SummaryThe spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin’s DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.

Most of the millions of single-nucleotide polymorphisms (SNPs) in the human genome are non-coding, and many overlap with putative regulatory elements. Genome-wide association studies (GWAS) have linked many of these SNPs to human traits or to gene expression levels, but rarely with sufficient resolution to identify the causal SNPs. Functional screens based on reporter assays have previously been of insufficient throughput to test the vast space of SNPs for possible effects on regulatory element activity. Here, we leveraged the throughput and resolution of the SuRE reporter technology to survey the effect of 5.9 million SNPs, including 57% of the known common SNPs, on enhancer and promoter activity. We identified more than 30,000 SNPs that alter the activity of putative regulatory elements, partially in a cell-type specific manner. Integration of this dataset with GWAS results may help pinpoint SNPs that underlie human traits.

Summary DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.