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.

We have identified 52 novel human cadherin-like genes organized into three closely linked clusters. Comparison of the genomic DNA sequences with those of representative cDNAs reveals a striking genomic organization similar to that of immunoglobulin and T cell receptor gene clusters. The N-terminal extracellular and transmembrane domains of each cadherin protein are encoded by a distinct and unusually large exon. These exons are organized in a tandem array. By contrast, the C-terminal cytoplasmic domain of each protein is identical and is encoded by three small exons located downstream from the cluster of N-terminal exons. This unusual organization has interesting implications regarding the molecular code required to establish complex networks of neuronal connections in the brain and the mechanisms of cell-specific cadherin-like gene expression.

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...