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De-novo identification of PPARgamma/RXR binding sites and direct targets during adipogenesis.

Hamza MS, Pott S, Vega VB, Thomsen JS, Kandhadayar GS, Ng PW, Chiu KP, Pettersson S, Wei CL, Ruan Y, Liu ET - PLoS ONE (2009)

Bottom Line: Our results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation.Additionally, we find that PPARgamma/RXR association is enriched within the proximity of the 5' region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARgamma as the master transcriptional regulator of adipogenesis.Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARgamma/RXR is in the induction of genes.

View Article: PubMed Central - PubMed

Affiliation: Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore.

ABSTRACT

Background: The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARgamma have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARgamma binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARgamma binding sites in 3T3-L1 preadipocyte cells.

Methodology/principal findings: Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARgamma and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPARgamma and RXR and that they are functionally capable of driving PPARgamma specific transcription. Our results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARgamma/RXR association is enriched within the proximity of the 5' region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARgamma as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARgamma/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPARgamma direct targets that have not been previously reported to promote adipogenic differentiation.

Conclusions/significance: We have identified in a genome-wide manner the binding sites of PPARgamma and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPARgamma function in the context of adipocyte differentiation.

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Related in: MedlinePlus

Association of PPARγ∶RXR heterosites with differentially expressed genes.The majority of genes identified as direct PPARγ target genes are induced during adipogenesis. Genes differentially expressed during adipogenesis that are associated with a PPARγ∶RXR heterosite were called high confidence PPARγ target genes. Those with a PPARγ or RXR monosite were labeled as medium confidence targets. Expression profiles of high confidence, medium confidence, and no target genes are shown as heatmaps (day-2-6). Displayed is the fold difference of cells treated with PPARγ specific siRNA over non-targeting siRNA. Values representing the average of 3 biological replicates per conditions. Average fold difference of expression levels between cells treated with PPARγ specific siRNA over non-targeting siRNA was plotted for high confidence, medium confidence, and no target genes was plotted across the timepoints.
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pone-0004907-g005: Association of PPARγ∶RXR heterosites with differentially expressed genes.The majority of genes identified as direct PPARγ target genes are induced during adipogenesis. Genes differentially expressed during adipogenesis that are associated with a PPARγ∶RXR heterosite were called high confidence PPARγ target genes. Those with a PPARγ or RXR monosite were labeled as medium confidence targets. Expression profiles of high confidence, medium confidence, and no target genes are shown as heatmaps (day-2-6). Displayed is the fold difference of cells treated with PPARγ specific siRNA over non-targeting siRNA. Values representing the average of 3 biological replicates per conditions. Average fold difference of expression levels between cells treated with PPARγ specific siRNA over non-targeting siRNA was plotted for high confidence, medium confidence, and no target genes was plotted across the timepoints.

Mentions: To examine this more closely, we collapsed the array probe sets onto genes and classified a gene as target if it is both regulated during adipogenesis and has at least one binding site (PPARγ and RXR) within 5 kb of the gene's TSS. Since it is highly possible that binding sites farther away from the TSS may also regular its proximate gene, these criteria are likely to be more stringent. We identified 75 high confidence target genes as defined by association with heterosites and 180 lower confidence targets defined by association with monosites (Table S2). Further, we assessed their expression dynamics after PPARγ activation (Figure 5). Our analysis revealed that high confidence targets were induced at significantly higher levels than lower confidence target genes. Genes induced by PPARγ are more commonly associated with RXR/PPARγ binding sites in proximity of the TSS (P = 2.57E-07 to P = 1.91E-38) (Table 1). No significant association is seen with genes repressed by PPARγ activation.


De-novo identification of PPARgamma/RXR binding sites and direct targets during adipogenesis.

Hamza MS, Pott S, Vega VB, Thomsen JS, Kandhadayar GS, Ng PW, Chiu KP, Pettersson S, Wei CL, Ruan Y, Liu ET - PLoS ONE (2009)

Association of PPARγ∶RXR heterosites with differentially expressed genes.The majority of genes identified as direct PPARγ target genes are induced during adipogenesis. Genes differentially expressed during adipogenesis that are associated with a PPARγ∶RXR heterosite were called high confidence PPARγ target genes. Those with a PPARγ or RXR monosite were labeled as medium confidence targets. Expression profiles of high confidence, medium confidence, and no target genes are shown as heatmaps (day-2-6). Displayed is the fold difference of cells treated with PPARγ specific siRNA over non-targeting siRNA. Values representing the average of 3 biological replicates per conditions. Average fold difference of expression levels between cells treated with PPARγ specific siRNA over non-targeting siRNA was plotted for high confidence, medium confidence, and no target genes was plotted across the timepoints.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654672&req=5

pone-0004907-g005: Association of PPARγ∶RXR heterosites with differentially expressed genes.The majority of genes identified as direct PPARγ target genes are induced during adipogenesis. Genes differentially expressed during adipogenesis that are associated with a PPARγ∶RXR heterosite were called high confidence PPARγ target genes. Those with a PPARγ or RXR monosite were labeled as medium confidence targets. Expression profiles of high confidence, medium confidence, and no target genes are shown as heatmaps (day-2-6). Displayed is the fold difference of cells treated with PPARγ specific siRNA over non-targeting siRNA. Values representing the average of 3 biological replicates per conditions. Average fold difference of expression levels between cells treated with PPARγ specific siRNA over non-targeting siRNA was plotted for high confidence, medium confidence, and no target genes was plotted across the timepoints.
Mentions: To examine this more closely, we collapsed the array probe sets onto genes and classified a gene as target if it is both regulated during adipogenesis and has at least one binding site (PPARγ and RXR) within 5 kb of the gene's TSS. Since it is highly possible that binding sites farther away from the TSS may also regular its proximate gene, these criteria are likely to be more stringent. We identified 75 high confidence target genes as defined by association with heterosites and 180 lower confidence targets defined by association with monosites (Table S2). Further, we assessed their expression dynamics after PPARγ activation (Figure 5). Our analysis revealed that high confidence targets were induced at significantly higher levels than lower confidence target genes. Genes induced by PPARγ are more commonly associated with RXR/PPARγ binding sites in proximity of the TSS (P = 2.57E-07 to P = 1.91E-38) (Table 1). No significant association is seen with genes repressed by PPARγ activation.

Bottom Line: Our results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation.Additionally, we find that PPARgamma/RXR association is enriched within the proximity of the 5' region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARgamma as the master transcriptional regulator of adipogenesis.Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARgamma/RXR is in the induction of genes.

View Article: PubMed Central - PubMed

Affiliation: Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore.

ABSTRACT

Background: The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARgamma have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARgamma binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARgamma binding sites in 3T3-L1 preadipocyte cells.

Methodology/principal findings: Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARgamma and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPARgamma and RXR and that they are functionally capable of driving PPARgamma specific transcription. Our results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARgamma/RXR association is enriched within the proximity of the 5' region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARgamma as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARgamma/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPARgamma direct targets that have not been previously reported to promote adipogenic differentiation.

Conclusions/significance: We have identified in a genome-wide manner the binding sites of PPARgamma and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPARgamma function in the context of adipocyte differentiation.

Show MeSH
Related in: MedlinePlus