Tandem Directional CTCF Sites Balance Protocadherin Promoter Usage

Abstract

CTCF is a key insulator-binding protein and mammalian genomes contain numerous CTCFbinding sites (CBSs), many of which are organized in tandem arrays. Here we provide direct evidence that CBSs, if located between enhancers and promoters in the Pcdh and -globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation and experimental capture revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter usage. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation.2 22]. Insertion, mutation, deletion, inversion, or duplication of CBS elements alters chromatin topology and gene expression [12, 14-16, 18, 22-24]. Emerging evidence suggests that spatial control of genome topology by CTCF/Cohesin regulates gene expression; however, how numerous CBS elements in mammalian genomes function as insulators to control proper promoter activation and balanced usage remains obscure. RESULTS Exogenous CTCF Sites as Protocadherin InsulatorsSimilar to the enormous diversity of DSCAM1 proteins in Drosophila, combinatorial cisand trans-interactions between mammalian clustered cell-surface protocadherin (Pcdh) proteins, encoded by the three closely-linked gene clusters (, , and ), endow individual neurons with a unique identity code and specific self-recognition module, which are required for neuronal migration, dendrite self-avoidance, and axon tiling in the brain [25][26][27][28][29][30][31]. The human Pcdh cluster contains 13 highly-similar, tandem-arrayed, unusually-large "alternate" variable exons (1-13) and 2 divergent "ubiquitous" variable exons (c1-c2), followed by 3 downstream small constant exons ( Figure 1A), reminiscent of the variable and constant genome organizations of immunoglobulin (Ig) and T-cell receptor (Tcr) clusters [25,32]. Each of the 13 "alternate" variable exons (1-13) carries its own promoter, which is flanked by two forward-oriented CBS (CSE and eCBS) elements ( Figure 1A). However, the c1 "ubiquitous" promoter carries only one forward-oriented CBS and the c2 promoter has no CBS element ( Figure 1A). Two distal Pcdh enhancers, HS7 and HS5-1, are located downstream, and one of which, HS5-1, is flanked by two reverse-oriented CBS (HS5-1a and HS5-1b) elements [33,34]. Multiple longdistance chromatin interactions between these enhancers and Pcdh target promoters form a transcription hub and determine the promoter choice [34,35]. We performed single-cell RNAseq of mouse cortical neurons and found members of the Pcdh clu...

Genetic evidence for asymmetric blocking of higher-order chromatin structure by CTCF/cohesin

“…Specially, one type of genetic elements, known as insulators, plays a delicate role to ensure proper activation of target promoters by distal enhancers (Huang and Wu, 2016). In mammals, the most prominent insulator-binding protein is CCCTC binding factor (CTCF), an architectural protein with 11 zinc-fingers essential for 3D genome organization (Yin et al, 2017; Wu et al, 2019).…”

“…CTCF dynamically and directionally binds to hundreds of thousands genomic sites and this binding is pivotal for its multivalent role in many cellular and developmental processes (Guo et al, 2015; Yin et al, 2017). In particular, CTCF, in collaboration with its associated cohesin complex, determines V(D)J recombination of the Bcr (B-cell receptor) and Tcr (T-cell receptor) gene clusters in the immune system (Jain et al, 2018; Wu et al, 2019) and promoter choice of the protocadherin ( Pcdh ) gene clusters in the nervous system (Guo et al, 2012; Mountoufaris et al, 2018; Canzio et al, 2019; Wu et al, 2019). The clustered Pcdh genes have been used as model genes for investigating mechanisms of higher-order chromatin folding (Fig.…”