Tissue-specific CTCF/Cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo (ATAC-Seq)
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ABSTRACT: The genome is organized via CTCF/cohesin binding sites, which partition chromosomes into 1-5Mb topologically associated domains (TADs), and further into smaller contact sub-domains within TADs (sub-TADs; 40-1000kb). Here we examined in vivo an ~80kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ~1Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF/cohesin sites which are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF/cohesin boundary extends the sub-TAD to the adjacent upstream CTCF/cohesin binding site. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF/cohesin boundaries in this sub-TAD regulate both directionality and specificity of enhancer interactions with surrounding promoters.
Project description:The genome is organized via CTCF/cohesin binding sites, which partition chromosomes into 1-5Mb topologically associated domains (TADs), and further into smaller contact sub-domains within TADs (sub-TADs; 40-1000kb). Here we examined in vivo an ~80kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ~1Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF/cohesin sites which are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF/cohesin boundary extends the sub-TAD to the adjacent upstream CTCF/cohesin binding site. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF/cohesin boundaries in this sub-TAD regulate both directionality and specificity of enhancer interactions with surrounding promoters.
Project description:The genome is organized via CTCF/cohesin binding sites, which partition chromosomes into 1-5Mb topologically associated domains (TADs), and further into smaller contact sub-domains within TADs (sub-TADs; 40-1000kb). Here we examined in vivo an ~80kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ~1Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF/cohesin sites which are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF/cohesin boundary extends the sub-TAD to the adjacent upstream CTCF/cohesin binding site. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF/cohesin boundaries in this sub-TAD regulate both directionality and specificity of enhancer interactions with surrounding promoters.
Project description:The genome is organized via CTCF/cohesin binding sites, which partition chromosomes into 1-5Mb topologically associated domains (TADs), and further into smaller contact sub-domains within TADs (sub-TADs; 40-1000kb). Here we examined in vivo an ~80kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ~1Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF/cohesin sites which are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF/cohesin boundary extends the sub-TAD to the adjacent upstream CTCF/cohesin binding site. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF/cohesin boundaries in this sub-TAD regulate both directionality and specificity of enhancer interactions with surrounding promoters.
Project description:Mammalian 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.
Project description:Mammalian 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.
Project description:Mammalian 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.
Project description:Mammalian 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.
Project description:We have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation. We found that, similarly to mammalian genome, the chicken genome is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation.
Project description:The mammalian genome is sequentially partitioned from chromosomes into looped chromatin regions termed Topologically Associating Domains (TADs), and then into numerous smaller sub-TAD loops, each bounded and insulated by CTCF and Cohesin. The sub-TADs encompass enhancers, promoters and often multiple genes but whether promoter-enhancer interactions and gene regulation are broadly restricted to within the sub-TAD loops has not been functionally determined. Here we identify “Gene Unit Sub-TADs” or GUSTs as a class of sub-TADs that confine promoter-enhancer interactions and demarcate functional gene regulatory regions. We depleted Estrogen-related receptor β (Esrrb), which binds to the Mediator co-activator complex, to impair the activity of enhancers controlling 245 mouse embryonic stem cell genes. We find that most Esrrb-responsive enhancers lack significant Cohesin binding but instead correlate with Mediator binding. Esrrb depletion causes reduced Mediator binding, decreased nascent RNA expression and diminished promoter-enhancer looping. In 88% of the cases examined, the effects of Esrrb depletion are restricted to enhancers and target genes within GUSTs. In some GUSTs, active genes lay alongside inactive ones but are distinguished by their proximal promoter chromatin accessibility, explaining further the specificity of enhancer-promoter interactions within GUSTs. Our data indicate that GUSTs represent functional gene regulons in mammalian genomes.
Project description:In addition to mediating sister chromatid cohesion, cohesin plays a central role in DNA looping and segmentation of the genome into contact domains (TADs). Two variant cohesin complexes that contain either STAG/SA1 or SA2 are present in all cell types. Here we addressed their specific contribution to genome architecture in non-transformed human cells. We found that cohesin-SA1 drives stacking of cohesin rings at CTCF-bound sites and thereby contributes to the stabilization and preservation of TAD boundaries. In contrast, a more dynamic cohesin-SA2 promotes cell type-specific contacts between enhancers and promoters within TADs independently of CTCF. SA2 loss, a condition frequently observed in cancer cells, results in increased intra-TAD interactions, likely altering the expression of key cell identity genes.