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1.
Res Sq ; 2024 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-39070636

RESUMEN

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.

2.
bioRxiv ; 2024 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-38370764

RESUMEN

Here we used a series of CTCF mutations to explore CTCF's relationship with chromatin and its contribution to gene regulation. CTCF's impact depends on the genomic context of bound sites and the unique binding properties of WT and mutant CTCF proteins. Specifically, CTCF's signal strength is linked to changes in accessibility, and the ability to block cohesin is linked to its binding stability. Multivariate modelling reveals that both CTCF and accessibility contribute independently to cohesin binding and insulation, however CTCF signal strength has a stronger effect. CTCF and chromatin have a bidirectional relationship such that at CTCF sites, accessibility is reduced in a cohesin-dependent, mutant specific fashion. In addition, each mutant alters TF binding and accessibility in an indirect manner, changes which impart the most influence on rewiring transcriptional networks and the cell's ability to be reprogrammed. Collectively, the mutant perturbations provide a rich resource for determining CTCF's site-specific effects.

3.
Mol Cell ; 83(9): 1377-1392.e6, 2023 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-37146570

RESUMEN

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.


Asunto(s)
Cromatina , Cromosomas , Animales , Ratones , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Cromosomas/genética , Cromosomas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mamíferos/metabolismo
4.
Cell Syst ; 14(4): 247-251, 2023 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-37080160

RESUMEN

What new questions can we ask about transcriptional regulation given recent developments in large-scale approaches?


Asunto(s)
Regulación de la Expresión Génica , Regulación de la Expresión Génica/genética
5.
Nat Rev Genet ; 24(2): 73-85, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36180596

RESUMEN

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.


Asunto(s)
Cromatina , Cromosomas , Cromosomas/genética , Regulación de la Expresión Génica , Genoma , Proteínas de Ciclo Celular/genética
6.
Nat Genet ; 54(12): 1907-1918, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36471076

RESUMEN

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.


Asunto(s)
Cromosomas , Cromosomas/genética
7.
Development ; 149(9)2022 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-35502750

RESUMEN

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 245 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.


Asunto(s)
ARN Largo no Codificante , Inactivación del Cromosoma X , Animales , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Cromatina , Comunicación , Expresión Génica , Genoma , Ratones , ARN Largo no Codificante/genética , Inactivación del Cromosoma X/genética
8.
Mol Cell ; 82(1): 10-12, 2022 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-34995506

RESUMEN

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.


Asunto(s)
ARN Largo no Codificante , Inactivación del Cromosoma X , Genómica , ARN Largo no Codificante/genética , ARN no Traducido , Cromosoma X , Inactivación del Cromosoma X/genética
10.
Mol Cell ; 81(15): 3043-3045, 2021 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-34358457

RESUMEN

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.


Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , ADN/genética , Humanos , Cohesinas
11.
Nat Commun ; 12(1): 4856, 2021 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-34381034

RESUMEN

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.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Reprogramación Celular , Células Madre Totipotentes/citología , Animales , Sitios de Unión , Factor de Unión a CCCTC/genética , Muerte Celular , Daño del ADN , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Células Madre Totipotentes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
12.
Nat Commun ; 11(1): 5612, 2020 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-33154377

RESUMEN

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.


Asunto(s)
Factor de Unión a CCCTC/química , Factor de Unión a CCCTC/metabolismo , Cromosomas de los Mamíferos/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Factor de Unión a CCCTC/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Cricetinae , Drosophila , Ratones , Mutación , Motivos de Nucleótidos , Unión Proteica , Relación Estructura-Actividad , Cohesinas
13.
Nat Genet ; 52(10): 1003-1004, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32999490
14.
Nat Genet ; 52(11): 1151-1157, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33077913

RESUMEN

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.


Asunto(s)
Cromatina/química , Células Madre Embrionarias/ultraestructura , Dominios Proteicos , Animales , Diferenciación Celular/genética , Línea Celular , Pintura Cromosómica , Drosophila/genética , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Conformación Molecular , Nanoestructuras , Microscopía Nuclear
15.
Cell Stem Cell ; 27(3): 359-360, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32888424

RESUMEN

COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.


Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Investigadores , Animales , COVID-19 , Humanos , Pandemias , SARS-CoV-2 , Células Madre/citología
16.
17.
Genome Biol ; 21(1): 108, 2020 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-32393311

RESUMEN

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.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Animales , Células Madre Embrionarias , Femenino , Humanos , Masculino , Ratones
18.
Mol Cell ; 77(2): 352-367.e8, 2020 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-31759823

RESUMEN

cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.


Asunto(s)
Secuencia Conservada/genética , ARN Largo no Codificante/genética , Cromosoma X/genética , Animales , Línea Celular , Elementos de Facilitación Genéticos/genética , Ratones , Regiones Promotoras Genéticas/genética , ARN sin Sentido/genética , Elementos Silenciadores Transcripcionales/genética , Transcripción Genética/genética
19.
Elife ; 82019 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-31599722

RESUMEN

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.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Replicación del ADN , Células Madre Embrionarias/fisiología , Mitosis , Nucleosomas/metabolismo , Animales , Células Cultivadas , Regulación de la Expresión Génica , Ratones , Activación Transcripcional
20.
Mol Cell ; 76(3): 412-422.e5, 2019 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-31522988

RESUMEN

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.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , ARN/metabolismo , Animales , Sitios de Unión , Factor de Unión a CCCTC/química , Factor de Unión a CCCTC/genética , Línea Celular , Cromatina/química , Cromatina/genética , Ratones , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN/química , ARN/genética , Relación Estructura-Actividad , Transcripción Genética , Dedos de Zinc
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