Molecular mechanism of directional CTCF recognition of a diverse range of genomic sites

Yin, Maolu; Wang, Jiuyu; Wang, Min; Li, Xinmei; Zhang, Mo; Wu, Qiang; Wang, Yanli

doi:10.1038/cr.2017.131

Cited by 105 publications

(118 citation statements)

References 57 publications

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“…Second, the irregular nature of extended motifs like those seen for ZFY, where each finger does not appear to interact with the 'expected' three bases, can also inhibit motif detection when searches are guided by motif prediction methods. The CTCF upstream motif, which is outside the border of the predicted motif, was recently shown to be conferred by some irregular structural configuration [59]. A visual comparison between the predicted motifs and ChIP-seq results for 131 human ZFPs with reported motifs suggests that such irregular motifs may be quite common: for example, ZNF140, ZNF324, and ZNF449 all likely contain some irregular motifs with fewer than three basepairs for some internal fingers (Supplemental Figure S3).…”

Section: Discussionmentioning

confidence: 94%

Quantitative analysis of ZFY and CTCF reveals dependent recognition of tandem zinc finger proteins

Zuo

Billings

Walker

et al. 2019

Preprint

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The human genome has more than 800 C2H2 Zinc Finger-containing genes, and many of them are composed of long tandem arrays of zinc fingers. Current Zinc Finger Protein (ZFP) motif prediction models assume longer finger arrays correspond to longer DNA-binding motifs and higher specificity. However, recent experimental efforts to identify ZFP binding sites in vivo contradict this assumption with many having short reported motifs. Using Zinc Finger Y (ZFY), which has 13 ZFs, we quantitatively characterize its DNA binding specificity with several complementary methods, including Affinity-seq, HT-SELEX, Spec-seq and fluorescence anisotropy. Besides the previously identified core motif GGCCT recognized by fingers 12-13, we find a novel secondary irregular motif recognized by accessory fingers. Via high-throughput energy measurements and two-color anisotropy, we establish that this secondary motif contributes to binding and recognition in a non-independent manner, increasing overall affinity only in the presence of the core recognition site. Through additional experimental and iterative computational analysis of CTCF and ZNF343, we further establish that this non-independent recognition between core and secondary motifs could be a general mechanism for tandem zinc finger proteins. These results establish that better motif discovery methods that consider the intrinsic properties of tandem zinc fingers including irregular motif structure, variable spacing and non-independent recognition are essential to improve prediction of ZFP recognition, occupancies, and effects on downstream gene expression in vivo.

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Section: Discussionmentioning

confidence: 94%

Quantitative analysis of ZFY and CTCF reveals dependent recognition of tandem zinc finger proteins

Zuo

Billings

Walker

et al. 2019

Preprint

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“…The plasmid backbone contains a puromycin-resistance gene which is suitable for puromycin selection. In addition, we chose the sgRNA sequence to target module 2 and module 3 of CTCF sites according to the molecular structures of CTCF-DNA complexes [56,57]. The sgRNA expression plasmids were constructed by annealing two overlapping primers and inserting the annealed dsDNA into the plasmid backbone as previously described [37].…”

Section: Targeted Blocking Of Ctcf Sites By Dcas9mentioning

confidence: 99%

“…The first one is based on ChIP-seq experimental data for CTCF occupancy. The second one is based on dynamic interactions between CTCF and its genomic target sites [9,10,56,57].…”

Section: D Lattice Loop Extrusionmentioning

confidence: 99%

“…It was recently reported that CTCF binding to dsDNA is much more dynamic than cohesin and that the residence time of cohesin on DNA fiber is at least 10-fold more than CTCF [9]. The dynamic binding of CTCF to oriented CTCF sites provides hindrance for cohesin sliding [9,10,56,57]. In this scenario, the permeability is calculated as follows.…”

Section: Estimation Of Permeability Of Bins Based On Ctcf Chipseq Datamentioning

confidence: 99%

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Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection

Jia

et al. 2020

Genome Biol

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114

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Background: CTCF is a key insulator-binding protein, and mammalian genomes contain numerous CTCF sites, many of which are organized in tandem. Results: Using CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CTCF sites, if located between enhancers and promoters in the protocadherin (Pcdh) and β-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CTCF sites or not. Moreover, computational simulation in silico and genetic deletions in vivo as well as dCas9 blocking in vitro revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem CTCF sites in the Pcdh and immunoglobulin heavy chain (Igh) clusters. Furthermore, CTCF insulators promote, counter-intuitively, long-range chromatin interactions with distal directional CTCF sites, consistent with the cohesin "loop extrusion" model. Finally, gene expression levels are negatively correlated with CTCF insulators located between enhancers and promoters on a genome-wide scale. Thus, single CTCF insulators ensure proper enhancer insulation and promoter activation while tandem CTCF topological insulators determine balanced spatial contacts and promoter choice.Conclusions: These findings have interesting implications on the role of topological chromatin insulators in 3D genome folding and developmental gene regulation.

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“…We use FIMO (version 5.0.1) to call putative CBSs in both orientations of the hg19 human genome assembly by default parameters with CTCF binding position weight matrices (PWM), which define the probability , for the observed nucleotides ∈ {A, C, G, T} at position ∈ [1,42] of the CTCF motif [12,52].…”

Section: Define Insulator and Its Strengthmentioning

confidence: 99%

Tandem Directional CTCF Sites Balance Protocadherin Promoter Usage

Guo

et al. 2019

Preprint

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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...

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Molecular mechanism of directional CTCF recognition of a diverse range of genomic sites

Cited by 105 publications

References 57 publications

Quantitative analysis of ZFY and CTCF reveals dependent recognition of tandem zinc finger proteins

Quantitative analysis of ZFY and CTCF reveals dependent recognition of tandem zinc finger proteins

Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection

Tandem Directional CTCF Sites Balance Protocadherin Promoter Usage

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