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.

Chromatin Structure and Transcription of the Human Alpha Globin Locus during Erythroid Differentation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3575-3575
Author(s):  
Milind C Mahajan ◽  
Subhradip Karmakar ◽  
Sherman M. Weissman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Ctcf Binding ◽  
Gene Promoters ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Pol Ii ◽  
Cd34 Cells ◽  
Alpha Globin ◽  
Globin Promoter

Abstract The human alpha globin genes are controlled by DNase hypersensitive sites (HS) HS-4, HS-8, HS-10, HS-33 and HS-40 upstream of the ζ gene. Among these, HS40 functions as a strong enhancer of the alpha like genes. The alpha globin genes are situated amidst actively transcribing genes, but are transcriptionally silent in non-erythroid cells including hematopoietic progenitor cells We have undertaken an analysis of the chromatin structure of the alpha globin locus, recruitment of transcription factors, and the transcriptional activity of the locus in CD34+ hematopoietic progenitor cells and upon their differentiation into erythroid cells. Chromatin immunoprecipitation (ChIP) followed by PCR analysis of all the regulatory and structural segments of the α-globin locus were performed using antibodies against chemically modified tails of histone H3, the insulator binding factor CTCF, transcription factors such as GATA-1 and NF-E2, and Pol II. Both H3Me2K4 and H3AcK9 modifications were present at HS48 and HS33 in CD34+ cells and substantially increase when these cells are differentiated into erythroid lineage. At the HS40 region, these modifications were present at a low level in CD34+ cells and did not change during erythroid differentiation. Among the α-like gene promoters, we find these modifications at the Mu and theta gene promoters in CD34+ cells and they increase during erythropoiesis. These modifications were absent at the zeta gene promoter consistent with the inactivity of this gene during definitive erythropoiesis. Overall the dominant HS40 enhancer possesses moderate levels of H3Me2K4 and H3AcK9 modifications, and its cognate major a-globin promoter is devoid of these modifications in CD34+ cells even when these cells are differentiated into erythroid lineage. The entire α-globin locus including the HS enhancer regions and a-like gene promoters did not contain the unphosphorylated (initiation) form of Pol II recruitment in CD34+ cells. When these cells differentiated into the erythroid lineage, Pol II was recruited at the HS40 and HS48 regions and at the Mu and theta promoters. Rearrangement of the CTCF binding sites at the α-globin locus occurs during differentiation of CD34+ cells into the erythroid lineage. In CD34+ cells, as in HeLa cells, the α-globin genes are flanked by multiple CTCF binding events at the 5′ and 3′ ends of the locus. At the 5′ end of the locus, the HS40 and HS48 sequences were surrounded by four CTCF binding sites at HS33, HS46, HS55 and HS90. At the 3′ end of the locus CTCF was observed at the theta globin promoter and at the 3′ end of the theta globin gene. Upon differentiation of the CD34+ cells into the erythroid pathway, CTCF recruitment is significantly reduced at HS90 and HS46 sequences, while the sites at HS55 and HS33 show increased CTCF binding. Thus, in contrast to the CD34+ cells, the HS40 and HS48 sequences are y flanked by two CTCF recruitment sites in erythroid cells. Such a differential placement of CTCF binding sites suggests that differential interaction among CTCF sites may regulate the effects of the HS-40 enhancer. In erythroid cells, a strong HS40 enhancer formed by virtue of the recruitment of the enhancer factors can overcome blocking by the downstream flanking CTCF site and this might be mediated by specific interactions between the two flanking insulators. The CTCF binding at the 3′ end of the theta globin gene is abolished during erythropoiesis of CD34+ cells. However, the recruitment of CTCF at the theta globin promoter is unchanged suggesting that the theta globin may be insulated by the influence of the α-globin enhancer sequences. We have detected transcripts from parts of the theta and zeta genes and intergenic regions in HeLa, NB4 and 06990 lymphoblastoid cells and primary erythroid cells in culture. The transcription of the locus was localized to certain regions, suggesting that there may be unappreciated transcriptional regulatory elements within the locus.

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 The -175T----C mutation increases promoter strength in erythroid cells: correlation with evolutionary conservation of binding sites for two trans-acting factors

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 756-761 ◽  
Author(s):  
DL Gumucio ◽  
WK Lockwood ◽  
JL Weber ◽  
AM Saulino ◽  
K Delgrosso ◽  
...  
Keyword(s):  
Binding Site ◽  
Point Mutation ◽  
Binding Sites ◽  
Globin Gene ◽  
Evolutionary Conservation ◽  
Single Point Mutation ◽  
Promoter Strength ◽  
Globin Genes ◽  
Gamma Globin ◽  
Specific Distribution

Abstract A point mutation at position -175 has been detected in Agamma as well as Ggamma globin genes in individuals with hereditary persistence of fetal hemoglobin (HPFH). To prove that this single point mutation results in increased promoter strength, we transfected erythroid and nonerythroid cell lines with constructs containing normal and mutant promoters linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Differences in transfection efficiency were controlled by cotransfection of pRSVgpt. In K562 erythroleukemia cells, the -175 HPFH promoter directed three- to fourfold more CAT activity than its wild type counterpart. However, in HeLa cells the two promoters were similar in strength. The -195 to -165 region of the gamma-globin promoter contains binding sites for two proteins: a ubiquitously distributed octamer binding protein, OBP, and the erythroid-specific protein, GF-1. We find that while the GF-1 binding site is highly conserved among related primate gamma-globin genes, the octamer binding site is not. The evolutionary conservation of GF-1 as well as its erythroid-specific distribution suggest that this protein is important in gamma-globin gene expression. A role for OBP in the regulation of gamma-globin, if any, must have arisen recently in primate evolution.

