The KDM5 family is required for activation of pro-proliferative cell cycle genes during adipocyte differentiation.

The KDM5 family of histone demethylases removes the H3K4 tri-methylation (H3K4me3) mark frequently found at promoter regions of actively transcribed genes and is therefore generally considered to contribute to corepression. In this study, we show that knockdown (KD) of all expressed members of the KDM5 family in white and brown preadipocytes leads to deregulated gene expression and blocks differentiation to mature adipocytes. KDM5 KD leads to a considerable increase in H3K4me3 at promoter regions; however, these changes in H3K4me3 have a limited effect on gene expression per se. By contrast, genome-wide analyses demonstrate that KDM5A is strongly enriched at KDM5-activated promoters, which generally have high levels of H3K4me3 and are associated with highly expressed genes. We show that KDM5-activated genes include a large set of cell cycle regulators and that the KDM5s are necessary for mitotic clonal expansion in 3T3-L1 cells, indicating that KDM5 KD may interfere with differentiation in part by impairing proliferation. Notably, the demethylase activity of KDM5A is required for activation of at least a subset of pro-proliferative cell cycle genes. In conclusion, the KDM5 family acts as dual modulators of gene expression in preadipocytes and is required for early stage differentiation and activation of pro-proliferative cell cycle genes.

Genome-wide profiling of peroxisome proliferator-activated receptor γ in primary epididymal, inguinal, and brown adipocytes reveals depot-selective binding correlated with gene expression.

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation and function. We and others have previously mapped PPARγ binding at a genome-wide level in murine and human adipocyte cell lines and in primary human adipocytes. However, little is known about how binding patterns of PPARγ differ between brown and white adipocytes and among different types of white adipocytes. Here we have employed chromatin immunoprecipitation combined with deep sequencing to map and compare PPARγ binding in in vitro differentiated primary mouse adipocytes isolated from epididymal, inguinal, and brown adipose tissues. While these PPARγ binding profiles are overall similar, there are clear depot-selective binding sites. Most PPARγ binding sites previously mapped in 3T3-L1 adipocytes can also be detected in primary adipocytes, but there are a large number of PPARγ binding sites that are specific to the primary cells, and these tend to be located in closed chromatin regions in 3T3-L1 adipocytes. The depot-selective binding of PPARγ is associated with highly depot-specific gene expression. This indicates that PPARγ plays a role in the induction of genes characteristic of different adipocyte lineages and that preadipocytes from different depots are differentially preprogrammed to permit PPARγ lineage-specific recruitment even when differentiated in vitro.

Cross-species ChIP-seq studies provide insights into regulatory strategies of PPARγ in adipocytes.

Three recent studies have investigated interspecies retention of binding sites of peroxisome proliferator-activated receptor γ (PPARγ), the master regulator of adipocyte differention, between mouse and human adipocytes. Here we discuss the major findings and demonstrate that retention of binding events is highly context-dependent.

Gene program-specific regulation of PGC-1{alpha} activity.

Peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1 α (PGC-1α) activation coordinates induction of the hepatic fasting response through coactivation of numerous transcription factors and gene programs. In the June 15, 2011, issue of Genes & Development, Lustig and colleagues (pp. 1232-1244) demonstrated that phosphorylation of PGC-1α by the p70 ribosomal protein S6 kinase 1 (S6K1) specifically interfered with the interaction between PGC-1α and HNF4α in liver and blocked the coactivation of the gluconeogenic target genes. This demonstrates how independent fine-tuning of gene programs coregulated by the same coactivator can be obtained.

Cross species comparison of C/EBPα and PPARγ profiles in mouse and human adipocytes reveals interdependent retention of binding sites.

BACKGROUND: The transcription factors peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key transcriptional regulators of adipocyte differentiation and function. We and others have previously shown that binding sites of these two transcription factors show a high degree of overlap and are associated with the majority of genes upregulated during differentiation of murine 3T3-L1 adipocytes. RESULTS: Here we have mapped all binding sites of C/EBPα and PPARγ in human SGBS adipocytes and compared these with the genome-wide profiles from mouse adipocytes to systematically investigate what biological features correlate with retention of sites in orthologous regions between mouse and human. Despite a limited interspecies retention of binding sites, several biological features make sites more likely to be retained. First, co-binding of PPARγ and C/EBPα in mouse is the most powerful predictor of retention of the corresponding binding sites in human. Second, vicinity to genes highly upregulated during adipogenesis significantly increases retention. Third, the presence of C/EBPα consensus sites correlate with retention of both factors, indicating that C/EBPα facilitates recruitment of PPARγ. Fourth, retention correlates with overall sequence conservation within the binding regions independent of C/EBPα and PPARγ sequence patterns, indicating that other transcription factors work cooperatively with these two key transcription factors. CONCLUSIONS: This study provides a comprehensive and systematic analysis of what biological features impact on retention of binding sites between human and mouse. Specifically, we show that the binding of C/EBPα and PPARγ in adipocytes have evolved in a highly interdependent manner, indicating a significant cooperativity between these two transcription factors.

Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes.

We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) induced inflammation and insulin resistance in primary human adipocytes by activating nuclear factor kappaB (NFkappaB) and extracellular signal-related kinase (ERK) signaling. In this study, we demonstrated that the initial increase in intracellular calcium ([Ca2+]i) mediated by 10,12 CLA was attenuated by TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), by BAPTA, an intracellular calcium chelator, and by D609, a phospholipase C (PLC) inhibitor. Moreover, BAPTA, TMB-8, and D609 attenuated 10,12 CLA-mediated production of reactive oxygen species (ROS), activation of ERK1/2 and cJun-NH2-terminal kinase (JNK), and induction of inflammatory genes. 10,12 CLA-mediated binding of NFkappaB to the promoters of interleukin (IL)-8 and cyclooxygenase (COX)-2 and induction of calcium-calmodulin kinase II (CaMKII) beta were attenuated by TMB-8. KN-62, a CaMKII inhibitor, also suppressed 10,12 CLA-mediated ROS production and ERK1/2 and JNK activation. Additionally, KN-62 attenuated 10,12 CLA induction of inflammatory and integrated stress response genes, increase in prostaglandin F2alpha, and suppression of peroxisome proliferator activated receptor gamma protein levels and insulin-stimulated glucose uptake. These data suggest that 10,12 CLA increases inflammation and insulin resistance in human adipocytes, in part by increasing [Ca2+]i levels, particularly calcium from the ER.