Molecular basis of CTCF binding polarity in genome folding

Abstract Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns.

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Molecular basis of CTCF binding polarity in genome folding

10.1101/2019.12.13.876177 ◽

2019 ◽

Cited By ~ 4

Author(s):

Elphège P. Nora ◽

Laura Caccianini ◽

Geoffrey Fudenberg ◽

Vasumathi Kameswaran ◽

Abigail Nagle ◽

...

Keyword(s):

Single Molecule ◽

Binding Sites ◽

Molecular Basis ◽

Live Imaging ◽

Ctcf Binding ◽

Dna Loops ◽

Genomic Distribution ◽

Chromatin Loops ◽

Topologically Associating Domains ◽

N Terminus

SummaryCurrent models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin proteins (Merkenschlager & Nora, 2016; Fudenberg, Abdennur, Imakaev, Goloborodko, & Mirny, 2017). While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize DNA loops (de Wit et al., 2015; Guo et al., 2015; Rao et al., 2014; Vietri Rudan et al., 2015), the molecular basis of this polarity remains mysterious. Here we report that CTCF positions cohesin but does not control its overall binding or dynamics on chromatin by single molecule live imaging. Using an inducible complementation system, we found that CTCF mutants lacking the N-terminus cannot insulate TADs properly, despite normal binding. Cohesin remained at CTCF sites in this mutant, albeit with reduced enrichment. Given that the orientation of the CTCF motif presents the CTCF N-terminus towards cohesin as it translocates from the interior of TADs, these observations provide a molecular explanation for how the polarity of CTCF binding sites determines the genomic distribution of chromatin loops.

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Systematic Chromatin Architecture Analysis in Xenopus tropicalis Reveals Conserved Three-Dimensional Folding Principles of Vertebrate Genomes

10.1101/2020.04.02.021378 ◽

2020 ◽

Cited By ~ 2

Author(s):

Longjian Niu ◽

Wei Shen ◽

Zhaoying Shi ◽

Na He ◽

Jing Wan ◽

...

Keyword(s):

Binding Sites ◽

Three Dimensional ◽

Xenopus Tropicalis ◽

Ctcf Binding ◽

Interphase Nuclei ◽

Chromosome Architecture ◽

Topologically Associating Domains ◽

Genome Activation ◽

Zygotic Genome ◽

Architecture Analysis

ABSTRACTMetazoan genomes are folded into 3D structures in interphase nuclei. However, the molecular mechanism remains unknown. Here, we show that topologically associating domains (TADs) form in two waves during Xenopus tropicalis embryogenesis, first at zygotic genome activation and then as the expression of CTCF and Rad21 is elevated. We also found TAD structures continually change for at least three times during development. Surprisingly, the directionality index is preferentially stronger on one side of TADs where orientation-biased CTCF and Rad21 binding are observed, a conserved pattern that is found in human cells as well. Depletion analysis revealed CTCF, Rad21, and RPB1, a component of RNAPII, are required for the establishment of TADs. Overall, our work shows that Xenopus is a powerful model for chromosome architecture analysis. Furthermore, our findings indicate that cohesin-mediated extrusion may anchor at orientation-biased CTCF binding sites, supporting a CTCF-anchored extrusion model as the mechanism for TAD establishment.

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CTCF: the protein, the binding partners, the binding sites and their chromatin loops

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2012.0369 ◽

2013 ◽

Vol 368 (1620) ◽

pp. 20120369 ◽

Cited By ~ 108

Author(s):

Sjoerd Johannes Bastiaan Holwerda ◽

Wouter de Laat

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Rna Polymerase Ii ◽

Binding Sites ◽

Ctcf Binding ◽

Chromatin Domain ◽

Gene Promoters ◽

Tissue Specific ◽

Chromatin Loops ◽

Exact Function

CTCF has it all. The transcription factor binds to tens of thousands of genomic sites, some tissue-specific, others ultra-conserved. It can act as a transcriptional activator, repressor and insulator, and it can pause transcription. CTCF binds at chromatin domain boundaries, at enhancers and gene promoters, and inside gene bodies. It can attract many other transcription factors to chromatin, including tissue-specific transcriptional activators, repressors, cohesin and RNA polymerase II, and it forms chromatin loops. Yet, or perhaps therefore, CTCF's exact function at a given genomic site is unpredictable. It appears to be determined by the associated transcription factors, by the location of the binding site relative to the transcriptional start site of a gene, and by the site's engagement in chromatin loops with other CTCF-binding sites, enhancers or gene promoters. Here, we will discuss genome-wide features of CTCF binding events, as well as locus-specific functions of this remarkable transcription factor.

