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

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.