scholarly journals Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes

2015 ◽  
Vol 112 (47) ◽  
pp. E6456-E6465 ◽  
Author(s):  
Adrian L. Sanborn ◽  
Suhas S. P. Rao ◽  
Su-Chen Huang ◽  
Neva C. Durand ◽  
Miriam H. Huntley ◽  
...  
Keyword(s):  
Genome Editing ◽  
Binding Sites ◽  
Physical Structure ◽  
Ctcf Binding ◽  
Binding Motifs ◽  
Physical Simulations ◽  
Chromatin Folding ◽  
Mammalian Genomes ◽  
Single Base Pair ◽  
Fractal Globule

We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic “tension globule.” In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

scholarly journals CTCF Binding Sites in the Herpes Simplex Virus 1 Genome Display Site-Specific CTCF Occupation, Protein Recruitment, and Insulator Function

2018 ◽  
Vol 92 (8) ◽  
pp. e00156-18 ◽  
Author(s):  
Shannan D. Washington ◽  
Farhana Musarrat ◽  
Monica K. Ertel ◽  
Gregory L. Backes ◽  
Donna M. Neumann
Keyword(s):  
Herpes Simplex ◽  
Binding Sites ◽  
Ctcf Binding ◽  
Herpes Simplex Virus 1 ◽  
Binding Motifs ◽  
Site Specific ◽  
Specific Manner ◽  
Simplex Virus ◽  
Protein Recruitment ◽  
Hsv 1

ABSTRACTThere are seven conserved CTCF binding domains in the herpes simplex virus 1 (HSV-1) genome. These binding sites individually flank the latency-associated transcript (LAT) and the immediate early (IE) gene regions, suggesting that CTCF insulators differentially control transcriptional domains in HSV-1 latency. In this work, we show that two CTCF binding motifs in HSV-1 display enhancer blocking in a cell-type-specific manner. We found that CTCF binding to the latent HSV-1 genome was LAT dependent and that the quantity of bound CTCF was site specific. Following reactivation, CTCF eviction was dynamic, suggesting that each CTCF site was independently regulated. We explored whether CTCF sites recruit the polycomb-repressive complex 2 (PRC2) to establish repressive domains through a CTCF-Suz12 interaction and found that Suz12 colocalized to the CTCF insulators flanking the ICP0 and ICP4 regions and, conversely, was removed at early times postreactivation. Collectively, these data support the idea that CTCF sites in HSV-1 are independently regulated and may contribute to lytic-latent HSV-1 control in a site-specific manner.IMPORTANCEThe role of chromatin insulators in DNA viruses is an area of interest. It has been shown in several beta- and gammaherpesviruses that insulators likely control the lytic transcriptional profile through protein recruitment and through the formation of three-dimensional (3D) chromatin loops. The ability of insulators to regulate alphaherpesviruses has been understudied to date. The alphaherpesvirus HSV-1 has seven conserved insulator binding motifs that flank regions of the genome known to contribute to the establishment of latency. Our work presented here contributes to the understanding of how insulators control transcription of HSV-1.

scholarly journals Co-opted transposons help perpetuate conserved higher-order chromosomal structures

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Mayank NK Choudhary ◽  
Ryan Z. Friedman ◽  
Julia T. Wang ◽  
Hyo Sik Jang ◽  
Xiaoyu Zhuo ◽  
...  
Keyword(s):  
Binding Sites ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Genome Architecture ◽  
Mutational Signatures ◽  
Domain Structures ◽  
Domain Boundaries ◽  
Architectural Proteins ◽  
Mammalian Genomes ◽  
Species Specific