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

2020 ◽  
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.

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 Human β-Globin Locus Control Region HS5Contains CTCF- and Developmental Stage-Dependent Enhancer-BlockingActivity in ErythroidCells

2003 ◽  
Vol 23 (24) ◽  
pp. 8946-8952 ◽  
Author(s):  
Keiji Tanimoto ◽  
Akiko Sugiura ◽  
Akane Omori ◽  
Gary Felsenfeld ◽  
James Douglas Engel ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Control Region ◽  
Yeast Artificial Chromosome ◽  
Globin Gene ◽  
Artificial Chromosome ◽  
Locus Control Region ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Globin Genes ◽  
Mechanistic Basis

ABSTRACT The human β-globin locus contains five developmentally regulatedβ -type globin genes. All five genes depend on the locus control region (LCR), located at the 5′ end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human β-globin yeast artificial chromosome transgenic mice and observed reduced expression of all theβ -type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancer-blocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the ε-globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the β-globin gene.

scholarly journals CCCTC-Binding Factor Acts as a Heterochromatin Barrier on Herpes Simplex Viral Latent Chromatin and Contributes to Poised Latent Infection

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Jennifer S. Lee ◽  
Priya Raja ◽  
Dongli Pan ◽  
Jean M. Pesola ◽  
Donald M. Coen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Binding Sites ◽  
Latent Infection ◽  
Ctcf Binding ◽  
Herpes Simplex Virus 1 ◽  
Binding Factor ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTHerpes simplex virus 1 (HSV-1) establishes latent infection in neurons via a variety of epigenetic mechanisms that silence its genome. The cellular CCCTC-binding factor (CTCF) functions as a mediator of transcriptional control and chromatin organization and has binding sites in the HSV-1 genome. We constructed an HSV-1 deletion mutant that lacked a pair of CTCF-binding sites (CTRL2) within the latency-associated transcript (LAT) coding sequences and found that loss of these CTCF-binding sites did not alter lytic replication or levels of establishment of latent infection, but their deletion reduced the ability of the virus to reactivate from latent infection. We also observed increased heterochromatin modifications on viral chromatin over theLATpromoter and intron. We therefore propose that CTCF binding at theCTRL2sites acts as a chromatin insulator to keep viral chromatin in a form that is poised for reactivation, a state which we call poised latency.IMPORTANCEHerpes simplex virus 1 (HSV-1) is a human pathogen that persists for the lifetime of the host as a result of its ability to establish latent infection within sensory neurons. The mechanism by which HSV-1 transitions from the lytic to latent infection program is largely unknown; however, HSV-1 is able to coopt cellular silencing mechanisms to facilitate the suppression of lytic gene expression. Here, we demonstrate that the cellular CCCTC-binding factor (CTCF)-binding site within the latency associated transcript (LAT) region is critical for the maintenance of a specific local chromatin structure. Additionally, loss of CTCF binding has detrimental effects on the ability to reactivate from latent infection. These results argue that CTCF plays a critical role in epigenetic regulation of viral gene expression to establish and/or maintain a form of latent infection that can reactivate efficiently.

scholarly journals Molecular basis of CTCF binding polarity in genome folding

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Elphège P. Nora ◽  
Laura Caccianini ◽  
Geoffrey Fudenberg ◽  
Kevin So ◽  
Vasumathi Kameswaran ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Molecular Basis ◽  
Live Imaging ◽  
Ctcf Binding ◽  
Chromatin Loops ◽  
Topologically Associating Domains ◽  
N Terminus

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.

Functional and comparative analysis of globin loci in pufferfish and humans

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2842-2849 ◽  
Author(s):  
Nynke Gillemans ◽  
Tara McMorrow ◽  
Rita Tewari ◽  
Albert W. K. Wai ◽  
Carola Burgtorf ◽  
...  
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Cell Types ◽  
Specific Cell ◽  
Fish Analysis ◽  
Globin Genes ◽  
Site Mapping ◽  
Physical Linkage ◽  
Human And Mouse

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing α- and β-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show thatFugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only α-globin genes (α-locus), whereas the other also contains a β-globin gene (αβ-locus). This is the first poikilothermic species analyzed in which the physical linkage of the α- and β-globin genes has been uncoupled, supporting a model in which the separation of the α- and β-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu αβ-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian β-globin clusters, may not be present in the Fugu αβ-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types.

scholarly journals Molecular basis of CTCF binding polarity in genome folding

2019 ◽  
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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