RESUMO
Although only a fraction of CTCF motifs are bound in any cell type, and approximately half of the occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique properties of each protein, but also on the genomic context of bound sites. Our studies also highlight the reciprocal relationship between CTCF and chromatin, demonstrating that the unique binding properties of WT and mutant proteins have a distinct impact on accessibility, TF binding, cohesin overlap, chromatin interactivity and gene expression programs, providing insight into their cancer and brain related effects.
RESUMO
Although only a fraction of CTCF motifs are bound in any cell type, and approximately half of the occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique properties of each protein, but also on the genomic context of bound sites. Our studies also highlight the reciprocal relationship between CTCF and chromatin, demonstrating that the unique binding properties of WT and mutant proteins have a distinct impact on accessibility, TF binding, cohesin overlap, chromatin interactivity and gene expression programs, providing insight into their cancer and brain related effects.
RESUMO
Although population-level analyses revealed significant roles for CTCF and cohesin in mammalian genome organization, their contributions at the single-cell level remain incompletely understood. Here, we used a super-resolution microscopy approach to measure the effects of removal of CTCF or cohesin in mouse embryonic stem cells. Single-chromosome traces revealed cohesin-dependent loops, frequently stacked at their loop anchors forming multi-way contacts (hubs), bridging across TAD boundaries. Despite these bridging interactions, chromatin in intervening TADs was not intermixed, remaining separated in distinct loops around the hub. At the multi-TAD scale, steric effects from loop stacking insulated local chromatin from ultra-long range (>4 Mb) contacts. Upon cohesin removal, the chromosomes were more disordered and increased cell-cell variability in gene expression. Our data revise the TAD-centric understanding of CTCF and cohesin and provide a multi-scale, structural picture of how they organize the genome on the single-cell level through distinct contributions to loop stacking.
Assuntos
Cromatina , Cromossomos , Animais , Camundongos , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mamíferos/metabolismoRESUMO
What new questions can we ask about transcriptional regulation given recent developments in large-scale approaches?
Assuntos
Regulação da Expressão Gênica , Regulação da Expressão Gênica/genéticaRESUMO
Chromatin folds into dynamic loops that often span hundreds of kilobases and physically wire distant loci together for gene regulation. These loops are continuously created, extended and positioned by structural maintenance of chromosomes (SMC) protein complexes, such as condensin and cohesin, and their regulators, including CTCF, in a highly dynamic process known as loop extrusion. Genetic loss of extrusion factors is lethal, complicating their study. Inducible protein degradation technologies enable the depletion of loop extrusion factors within hours, leading to the rapid reconfiguration of chromatin folding. Here, we review how these technologies have changed our understanding of genome organization, upsetting long-held beliefs on its role in transcription. Finally, we examine recent models that attempt to reconcile observations after chronic versus acute perturbations, and discuss future developments in this rapidly developing field of research.
Assuntos
Cromatina , Cromossomos , Cromossomos/genética , Regulação da Expressão Gênica , Genoma , Proteínas de Ciclo Celular/genéticaRESUMO
In mammals, interactions between sequences within topologically associating domains enable control of gene expression across large genomic distances. Yet it is unknown how frequently such contacts occur, how long they last and how they depend on the dynamics of chromosome folding and loop extrusion activity of cohesin. By imaging chromosomal locations at high spatial and temporal resolution in living cells, we show that interactions within topologically associating domains are transient and occur frequently during the course of a cell cycle. Interactions become more frequent and longer in the presence of convergent CTCF sites, resulting in suppression of variability in chromosome folding across time. Supported by physical models of chromosome dynamics, our data suggest that CTCF-anchored loops last around 10 min. Our results show that long-range transcriptional regulation might rely on transient physical proximity, and that cohesin and CTCF stabilize highly dynamic chromosome structures, facilitating selected subsets of chromosomal interactions.
Assuntos
Cromossomos , Cromossomos/genéticaRESUMO
The interplay between the topological organization of the genome and the regulation of gene expression remains unclear. Depletion of molecular factors (e.g. CTCF) underlying topologically associating domains (TADs) leads to modest alterations in gene expression, whereas genomic rearrangements involving TAD boundaries disrupt normal gene expression and can lead to pathological phenotypes. Here, we targeted the TAD neighboring that of the noncoding transcript Xist, which controls X-chromosome inactivation. Inverting 245â kb within the TAD led to expected rearrangement of CTCF-based contacts but revealed heterogeneity in the 'contact' potential of different CTCF sites. Expression of most genes therein remained unaffected in mouse embryonic stem cells and during differentiation. Interestingly, expression of Xist was ectopically upregulated. The same inversion in mouse embryos led to biased Xist expression. Smaller inversions and deletions of CTCF clusters led to similar results: rearrangement of contacts and limited changes in local gene expression, but significant changes in Xist expression in embryos. Our study suggests that the wiring of regulatory interactions within a TAD can influence the expression of genes in neighboring TADs, highlighting the existence of mechanisms of inter-TAD communication.
