Peroxisome proliferator-activated receptor ␥(PPAR␥), a nuclear receptor and the target of anti-diabetic thiazolinedione drugs, is known as the master regulator of adipocyte biology. Although it regulates hundreds of adipocyte genes, PPAR␥ binding to endogenous genes has rarely been demonstrated. Here, utilizing chromatin immunoprecipitation (ChIP) coupled with whole genome tiling arrays, we identified 5299 genomic regions of PPAR␥ binding in mouse 3T3-L1 adipocytes. The consensus PPAR␥/RXR␣ "DR-1"-binding motif was found at most of the sites, and ChIP for RXR␣ showed colocalization at nearly all locations tested. Bioinformatics analysis also revealed CCAAT/enhancer-binding protein (C/EBP)-binding motifs in the vicinity of most PPAR␥-binding sites, and genome-wide analysis of C/EBP␣ binding demonstrated that it localized to3350 of the locations bound by PPAR␥. Importantly, most genes induced in adipogenesis were bound by both PPAR␥ and C/EBP␣, while very few were PPAR␥-specific. C/EBP␤ also plays a role at many of these genes, such that both C/EBP␣ and ␤ are required along with PPAR␥ for robust adipocyte-specific gene expression. Thus, PPAR␥ and C/EBP factors cooperatively orchestrate adipocyte biology by adjacent binding on an unanticipated scale.[Keywords: PPAR␥; C/EBP; adipocyte; genome wide; ChIP-chip] Supplemental material is available at http://www.genesdev.org.

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The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia. However, the normal function of DOT1L in mammalian gene regulation is poorly understood. Here we report that DOT1L recruitment is ubiquitously coupled with active transcription in diverse mammalian cell types. DOT1L preferentially occupies the proximal transcribed region of active genes, correlating with enrichment of H3K79 di-and trimethylation. Furthermore, Dot1l mutant fibroblasts lacked H3K79 di-and trimethylation at all sites examined, indicating that DOT1L is the sole enzyme responsible for these marks. Importantly, we identified chromatin immunoprecipitation (ChIP) assay conditions necessary for reliable H3K79 methylation detection. ChIP-chip tiling arrays revealed that levels of all degrees of genic H3K79 methylation correlate with mRNA abundance and dynamically respond to changes in gene activity. Conversion of H3K79 monomethylation into di-and trimethylation correlated with the transition from low-to high-level gene transcription. We also observed enrichment of H3K79 monomethylation at intergenic regions occupied by DNA-binding transcriptional activators. Our findings highlight several similarities between the patterning of H3K4 methylation and that of H3K79 methylation in mammalian chromatin, suggesting a widespread mechanism for parallel or sequential recruitment of DOT1L and MLL to genes in their normal "on" state.Histone lysine methylation encodes genomic functions into the chemical state of nucleosomes (38). The collective actions of lysine methyltransferase and lysine demethylase enzymes maintain a landscape of steady-state methylation of histones around which eukaryotic DNA is packaged. Histone methylation can facilitate or abrogate a variety of protein-protein interactions occurring along the chromatin fiber, thus permitting stable regulation over localized regions of the genome. Several recent high-throughput descriptions of histone lysine methylation across mammalian genomes have documented the pervasiveness of this form of epigenetic organization (2, 15, 23). However, the full biological significance of most histone lysine methylation pathways in mammals has yet to be revealed.Methylation of histone H3 at lysine 79 (H3K79) is conserved among most eukaryotic species. In budding yeast, nearly 90% of histone H3 bears monomethylation (H3K79me1), dimethylation (H3K79me2), or trimethylation (H3K79me3) at lysine 79, all catalyzed exclusively by the histone methyltransferase Dot1 (27, 46). H3K79 methylation is widely distributed across the euchromatic yeast genome but markedly depleted at heterochromatic mating-type, ribosomal DNA, and telomeric loci (26,30). Genes in these regions are controlled by silent information regulator (SIR) proteins, which can bind nucleosomes and silence transcription (reviewed in reference 33). Genetic, as well as biochemical, evidence suggests a mutual antagonism between H3K79 methylation by D...

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors which functions as a master regulator of adipocyte differentiation and metabolism. Here we review recent breakthroughs in the understanding of PPARγ gene regulation and function in a chromatin context. It is now clear that multiple transcription factors team up to induce PPARγ during adipogenesis, and that other transcription factors cooperate with PPARγ to ensure adipocyte-specific genomic binding and function. We discuss how this differs in other PPARγ-expressing cells such as macrophages, and how these genome-wide mechanisms are preserved across species despite modest conservation of specific binding sites. These emerging considerations inform our understanding of PPARγ function as well as adipocyte development and physiology.

The transcriptional mechanisms by which temporary exposure to developmental signals instigates adipocyte differentiation are unknown. During early adipogenesis, we find transient enrichment of the glucocorticoid receptor (GR), CCAAT/enhancer-binding protein b (CEBPb), p300, mediator subunit 1, and histone H3 acetylation near genes involved in cell proliferation, development, and differentiation, including the gene encoding the master regulator of adipocyte differentiation, peroxisome proliferator-activated receptor g2 (PPARg2). Occupancy and enhancer function are triggered by adipogenic signals, and diminish upon their removal. GR, which is important for adipogenesis but need not be active in the mature adipocyte, functions transiently with other enhancer proteins to propagate a new program of gene expression that includes induction of PPARg2, thereby providing a memory of the earlier adipogenic signal. Thus, the conversion of preadipocyte to adipocyte involves the formation of an epigenomic transition state that is not observed in cells at the beginning or end of the differentiation process. Transcription factors control cell fate decisions by programming the expression of large gene sets, often through mechanisms impacting the post-translational modification of histone proteins in chromatin (Allis et al. 2007). Genome-wide changes to histone modification patterns occur as cells differentiate, such that distinct epigenomes are present in different cell types (Bernstein et al. 2007). A cell's epigenome is thought to be involved in establishing its identity, yet whether histone modifications transmit the memory of a given cell state or implement the memory once it is transmitted by a distinct mechanism is currently unclear.The epigenomic factors controlling adipocyte cell differentiation are not known. Adipocytes, the major fatcontaining component of adipose tissue, are terminally differentiated cells derived from mesenchymal precursors (Ailhaud et al. 1992). Study of adipocyte precursors in vivo is difficult because adipose tissue in rodents is undetectable macroscopically until shortly before birth.Thus, most of what is known about the molecular basis of adipocyte differentiation derives from tissue culture models, one of the best characterized being the mouse 3T3-L1 model Kehinde 1975, 1976). Temporary exposure to a mix of insulin, glucocorticoid, and an inducer of cAMP signaling triggers adipogenesis, changing the expression of hundreds of genes, including a variety of transcription factors that regulate one another as well as structural genes (Farmer 2006). One of these encodes peroxisome proliferator-activated receptor g (PPARg), a sequence-specific transcription factor that is necessary (Barak et al. 1999;Kubota et al. 1999;Rosen et al. 1999) and sufficient (Tontonoz et al. 1994;Hu et al. 1995;Shao and Lazar 1997) for adipogenesis, and is hence termed the master regulator (Rosen et al. 2002). CCAAT/ enhancer-binding protein (CEBP) transcriptional regulators also play a major role, and each can induce adip...