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1.
Development ; 144(4): 567-579, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28087629

RESUMO

Jmjd2 H3K9 demethylases cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. Here, we reveal that Jmjd2c facilitates this process by stabilising the assembly of mediator-cohesin complexes at lineage-specific enhancers. Functionally, we show that Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while Jmjd2c-knockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a (also known as Ehmt2), independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and further destabilises loading of the mediator and cohesin components Med1 and Smc1a at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c and G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with an impact on timely gene activation.


Assuntos
Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos , Histona-Lisina N-Metiltransferase/fisiologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Animais , Proteínas de Ciclo Celular/fisiologia , Diferenciação Celular , Linhagem da Célula , Proteínas Cromossômicas não Histona/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Histonas/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Ligação Proteica , Análise de Sequência de RNA , Coesinas
2.
Cytoskeleton (Hoboken) ; 81(8): 393-408, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38682753

RESUMO

The platelet integrin αIIbß3 undergoes long-range conformational transitions between bent and extended conformations to regulate platelet aggregation during hemostasis and thrombosis. However, how exactly αIIbß3 transitions between conformations remains largely elusive. Here, we studied how transitions across bent and extended-closed conformations of αIIbß3 integrin are regulated by effective interactions between its functional domains. We first carried out µs-long equilibrium molecular dynamics (MD) simulations of full-length αIIbß3 integrins in bent and intermediate conformations, the latter characterized by an extended headpiece and closed legs. Then, we built heterogeneous elastic network models, perturbed inter-domain interactions, and evaluated their relative contributions to the energy barriers between conformations. Results showed that integrin extension emerges from: (i) changes in interfaces between functional domains; (ii) allosteric coupling of the head and upper leg domains with flexible lower leg domains. Collectively, these results provide new insights into integrin conformational activation based on short- and long-range interactions between its functional domains and highlight the importance of the lower legs in the regulation of integrin allostery.


Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Domínios Proteicos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Humanos , Simulação de Dinâmica Molecular
3.
Nat Commun ; 14(1): 3242, 2023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-37277355

RESUMO

Enhancer-promoter interactions preferentially occur within boundary-insulated topologically associating domains (TADs), limiting inter-TAD interactions. Enhancer clusters in linear proximity, termed super-enhancers (SEs), ensure high target gene expression levels. Little is known about SE topological regulatory impact during craniofacial development. Here, we identify 2232 genome-wide putative SEs in mouse cranial neural crest cells (CNCCs), 147 of which target genes establishing CNCC positional identity during face formation. In second pharyngeal arch (PA2) CNCCs, a multiple SE-containing region, partitioned into Hoxa Inter-TAD Regulatory Element 1 and 2 (HIRE1 and HIRE2), establishes long-range inter-TAD interactions selectively with Hoxa2, that is required for external and middle ear structures. HIRE2 deletion in a Hoxa2 haploinsufficient background results in microtia. HIRE1 deletion phenocopies the full homeotic Hoxa2 knockout phenotype and induces PA3 and PA4 CNCC abnormalities correlating with Hoxa2 and Hoxa3 transcriptional downregulation. Thus, SEs can overcome TAD insulation and regulate anterior Hoxa gene collinear expression in a CNCC subpopulation-specific manner during craniofacial development.


Assuntos
Crista Neural , Sequências Reguladoras de Ácido Nucleico , Camundongos , Animais , Crista Neural/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Crânio/metabolismo , Cromatina/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo
4.
Nat Genet ; 53(3): 379-391, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33603234

RESUMO

Rapid cellular responses to environmental stimuli are fundamental for development and maturation. Immediate early genes can be transcriptionally induced within minutes in response to a variety of signals. How their induction levels are regulated and their untimely activation by spurious signals prevented during development is poorly understood. We found that in developing sensory neurons, before perinatal sensory-activity-dependent induction, immediate early genes are embedded into a unique bipartite Polycomb chromatin signature, carrying active H3K27ac on promoters but repressive Ezh2-dependent H3K27me3 on gene bodies. This bipartite signature is widely present in developing cell types, including embryonic stem cells. Polycomb marking of gene bodies inhibits mRNA elongation, dampening productive transcription, while still allowing for fast stimulus-dependent mark removal and bipartite gene induction. We reveal a developmental epigenetic mechanism regulating the rapidity and amplitude of the transcriptional response to relevant stimuli, while preventing inappropriate activation of stimulus-response genes.