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Capturing the complexity of topologically associating domains through multi-feature optimization

10.1101/2021.01.04.425264 ◽

2021 ◽

Author(s):

Natalie Sauerwald ◽

Carl Kingsford

Keyword(s):

Binding Sites ◽

Three Dimensional ◽

Replication Timing ◽

Biological Properties ◽

Ctcf Binding ◽

Dimensional Structure ◽

Biological Targets ◽

Topologically Associating Domains ◽

Contact Frequency ◽

Algorithmic Framework

AbstractThe three-dimensional structure of human chromosomes is tied to gene regulation and replication timing, but there is still a lack of consensus on the computational and biological definitions for chromosomal substructures such as topologically associating domains (TADs). TADs are described and identified by various computational properties leading to different TAD sets with varying compatibility with biological properties such as boundary occupancy of structural proteins. We unify many of these computational and biological targets into one algorithmic framework that jointly maximizes several computational TAD definitions and optimizes TAD selection for a quantifiable biological property. Using this framework, we explore the variability of TAD sets optimized for six different desirable properties of TAD sets: high occupancy of CTCF, RAD21, and H3K36me3 at boundaries, reproducibility between replicates, high intra- vs inter-TAD difference in contact frequencies, and many CTCF binding sites at boundaries. The compatibility of these biological targets varies by cell type, and our results suggest that these properties are better reflected as subpopulations or families of TADs rather than a singular TAD set fitting all TAD definitions and properties. We explore the properties that produce similar TAD sets (reproducibility and inter- vs intra-TAD difference, for example) and those that lead to very different TADs (such as CTCF binding sites and inter- vs intra-TAD contact frequency difference).

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The formation of chromatin domains: a new model

10.1101/260323 ◽

2018 ◽

Cited By ~ 1

Author(s):

Giorgio Bernardi

Keyword(s):

Dna Sequences ◽

Binding Sites ◽

Ctcf Binding ◽

Second Step ◽

Essential Factor ◽

Chromatin Domains ◽

Topologically Associating Domains ◽

Wavy Structure ◽

Architectural Proteins ◽

Nucleosome Density

In spite of the recent advances in the field of chromatin architecture1,2, the formation mechanism of chromatin domains, TADs, the topologically associating domains, and LADs, the lamina associated domains, is still an open problem. While previous models only dealt with TADs and essentially relied on the architectural proteins CTCF and cohesin, the model presented here concerns both TADs and LADs and is primarily based on the corresponding DNA sequences, the GC-rich and GC-poor isochores, more specifically on their newly discovered 3-D structures. Indeed, the compositionally homogeneous GC-poor isochores were shown to be locally stiff because of the presence of interspersed oligo- Adenines4,5, whereas the compositionally heterogeneous GC-rich isochores were found to be peak-shaped and characterized by increasing gradients of GC and of interspersed oligo- Guanines. In LADs, oligo-Adenines induce local nucleosome depletions4,5 that are responsible for a wavy structure well adapted for interaction with the lamina. In TADs, the increasing GC levels and increasing oligo-Guanines of the isochore peaks are responsible for a decreasing nucleosome density5,6, a decreasing supercoiling7 and an increasing accessibility8. These factors mould the loops of “primary TADs”, that lack self-interactions since they are CTCF/cohesin-free, yet transcriptionally functional structures9-11. This “moulding step” is followed by a second step, in which the cohesin rings bind to the tips of the “primary TADs” and slide down the loops. This process is very likely due to Scc2/Nipbl, an essential factor not only for loading cohesin, but also for stimulating its translocation12 and its ATPase activity13. This “sliding step” creates self-interactions in the loops and stops at the CTCF binding sites located at the base of the loops that are thus closed and insulated.

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A functional overlap between actively transcribed genes and chromatin boundary elements

10.1101/2020.07.01.182089 ◽

2020 ◽

Cited By ~ 2

Author(s):

Caroline L Harrold ◽

Matthew E Gosden ◽

Lars L P Hanssen ◽

Rosa J Stolper ◽

Damien J Downes ◽

...

Keyword(s):

