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1.
Cell ; 184(14): 3599-3611, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34146479

RESUMO

Eukaryotic DNA-binding proteins operate in the context of chromatin, where nucleosomes are the elementary building blocks. Nucleosomal DNA is wrapped around a histone core, thereby rendering a large fraction of the DNA surface inaccessible to DNA-binding proteins. Nevertheless, first responders in DNA repair and sequence-specific transcription factors bind DNA target sites obstructed by chromatin. While early studies examined protein binding to histone-free DNA, it is only now beginning to emerge how DNA sequences are interrogated on nucleosomes. These readout strategies range from the release of nucleosomal DNA from histones, to rotational/translation register shifts of the DNA motif, and nucleosome-specific DNA binding modes that differ from those observed on naked DNA. Since DNA motif engagement on nucleosomes strongly depends on position and orientation, we argue that motif location and nucleosome positioning co-determine protein access to DNA in transcription and DNA repair.


Assuntos
Cromatina/genética , Genoma , Animais , Humanos , Modelos Biológicos , Nucleossomos/metabolismo , Motivos de Nucleotídeos/genética , Fatores de Transcrição/metabolismo
2.
Cell ; 184(16): 4237-4250.e19, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34297924

RESUMO

The organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of "minimalist" histones in archaea and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones are essential for viral infectivity, localize to cytoplasmic viral factories after virus infection, and ultimately are found in the mature virions. Cryogenic electron microscopy (cryo-EM) structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of "organisms" that require nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses.


Assuntos
Vírus de DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Amoeba/virologia , Corantes Fluorescentes/metabolismo , Histonas/química , Modelos Moleculares , Proteômica , Vírion/metabolismo
3.
Cell ; 184(2): 352-369.e23, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33357448

RESUMO

Repetitive elements (REs) compose ∼50% of the human genome and are normally transcriptionally silenced, although the mechanism has remained elusive. Through an RNAi screen, we identified FBXO44 as an essential repressor of REs in cancer cells. FBXO44 bound H3K9me3-modified nucleosomes at the replication fork and recruited SUV39H1, CRL4, and Mi-2/NuRD to transcriptionally silence REs post-DNA replication. FBXO44/SUV39H1 inhibition reactivated REs, leading to DNA replication stress and stimulation of MAVS/STING antiviral pathways and interferon (IFN) signaling in cancer cells to promote decreased tumorigenicity, increased immunogenicity, and enhanced immunotherapy response. FBXO44 expression inversely correlated with replication stress, antiviral pathways, IFN signaling, and cytotoxic T cell infiltration in human cancers, while a FBXO44-immune gene signature correlated with improved immunotherapy response in cancer patients. FBXO44/SUV39H1 were dispensable in normal cells. Collectively, FBXO44/SUV39H1 are crucial repressors of RE transcription, and their inhibition selectively induces DNA replication stress and viral mimicry in cancer cells.


Assuntos
Replicação do DNA/genética , Proteínas F-Box/metabolismo , Neoplasias/genética , Sequências Repetitivas de Ácido Nucleico/genética , Adulto , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunidade , Interferons/metabolismo , Lisina/metabolismo , Masculino , Metilação , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Neoplasias/imunologia , Nucleossomos/metabolismo , Transdução de Sinais , Transcrição Gênica , Resultado do Tratamento
4.
Cell ; 183(3): 802-817.e24, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33053319

RESUMO

Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.


Assuntos
Doença , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Montagem e Desmontagem da Cromatina , Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Doença/genética , Humanos , Mutação de Sentido Incorreto/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Cell ; 182(1): 127-144.e23, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32502394

RESUMO

Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. Here, we show that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.


