CTCF/cohesin-mediated DNA looping is required for protocadherin α promoter choice

Abstract

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...

CTCF-mediated topological boundaries during development foster appropriate gene regulation

“…Within this hierarchical organization, conserved TAD and sub-TAD structures frame the majority of enhancer-promoter interactions (Dekker et al 2013;Tang et al 2015;Dekker and Mirny 2016). The CCCTCbinding factor (CTCF) regulates each nested level of genomic organization (Guo et al 2015;Hnisz et al 2016), from local enhancer-promoter looping events within the protocadherin clusters to broader genomic neighborhoods and TADs (Guo et al 2012;Rao et al 2014).…”

Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes

“…However, the mechanism of glucocorticoid-induced DNA looping is not clear. One DNA binding protein that can induce DNA looping is CTCF (10,26,38,44). Cohesin has been shown to act with CTCF to induce DNA looping (5,6).…”

Regulation of the Protocadherin Celsr3 Gene and Its Role in Globus Pallidus Development and Connectivity

“…In particular, the clustered Pcdh genes are organized into variable and constant regions and generate enormous neural diversity by stochastic promoter choice combined with alternative pre-mRNA splicing (51)(52)(53). Recently, the neural expression of the clustered Pcdh genes was found to be controlled by both CTCF and NRSF (30,38,54,55). In particular, the CTCF/ cohesin complex plays a pivotal role in the promoter choice of the clustered Pcdh genes by mediating specific DNA looping interactions between enhancers and selective variable promoters (30).…”

“…For the knockdown of NRSF and Celsr3, we screened three shRNAs for each gene and found one with high efficiency. The CTCF and RAD21 knockdown plasmids were previously reported (30).…”