Binding Sites ◽

Globin Gene ◽

Boundary Elements ◽

Ctcf Binding ◽

Chromatin Loop ◽

Globin Genes ◽

Topologically Associating Domains ◽

Binding Factor ◽

Mammalian Genomes ◽

Chromatin Boundary

AbstractMammalian genomes are subdivided into large (50-2000 kb) regions of chromatin referred to as Topologically Associating Domains (TADs or sub-TADs). Chromatin within an individual TAD contacts itself more frequently than with regions in surrounding TADs thereby directing enhancer-promoter interactions. In many cases, the borders of TADs are defined by convergently orientated boundary elements associated with CCCTC-binding factor (CTCF), which stabilises the cohesin complex on chromatin and prevents its translocation. This delimits chromatin loop extrusion which is thought to underlie the formation of TADs. However, not all CTCF-bound sites act as boundaries and, importantly, not all TADs are flanked by convergent CTCF sites. Here, we examined the CTCF binding sites within a ∼70 kb sub-TAD containing the duplicated mouse α-like globin genes and their five enhancers (5’-R1-R2-R3-Rm-R4-α1-α2-3’). The 5’ border of this sub-TAD is defined by a pair of CTCF sites. Surprisingly, we show that deletion of the CTCF binding sites within and downstream of the α-globin locus leaves the sub-TAD largely intact. The predominant 3’ border of the sub-TAD is defined by a steep reduction in contacts: this corresponds to the transcribed α2-globin gene rather than the CTCF sites at the 3’-end of the sub-TAD. Of interest, the almost identical α1- and α2-globin genes interact differently with the enhancers, resulting in preferential expression of the proximal α1-globin gene which behaves as a partial boundary between the enhancers and the distal α2-globin gene. Together, these observations provide direct evidence that actively transcribed genes can behave as boundary elements.Significance StatementMammalian genomes are complex, organised 3D structures, partitioned into Topologically Associating Domains (TADs): chromatin regions that preferentially self-interact. These chromatin interactions are thought to be driven by a mechanism that continuously extrudes chromatin loops, forming structures delimited by chromatin boundary elements and reflecting the activity of enhancers and promoters. Boundary elements bind architectural proteins such as CCCTC-binding factor (CTCF). Previously, an overlap between the functional roles of enhancers and promoters has been shown. However, whether there is overlap between enhancers/promoters and boundary elements is not known. Here, we show that actively transcribed genes can also behave as boundary elements, similar to CTCF boundaries. In both cases, multi-protein complexes bound to these regions may stall the process of chromatin loop extrusion.

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Chromatin topology and the timing of enhancer function at theHoxDlocus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2015083117 ◽

2020 ◽

Vol 117 (49) ◽

pp. 31231-31241

Author(s):

Eddie Rodríguez-Carballo ◽

Lucille Lopez-Delisle ◽

Andréa Willemin ◽

Leonardo Beccari ◽

Sandra Gitto ◽

...

Keyword(s):

Binding Sites ◽

Target Genes ◽

Gene Activation ◽

Ctcf Binding ◽

Topologically Associating Domains ◽

Regulatory Signal ◽

Key Factor ◽

Enhancer Function ◽

Natural Target ◽

Enhancer Sequences

TheHoxDgene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different subgroups ofHoxdgenes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct topologically associating domains (TADs) flankingHoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory submodules. To understand the importance of this particular regulatory topology to controlHoxdgene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites, or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation ofHoxdgenes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancer sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavorable orientation of CTCF sites.

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CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911708117 ◽

2020 ◽

Vol 117 (4) ◽

pp. 2020-2031 ◽

Cited By ~ 21

Author(s):

Elena M. Pugacheva ◽

Naoki Kubo ◽

Dmitri Loukinov ◽

Md Tajmul ◽

Sungyun Kang ◽

...

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Mammalian Cells ◽

Zinc Fingers ◽

Complex Function ◽

Ctcf Binding ◽

Loop Formation ◽

Dna Binding Sites ◽

3D Geometry ◽

N Terminus

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF–BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF–DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.

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A Modified Autonomous En Transposon in Maize (Zea mays L.) Elicits a Differential Response of Reporter Alleles

Genetics ◽

10.1093/genetics/147.3.1329 ◽

1997 ◽

Vol 147 (3) ◽

pp. 1329-1338

Author(s):

Peter A Peterson

Keyword(s):

Molecular Structure ◽

Binding Sites ◽

Molecular Basis ◽

Zea Mays L ◽

Phenotypic Expression ◽

Terminal Inverted Repeat ◽

Coding Sequences ◽

Defective Element ◽

Related Element ◽

Unstable Genes

Transposable elements in maize are composed of a defined molecular structure that includes coding sequences, determiners of functionality and ordered terminal motifs that provide binding sites for transposase proteins. Alterations in these components change the phenotypic expression of unstable genes with transposon inserts. The molecular basis for the altered timing and frequency of transposition as determined by the size and number of spots on kernels or stripes on leaves has generally been described for defective inserts in genes. Most differential patterns can be ascribed to alterations in the terminal motifs of the reporter allele structure that supplies a substrate (terminal inverted repeat motifs) for transposase activity. For autonomously functioning alleles, the explanations for changes in phenotype are not so clear. In this report, an En-related element identified as F-En is described that shares with En the recognition of a specific defective element c1(mr)888104 but differs from En in that this F-En element does not recognize the canonical c1(mr) elements that are recognized by En. Evidence is provided suggesting that F-En does not recognize other En/Spm-related defective elements, some of whose sequences are known. This modified En arose from a c1-m autonomously mutating En allele.

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