PPARγ lipodystrophy mutants reveal intermolecular interactions required for enhancer activation.

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipocyte differentiation, and mutations that interfere with its function cause lipodystrophy. PPARγ is a highly modular protein, and structural studies indicate that PPARγ domains engage in several intra- and inter-molecular interactions. How these interactions modulate PPARγ's ability to activate target genes in a cellular context is currently poorly understood. Here we take advantage of two previously uncharacterized lipodystrophy mutations, R212Q and E379K, that are predicted to interfere with the interaction of the hinge of PPARγ with DNA and with the interaction of PPARγ ligand binding domain (LBD) with the DNA-binding domain (DBD) of the retinoid X receptor, respectively. Using biochemical and genome-wide approaches we show that these mutations impair PPARγ function on an overlapping subset of target enhancers. The hinge region-DNA interaction appears mostly important for binding and remodelling of target enhancers in inaccessible chromatin, whereas the PPARγ-LBD:RXR-DBD interface stabilizes the PPARγ:RXR:DNA ternary complex. Our data demonstrate how in-depth analyses of lipodystrophy mutants can unravel molecular mechanisms of PPARγ function.

Integrative Genomics Outlines a Biphasic Glucose Response and a ChREBP-RORγ Axis Regulating Proliferation in ß Cells.

Glucose is an important inducer of insulin secretion, but it also stimulates long-term adaptive changes in gene expression that can either promote or antagonize the proliferative potential and function of ß cells. Here, we have generated time-resolved profiles of enhancer and transcriptional activity in response to glucose in the INS-1E pancreatic ß cell line. Our data outline a biphasic response with a first transcriptional wave during which metabolic genes are activated, and a second wave where cell-cycle genes are activated and ß cell identity genes are repressed. The glucose-sensing transcription factor ChREBP directly activates first wave enhancers, whereas repression and activation of second wave enhancers are indirect. By integrating motif enrichment within late-regulated enhancers with expression profiles of the associated transcription factors, we have identified multiple putative regulators of the second wave. These include RORγ, the activity of which is important for glucose-induced proliferation of both INS-1E and primary rat ß cells.

Peroxisome proliferator-activated receptor γ and C/EBPα synergistically activate key metabolic adipocyte genes by assisted loading.

Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key activators of adipogenesis. They mutually induce the expression of each other and have been reported to cooperate in activation of a few adipocyte genes. Recently, genome-wide profiling revealed a high degree of overlap between PPARγ and C/EBPα binding in adipocytes, suggesting that cooperativeness could be mediated through common binding sites. To directly investigate the interplay between PPARγ and C/EBPα at shared binding sites, we established a fibroblastic model system in which PPARγ and C/EBPα can be independently expressed. Using RNA sequencing, we demonstrate that coexpression of PPARγ and C/EBPα leads to synergistic activation of many key metabolic adipocyte genes. This is associated with extensive C/EBPα-mediated reprogramming of PPARγ binding and vice versa in the vicinity of these genes, as determined by chromatin immunoprecipitation combined with deep sequencing. Our results indicate that this is at least partly mediated by assisted loading involving chromatin remodeling directed by the leading factor. In conclusion, we report a novel mechanism by which the key adipogenic transcription factors, PPARγ and C/EBPα, cooperate in activation of the adipocyte gene program.