Abstract Background Transposable elements (TEs) make up half of mammalian genomes and shape genome regulation by harboring binding sites for regulatory factors. These include binding sites for architectural proteins, such as CTCF, RAD21, and SMC3, that are involved in tethering chromatin loops and marking domain boundaries. The 3D organization of the mammalian genome is intimately linked to its function and is remarkably conserved. However, the mechanisms by which these structural intricacies emerge and evolve have not been thoroughly probed. Results Here, we show that TEs contribute extensively to both the formation of species-specific loops in humans and mice through deposition of novel anchoring motifs, as well as to the maintenance of conserved loops across both species through CTCF binding site turnover. The latter function demonstrates the ability of TEs to contribute to genome plasticity and reinforce conserved genome architecture as redundant loop anchors. Deleting such candidate TEs in human cells leads to the collapse of conserved loop and domain structures. These TEs are also marked by reduced DNA methylation and bear mutational signatures of hypomethylation through evolutionary time. Conclusions TEs have long been considered a source of genetic innovation. By examining their contribution to genome topology, we show that TEs can contribute to regulatory plasticity by inducing redundancy and potentiating genetic drift locally while conserving genome architecture globally, revealing a paradigm for defining regulatory conservation in the noncoding genome beyond classic sequence-level conservation.

scholarly journals Defining the relative and combined contribution of CTCF and CTCFL to genomic regulation

2019 ◽  
Author(s):  
Mayilaadumveettil Nishana ◽  
Caryn Ha ◽  
Javier Rodriguez-Hernaez ◽  
Ali Ranjbaran ◽  
Erica Chio ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Binding Sites ◽  
Ctcf Binding ◽  
Chromosome Organization ◽  
Chimeric Proteins ◽  
Loop Formation ◽  
Cellular Functions ◽  
Intergenic Regions ◽  
Chromatin Folding ◽  
The Impact

BackgroundUbiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL is normally only present in testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription.ResultsWe demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites, to regulate transcription, chromatin looping and insulation.ConclusionThis study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

scholarly journals ZCCHC8 is required for the degradation of pervasive transcripts originating from multiple genomic regulatory features

2021 ◽  
Author(s):  
Joshua W. Collins ◽  
Daniel Martin ◽  
Shaohe Wang ◽  
Kenneth M. Yamada ◽  
Keyword(s):  
Binding Sites ◽  
Rna Binding ◽  
Ctcf Binding ◽  
Open Chromatin ◽  
Transcription Start Sites ◽  
Non Coding Rna ◽  
Mammalian Genomes ◽  
Nuclear Exosome ◽  
Flanking Regions ◽  
Pervasive Transcription

ABSTRACTThe vast majority of mammalian genomes are transcribed as non-coding RNA in what is referred to as “pervasive transcription.” Recent studies have uncovered various families of non-coding RNA transcribed upstream of transcription start sites. In particular, highly unstable promoter upstream transcripts known as PROMPTs have been shown to be targeted for exosomal degradation by the nuclear exosome targeting complex (NEXT) consisting of the RNA helicase MTR4, the zinc-knuckle scaffold ZCCHC8, and the RNA binding protein RBM7. Here, we report that in addition to its known RNA substrates, ZCCHC8 is required for the targeted degradation of pervasive transcripts produced at CTCF binding sites, open chromatin regions, promoters, promoter flanking regions, and transcription factor binding sites. Additionally, we report that a significant number of RIKEN cDNAs and predicted genes display the hallmarks of PROMPTs and are also substrates for ZCCHC8 and/or NEXT complex regulation suggesting these are unlikely to be functional genes. Our results suggest that ZCCHC8 and/or the NEXT complex may play a larger role in the global regulation of pervasive transcription than previously reported.

scholarly journals AddTag, a two-step approach with supporting software package that facilitates CRISPR/Cas-mediated precision genome editing

2021 ◽  
Author(s):  
Thaddeus D Seher ◽  
Namkha Nguyen ◽  
Diana Ramos ◽  
Priyanka Bapat ◽  
Clarissa J Nobile ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Genome Editing ◽  
Binding Sites ◽  
Software Package ◽  
Gene Editing ◽  
Target Site ◽  
Genomic Features ◽  
Single Base ◽  
Gene Complementation ◽  
Single Base Pair

