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.

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...

The nuclear receptor peroxisome proliferator activator receptor ␥ (PPAR␥) is the target of antidiabetic thiazolidinedione drugs, which improve insulin resistance but have side effects that limit widespread use. PPAR␥ is required for adipocyte differentiation, but it is also expressed in other cell types, notably macrophages, where it influences atherosclerosis, insulin resistance, and inflammation. A central question is whether PPAR␥ binding in macrophages occurs at genomic locations the same as or different from those in adipocytes. Here, utilizing chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we demonstrate that PPAR␥ cistromes in mouse adipocytes and macrophages are predominantly cell type specific. In thioglycolate-elicited macrophages, PPAR␥ colocalizes with the hematopoietic transcription factor PU.1 in areas of open chromatin and histone acetylation, near a distinct set of immune genes in addition to a number of metabolic genes shared with adipocytes. In adipocytes, the macrophage-unique binding regions are marked with repressive histone modifications, typically associated with local chromatin compaction and gene silencing. PPAR␥, when introduced into preadipocytes, bound only to regions depleted of repressive histone modifications, where it increased DNA accessibility, enhanced histone acetylation, and induced gene expression. Thus, the cell specificity of PPAR␥ function is regulated by cell-specific transcription factors, chromatin accessibility, and histone marks. Our data support the existence of an epigenomic hierarchy in which PPAR␥ binding to cell-specific sites not marked by repressive marks opens chromatin and leads to local activation marks, including histone acetylation.Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a nuclear receptor that regulates essential aspects of adipocyte biology, including insulin sensitivity, lipogenesis, and survival, and is the target of anti-diabetic thiazolidinedione drugs (39, 69). Recent genome-wide studies of adipocytes (40,53) have demonstrated that PPAR␥ localizes preferentially to lipid and carbohydrate metabolism genes, many of which are downregulated by PPAR␥ knockdown (62). It has also become apparent that in vivo PPAR␥ binding occurs predominantly as a heterodimer with retinoid X receptor (RXR) at direct repeats of the sequence AGGTCA separated by a single base pair, i.e., DR1 elements, as predicted by in vitro studies and a small number of previously known target genes (61). Furthermore, CCAAT/enhancer-binding proteins (C/EBPs) were found to colocalize with PPAR␥ at the majority of its binding sites and to have cooperative effects on target gene transcription (40).The two isoforms of PPAR␥, ␥1 and ␥2, are transcribed from alternative start sites and are most abundant in adipocytes, which require PPAR␥ for differentiation (39, 69), although other cell types express lower levels of the ␥1 isoform (10, 72). Among these, macrophages have garnered much attention for their ability to affect metabolism in a number of tissues...

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Pulmonary artery pulsatility index was independently associated with survival in PAH, highlighting the utility of PAPi in combination with other key measures for risk stratification in this population.