Assuntos
Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Precoces , Proteínas do Grupo Polycomb/genética , Animais , Cromatina/metabolismo , Células-Tronco Embrionárias/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Histonas/metabolismo , Camundongos Transgênicos , Mutação , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rombencéfalo/efeitos dos fármacos , Rombencéfalo/embriologia , Células Receptoras Sensoriais/fisiologia
5.
Nat Cell Biol ; 21(7): 911-912, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31097792

RESUMO

In the version of the article originally published, extra lines were displayed in Fig. 7. Fig. 7a contained a solid black line that extended into panel b, and Fig. 7c contained two extra scale bars on the left. These have been removed from the figure. The errors have been corrected in the HTML and PDF versions of the article.

6.
Nat Cell Biol ; 21(5): 568-578, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036938

RESUMO

The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time-resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between 'serum' and '2i' states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of transcription factors and allele-specific enhancer analysis reveal an essential role for Esrrb in H3K27 acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR-Cas9 in a hybrid ESC line restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by transcription factor binding and epigenetic marking in a hardwired network of chromatin interactions.


Assuntos
Cromatina/genética , Epigênese Genética , Células-Tronco Embrionárias Murinas/metabolismo , Receptores de Estrogênio/genética , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Elementos Facilitadores Genéticos , Histonas/genética , Camundongos , Células-Tronco Pluripotentes , Regiões Promotoras Genéticas , Transcriptoma/genética
7.
Nat Commun ; 8: 14418, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28195176

RESUMO

Genome-wide association studies have identified a great number of non-coding risk variants for colorectal cancer (CRC). To date, the majority of these variants have not been functionally studied. Identification of allele-specific transcription factor (TF) binding is of great importance to understand regulatory consequences of such variants. A recently developed proteome-wide analysis of disease-associated SNPs (PWAS) enables identification of TF-DNA interactions in an unbiased manner. Here we perform a large-scale PWAS study to comprehensively characterize TF-binding landscape that is associated with CRC, which identifies 731 allele-specific TF binding at 116 CRC risk loci. This screen identifies the A-allele of rs1800734 within the promoter region of MLH1 as perturbing the binding of TFAP4 and consequently increasing DCLK3 expression through a long-range interaction, which promotes cancer malignancy through enhancing expression of the genes related to epithelial-to-mesenchymal transition.


Assuntos
Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Progressão da Doença , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Alelos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA , Quinases Semelhantes a Duplacortina , Epigênese Genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Proteína 1 Homóloga a MutL/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Proteoma , Proteômica , Fatores de Transcrição
8.
Cell Stem Cell ; 17(6): 748-757, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26637943

RESUMO

Serum-to-2i interconversion of mouse embryonic stem cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum-grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon their further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary, but not sufficient, to establish these interactions, as confirmed by Capture Hi-C on Eed(-/-) serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation.


Assuntos
Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas , Animais , Diferenciação Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/genética , Cromatina/metabolismo , Desoxirribonuclease I/metabolismo , Células-Tronco Embrionárias/citologia , Genes Homeobox , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Domínios Proteicos
9.
Sci Rep ; 5: 9824, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25965993

RESUMO

The conserved histone methyltransferase Dot1 establishes an H3K79 methylation pattern consisting of mono-, di- and trimethylation states on histone H3 via a distributive mechanism. This mechanism has been shown to be important for the regulation of the different H3K79 methylation states in yeast. Dot1 enzymes in yeast, Trypanosoma brucei (TbDot1A and TbDot1B, which methylate H3K76) and human (hDot1L) generate very divergent methylation patterns. To understand how these species-specific methylation patterns are generated, the methylation output of the Dot1 enzymes was compared by expressing them in yeast at various expression levels. Computational simulations based on these data showed that the Dot1 enzymes have highly distinct catalytic properties, but share a distributive mechanism. The mechanism of methylation and the distinct rate constants have implications for the regulation of H3K79/K76 methylation. A mathematical model of H3K76 methylation during the trypanosome cell cycle suggests that temporally-regulated consecutive action of TbDot1A and TbDot1B is required for the observed regulation of H3K76 methylation states.


Assuntos
Ciclo Celular/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Trypanosoma brucei brucei/enzimologia , Catálise , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Histonas/genética , Humanos , Metilação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie , Trypanosoma brucei brucei/genética
10.
Genome Biol ; 16: 149, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26235224

RESUMO

BACKGROUND: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. RESULTS: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. CONCLUSIONS: The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome.


Assuntos
Inativação Gênica , Inativação do Cromossomo X , Alelos , Animais , Cromatina/química , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Camundongos , Células-Tronco Neurais/metabolismo , Análise de Sequência de RNA
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