Chromatin topology and the timing of enhancer function at theHoxDlocus

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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Chromatin topology and the timing of enhancer function at the hoxd locus

10.1101/2020.07.12.199109 ◽

2020 ◽

Cited By ~ 1

Author(s):

Eddie Rodríguez-Carballo ◽

Lucille Lopez-Delisle ◽

Andréa Willemin ◽

Leonardo Beccari ◽

Sandra Gitto ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Gene Activation ◽

Ctcf Binding ◽

Regulatory Sequences ◽

Promoter Recognition ◽

Key Factor ◽

Natural Target ◽

Enhancer Sequences ◽

The Impact

ABSTRACTThe HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different sub-groups of Hoxd genes 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 TADs flanking HoxD, 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 sub-modules. To understand the importance of this particular regulatory topology to control Hoxd gene 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 of Hoxd genes 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 enhancers 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 unfavourable orientation of CTCF sites.SIGNIFICANCE STATEMENTMany genes important for vertebrate development are surrounded by series of remote enhancer sequences. Such regulatory landscapes and their target genes are usually located within the same chromatin domains, which appears to constrain the action of these regulatory sequences and hence to facilitate enhancer-promoter recognition and gene expression. We used the HoxD locus to assess the impact of modifying the regulatory topology upon gene activation in space and time. A series of chromosomal re-arrangements involving deletions and inversions reveals that the enhancer topology plays a role in the timing of gene activation. However, gene expression was often recovered, subsequently, illustrating the intrinsic capacity of some enhancers to find their target promoters despite an apparently adverse chromatin topology.

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Low-affinity transcription factor binding sites shape morphogen responses and enhancer evolution

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0018 ◽

2013 ◽

Vol 368 (1632) ◽

pp. 20130018 ◽

Cited By ~ 67

Author(s):

Andrea I. Ramos ◽

Scott Barolo

Keyword(s):

Transcription Factor ◽

Binding Affinity ◽

Binding Sites ◽

Target Genes ◽

Gene Activation ◽

Transcriptional Response ◽

Morphogen Gradients ◽

Weak Binding ◽

Initial Hypothesis

In the era of functional genomics, the role of transcription factor (TF)–DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. Here, we investigate the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients . We noted that enhancers of several Hh-responsive Drosophila genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to our initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of dpp , wingless and stripe , by transplanting optimal sites from the patched gene, did not result in ectopic responses to Hh signalling. Instead, we found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, we observed patterning defects in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. We propose that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes.

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An H3K4me3 reader, BAP18 as an adaptor of COMPASS-like core subunits co-activates ERα action and associates with the sensitivity of antiestrogen in breast cancer

Nucleic Acids Research ◽

10.1093/nar/gkaa787 ◽

2020 ◽

Vol 48 (19) ◽

pp. 10768-10784

Author(s):

Ge Sun ◽

Chunyu Wang ◽

Shengli Wang ◽

Hongmiao Sun ◽

Kai Zeng ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Progression ◽

Binding Sites ◽

Estrogen Receptor Alpha ◽

Target Genes ◽

Regulatory Element ◽

Therapy Resistance ◽

Ctcf Binding ◽

Promoter Regions ◽

Positive Breast Cancer

Abstract Estrogen receptor alpha (ERα) signaling pathway is essential for ERα-positive breast cancer progression and endocrine therapy resistance. Bromodomain PHD Finger Transcription Factor (BPTF) associated protein of 18kDa (BAP18) has been recognized as a crucial H3K4me3 reader. However, the whole genomic occupation of BAP18 and its biological function in breast cancer is still elusive. Here, we found that higher expression of BAP18 in ERα-positive breast cancer is positively correlated with poor prognosis. ChIP-seq analysis further demonstrated that the half estrogen response elements (EREs) and the CCCTC binding factor (CTCF) binding sites are the significant enrichment sites found in estrogen-induced BAP18 binding sites. Also, we provide the evidence to demonstrate that BAP18 as a novel co-activator of ERα is required for the recruitment of COMPASS-like core subunits to the cis-regulatory element of ERα target genes in breast cancer cells. BAP18 is recruited to the promoter regions of estrogen-induced genes, accompanied with the enrichment of the lysine 4-trimethylated histone H3 tail (H3K4me3) in the presence of E2. Furthermore, BAP18 promotes cell growth and associates the sensitivity of antiestrogen in ERα-positive breast cancer. Our data suggest that BAP18 facilitates the association between ERα and COMPASS-like core subunits, which might be an essential epigenetic therapeutic target for breast cancer.

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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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Luminal A Breast Cancer Co-expression Network: Structural and Functional Alterations

Frontiers in Genetics ◽

10.3389/fgene.2021.629475 ◽

2021 ◽

Vol 12 ◽

Author(s):

Diana García-Cortés ◽

Enrique Hernández-Lemus ◽

Jesús Espinal-Enríquez

Keyword(s):

