Chromatin accessibility classification of TAD boundaries discloses new chromatin insulators (Hi-C 2)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE268028 | GEO | 2026/07/27
REPOSITORIES: GEO
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Project description:Perturbation of domain boundaries causes restructuration of domains and gene expression misregulation. Structural proteins such as CTCF and the cohesin complex have been identified enriched at domain boundaries. However, we still lack a complete understanding of the function of boundaries and what makes one boundary more effective at insulating genomic interactions than others. To understand how domains are structured, we experimentally classified boundaries according to their accessibility degree as a proxy of protein occupancy in K562 human cells. To determine if accessibility indeed reflects more robust boundaries and domains, we experimentally explored whether domain boundaries exhibit different degrees of accessibility in K562 cell line. To do so, we performed Hi-C experiments varying the incubation time of digestion with the restriction enzyme. The incorporation of this digestion time course into the Hi-C enables us to directly assess boundary accessibility and TAD formation from the same experiments.
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Project description:The three-dimensional (3D) chromatin organization within the nucleus is essential for regulating gene expression by facilitating interactions between cis-regulatory elements while insulating domains to restrict non-specific interactions. CTCF and cohesin, key architectural proteins, form topologically associated domains (TADs) that play a significant role in this spatial organization. While previous studies have demonstrated that TAD boundaries function to insulate genes from inappropriate interactions with distal enhancers or silencers, the functional impact of these boundaries on gene expression remains highly complex and context dependent. Here, we investigate the topological organization of the Notch1 locus, a gene involved in cell fate determination and cancer, to understand the regulatory impact of TAD boundaries on gene expression. We identified a highly conserved topological domain surrounding Notch1, with CTCF and cohesin binding at both 5' and 3' boundaries. Deletion of these boundary elements via CRISPR-Cas9 led to a significant increase in Notch1 expression, as well as enhanced expression of neighboring non-coding RNAs (Nalt1 and Linc01451) within the domain. Disruption of these boundaries also resulted in altered cell proliferation and colony formation. These effects correlated with altered enhancer-promoter interactions within the domain, especially involving a key enhancer located within the Linc01451 gene. Cross-species analyses revealed that the Notch1 domain and its boundary organization are conserved in mice, where perturbations similarly influenced Notch1 expression and cellular phenotypes in mouse embryonic stem cells (mESCs) but had minimal effects in murine erythroleukemia (MEL) cells. Together, these findings suggest that CTCF-dependent topological boundaries play a critical role in insulating the Notch1 locus and maintaining precise expression levels in a chromatin context- dependent manner, highlighting a regulatory mechanism that may extend to other lineage-specific and disease-relevant genes.
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Project description:Animal genomes fold into contact domains defined by enhanced internal contact frequencies with debated functions in establishing independent gene regulatory domains. A large fraction of contact domains in mammals are formed by stalling of chromosomal loop-extruding cohesin by CTCF at domain boundaries. 90% of domain boundaries in Drosophila form CTCF-independently, and other proteins were proposed to form chromosomal loops with dual functions of segregating promoters from inappropriate regulatory elements and connecting distal regulatory elements to their correct targets. Here, we genetically ablate the ubiquitous boundary-associated factor Cp190 and assess impacts on genome folding and transcriptional regulation in embryos. Our results reveal that Cp190 is a major factor required for contact domain boundary formation and gene insulation in Drosophila.
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Project description:Chromatin domain boundaries delimited by CTCF motifs have been shown to restrict the range of enhancers. However, disruption of domain structure often results in mild gene expression changes, and predicting the impact of boundary rearrangements on animal development remains challenging. By targeting clusters of CTCF motifs in a domain with three FGF genes—Fgf3, Fgf4, and Fgf15—we tested whether gene regulation and mouse development may be particularly sensitive to disruption of chromatin domains with multiple developmental regulators. Deletion of a cluster of four CTCF motifs that defines the centromeric boundary of this domain, resulted in ectopic interactions of the FGF genes with brain enhancers located across the deleted boundary, and a strong induction of FGF expression that led to perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary loss was enough to cause these fully penetrant phenotypes, and strikingly, loss of the single CTCF motif within the cluster that is oriented towards the brain enhancers, was sufficient to induce ectopic FGF expression and cause encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF-mediated boundaries of this domain nor to all developmental processes controlled by these three FGF genes. Although precise control of FGF4 levels is essential to regulate blastocyst development, none of our structural perturbations affected implantation. In fact, deletion of a CTCF boundary between Fgf3 and Fgf4, showed that the ability of these neighboring genes to have fully divergent expression patterns in blastocysts is achieved through CTCF-independent mechanisms and relies on remarkable enhancer-promoter specificity in vivo. Our work highlights how small sequence variants at certain domain boundaries can have a surprisingly outsized phenotypic effect and provides important insight into how different developmental contexts affect phenotypic sensitivity to perturbation of chromatin structure.
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Project description:Chromatin domain boundaries delimited by CTCF motifs have been shown to restrict the range of enhancers. However, disruption of domain structure often results in mild gene expression changes, and predicting the impact of boundary rearrangements on animal development remains challenging. By targeting clusters of CTCF motifs in a domain with three FGF genes—Fgf3, Fgf4, and Fgf15—we tested whether gene regulation and mouse development may be particularly sensitive to disruption of chromatin domains with multiple developmental regulators. Deletion of a cluster of four CTCF motifs that defines the centromeric boundary of this domain, resulted in ectopic interactions of the FGF genes with brain enhancers located across the deleted boundary, and a strong induction of FGF expression that led to perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary loss was enough to cause these fully penetrant phenotypes, and strikingly, loss of the single CTCF motif within the cluster that is oriented towards the brain enhancers, was sufficient to induce ectopic FGF expression and cause encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF-mediated boundaries of this domain nor to all developmental processes controlled by these three FGF genes. Although precise control of FGF4 levels is essential to regulate blastocyst development, none of our structural perturbations affected implantation. In fact, deletion of a CTCF boundary between Fgf3 and Fgf4, showed that the ability of these neighboring genes to have fully divergent expression patterns in blastocysts is achieved through CTCF-independent mechanisms and relies on remarkable enhancer-promoter specificity in vivo. Our work highlights how small sequence variants at certain domain boundaries can have a surprisingly outsized phenotypic effect and provides important insight into how different developmental contexts affect phenotypic sensitivity to perturbation of chromatin structure.