Assuntos
RNA Longo não Codificante , Inativação do Cromossomo X , Animais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina , Comunicação , Expressão Gênica , Genoma , Camundongos , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genéticaRESUMO
Gjaltema et al. (2021) perform systematic screens to identify the long-sought cis-regulatory elements of Xist. They discover that distal elements give Xist a boost as cells exit pluripotency, while proximal elements restrict Xist expression to cells with two X chromosomes.
Assuntos
RNA Longo não Codificante , Inativação do Cromossomo X , Genômica , RNA Longo não Codificante/genética , RNA não Traduzido , Cromossomo X , Inativação do Cromossomo X/genéticaAssuntos
Fator de Ligação a CCCTC/genética , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário/genética , Peixe-Zebra/embriologia , Animais , Fator de Ligação a CCCTC/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrião não Mamífero , Feminino , Técnicas de Inativação de Genes , Camundongos , Camundongos Knockout , Oócitos/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Totipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Reprogramação Celular , Células-Tronco Totipotentes/citologia , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Morte Celular , Dano ao DNA , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Células-Tronco Totipotentes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Neguembor et al. (2021) use super-resolution microscopy to illuminate genome packaging inside the cell nucleus. They discover that transcription and topoisomerases protect chromatin from collapsing in a crumpled state refractory to DNA loop extrusion by cohesin proteins.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , DNA/genética , Humanos , CoesinasRESUMO
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.
Assuntos
Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Cromossomos de Mamíferos/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Cricetinae , Drosophila , Camundongos , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Relação Estrutura-Atividade , CoesinasRESUMO
The genome folds into a hierarchy of three-dimensional structures within the nucleus. At the sub-megabase scale, chromosomes form topologically associating domains (TADs)1-4. However, how TADs fold in single cells is elusive. Here, we reveal TAD features inaccessible to cell population analysis by using super-resolution microscopy. TAD structures and physical insulation associated with their borders are variable between individual cells, yet chromatin intermingling is enriched within TADs compared to adjacent TADs in most cells. The spatial segregation of TADs is further exacerbated during cell differentiation. Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms: cohesin generates chromatin contacts and intermingling while CTCF prevents inter-TAD contacts. Furthermore, TADs are subdivided into discrete nanodomains, which persist in cells depleted of CTCF or cohesin, whereas disruption of nucleosome contacts alters their structural organization. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale.
Assuntos
Cromatina/química , Células-Tronco Embrionárias/ultraestrutura , Domínios Proteicos , Animais , Diferenciação Celular/genética , Linhagem Celular , Coloração Cromossômica , Drosophila/genética , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Conformação Molecular , Nanoestruturas , Microscopia NuclearRESUMO
An amendment to this paper has been published and can be accessed via the original article.
RESUMO
BACKGROUND: Ubiquitously 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 the 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. RESULTS: We 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. CONCLUSION: This 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.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Animais , Células-Tronco Embrionárias , Feminino , Humanos , Masculino , CamundongosRESUMO
The access of Transcription Factors (TFs) to their cognate DNA binding motifs requires a precise control over nucleosome positioning. This is especially important following DNA replication and during mitosis, both resulting in profound changes in nucleosome organization over TF binding regions. Using mouse Embryonic Stem (ES) cells, we show that the TF CTCF displaces nucleosomes from its binding site and locally organizes large and phased nucleosomal arrays, not only in interphase steady-state but also immediately after replication and during mitosis. Correlative analyses suggest this is associated with fast gene reactivation following replication and mitosis. While regions bound by other TFs (Oct4/Sox2), display major rearrangement, the post-replication and mitotic nucleosome positioning activity of CTCF is not unique: Esrrb binding regions are also characterized by persistent nucleosome positioning. Therefore, selected TFs such as CTCF and Esrrb act as resilient TFs governing the inheritance of nucleosome positioning at regulatory regions throughout the cell-cycle.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Replicação do DNA , Células-Tronco Embrionárias/fisiologia , Mitose , Nucleossomos/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Camundongos , Ativação TranscricionalRESUMO
The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms by which CTCF dynamically contributes to genome organization are not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs, promoting its self-association. In this regard, we now report two independent features that disrupt CTCF association with chromatin: inhibition of transcription and disruption of CTCF-RNA interactions through mutations of 2 of its 11 zinc fingers that are not required for CTCF binding to its cognate DNA site: zinc finger 1 (ZF1) or zinc finger 10 (ZF10). These mutations alter gene expression profiles as CTCF mutants lose their ability to form chromatin loops and thus the ability to insulate chromatin domains and to mediate CTCF long-range genomic interactions. Our results point to the importance of CTCF-mediated RNA interactions as a structural component of genome organization.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , RNA/metabolismo , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/genética , Linhagem Celular , Cromatina/química , Cromatina/genética , Camundongos , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA/química , RNA/genética , Relação Estrutura-Atividade , Transcrição Gênica , Dedos de ZincoRESUMO
The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other's TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Longo não Codificante/genética , Inativação do Cromossomo X , Animais , Diferenciação Celular/genética , Expressão Ectópica do Gene , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Inativação Gênica , Loci Gênicos , Masculino , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Inversão de Sequência , Transcrição GênicaRESUMO
The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.