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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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Capacity for adipocyte differentiation after siRNA treatment of selected PPARγ direct target genes.Undifferentiated 3T3-L1 cells were transfected with siRNAs that targeted genes that were selected on the basis of ChIP-PET binding for both PPARγ and RXR, and expression data. Transfected cells were brought to confluence and then treated with IBMX/DEX for 48 hrs, and then stimulated with insulin and rosiglitazone. Cells were stained after 4 days of treatment using Oil Red O stain, and quantified for lipid accumulation using a spectrophometer set at an absorbance of 540 nM. Percentage of adipogenesis, as measured by Oil Red O staining, was compared for each siRNA target to that of the non-transfected differentiated cells (set at 100% adipogenesis). Non-targeting siRNA served as a control, while siRNA targeting PPARγ served as a threshold for adipogenesis inhibition. A gene target was considered affecting lipid accumulation if it did not produce significantly higher lipid accumulation (one-tailed t-test p>0.2) than that of the PPARγ KD. Starred bars represent genes that meet the criteria. Confluent fibroblasts, as well as differentiated 3T3-L1 cells, that were not transfected with siRNA also served as controls to compare the effects of non-targeting siRNA on adipocyte differentiation. Results indicate two biological replicates, each of which was transfected with siRNA to a final concentration of 50 nM.
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pone-0004907-g007: Capacity for adipocyte differentiation after siRNA treatment of selected PPARγ direct target genes.Undifferentiated 3T3-L1 cells were transfected with siRNAs that targeted genes that were selected on the basis of ChIP-PET binding for both PPARγ and RXR, and expression data. Transfected cells were brought to confluence and then treated with IBMX/DEX for 48 hrs, and then stimulated with insulin and rosiglitazone. Cells were stained after 4 days of treatment using Oil Red O stain, and quantified for lipid accumulation using a spectrophometer set at an absorbance of 540 nM. Percentage of adipogenesis, as measured by Oil Red O staining, was compared for each siRNA target to that of the non-transfected differentiated cells (set at 100% adipogenesis). Non-targeting siRNA served as a control, while siRNA targeting PPARγ served as a threshold for adipogenesis inhibition. A gene target was considered affecting lipid accumulation if it did not produce significantly higher lipid accumulation (one-tailed t-test p>0.2) than that of the PPARγ KD. Starred bars represent genes that meet the criteria. Confluent fibroblasts, as well as differentiated 3T3-L1 cells, that were not transfected with siRNA also served as controls to compare the effects of non-targeting siRNA on adipocyte differentiation. Results indicate two biological replicates, each of which was transfected with siRNA to a final concentration of 50 nM.

Mentions: To assess the function of selected direct PPARγ targets during adipogenesis, we carried out gene knockdown experiments using siRNA in 3T3-L1 cells. Adipogenic potential of the selected target genes was evaluated by quantifying the reduction of lipid accumulation in differentiated adipocytes in the absence of the respective gene. Evaluated genes were selected from the high confidence target genes defined by a transcriptional unit with heterosites within 5 kb of their TSS and showing differential expression pattern during adipogenesis. After Oil Red O staining, lipid accumulation in cells was measured using a spectrometer set at 540 nm. Level of lipid accumulation inhibition ascribed to PPARγ KD was used as the positive control (40% reduction compared with untreated adipocytes). A knockdown was considered affecting lipid accumulation if it did not produce significantly higher lipid accumulation (one-tailed t-test p>0.02) than that of PPARγ KD. The level of lipid accumulation of untransfected cells undergoing adipogenesis after chemical stimulation was taken arbitrarily to exhibit 100% adipogenesis. Whereas non-targeting siRNA had no effect on the adipogenic phenotype, 6 of the 20 (30%) tested siRNAs showed a reduction of lipid accumulation similar to that of PPARγ knockdown. The knockdowns that were biologically effective were Pim3, Mnk2, Agt, Fsp27, Smaf1(adipogenin) and Pdzrn3, while other targets showed no or very modest levels of inhibition of lipid accumulation (Figure 7). These affects on lipogenesis are not due to non specific cellular toxicity (data not shown).


