Transcription factors have recently been shown to colocalize in hotspot regions of the genome, which are further clustered into super-enhancers. However, the detailed molecular organization of transcription factors at hotspot regions is poorly defined. Here, we have used digital genomic footprinting to precisely define factor localization at a genome-wide level during the early phase of 3T3-L1 adipocyte differentiation, which allows us to obtain detailed molecular insight into how transcription factors target hotspots. We demonstrate the formation of ATF-C/EBP heterodimers at a composite motif on chromatin, and we suggest that this may be a general mechanism for integrating external signals on chromatin. Furthermore, we find evidence of extensive recruitment of transcription factors to hotspots through alternative mechanisms not involving their known motifs and demonstrate that these alternative binding events are functionally important for hotspot formation and activity. Taken together, these findings provide a framework for understanding transcription factor cooperativity in hotspots.
The three dimensional folding of mammalian genomes is cell type specific and difficult to alter suggesting that it is an important component of gene regulation. However, given the multitude of chromatin‐associating factors, the mechanisms driving the colocalization of active chromosomal domains and the role of this organization in regulating the transcription program in adipocytes are not clear. Analysis of genome‐wide chromosomal associations revealed cell type‐specific spatial clustering of adipogenic genes in 3T3‐L1 cells. Time course analysis demonstrated that the adipogenic ‘hub’, sampled by PPAR γ and Lpin1 , undergoes orchestrated reorganization during adipogenesis. Coupling the dynamics of genome architecture with multiple chromatin datasets indicated that among all the transcription factors (TFs) tested, RXR is central to genome reorganization at the beginning of adipogenesis. Interestingly, at the end of differentiation, the adipogenic hub was shifted to an H3K27me3‐repressive environment in conjunction with attenuation of gene transcription. We propose a stage‐specific hierarchy for the activity of TFs contributing to the establishment of an adipogenic genome architecture that brings together the adipogenic genetic program. In addition, the repositioning of this network in a H3K27me3‐rich environment at the end of differentiation may contribute to the stabilization of gene transcription levels and reduce the developmental plasticity of these specialized cells. Database All sequence data reported in this paper have been deposited at GEO ( http://www.ncbi.nlm.nih.gov/geo/ ) ( GSE92475 )
Adipocyte differentiation is controlled by a tightly regulated transcriptional cascade in which PPARγ and members of the C/EBP family are key players. Here we review the roles of PPARγ and C/EBPs in adipocyte differentiation with emphasis on the recently published genome‐wide binding profiles for PPARγ and C/EBPα. Interestingly, these analyses show that PPARγ and C/EBPα binding sites are associated with most genes that are induced during adipogenesis suggesting direct activation of many more adipocyte genes than previously anticipated. Furthermore, an extensive overlap between the C/EBPα and PPARγ cistromes indicate a hitherto unrecognized direct crosstalk between these transcription factors. As more genome‐wide data emerge in the future, this crosstalk will likely be found to include several other adipogenic transcription factors.
The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of adipocyte differentiation in vivo and ex vivo and has been shown to control the expression of several adipocyte-specific genes. In this study, we used chromatin immunoprecipitation combined with deep sequencing to generate genome-wide maps of PPARγ and retinoid X receptor (RXR)-binding sites, and RNA polymerase II (RNAPII) occupancy at very high resolution throughout adipocyte differentiation of 3T3-L1 cells. We identify >5000 high-confidence shared PPARγ:RXR-binding sites in adipocytes and show that during early stages of differentiation, many of these are preoccupied by non-PPARγ RXR-heterodimers. Different temporal and compositional patterns of occupancy are observed. In addition, we detect co-occupancy with members of the C/EBP family. Analysis of RNAPII occupancy uncovers distinct clusters of similarly regulated genes of different biological processes. PPARγ:RXR binding is associated with the majority of induced genes, and sites are particularly abundant in the vicinity of genes involved in lipid and glucose metabolism. Our analyses represent the first genome-wide map of PPARγ:RXR target sites and changes in RNAPII occupancy throughout adipocyte differentiation and indicate that a hitherto unrecognized high number of adipocyte genes of distinctly regulated pathways are directly activated by PPARγ:RXR.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