Assuntos
Histonas/metabolismo , Lisina/metabolismo , Ativação Transcricional/genética , Acetilação , Animais , Sequência de Bases , Segregação de Cromossomos/genética , Sequência Conservada , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Evolução Molecular , Feminino , Genoma , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Masculino , Mamíferos/genética , Camundongos , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Oócitos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Cromossomo X/metabolismo , Zigoto/metabolismo
6.
Cell ; 176(3): 520-534.e25, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30661750

RESUMO

Elucidating the global and local rules that govern genome-wide, hierarchical chromatin architecture remains a critical challenge. Current high-throughput chromosome conformation capture (Hi-C) technologies have identified large-scale chromatin structural motifs, such as topologically associating domains and looping. However, structural rules at the smallest or nucleosome scale remain poorly understood. Here, we coupled nucleosome-resolved Hi-C technology with simulated annealing-molecular dynamics (SA-MD) simulation to reveal 3D spatial distributions of nucleosomes and their genome-wide orientation in chromatin. Our method, called Hi-CO, revealed distinct nucleosome folding motifs across the yeast genome. Our results uncovered two types of basic secondary structural motifs in nucleosome folding: α-tetrahedron and ß-rhombus analogous to α helix and ß sheet motifs in protein folding. Using mutants and cell-cycle-synchronized cells, we further uncovered motifs with specific nucleosome positioning and orientation coupled to epigenetic features at individual loci. By illuminating molecular-level structure-function relationships in eukaryotic chromatin, our findings establish organizational principles of nucleosome folding.


Assuntos
Cromatina/ultraestrutura , Nucleossomos/ultraestrutura , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Cromossomos/metabolismo , Cromossomos/ultraestrutura , Nucleossomos/genética , Nucleossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sítio de Iniciação de Transcrição
7.
Cell ; 176(6): 1490-1501.e12, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30765112

RESUMO

Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Animais , Domínio Catalítico , Cromatina/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/ultraestrutura , Histonas/química , Histonas/genética , Humanos , Metilação , Modelos Moleculares , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Receptor Cross-Talk , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Xenopus laevis
8.
Cell ; 178(3): 624-639.e19, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348889

RESUMO

Recent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. Mammals, unlike budding yeast, depend on the histone H3 variant, CENP-A, to epigenetically specify the location of the centromere-the locus essential for chromosome segregation. Prior human artificial chromosomes (HACs) required large arrays of centromeric α-satellite repeats harboring binding sites for the DNA sequence-specific binding protein, CENP-B. We report the development of a type of HAC that functions independently of these constraints. Formed by an initial CENP-A nucleosome seeding strategy, a construct lacking repetitive centromeric DNA formed several self-sufficient HACs that showed no uptake of genomic DNA. In contrast to traditional α-satellite HAC formation, the non-repetitive construct can form functional HACs without CENP-B or initial CENP-A nucleosome seeding, revealing distinct paths to centromere formation for different DNA sequence types. Our developments streamline the construction and characterization of HACs to facilitate mammalian synthetic genome efforts.


Assuntos
Centrômero/metabolismo , Cromossomos Artificiais Humanos/metabolismo , DNA Satélite/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Centrômero/genética , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteína B de Centrômero/deficiência , Proteína B de Centrômero/genética , Proteína B de Centrômero/metabolismo , Epigênese Genética , Humanos , Nucleossomos/química , Nucleossomos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
9.
Cell ; 179(6): 1342-1356.e23, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31759698

RESUMO

Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Mutação/genética , Nucleossomos/metabolismo , Proteína SMARCB1/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Elementos Facilitadores Genéticos/genética , Feminino , Genoma Humano , Células HEK293 , Células HeLa , Heterozigoto , Humanos , Masculino , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Proteína SMARCB1/química , Proteína SMARCB1/metabolismo
10.
Cell ; 179(4): 953-963.e11, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31675501

RESUMO

Chromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. However, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication, is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at desired loci in mouse embryonic stem cells so that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment of the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Epigênese Genética , Nucleossomos/genética , Animais , Diferenciação Celular/genética , Divisão Celular/genética , Linhagem da Célula/genética , Replicação do DNA/genética , Histonas/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional/genética
11.
Cell ; 178(2): 302-315.e23, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31299200