Abstract CRISPR/Cas-induced genome editing is a powerful tool for genetic engineering, however targeting constraints limit which loci are editable with this method. Since the length of a DNA sequence impacts the likelihood it overlaps a unique target site, precision editing of small genomic features with CRISPR/Cas remains an obstacle. We introduce a two-step genome editing strategy that virtually eliminates CRISPR/Cas targeting constraints and facilitates precision genome editing of elements as short as a single base-pair at virtually any locus in any organism that supports CRISPR/Cas-induced genome editing. Our two-step approach first replaces the locus of interest with an “AddTag” sequence, which is subsequently replaced with any engineered sequence, and thus circumvents the need for direct overlap with a unique CRISPR/Cas target site. In this study, we demonstrate the feasibility of our approach by editing transcription factor binding sites within Candida albicans that could not be targeted directly using the traditional gene editing approach. We also demonstrate the utility of the AddTag approach for combinatorial genome editing and gene complementation analysis, and we present a software package that automates the design of AddTag editing.

scholarly journals A functional overlap between actively transcribed genes and chromatin boundary elements

2020 ◽  
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.

scholarly journals CTCF Mediates Dosage and Sequence-context-dependent Transcriptional Insulation through Formation of Local Chromatin Domains

2020 ◽  
Author(s):  
Hui Huang ◽  
Quan Zhu ◽  
Adam Jussila ◽  
Yuanyuan Han ◽  
Bogdan Bintu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Binding Sites ◽  
Imaging Techniques ◽  
Critical Role ◽  
Embryonic Stem ◽  
Ctcf Binding ◽  
Chromatin Domain ◽  
Chromatin Domains ◽  
Binding Motifs ◽  
Binding Factor

AbstractInsulators play a critical role in spatiotemporal gene expression in metazoans by separating active and repressive chromatin domains and preventing inappropriate enhancer-promoter contacts. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here, we explore the sequence requirements of CTCF-mediated transcriptional insulation with the use of a sensitive insulator reporter assay in mouse embryonic stem cells. We find that insulation potency depends on the number of CTCF binding sites in tandem. Furthermore, CTCF-mediated insulation is dependent on DNA sequences flanking its core binding motifs, and CTCF binding sites at topologically associating domain(TAD) boundaries are more likely to function as insulators than those outside TAD boundaries, independent of binding strength. Using chromosomal conformation capture assays and high-resolution chromatin imaging techniques, we demonstrate that insulators form local chromatin domain boundaries and reduce enhancer-promoter contacts. Taken together, our results provide strong genetic, molecular, and structural evidence connecting chromatin topology to the action of insulators in the mammalian genome.

scholarly journals Evolution of mouse circadian enhancers from transposable elements

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Julius Judd ◽  
Hayley Sanderson ◽  
Cédric Feschotte
Keyword(s):  
Gene Expression ◽  
Transposable Elements ◽  
Binding Sites ◽  
Mouse Liver ◽  
Integration Site ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Circadian Gene ◽  
Binding Motifs ◽  
Reporter Assays

Abstract Background Transposable elements are increasingly recognized as a source of cis-regulatory variation. Previous studies have revealed that transposons are often bound by transcription factors and some have been co-opted into functional enhancers regulating host gene expression. However, the process by which transposons mature into complex regulatory elements, like enhancers, remains poorly understood. To investigate this process, we examined the contribution of transposons to the cis-regulatory network controlling circadian gene expression in the mouse liver, a well-characterized network serving an important physiological function. Results ChIP-seq analyses reveal that transposons and other repeats contribute ~ 14% of the binding sites for core circadian regulators (CRs) including BMAL1, CLOCK, PER1/2, and CRY1/2, in the mouse liver. RSINE1, an abundant murine-specific SINE, is the only transposon family enriched for CR binding sites across all datasets. Sequence analyses and reporter assays reveal that the circadian regulatory activity of RSINE1 stems from the presence of imperfect CR binding motifs in the ancestral RSINE1 sequence. These motifs matured into canonical motifs through point mutations after transposition. Furthermore, maturation occurred preferentially within elements inserted in the proximity of ancestral CR binding sites. RSINE1 also acquired motifs that recruit nuclear receptors known to cooperate with CRs to regulate circadian gene expression specifically in the liver. Conclusions Our results suggest that the birth of enhancers from transposons is predicated both by the sequence of the transposon and by the cis-regulatory landscape surrounding their genomic integration site.

