will contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States.
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