Qiang Wu

The actin polymerization machinery, comprising the ARP2/3 complex and its activators, the WASP family proteins, has been implicated in regulating a broad spectrum of nuclear processes, such as transcriptional regulation and nuclear organization. Here, using clustered protocadherin ( cPcdh ) and β‐globin genes as model systems, we showed that WAVE2 , a member of the WASP family, regulates chromatin organization by maintaining heterochromatin dynamics. Specifically, by CRISPR DNA‐fragment editing, in conjunction with integrated analyses of ChIP‐seq, MeDIP‐seq, ATAC‐seq, QHR‐4C, and RNA‐seq, we showed that deposition of H3K9me3, a key heterochromatin mark, is significantly decreased at the cPcdh locus upon WAVE2 deletion, concurrent with aberrant accumulation of CTCF/cohesin complex at promoter regions and spatial reorganization of chromatin architecture around the nucleolus. In addition, REST/NRSF exerts a similar heterochromatin‐dependent effect on the cPcdh locus. Finally, genetic and genomic data showed that WAVE2 regulates β‐globin gene expression by maintaining heterochromatin status. Together, our data suggested that WAVE2 and REST/NRSF regulate developmental expression of clustered genes in a heterochromatin‐dependent manner.

Zinc finger proteins (ZFPs or ZNFs) constitute the largest family of transcription factors in mammals; however, their regulatory mechanism remains largely elusive. Here we propose COP (C2H2-ZFP occupancy predictor), a deep learning-based heuristic screening tool that integrates DNA sequence with protein primary and secondary features to assess ZFP genomic enrichments. Applying COP to the mouse clustered protocadherin ( cPcdh ) gene locus, we identified dozens of C2H2-ZFPs potentially involved in CTCF-mediated gene regulation with Wiz (widely interspaced zinc finger-containing protein) having the highest number of 12 ZFs. We confirmed Wiz enrichments at all of the CTCF-binding site (CBS) elements across the three Pcdh clusters by Myc-tagging the endogenous Wiz gene. Genetic experiments revealed significant increases of expression levels of the cPcdh genes upon Wiz deletion in both neuronal cells in vitro and in mouse brain in vivo . Finally, integrated ChIP-seq, RNA-seq, and 4C-seq analyses demonstrated that Wiz regulates CTCF/cohesin occupancy and long-range enhancer-promoter contacts in a genomic-distance biased manner. Together, these findings reveal a key role for Wiz in coupling cohesin occupancy to long-range cPcdh regulation and highlight important functions of C2H2-ZFPs in enhancer-promoter interactions.

Chromosomal translocations originate from erroneous rejoining of DNA double-strand break (DSB) ends and mechanisms of their formation remain largely unknown. Here we reveal DSB end configuration and three-dimensional (3D) genome architecture cooperatively shape both translocation frequency and junctional indel pattern. By integrating multiplex CRISPR/Cas9 editing with an optimized translocation assay, we find that translocation frequency correlates with spatial proximity. In addition, templated insertions emerge as a defining structural feature of translocation junctions induced by Cas9-mediated staggered cleavage and confirmed by DISCOVER-seq. Interestingly, translocation frequency is correlated with cleavage geometry. Finally, engineered Cas9 variants with altered DSB end configurations enable programmable modulation of junctional indel patterns. These findings shed significant insights into mechanisms of recurrent chromosomal rearrangements in human diseases and have interesting implications in safe applications of genome editing.

The actin polymerization machinery, comprising the ARP2/3 complex and its activators, the WASP family proteins, has been implicated in regulating a broad spectrum of nuclear processes, such as transcriptional regulation and nuclear organization. Here, using clustered protocadherin ( cPcdh ) and β-globin genes as model systems, we showed that WAVE2 , a member of the WASP family, regulates chromatin organization by maintaining heterochromatin dynamics. Specifically, by CRISPR DNA-fragment editing, in conjunction with integrated analyses of ChIP-seq, MeDIP-seq, ATAC-seq, 4C-seq, and RNA-seq, we showed that deposition of H3K9me3, a key heterochromatin mark, is significantly decreased at the cPcdh locus upon WAVE2 deletion, concurrent with aberrant accumulation of CTCF/cohesin complex at promoter regions and spatial reorganization of chromatin architecture around nucleolus. In addition, REST/NRSF exerts a similar heterochromatindependent effect on the cPcdh locus. Finally, genetic and genomic data showed that WAVE2 regulates β-globin gene expression by maintaining heterochromatin status. Together our data suggested that WAVE2 and REST/NRSF regulate clustered gene expression in a heterochromatin-dependent manner.