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)

Capacity for adipocyte differentiation after siRNA treatment of selected PPARγ direct target genes.Undifferentiated 3T3-L1 cells were transfected with siRNAs that targeted genes that were selected on the basis of ChIP-PET binding for both PPARγ and RXR, and expression data. Transfected cells were brought to confluence and then treated with IBMX/DEX for 48 hrs, and then stimulated with insulin and rosiglitazone. Cells were stained after 4 days of treatment using Oil Red O stain, and quantified for lipid accumulation using a spectrophometer set at an absorbance of 540 nM. Percentage of adipogenesis, as measured by Oil Red O staining, was compared for each siRNA target to that of the non-transfected differentiated cells (set at 100% adipogenesis). Non-targeting siRNA served as a control, while siRNA targeting PPARγ served as a threshold for adipogenesis inhibition. A gene target was considered affecting lipid accumulation if it did not produce significantly higher lipid accumulation (one-tailed t-test p>0.2) than that of the PPARγ KD. Starred bars represent genes that meet the criteria. Confluent fibroblasts, as well as differentiated 3T3-L1 cells, that were not transfected with siRNA also served as controls to compare the effects of non-targeting siRNA on adipocyte differentiation. Results indicate two biological replicates, each of which was transfected with siRNA to a final concentration of 50 nM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004907-g007: Capacity for adipocyte differentiation after siRNA treatment of selected PPARγ direct target genes.Undifferentiated 3T3-L1 cells were transfected with siRNAs that targeted genes that were selected on the basis of ChIP-PET binding for both PPARγ and RXR, and expression data. Transfected cells were brought to confluence and then treated with IBMX/DEX for 48 hrs, and then stimulated with insulin and rosiglitazone. Cells were stained after 4 days of treatment using Oil Red O stain, and quantified for lipid accumulation using a spectrophometer set at an absorbance of 540 nM. Percentage of adipogenesis, as measured by Oil Red O staining, was compared for each siRNA target to that of the non-transfected differentiated cells (set at 100% adipogenesis). Non-targeting siRNA served as a control, while siRNA targeting PPARγ served as a threshold for adipogenesis inhibition. A gene target was considered affecting lipid accumulation if it did not produce significantly higher lipid accumulation (one-tailed t-test p>0.2) than that of the PPARγ KD. Starred bars represent genes that meet the criteria. Confluent fibroblasts, as well as differentiated 3T3-L1 cells, that were not transfected with siRNA also served as controls to compare the effects of non-targeting siRNA on adipocyte differentiation. Results indicate two biological replicates, each of which was transfected with siRNA to a final concentration of 50 nM.
Mentions: To assess the function of selected direct PPARγ targets during adipogenesis, we carried out gene knockdown experiments using siRNA in 3T3-L1 cells. Adipogenic potential of the selected target genes was evaluated by quantifying the reduction of lipid accumulation in differentiated adipocytes in the absence of the respective gene. Evaluated genes were selected from the high confidence target genes defined by a transcriptional unit with heterosites within 5 kb of their TSS and showing differential expression pattern during adipogenesis. After Oil Red O staining, lipid accumulation in cells was measured using a spectrometer set at 540 nm. Level of lipid accumulation inhibition ascribed to PPARγ KD was used as the positive control (40% reduction compared with untreated adipocytes). A knockdown was considered affecting lipid accumulation if it did not produce significantly higher lipid accumulation (one-tailed t-test p>0.02) than that of PPARγ KD. The level of lipid accumulation of untransfected cells undergoing adipogenesis after chemical stimulation was taken arbitrarily to exhibit 100% adipogenesis. Whereas non-targeting siRNA had no effect on the adipogenic phenotype, 6 of the 20 (30%) tested siRNAs showed a reduction of lipid accumulation similar to that of PPARγ knockdown. The knockdowns that were biologically effective were Pim3, Mnk2, Agt, Fsp27, Smaf1(adipogenin) and Pdzrn3, while other targets showed no or very modest levels of inhibition of lipid accumulation (Figure 7). These affects on lipogenesis are not due to non specific cellular toxicity (data not shown).

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