Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Cell-Specific Determinants of PPARg Function in Adipocytes and Macrophages

ABSTRACT: 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 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 the same or different genomic locations compared to adipocytes. Here, utilizing chromatin immunoprecipitation and high throughput sequencing (ChIP-seq), we demonstrate that PPAR cistromes in adipocytes and macrophages are predominantly cell type specific. In 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 cells that are neither macrophages nor adipocytes, 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 chromatin accessibility, histone marks, and transcription factors. Genomic occupancy profiled by high throughput sequencing (ChIP-seq) from mouse macrophages for PPARgamma, PU.1, C/EBPbeta, H3K9Ace; and from 3T3-L1 adipocytes for PPARgamma and H3K9Ace Masking file used to subtract input bias areas prior to generating final peak lists is linked below.

ORGANISM(S): Mus musculus

SUBMITTER: Mitchell Lazar

PROVIDER: E-GEOD-21314 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Cell-specific determinants of peroxisome proliferator-activated receptor gamma function in adipocytes and macrophages.

Lefterova Martina I MI Steger David J DJ Zhuo David D Qatanani Mohammed M Mullican Shannon E SE Tuteja Geetu G Manduchi Elisabetta E Grant Gregory R GR Lazar Mitchell A MA

Molecular and cellular biology 20100222 9

The nuclear receptor peroxisome proliferator activator receptor gamma (PPARgamma) is the target of antidiabetic thiazolidinedione drugs, which improve insulin resistance but have side effects that limit widespread use. PPARgamma 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 PPARgamma binding in macrophages occurs at genomic locat ...[more]

PMID: 20176806

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Project description:The winged helix protein FOXA2 and the nuclear receptor PPARg are highly conserved, regionally-expressed transcription factors that regulate networks of genes controlling complex metabolic functions. Cistrome analysis for FOXA2 in mouse liver and PPARg in mouse adipocytes has previously produced consensus binding sites that are nearly identical to those used by the factors in human cells. Despite this conservation of the canonical binding motif, we report here that the great majority of specific binding regions for FOXA2 in human liver and for PPARg in human adipocytes are not in the orthologous locations to the mouse genome. Nevertheless, gene-centric analysis reveals strong shared transcription factor occupancy near genes in tissue-specific metabolic pathways that are functionally conserved across species. Genes with only species-specific binding sites fail to show enrichment for these pathways. Thus, the biological functions of transcription factors that control specific metabolic functions are highly shared across species. Two TFs, FOXA2 and PPARg, were studied for genome-wide conservation of binding between mouse and human in specific tissues/cell-types (liver for FOXA2, adipocytes for PPARg). The number of replicates for each TF was chosen to obtain a comparable number of reads between the TFs and species. Human FOXA2 ChIP-seq was performed on two biological replicates of human liver samples, in three technical replicates each. Input DNA was also collected and sequenced from both biological samples. Mouse FOXA2 ChIP-seq was performed on four biological replicates of mouse liver samples. The ChIP and sequencing were repeated on two of these biological replicates to create technical replicates for additional sequence reads. Input DNA was sequenced from three additional mouse livers. Human PPARg ChIP-seq was performed on a human adipocyte cell-line (SGBS) differentiated in two replicate cultures. Input DNA was also collected and sequenced from one culture. Mouse PPARg ChIP-seq was performed on 3T3-L1 cells differentiated into adipocytes in culture in a single replicate, and this sequence data was pooled with existing data previously generated by the same lab, already available in GEO (GSE21314). A standard pool of input DNA sample sequence from multiple mouse tissue was used for analyzing the Mouse PPARg ChIP-seq data.

Project description:Recent meta-analyses of genome wide association studies (GWAS) have identified approximately 150 loci that are associated with coronary artery disease (CAD). To link the causal genes in these loci to functional transcriptional networks, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with human coronary artery smooth muscle cells (HCASMC) to investigate the cellular and molecular program downstream of one CAD associated transcription factor, TCF21. Analysis of the TCF21 downstream target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including growth factor binding (PDGF, VEGF), matrix interactions (“integrin binding,” “cell adhesion”), and smooth muscle contraction (“actin filament-based processes,” “actin cytoskeleton”). Motif searches of peak sequences confirmed the canonical E-box CAGCTG as the likely binding sequence for TCF21, but also identified bZip motifs that coordinate binding of AP1 family transcription factors. Follow-up ChIP-Seq studies verified enriched binding of bZip factors JUN and JUND to TCF21 target loci. Importantly, analysis of the representation of CAD genes among TCF21 target loci showed highly significant enrichment. Further, expression quantitative trait variation mapped to target genes of TCF21 were significantly enriched among variants with low P-values in the GWAS analyses, and single nucleotide polymorphisms within TCF21 peaks were shown to be in linkage disequilibrium with CAD-associated SNPs, suggesting a functional interaction between TCF21 binding and causal variants in other CAD disease loci. Thus, data and analyses presented here provide evidence for a transcriptional regulatory network that links TCF21 function in smooth muscle cells with a number of other CAD-associated genes, and suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology. We did ChIP-Seq on human coronary artery smooth muscle cells grown in SmGM-2 Smooth Muscle Growth Medium-2 including hEGF, insulin, hFGF-B and FBS, but without antibiotics (Lonza, #CC-3182. We did immunoprecipitations with two antibodies (Sigma HPA013189 and Abcam 49475). We conducted two biological replicates for each antibody, and also did an IgG control for these studies. For these experiments, sequencing was done on an Illumina GAII, single end reads. In separate set of experiments we performed ChIP-Seq for JUN (Ab sc-1694) and JUND (Ab sc-74) with the same HCASMC cells under the same culture conditions. For these studies we did one replicate for each antibody, and also included an IgG control. Sequencing for the JUN and JUND studies was paired ends, on an Illumina HiSeq machine.

Dataset Information

Cell-Specific Determinants of PPARg Function in Adipocytes and Macrophages

Publications

Cell-specific determinants of peroxisome proliferator-activated receptor gamma function in adipocytes and macrophages.

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