Zinc finger (ZF) proteins (ZFPs) constitute the largest family of transcription factors in mammals; however, their regulatory mechanism remains largely elusive. Here we propose COP (C2H2-ZFP occupancy predictor), a deep learning framework that integrates DNA sequence information with protein primary and secondary features to systematically predict ZFP genomic enrichments. Applying COP to the mouse clustered protocadherin ( cPcdh ) gene locus, we identified dozens of C2H2-ZFPs potentially involved in CTCF-mediated gene regulation with Wiz having the highest number of 12 ZFs. We confirmed Wiz enrichments at all of the CBS (CTCF-binding site) elements across the Pcdh clusters by Myc-tagging the endogenous Wiz gene. Genetic experiments revealed significant increases of expression levels of the cPcdh genes upon Wiz deletion in both neuronal cells in vitro and in mouse brain in vivo . Finally, integrated ChIP-seq, RNA-seq, and 4C-seq analyses demonstrated that Wiz regulates CTCF/cohesin occupancy and long-range enhancer-promoter contacts in a genomic-distance biased manner. Together, these findings have interesting implications regarding molecular mechanism of C2H2-ZFPs in cPcdh gene regulation.

CTCF is a central architectural protein that shapes 3D genome organization through sequence-specific DNA binding, but how DNA sequence quantitatively determines CTCF-binding strength remains poorly understood. Progress has been hampered by the lack of large, high-quality measurements of binding affinity. Here, we experimentally determine in vitro CTCF-binding affinity for 276,765 DNA sequences derived from the human genome, generating a comprehensive quantitative landscape of CTCF-DNA interactions. Leveraging these data, we develop DeepCTCF, a deep learning model that predicts CTCF-binding strength directly from DNA sequence and enables quantitative interpretation of CTCF motif grammar. Using this framework, we systematically dissect how specific sequence features modulate CTCF-binding affinity and generate quantitative predictions for disease-associated variants that alter CTCF binding. Together, this study defines general principles by which DNA sequence encodes CTCF-binding affinity and provides a quantitative framework for interpreting regulatory sequence variation.

Topologically associating domain (TAD) boundaries are the flanking edges of TADs, also known as insulated neighborhoods, within the 3D structure of genomes. A prominent feature of TAD boundaries in mammalian genomes is the enrichment of clustered CTCF sites often with mixed orientations, which can either block or facilitate enhancer–promoter (E‐P) interactions within or across distinct TADs, respectively. We will discuss recent progress in the understanding of fundamental organizing principles of the clustered CTCF insulator codes at TAD boundaries. Specifically, both inward‐ and outward‐oriented CTCF sites function as topological chromatin insulators by asymmetrically blocking improper TAD‐boundary‐crossing cohesin loop extrusion. In addition, boundary stacking and enhancer clustering facilitate long‐distance E‐P interactions across multiple TADs. Finally, we provide a unified mechanism for RNA‐mediated TAD boundary function via R‐loop formation for both insulation and facilitation. This mechanism of TAD boundary formation and insulation has interesting implications not only on how the 3D genome folds in the Euclidean nuclear space but also on how the specificity of E‐P interactions is developmentally regulated.

CTCF (CCCTC-binding factor) instructs three-dimensional (3D) genome folding by anchoring or forestalling cohesin loop extrusion, but the exact mechanism remains obscure. Here, using clustered protocadherins ( cPcdh ) as model genes, we report that CTCF assists or facilitates cohesin loop extrusion by enhancing its processivity. Specifically, we show that, compared with the Pcdh α and γ gene clusters, the Pcdhβ cluster is greatly affected upon CTCF ^Y226A/F228A mutation in the N-terminal domain. Given the long-range distance of the Pcdhβ cluster from the distal enhancer, this finding has interesting implications in CTCF regulation of cohesin processivity along the linear chromatin during DNA loop extrusion. In particular, the effect on cohesin processivity upon CTCF ^Y226A/F228A mutation is conspicuously similar to that of WAPL overexpression, suggesting that, in addition to the general view of blocking or forestalling cohesin, CTCF may actually enhance or facilitate cohesin loop extrusion during 3D genome folding. We conclude that CTCF enhances cohesin enrichments via the N-terminal YDF motif in clustered protocadherin genes in a genomic-distance biased manner.