RESUMO

Pathogenic and other cytoplasmic DNAs activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to induce inflammation via transcriptional activation by IRF3 and nuclear factor κB (NF-κB), but the functional consequences of exposing cGAS to chromosomes upon mitotic nuclear envelope breakdown are unknown. Here, we show that nucleosomes competitively inhibit DNA-dependent cGAS activation and that the cGAS-STING pathway is not effectively activated during normal mitosis. However, during mitotic arrest, low level cGAS-dependent IRF3 phosphorylation slowly accumulates without triggering inflammation. Phosphorylated IRF3, independently of its DNA-binding domain, stimulates apoptosis through alleviating Bcl-xL-dependent suppression of mitochondrial outer membrane permeabilization. We propose that slow accumulation of phosphorylated IRF3, normally not sufficient for inducing inflammation, can trigger transcription-independent induction of apoptosis upon mitotic aberrations. Accordingly, expression of cGAS and IRF3 in cancer cells makes mouse xenograft tumors responsive to the anti-mitotic agent Taxol. The Cancer Genome Atlas (TCGA) datasets for non-small cell lung cancer patients also suggest an effect of cGAS expression on taxane response.


Assuntos
Apoptose , DNA/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Fator Regulador 3 de Interferon/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Mitose , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Neoplasias/patologia , Nucleossomos/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Transdução de Sinais , Taxa de Sobrevida , Ativação Transcricional , Proteína bcl-X/metabolismo
12.
Annu Rev Cell Dev Biol ; 36: 1-34, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32822539

RESUMO

Gene transcription by RNA polymerase II (Pol II) is the first step in the expression of the eukaryotic genome and a focal point for cellular regulation during development, differentiation, and responses to the environment. Two decades after the determination of the structure of Pol II, the mechanisms of transcription have been elucidated with studies of Pol II complexes with nucleic acids and associated proteins. Here we provide an overview of the nearly 200 available Pol II complex structures and summarize how these structures have elucidated promoter-dependent transcription initiation, promoter-proximal pausing and release of Pol II into active elongation, and the mechanisms that Pol II uses to navigate obstacles such as nucleosomes and DNA lesions. We predict that future studies will focus on how Pol II transcription is interconnected with chromatin transitions, RNA processing, and DNA repair.


Assuntos
RNA Polimerase II/química , RNA Polimerase II/genética , Transcrição Gênica , Animais , Humanos , Modelos Moleculares , Mutagênese/genética , Nucleossomos/metabolismo
13.
Cell ; 174(5): 1127-1142.e19, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30078706

RESUMO

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.


Assuntos
Replicação do DNA , DNA Ribossômico/química , Nucleossomos/metabolismo , Poli dA-dT/química , Origem de Replicação , Motivos de Aminoácidos , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Instabilidade Cromossômica , Sítios Frágeis do Cromossomo , Fragilidade Cromossômica , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Saccharomyces cerevisiae , Schizosaccharomyces , Sítio de Iniciação de Transcrição , Transcrição Gênica
14.
Cell ; 172(5): 993-1006.e13, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29456083

RESUMO

The fate and function of epigenetic marks during the germline-to-embryo transition is a key issue in developmental biology, with relevance to stem cell programming and transgenerational inheritance. In zebrafish, DNA methylation patterns are programmed in transcriptionally quiescent cleavage embryos; paternally inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal. Here, we provide the mechanism by demonstrating that "Placeholder" nucleosomes, containing histone H2A variant H2A.Z(FV) and H3K4me1, virtually occupy all regions lacking DNA methylation in both sperm and cleavage embryos and reside at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with Placeholder become either active (H3K4me3) or silent (H3K4me3/K27me3). Notably, perturbations causing Placeholder loss confer DNA methylation accumulation, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally quiescent gametic and embryonic stages, an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNA methylation, poising parental genes for either gene-specific activation or facultative repression.


Assuntos
Reprogramação Celular/genética , Metilação de DNA/genética , Embrião não Mamífero/metabolismo , Células Germinativas/metabolismo , Nucleossomos/metabolismo , Animais , Histonas/metabolismo , Masculino , Mutação/genética , Espermatozoides/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
15.
Nat Rev Mol Cell Biol ; 21(9): 522-541, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32665685