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Project description:Chromatin domain boundaries delimited by CTCF motifs have been shown to restrict the range of enhancers. However, disruption of domain structure often results in mild gene expression changes, and predicting the impact of boundary rearrangements on animal development remains challenging. By targeting clusters of CTCF motifs in a domain with three FGF genes—Fgf3, Fgf4, and Fgf15—we tested whether gene regulation and mouse development may be particularly sensitive to disruption of chromatin domains with multiple developmental regulators. Deletion of a cluster of four CTCF motifs that defines the centromeric boundary of this domain, resulted in ectopic interactions of the FGF genes with brain enhancers located across the deleted boundary, and a strong induction of FGF expression that led to perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary loss was enough to cause these fully penetrant phenotypes, and strikingly, loss of the single CTCF motif within the cluster that is oriented towards the brain enhancers, was sufficient to induce ectopic FGF expression and cause encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF-mediated boundaries of this domain nor to all developmental processes controlled by these three FGF genes. Although precise control of FGF4 levels is essential to regulate blastocyst development, none of our structural perturbations affected implantation. In fact, deletion of a CTCF boundary between Fgf3 and Fgf4, showed that the ability of these neighboring genes to have fully divergent expression patterns in blastocysts is achieved through CTCF-independent mechanisms and relies on remarkable enhancer-promoter specificity in vivo. Our work highlights how small sequence variants at certain domain boundaries can have a surprisingly outsized phenotypic effect and provides important insight into how different developmental contexts affect phenotypic sensitivity to perturbation of chromatin structure.
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Project description:Chromatin domain boundaries delimited by CTCF motifs have been shown to restrict the range of enhancers. However, disruption of domain structure often results in mild gene expression changes, and predicting the impact of boundary rearrangements on animal development remains challenging. By targeting clusters of CTCF motifs in a domain with three FGF genes—Fgf3, Fgf4, and Fgf15—we tested whether gene regulation and mouse development may be particularly sensitive to disruption of chromatin domains with multiple developmental regulators. Deletion of a cluster of four CTCF motifs that defines the centromeric boundary of this domain, resulted in ectopic interactions of the FGF genes with brain enhancers located across the deleted boundary, and a strong induction of FGF expression that led to perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary loss was enough to cause these fully penetrant phenotypes, and strikingly, loss of the single CTCF motif within the cluster that is oriented towards the brain enhancers, was sufficient to induce ectopic FGF expression and cause encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF-mediated boundaries of this domain nor to all developmental processes controlled by these three FGF genes. Although precise control of FGF4 levels is essential to regulate blastocyst development, none of our structural perturbations affected implantation. In fact, deletion of a CTCF boundary between Fgf3 and Fgf4, showed that the ability of these neighboring genes to have fully divergent expression patterns in blastocysts is achieved through CTCF-independent mechanisms and relies on remarkable enhancer-promoter specificity in vivo. Our work highlights how small sequence variants at certain domain boundaries can have a surprisingly outsized phenotypic effect and provides important insight into how different developmental contexts affect phenotypic sensitivity to perturbation of chromatin structure.
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Project description:Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes¬—Fgf3, Fgf4, and Fgf15—that regulate several developmental processes¬. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain¬ resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes¬—for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, our work starts to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.
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2024-08-07 | GSE274230 | GEO
Project description:The genome is partitioned into Topologically Associating Domains (TADs). About half of the boundaries of these TADs exhibit transcriptional activity and are correlated with better TAD insulation. However, the association between these transcripts and TAD insulation, enhancer:promoter interactions and transcription of genes remains unknown. Here we investigate the functional roles of these bRNAs (boundary-RNA) in boundary insulation and consequent effects on enhancer-promoter interactions and TAD transcription genome-wide and more specifically on the disease relevant INK4a/ARF TAD. Using a series of CTCF site deletions and bRNA knockdown experiments at this TAD boundary, we show a direct association of CTCF with the bidirectional bRNAs where the loss of bRNA triggers the concomitant loss of: CTCF at the TAD boundary, its insulation, enhancer:promoter interactions and gene transcription within the targeted TAD. We used another series of enhancer deletions and CRISPRi on promoters within INK4a/ARF TAD to better understand the regulation and origins of bRNA itself. We observe that the enhancers interact with boundaries and positively regulate the bRNA transcription at TAD boundaries. In return, the bRNAs recruit/stabilize the CTCF even on weak motifs within these boundaries and supports CTCF binding in clusters, therefore enhancing TAD insulation. This favors the intra-TAD enhancer:promoter interactions and leads to robust gene transcription. Functionally, bRNAs are more stable than eRNAs and their knockdown exactly mimics the CTCF site deletion. Furthermore, transcribing boundaries exhibit high TAD transcription in TCGA tumour datasets. Together, these results show that active enhancers directly mediate better insulation of TADs by activating the transcription at TAD boundaries triggering CTCF clustering at the boundary which results in better insulation and favours robust intra-TAD enhancer:promoter interactions to activate the gene transcription.
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