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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

Binding regions of PPARγ and RXR are abundant but not unevenly dispersed.Genome-wide view of PPARγ and RXR binding regions, binned into 200 kbp blocks. No particular chromosomal concentration of bins containing PPARγ only (red), RXR only (green), or both (blue) was observed. Summary of the moPET counts for the PPARγ and RXR ChIP-PET libraries. Good binding regions were identified using aberration normalizing algorithm [25].
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pone-0004907-g002: Binding regions of PPARγ and RXR are abundant but not unevenly dispersed.Genome-wide view of PPARγ and RXR binding regions, binned into 200 kbp blocks. No particular chromosomal concentration of bins containing PPARγ only (red), RXR only (green), or both (blue) was observed. Summary of the moPET counts for the PPARγ and RXR ChIP-PET libraries. Good binding regions were identified using aberration normalizing algorithm [25].

Mentions: Overlapping PET fragments were clustered as before [23], [24] to form ChIP-PET clusters. The resultant ChIP-PET clusters were then stratified based on the maximum number of overlapping fragments within each cluster. For example, a non-overlapping fragment is labeled as a moPET1 cluster, and as before, these are considered background/noise [21], while a moPET3 cluster is one where the maximum number of PET fragments overlapping at any location within the cluster is three. (moPETn+ clusters is the set of moPETi clusters, where i≥n). Applying the adaptive thresholding method [25], 2953 PPARγ ChIP-PET clusters and 5142 RXR ChIP-PET clusters were found to be statistically significant (FDR of ≤0.01) and henceforth referred to as putative binding regions (Figure 2).


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)

Binding regions of PPARγ and RXR are abundant but not unevenly dispersed.Genome-wide view of PPARγ and RXR binding regions, binned into 200 kbp blocks. No particular chromosomal concentration of bins containing PPARγ only (red), RXR only (green), or both (blue) was observed. Summary of the moPET counts for the PPARγ and RXR ChIP-PET libraries. Good binding regions were identified using aberration normalizing algorithm [25].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004907-g002: Binding regions of PPARγ and RXR are abundant but not unevenly dispersed.Genome-wide view of PPARγ and RXR binding regions, binned into 200 kbp blocks. No particular chromosomal concentration of bins containing PPARγ only (red), RXR only (green), or both (blue) was observed. Summary of the moPET counts for the PPARγ and RXR ChIP-PET libraries. Good binding regions were identified using aberration normalizing algorithm [25].
Mentions: Overlapping PET fragments were clustered as before [23], [24] to form ChIP-PET clusters. The resultant ChIP-PET clusters were then stratified based on the maximum number of overlapping fragments within each cluster. For example, a non-overlapping fragment is labeled as a moPET1 cluster, and as before, these are considered background/noise [21], while a moPET3 cluster is one where the maximum number of PET fragments overlapping at any location within the cluster is three. (moPETn+ clusters is the set of moPETi clusters, where i≥n). Applying the adaptive thresholding method [25], 2953 PPARγ ChIP-PET clusters and 5142 RXR ChIP-PET clusters were found to be statistically significant (FDR of ≤0.01) and henceforth referred to as putative binding regions (Figure 2).

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