SUMMARY CTCF/cohesin play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters, and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. The findings reveal how 3D chromosome architecture can be encoded by genome sequence.

The closely linked human protocadherin (Pcdh) α, β, and γ gene clusters encode 53 distinct protein isoforms, which are expressed in a combinatorial manner to generate enormous diversity on the surface of individual neurons. This diversity is a consequence of stochastic promoter choice and alternative pre-mRNA processing. Here, we show that Pcdhα promoter choice is achieved by DNA looping between two downstream transcriptional enhancers and individual promoters driving the expression of alternate Pcdhα isoforms. In addition, we show that this DNA looping requires specific binding of the CTCF/cohesin complex to two symmetrically aligned binding sites in both the transcriptionally active promoters and in one of the enhancers. These findings have important implications regarding enhancer/promoter interactions in the generation of complex Pcdh cell surface codes for the establishment of neuronal identity and self-avoidance in individual neurons.T he clustered protocadherin (Pcdh) genes are expressed in the nervous system and organized into three closely linked clusters (α, β, and γ) (1-5). The human Pcdhα gene cluster contains 13 highly similar variable first exons (α1 to α13) arrayed in tandem and two more distantly related c-type variable first exons designated αc1 and αc2 (Fig. 1A). The variable first exons encode the extracellular, transmembrane, and juxtamembrane intracellular domains of the Pcdhα proteins. Each of these 15 variable first exons is cis-spliced to a single set of three downstream constant exons that encode a distal intracellular domain (1-3). The human β cluster is located downstream from the α cluster and contains a tandem array of 16 highly similar variable exons but with no constant exons, whereas the γ cluster contains 22 variable first exons arrayed in tandem and divided into three types (γa1 to γa12, γb1 to γb7, and γc3 to γc5) (Fig. 1D). As in the case of the α cluster, each of these 22 γ variable first exons is cis-spliced to a single set of three downstream constant exons, which are distinct from the α constant exons, to generate diverse γ mRNAs (1, 3, 6). Analyses of the α and γ transcripts have revealed that highly similar Pcdh alternate isoforms are expressed in a stochastic fashion, whereas all of the c-type divergent isoforms, αc1 and αc2 in the α cluster and γc3, γc4, and γc5 in the γ cluster, are expressed ubiquitously in all cells (1-3, 5, 7). Hereafter, we refer to the c-type genes as "ubiquitously expressed" in contrast to the "alternately expressed" Pcdh genes ( Fig. 1 A and D). A combination of stochastic activation of alternate promoters and constitutive activation of c-type ubiquitous promoters generates enormous single-cell diversity on the surface of individual neurons.Significant advances have been made in understanding the mechanisms by which individual neurons express distinct combinations of the clustered Pcdh genes (2, 3, 8-10). Two long-range cis-regulatory elements in the α cluster, HS5-1 and HS7 (hypersensitive sites 5-1 and 7), function as developmental and tissue...

Cohesin is important for 3D genome organization. Nevertheless, even the complete removal of cohesin has surprisingly little impact on steady-state gene transcription and enhancer activity. Here we show that cohesin is required for the core transcriptional response of primary macrophages to microbial signals, and for inducible enhancer activity that underpins inflammatory gene expression. Consistent with a role for inflammatory signals in promoting myeloid differentiation of hematopoietic stem and progenitor cells (HPSCs), cohesin mutations in HSPCs led to reduced inflammatory gene expression and increased resistance to differentiation-inducing inflammatory stimuli. These findings uncover an unexpected dependence of inducible gene expression on cohesin, link cohesin with myeloid differentiation, and may help explain the prevalence of cohesin mutations in human acute myeloid leukemia.

The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.