SummaryChromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.

Many genes determining cell identity are regulated by clusters of mediator-bound enhancer elements collectively referred to as super-enhancers. These have been proposed to manifest higher-order properties important in development and disease. Here, we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer singly and in informative combinations, we demonstrate that each constituent enhancer appears to act independently and in an additive fashion with respect to hematologic phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

The genome is organised via CTCF/Cohesin binding sites, which partition chromosomes into 1-5Mb topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ~80kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ~1Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF/cohesin sites which are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF/cohesin boundary extends the sub-TAD to adjacent upstream CTCF/cohesin binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF/cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.

Complex gene regulation is one of the key requirements for the evolution of higher eukaryotes. 1 In these organisms, many genes are regulated by enhancers that are 10 4 -10 6 base pairs (bp) distant from the promoter. Enhancer sequences usually contain multiple small transcription factor binding sites (typically ~10bp), and physical contact between the promoter and enhancer is thought to be required to modulate gene expression. 2 Current methods have extensively defined chromatin architecture at scales above 1 kb but until now it has not been possible to define physical contacts at the scale of the key proteins determining gene expression. Here we define the interactions between different classes of regulatory elements (enhancers, promoters and boundary elements) in unprecedented detail, using a novel chromosome conformation capture method (Micro Capture-C (MCC)), which allows physical contacts to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show, using base pair resolution plots of ligation junctions, that transcription factors generate a key component of the contacts between enhancers and promoters. Our data show that contacts from CTCF sites highly correlate with cooccupancy of cohesin and that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. We also find that promoters make the strongest contacts with both enhancers and CTCF sites and that while CTCF sites contact promoters strongly they only make weak contacts with enhancers. The highly punctate nature of the contacts is an unexpected finding because the current view is that physical contacts are constrained by much larger domains such as topological associated domains (TADs). 3 Our results support a model in which chromatin loop extrusion 4-6 is dependent on cohesin loading at active promoters and enhancers, explaining the formation of tissue-specific chromatin domains without changes in CTCF binding. The data suggest that a separate mechanism to loop extrusion underlies enhancer-/promoter contacts, which likely involves DNA binding proteins at enhancers and promoters. The unprecedented