ABSTRACT: We aimed to characterise genome-wide gene expression changes upon ZEB1 knockdown in 3T3-L1 cells at distinct timepoints of adipogenic differentiation (days 0 and 2), performing replicate experiments.
Project description:We aimed to characterise genome-wide ZEB1 binding pattern in 3T3-L1 cells at distinct timepoints of adipogenic differentiation (days -2, 0, 2 and 4). We performed replicate experiments at days -2 and 0 and single experiments at days 2 and 4.
Project description:Here we report, for the first time, the acute effects of the synthetic PPARγ agonist rosiglitazone on the transcriptional network of PPARγ in adipocytes. Treatment with Rosiglitazone for 1 hour leads to acute transcriptional activation as well as repression of a number of genes as determined by genome-wide RNA polymerase II occupancy. Unlike what has been shown for many other nuclear receptors, agonist treatment does not lead to major changes in the occurrence of PPARγ binding sites. However, rosiglitazone promotes PPARγ occupancy at many preexisting sites, and this is paralleled by increased occupancy of the mediator subunit MED1. The increase in PPARγ and MED1 binding is correlated with an increase in transcription of nearby genes indicating that rosiglitazone, in addition to activating the receptor, also promotes its association with DNA, and that this is causally linked to recruitment of mediator and activation of genes. Notably, both Rosiglitazone-activated and -repressed genes are induced during adipogenesis. However, Rosiglitazone-activated genes are markedly more associated with PPARγ than repressed genes and are highly dependent on PPARγ for expression in adipocytes. By contrast, repressed genes are associated with the other key adipocyte transcription factor CCAAT-Enhancer binding protein (C/EBPα), and their expression is more dependent on C/EBPα. This suggests that the relative occupancies of PPARγ and C/EBPα are critical for whether genes will be induced or repressed by PPARγ agonist. Examination of binding of PPARγ, C/EBPα, RNAPII, CBP and MED1 in mature 3T3-L1 adipocytes treated with 1 μM Rosiglitazone and/or 0.1% DMSO for 1 hour.
Project description:Adipogenesis is tightly controlled by a complex network of transcription factors acting at different stages of differentiation. Peroxisome proliferator-activated receptor gamma (PPAR gamma) and CCAAT/enhancer binding protein (C/EBP) family members are key regulators of this process. We have employed DNase I hypersensitive site analysis to investigate the genome-wide changes in chromatin structure that accompany the binding of adipogenic transcription factors. These analyses revealed a dramatic and dynamic modulation of the chromatin landscape during the first hours of adipocyte differentiation that coincides with cooperative binding of multiple early transcription factors (including glucocorticoid receptor, retinoid X receptor, Stat5a, C/EBPbeta and -delta) to transcription factor 'hotspots'. Our results demonstrate that C/EBPbeta marks a large number of these transcription factor 'hotspots' prior to induction of differentiation and chromatin remodeling and is required for their establishment. Furthermore, a subset of early remodeled C/EBP binding sites persists throughout differentiation and is later occupied by PPAR gamma , indicating that early C/EBP family members, in addition to their well established role in activation of PPAR gamma transcription, may act as pioneering factors for PPAR gamma binding. DNase I hypersensitive chromatin regions and transcription factor binding sites were identified at various time points of 3T3-L1 differentiation using DHS-seq and ChIP-seq, respectively.
Project description:We analyzed RING1B binding regions in 3T3-L1 cell lines transduced with the retroviral vector for V5-tagged Fbxl10 or its dF-box mutant. RING1B ChIP-seq in empty, Fbxl10-1, and dF-box mutant vector transduced 3T3-L1 preadipocytes, in duplicate, and V5-Fbxl10 ChIP-seq in Fbxl10 overexpressing 3T3-L1 cells
Project description:ChIP-seq of mouse embryonic fibroblast-adipose like cell line 3T3-L1 to identify binding sites of NCoR1 and SMRT following induction of differentiation, and RNA Pol-II after SMRT knock down
Project description:Here, we have used digital genomic footprinting to precisely define protein localization for several adipogenic transcription factors at a genome-wide level. In combination with ChIP-seq data, these analyses reveal novel molecular insight into the organization of transcription factors at hotpot regions, which provides a new framework for understanding transcription factor cooperativity on chromatin. Digital genomic footprinting and gene expression in 3T3-L1 pre-adipocytes by high throughput sequencing.
Project description:Lipid mobilization (lipolysis) in white adipose tissue (WAT) critically controls lipid turnover and adiposity in humans. While the acute regulation of lipolysis has been studied in detail, the transcriptional determinants of WAT lipolytic activity remain still largely unexplored. Here we show that the genetic inactivation of transcriptional co-factor transducin beta-like-related (TBLR) 1 blunts the lipolytic response of white adipocytes through the impairment of cAMP-dependent signal transduction. Indeed, mice lacking TBLR1 in adipocytes are defective in fasting-induced lipid mobilization and when placed on a high fat diet show aggravated adiposity, glucose intolerance and insulin resistance. TBLR1 levels are found to increase under lipolytic conditions in WAT of both human patients and mice, correlating with serum free fatty acids (FFA). As a critical regulator of WAT cAMP signaling and lipid mobilization, proper activity of TBLR1 in adipocytes may thus represent a critical molecular checkpoint for the prevention of metabolic dysfunction in subjects with obesity-related disorders. We used microarrays to identify global gene expression in 3T3-L1 adipocytes lacking TBLR1 and compared gene expression to control shRNA treated cells in both basal and isoproterenol stimulated states. We analyzed 12 RNA samples extracted from 3T3-L1 adipocytes that were treated with either control or TBLR1 specific shRNAs and with or without 10 µM isoproterenol for 3 hrs. Three replicates of each condition.
Project description:miRNA profiles of adipocyte-derived microvesicles (ADMs) on the Day 2-4 and Day 8-10 were compared. ADMs were prepared from the 48h-conditioned medium of 3T3-L1 adipocytes (Day 2-4 and Day 8-10) followed by RNA isolation.