Breast Cancer ◽

Network Topology ◽

Binding Sites ◽

Target Genes ◽

Molecular Subtype ◽

Differential Expression Analysis ◽

Ctcf Binding ◽

Luminal A ◽

Long Distance ◽

Transcriptomic Level

Luminal A is the most common breast cancer molecular subtype in women worldwide. These tumors have characteristic yet heterogeneous alterations at the genomic and transcriptomic level. Gene co-expression networks (GCNs) have contributed to better characterize the cancerous phenotype. We have previously shown an imbalance in the proportion of intra-chromosomal (cis-) over inter-chromosomal (trans-) interactions when comparing cancer and healthy tissue GCNs. In particular, for breast cancer molecular subtypes (Luminal A included), the majority of high co-expression interactions connect gene-pairs in the same chromosome, a phenomenon that we have called loss of trans- co-expression. Despite this phenomenon has been described, the functional implication of this specific network topology has not been studied yet. To understand the biological role that communities of co-expressed genes may have, we constructed GCNs for healthy and Luminal A phenotypes. Network modules were obtained based on their connectivity patterns and they were classified according to their chromosomal homophily (proportion of cis-/trans- interactions). A functional overrepresentation analysis was performed on communities in both networks to observe the significantly enriched processes for each community. We also investigated possible mechanisms for which the loss of trans- co-expression emerges in cancer GCN. To this end we evaluated transcription factor binding sites, CTCF binding sites, differential gene expression and copy number alterations (CNAs) in the cancer GCN. We found that trans- communities in Luminal A present more significantly enriched categories than cis- ones. Processes, such as angiogenesis, cell proliferation, or cell adhesion were found in trans- modules. The differential expression analysis showed that FOXM1, CENPA, and CIITA transcription factors, exert a major regulatory role on their communities by regulating expression of their target genes in other chromosomes. Finally, identification of CNAs, displayed a high enrichment of deletion peaks in cis- communities. With this approach, we demonstrate that network topology determine, to at certain extent, the function in Luminal A breast cancer network. Furthermore, several mechanisms seem to be acting together to avoid trans- co-expression. Since this phenomenon has been observed in other cancer tissues, a remaining question is whether the loss of long distance co-expression is a novel hallmark of cancer.

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Controlling gene activation by enhancers through a drug-inducible topological insulator

10.1101/534073 ◽

2019 ◽

Author(s):

Taro Tsujimura ◽

Osamu Takase ◽

Masahiro Yoshikawa ◽

Etsuko Sano ◽

Matsuhiko Hayashi ◽

...

Keyword(s):

Pluripotent Stem Cells ◽

Binding Sites ◽

Time Course ◽

Gene Activation ◽

Ctcf Binding ◽

Control Gene ◽

Expression Change ◽

Krab Domain ◽

Gene Enhancer ◽

Drug Induction

AbstractWhile regulation of gene-enhancer interaction is better understood, its application remains limited. Here, we reconstituted arrays of CTCF binding sites and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. We applied this to dissect MYC regulation in human pluripotent stem cells. Insertion of STITCH between MYC and the enhancer down-regulated MYC and affected its target transcriptome. Progressive mutagenesis of STITCH led to preferential escalation of the gene-enhancer interaction, corroborating the strong insulation ability of STITCH. The STITCH insertion altered epigenetic states around MYC. Time-course analysis by drug induction uncovered deposition and removal of H3K27me3 repressive marks follows and reflects, but does not precede and determine, the expression change. Thus the tool provided important insights in gene regulation, demonstrating its potency.

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Molecular basis of CTCF binding polarity in genome folding

Nature Communications ◽

10.1038/s41467-020-19283-x ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 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.

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Binding site profiles and N-terminal minor groove interactions of the master quorum-sensing regulator LuxR enable flexible control of gene activation and repression

Nucleic Acids Research ◽

10.1093/nar/gkab150 ◽

2021 ◽

Vol 49 (6) ◽

pp. 3274-3293

Author(s):

Jun Zhang ◽

Bing Liu ◽

Dan Gu ◽

Yuan Hao ◽

Mo Chen ◽

...

Keyword(s):

Quorum Sensing ◽

Binding Sites ◽

Target Genes ◽

Gene Activation ◽

Underlying Mechanism ◽

Minor Groove ◽

Median Number ◽

Collective Behaviors ◽

Flexible Control ◽

High Flexibility

Abstract LuxR is a TetR family master quorum sensing (QS) regulator activating or repressing expression of hundreds of genes that control collective behaviors in Vibrios with underlying mechanism unknown. To illuminate how this regulator controls expression of various target genes, we applied ChIP-seq and DNase I-seq technologies. Vibrio alginolyticus LuxR controls expression of ∼280 genes that contain either symmetric palindrome (repDNA) or asymmetric (actDNA) binding motifs with different binding profiles. The median number of LuxR binding sites for activated genes are nearly double for that of repressed genes. Crystal structures of LuxR in complex with the respective repDNA and actDNA motifs revealed a new mode of LuxR DNA binding that involves contacts of its N-terminal extension to the minor groove. The N-terminal contacts mediated by Arginine-9 and Arginine-11 differ when LuxR binds to repDNA vs actDNA, leading to higher binding affinity at repressed targets. Moreover, modification of LuxR binding sites, binding profiles, and N-terminal extension have important consequences on QS-regulated phenotypes. These results facilitate fundamental understanding of the high flexibility of mechanisms of LuxR control of gene activation and repression in Vibrio QS, which may facilitate to design QS inhibiting chemicals that interfere with LuxR regulation to effectively control pathogens.

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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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Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes

10.1101/085654 ◽

2016 ◽

Author(s):

Naoki Osato

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Sites ◽

Target Genes ◽

Functional Enrichment ◽

Expression Level ◽

Ctcf Binding ◽

Cellular Functions ◽

Median Expression ◽

Transcriptional Target

AbstractBackgroundTranscriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes.ResultsGene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5 – 60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes.ConclusionsHuman putative transcriptional target genes showed significant functional enrichments. Functional enrichments were related to the cellular functions. The normalized number of functional enrichments of human putative transcriptional target genes changed according to the criteria of enhancer-promoter assignments and correlated with the median expression level of the target genes. These analyses and characters of human putative transcriptional target genes would be useful to examine the criteria of enhancer-promoter assignments and to predict the novel mechanisms and factors such as DNA binding proteins and DNA sequences of enhancer-promoter interactions.

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