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.

Background-Angiotensin type 1 receptor (AT 1 R) blockers (ARB) have been shown to reduce the incidence of type 2 diabetes mellitus by an unknown molecular mechanism. The peroxisome proliferator-activated receptor-␥ (PPAR␥) is the central regulator of insulin and glucose metabolism improving insulin sensitivity. We investigated the regulation of PPAR␥ function by ARBs. Methods and Results-The ARBs irbesartan and telmisartan (10 mol/L) potently enhanced PPAR␥-dependent 3T3-L1 adipocyte differentiation associated with a significant increase in mRNA expression of the adipogenic marker gene adipose protein 2 (aP2), as measured by quantitative real-time polymerase chain reaction (irbesartan: 3.3Ϯ0.1-fold induction; telmisartan: 3.1Ϯ0.3-fold induction; both PϽ0.01). Telmisartan showed a more pronounced induction of aP2 expression in lower, pharmacologically relevant concentrations compared with the other ARBs. The ARB losartan enhanced aP2 expression only at high concentrations (losartan 100 mol/L: 3.6Ϯ0.3-fold induction; PϽ0.01), whereas eprosartan up to 100 mol/L had no significant effects. In transcription reporter assays, irbesartan and telmisartan (10 mol/L) markedly induced transcriptional activity of PPAR␥ by 3.4Ϯ0.9-fold and 2.6Ϯ0.6-fold (PϽ0.05), respectively, compared with 5.2Ϯ1.1-fold stimulation by the PPAR␥ ligand pioglitazone (10 mol/L). Irbesartan and telmisartan also induced PPAR␥ activity in an AT 1 R-deficient cell model (PC12W), demonstrating that these ARBs stimulate PPAR␥ activity independent of their AT 1 R blocking actions. Conclusions-The present study demonstrates that a specific subset of ARBs induces PPAR␥ activity, thereby promoting PPAR␥-dependent differentiation in adipocytes. 3 The underlying mechanism of the insulin-sensitizing/antidiabetic effect of ARBs is widely unknown.The nuclear hormone receptor peroxisome proliferator-activated receptor-␥ (PPAR␥) plays an important role in the regulation of insulin sensitivity. 4 Activated by its ligands such as prostaglandins or synthetic insulin-sensitizing thiazolidinediones/glitazones, PPAR␥ functions as a transcriptional regulator of multiple genes involved in glucose and lipid metabolism, thereby ameliorating type 2 diabetes. 4 To elucidate the underlying mechanisms of the antidiabetic effect of ARBs, we investigated the effects of different ARBs on PPAR␥ function in 3T3-L1 cells, an established cell model to study PPAR␥ function. Methods Cell CultureMouse 3T3-L1 preadipocytes were cultured and differentiated as previously described by using a standard differentiation mixture Semiquantitative RT-PCR and Quantitative Real-Time PCRReal-time polymerase chain reaction (PCR) was performed as previously described with an ABI 7000 sequence detection system for real-time PCR. 6 Mouse 18S ribosomal RNA for real-time PCR and hypoxanthine guanine phosphoribosyl transferase or ␤-actin for semiquantitative reverse transcription (RT)-PCR were chosen as endogenous controls (housekeeping genes). Transfection and Luciferase AssayTransient transfection and...

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...

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