RESUMO

Histones serve to both package and organize DNA within the nucleus. In addition to histone post-translational modification and chromatin remodelling complexes, histone variants contribute to the complexity of epigenetic regulation of the genome. Histone variants are characterized by a distinct protein sequence and a selection of designated chaperone systems and chromatin remodelling complexes that regulate their localization in the genome. In addition, histone variants can be enriched with specific post-translational modifications, which in turn can provide a scaffold for recruitment of variant-specific interacting proteins to chromatin. Thus, through these properties, histone variants have the capacity to endow specific regions of chromatin with unique character and function in a regulated manner. In this Review, we provide an overview of recent advances in our understanding of the contribution of histone variants to chromatin function in mammalian systems. First, we discuss new molecular insights into chaperone-mediated histone variant deposition. Next, we discuss mechanisms by which histone variants influence chromatin properties such as nucleosome stability and the local chromatin environment both through histone variant sequence-specific effects and through their role in recruiting different chromatin-associated complexes. Finally, we focus on histone variant function in the context of both embryonic development and human disease, specifically developmental syndromes and cancer.


Assuntos
Cromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Animais , DNA/metabolismo , Reparo do DNA/genética , Epigênese Genética/genética , Humanos , Chaperonas Moleculares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia
16.
Cell ; 171(2): 305-320.e24, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28985562

RESUMO

The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Genoma Humano , Proteínas Repressoras/metabolismo , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos , Código das Histonas , Humanos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fosfoproteínas/metabolismo , Coesinas
17.
Mol Cell ; 84(9): 1651-1666.e12, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38521066

RESUMO

Polycomb repressive complexes (PRCs) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells that are proposed to contribute to the maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using a reconstitution approach and single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. We find that the exact combination of PHC and CBX subunits determines condensate initiation, morphology, stability, and dynamics. Particularly, PHC2's polymerization activity influences condensate dynamics by promoting the formation of distinct domains that adhere to each other but do not coalesce. Live-cell imaging confirms CBX's role in condensate initiation and highlights PHC's importance for condensate stability. We propose that PRC1 composition can modulate condensate properties, providing crucial regulatory flexibility across developmental stages.


Assuntos
Proteínas de Ciclo Celular , Cromatina , Nucleossomos , Complexo Repressor Polycomb 1 , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genética , Cromatina/metabolismo , Cromatina/genética , Humanos , Nucleossomos/metabolismo , Nucleossomos/genética , Animais , Imagem Individual de Molécula
18.
Mol Cell ; 84(20): 3885-3898.e8, 2024 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-39303719

RESUMO

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit enhancer of zeste homolog 2 (EZH2) stimulates its activity by an unknown mechanism. Here, we show that human PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosteric trans-autoactivation via EED, suggesting a previously unknown mechanism mediating context-dependent activation of PRC2. Our work showcases the molecular mechanism of auto-modification-coupled dimerization in the regulation of chromatin-modifying complexes.


Assuntos
Cromatina , Proteína Potenciadora do Homólogo 2 de Zeste , Histonas , Complexo Repressor Polycomb 2 , Multimerização Proteica , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Humanos , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Histonas/genética , Metilação , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Regulação Alostérica , Células HEK293 , Ligação Proteica
20.
Mol Cell ; 84(18): 3455-3468.e6, 2024 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-39208807

RESUMO

Mammalian gene expression is controlled by transcription factors (TFs) that engage sequence motifs in a chromatinized genome, where nucleosomes can restrict DNA access. Yet, how nucleosomes affect individual TFs remains unclear. Here, we measure the ability of over one hundred TF motifs to recruit TFs in a defined chromosomal locus in mouse embryonic stem cells. This identifies a set sufficient to enable the binding of TFs with diverse tissue specificities, functions, and DNA-binding domains. These chromatin-competent factors are further classified when challenged to engage motifs within a highly phased nucleosome. The pluripotency factors OCT4-SOX2 preferentially engage non-nucleosomal and entry-exit motifs, but not nucleosome-internal sites, a preference that also guides binding genome wide. By contrast, factors such as BANP, REST, or CTCF engage throughout, causing nucleosomal displacement. This supports that TFs vary widely in their sensitivity to nucleosomes and that genome access is TF specific and influenced by nucleosome position in the cell.


Assuntos
Células-Tronco Embrionárias Murinas , Nucleossomos , Fatores de Transcrição , Nucleossomos/metabolismo , Nucleossomos/genética , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco Embrionárias Murinas/metabolismo , Sítios de Ligação , Ligação Proteica , Genoma/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Cromatina/metabolismo , Cromatina/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Montagem e Desmontagem da Cromatina
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