Abstract P531: FOXO3 , a Cardiovascular Protector, is at the Hub of a 46-gene Cell Resilience “Gene Factory” on Human Chromosome 6

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Brian J Morris ◽  
Timothy A Donlon ◽  
Randi Chen ◽  
Kamal H Masaki ◽  
Richard C Allsopp ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Cell Lines ◽  
Binding Sites ◽  
Healthy Aging ◽  
Nutrient Sensing ◽  
Physical Contact ◽  
Ctcf Binding ◽  
Lymphoblastoid Cell Lines ◽  
Chromosome 6 ◽  
Disease Etiology

The transcription factor FoxO3 regulates multiple genes involved in cell resilience. We have previously implicated variation in non-coding DNA of the FoxO3 gene ( FOXO3 ) with lower blood pressure, reduced inflammation, less hypertension, reduced coronary heart disease mortality, and longevity. The aim of the present study was to determine transcriptional, genetic and genomic mechanisms involving FOXO3 . By DNA sequencing of chromosome 6q21 in lymphoblastoid cell lines of 95 men who had survived to ≥ 95 years of age we identified 110 FOXO3 single nucleotide polymorphisms (SNPs). Thirteen SNPs were at binding sites for 18 transcription factors. Those SNPs appeared to be in physical contact, via RNA polymerase II binding chromatin looping, with sites in the FOXO3 promoter, and likely function together as a cis -regulatory unit. At the chromosome level, FOXO3 was located at the center of a 7.3 Mb 46-gene chromatin domain flanked by gene deserts. We identified distant contact points between FOXO3 and these 46 neighboring genes, through long-range physical contacts via CCCTC-binding factor zinc finger protein (CTCF) binding sites. The genes in this “archipelago” of neighbourhood genes mediate a similar repertoire of functions as FoxO3, including stress resistance, nutrient sensing, cell proliferation, autophagy, apoptosis and stem cell maintenance. The 7.3 Mb gene domain was highly conserved across species, indicating evolutionary importance. We believe that FOXO3 serves as the hub for an “interactome” involved in healthy aging, including cardiovascular disease reduction, in those with favorable FOXO3 genotypes. In support, we found that cellular stress (H 2 O 2 ) could stimulate FOXO3 expression in 20 lymphoblastoid cell lines, being 3-fold stronger for those with a favorable FOXO3 genotype. In FISH experiments, stress-induced activation of FOXO3 caused it to move towards its neighboring genes as suggested by our genomic data. In conclusion, we have shown, for the first time, that FOXO3 is at the central hub of a gene network on chromosome 6 involved in cell protection and healthy aging. The concept of “gene factories” may apply more broadly to genome and genetic mechanisms involved in cardiovascular disease etiology.

scholarly journals Functions of Gtf2i and Gtf2ird1 in the developing brain: transcription, DNA-binding, and long term behavioral consequences

2019 ◽  
Author(s):  
Nathan D. Kopp ◽  
Kayla R. Nygaard ◽  
Katherine B. McCullough ◽  
Susan E. Maloney ◽  
Harrison W. Gabel ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Conditioned Fear ◽  
Developing Brain ◽  
Ctcf Binding ◽  
Microdeletion Syndrome ◽  
Behavioral Phenotypes ◽  
Marble Burying ◽  
And Behavior

AbstractGtf2ird1 and Gtf2i may mediate aspects of the cognitive and behavioral phenotypes of Williams Syndrome (WS) – a microdeletion syndrome encompassing these transcription factors (TFs). Knockout mouse models of each TF show behavioral phenotypes. Here we identify their genomic binding sites in the developing brain, and test for additive effects of their mutation on transcription and behavior. Both TFs target constrained chromatin modifier and synaptic protein genes, including a significant number of ASD genes. They bind promoters, strongly overlap CTCF binding and TAD boundaries, and moderately overlap each other, suggesting epistatic effects. We used single and double mutants to test whether mutating both TFs will modify transcriptional and behavioral phenotypes of single Gtf2ird1 mutants. Despite little difference in DNA-binding and transcriptome-wide expression, Gtf2ird1 mutation caused balance, marble burying, and conditioned fear phenotypes. However, mutating Gtf2i in addition to Gtf2ird1 did not further modify transcriptomic or most behavioral phenotypes, suggesting Gtf2ird1 mutation alone is sufficient.

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