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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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3T3L1 cells as model system for adipocyte differentiation.Undifferentiated 3T3-L1 fibroblast undergoes adipogenesis upon treatment with Dexamethasone and IBMX, followed by Insulin and Rosiglitazone after two days. Adipogenesis was completely abrogated by PPARγ specific siRNA. a) Adipogenesis was monitored by Western blot for induction of adipocyte-specific marker genes. Beta-actin served as loading control. b) accumulation of lipid droplets in differentiated 3T3-L1 cells was visualized by OilRed-O staining c) Schematic outline of the experimental setup: 3T3-L1 cell were treated with Dexamethasone and IBMX at confluency 2 days before stimulation with Rosiglitazone and Insulin. Samples for ChIP were taken at day 4. Samples for expression analysis were taken at all time points indicated. For each time point cells treated with PPARγ specific and control siRNA prior to the differentiation protocol were collected in 3 biological replicates. Microarray data was submitted to the GEO repository under GEO Submission (GSE12929).
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pone-0004907-g001: 3T3L1 cells as model system for adipocyte differentiation.Undifferentiated 3T3-L1 fibroblast undergoes adipogenesis upon treatment with Dexamethasone and IBMX, followed by Insulin and Rosiglitazone after two days. Adipogenesis was completely abrogated by PPARγ specific siRNA. a) Adipogenesis was monitored by Western blot for induction of adipocyte-specific marker genes. Beta-actin served as loading control. b) accumulation of lipid droplets in differentiated 3T3-L1 cells was visualized by OilRed-O staining c) Schematic outline of the experimental setup: 3T3-L1 cell were treated with Dexamethasone and IBMX at confluency 2 days before stimulation with Rosiglitazone and Insulin. Samples for ChIP were taken at day 4. Samples for expression analysis were taken at all time points indicated. For each time point cells treated with PPARγ specific and control siRNA prior to the differentiation protocol were collected in 3 biological replicates. Microarray data was submitted to the GEO repository under GEO Submission (GSE12929).

Mentions: We used the well established 3T3-L1 cell line as a model system for adipocyte differentiation. Following a protocol schematically outlined in Figure 1C, 3T3-L1 cells were differentiated into adipocytes 4 days post treatment with dexamethasone and IBMX, followed by insulin and rosiglitazone after two days. Differentiation was verified both by western blot for adipocyte specific markers and accumulation of lipids within the cells as detected by Oil-Red O staining. 3T3-L1 cell transfected with PPARγ specific siRNA prior to differentiation completely abolished this process (Figures 1 A, B).


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)

3T3L1 cells as model system for adipocyte differentiation.Undifferentiated 3T3-L1 fibroblast undergoes adipogenesis upon treatment with Dexamethasone and IBMX, followed by Insulin and Rosiglitazone after two days. Adipogenesis was completely abrogated by PPARγ specific siRNA. a) Adipogenesis was monitored by Western blot for induction of adipocyte-specific marker genes. Beta-actin served as loading control. b) accumulation of lipid droplets in differentiated 3T3-L1 cells was visualized by OilRed-O staining c) Schematic outline of the experimental setup: 3T3-L1 cell were treated with Dexamethasone and IBMX at confluency 2 days before stimulation with Rosiglitazone and Insulin. Samples for ChIP were taken at day 4. Samples for expression analysis were taken at all time points indicated. For each time point cells treated with PPARγ specific and control siRNA prior to the differentiation protocol were collected in 3 biological replicates. Microarray data was submitted to the GEO repository under GEO Submission (GSE12929).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654672&req=5

pone-0004907-g001: 3T3L1 cells as model system for adipocyte differentiation.Undifferentiated 3T3-L1 fibroblast undergoes adipogenesis upon treatment with Dexamethasone and IBMX, followed by Insulin and Rosiglitazone after two days. Adipogenesis was completely abrogated by PPARγ specific siRNA. a) Adipogenesis was monitored by Western blot for induction of adipocyte-specific marker genes. Beta-actin served as loading control. b) accumulation of lipid droplets in differentiated 3T3-L1 cells was visualized by OilRed-O staining c) Schematic outline of the experimental setup: 3T3-L1 cell were treated with Dexamethasone and IBMX at confluency 2 days before stimulation with Rosiglitazone and Insulin. Samples for ChIP were taken at day 4. Samples for expression analysis were taken at all time points indicated. For each time point cells treated with PPARγ specific and control siRNA prior to the differentiation protocol were collected in 3 biological replicates. Microarray data was submitted to the GEO repository under GEO Submission (GSE12929).
Mentions: We used the well established 3T3-L1 cell line as a model system for adipocyte differentiation. Following a protocol schematically outlined in Figure 1C, 3T3-L1 cells were differentiated into adipocytes 4 days post treatment with dexamethasone and IBMX, followed by insulin and rosiglitazone after two days. Differentiation was verified both by western blot for adipocyte specific markers and accumulation of lipids within the cells as detected by Oil-Red O staining. 3T3-L1 cell transfected with PPARγ specific siRNA prior to differentiation completely abolished this process (Figures 1 A, B).

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