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/genéticaRESUMO
CBP/p300 are transcription co-activators whose binding is a signature of enhancers, cis-regulatory elements that control patterns of gene expression in multicellular organisms. Active enhancers produce bi-directional enhancer RNAs (eRNAs) and display CBP/p300-dependent histone acetylation. Here, we demonstrate that CBP binds directly to RNAs in vivo and in vitro. RNAs bound to CBP in vivo include a large number of eRNAs. Using steady-state histone acetyltransferase (HAT) assays, we show that an RNA binding region in the HAT domain of CBP-a regulatory motif unique to CBP/p300-allows RNA to stimulate CBP's HAT activity. At enhancers where CBP interacts with eRNAs, stimulation manifests in RNA-dependent changes in the histone acetylation mediated by CBP, such as H3K27ac, and by corresponding changes in gene expression. By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active enhancers, which, in turn, is required for regulation of target genes.
Assuntos
Histona Acetiltransferases/metabolismo , RNA não Traduzido/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Linhagem Celular , Elementos Facilitadores Genéticos , Fibroblastos/metabolismo , Histonas/metabolismo , CamundongosRESUMO
Social insects are emerging models to study how gene regulation affects behavior because their colonies comprise individuals with the same genomes but greatly different behavioral repertoires. To investigate the molecular mechanisms that activate distinct behaviors in different castes, we exploit a natural behavioral plasticity in Harpegnathos saltator, where adult workers can transition to a reproductive, queen-like state called gamergate. Analysis of brain transcriptomes during the transition reveals that corazonin, a neuropeptide homologous to the vertebrate gonadotropin-releasing hormone, is downregulated as workers become gamergates. Corazonin is also preferentially expressed in workers and/or foragers from other social insect species. Injection of corazonin in transitioning Harpegnathos individuals suppresses expression of vitellogenin in the brain and stimulates worker-like hunting behaviors, while inhibiting gamergate behaviors, such as dueling and egg deposition. We propose that corazonin is a central regulator of caste identity and behavior in social insects.
Assuntos
Formigas/metabolismo , Proteínas de Insetos/metabolismo , Neuropeptídeos/metabolismo , Animais , Formigas/genética , Formigas/crescimento & desenvolvimento , Comportamento Animal , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Comportamento SocialRESUMO
Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.
Assuntos
Formigas/crescimento & desenvolvimento , Formigas/genética , Proteínas de Insetos/genética , Receptores Odorantes/genética , Comportamento Social , Sequência de Aminoácidos , Animais , Formigas/anatomia & histologia , Formigas/fisiologia , Antenas de Artrópodes/anatomia & histologia , Antenas de Artrópodes/metabolismo , Sequência de Bases , Comportamento Animal , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Técnicas de Inativação de Genes , Proteínas de Insetos/química , Masculino , Mutação , Feromônios/metabolismo , Receptores Odorantes/químicaRESUMO
Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.
Assuntos
Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Coesinas , Proteínas de Ligação a DNA , Animais , Cromatina/metabolismo , Cromatina/genética , Camundongos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neurônios Motores/metabolismo , Diferenciação Celular , Dedos de Zinco , Humanos , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genéticaRESUMO
Erk1/2 activation contributes to mouse ES cell pluripotency. We found a direct role of Erk1/2 in modulating chromatin features required for regulated developmental gene expression. Erk2 binds to specific DNA sequence motifs typically accessed by Jarid2 and PRC2. Negating Erk1/2 activation leads to increased nucleosome occupancy and decreased occupancy of PRC2 and poised RNAPII at Erk2-PRC2-targeted developmental genes. Surprisingly, Erk2-PRC2-targeted genes are specifically devoid of TFIIH, known to phosphorylate RNA polymerase II (RNAPII) at serine-5, giving rise to its initiated form. Erk2 interacts with and phosphorylates RNAPII at its serine 5 residue, which is consistent with the presence of poised RNAPII as a function of Erk1/2 activation. These findings underscore a key role for Erk1/2 activation in promoting the primed status of developmental genes in mouse ES cells and suggest that the transcription complex at developmental genes is different than the complexes formed at other genes, offering alternative pathways of regulation.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/metabolismo , Animais , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nucleossomos/metabolismo , Fosforilação , Complexo Repressor Polycomb 2/metabolismoRESUMO
The heterogeneous family of complexes comprising Polycomb repressive complex 1 (PRC1) is instrumental for establishing facultative heterochromatin that is repressive to transcription. However, two PRC1 species, ncPRC1.3 and ncPRC1.5, are known to comprise novel components, AUTS2, P300, and CK2, that convert this repressive function to that of transcription activation. Here, we report that individuals harboring mutations in the HX repeat domain of AUTS2 exhibit defects in AUTS2 and P300 interaction as well as a developmental disorder reflective of Rubinstein-Taybi syndrome, which is mainly associated with a heterozygous pathogenic variant in CREBBP/EP300. Moreover, the absence of AUTS2 or mutation in its HX repeat domain gives rise to misregulation of a subset of developmental genes and curtails motor neuron differentiation of mouse embryonic stem cells. The transcription factor nuclear respiratory factor 1 (NRF1) has a novel and integral role in this neurodevelopmental process, being required for ncPRC1.3 recruitment to chromatin.
Assuntos
Encéfalo/metabolismo , Proteína de Ligação a CREB/genética , Proteínas do Citoesqueleto/metabolismo , Proteína p300 Associada a E1A/genética , Células-Tronco Embrionárias/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Cromatina/química , Feminino , Genômica , Células HEK293 , Heterozigoto , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Ligação Proteica , Domínios Proteicos , Proteômica , Ativação TranscricionalRESUMO
Major advances in the chromatin and epigenetics fields have uncovered the importance of core histones, histone variants and their post-translational modifications (PTMs) in modulating chromatin structure. However, an acutely understudied related feature of chromatin structure is the role of linker histone H1. Previous assumptions of the functional redundancy of the 11 nonallelic H1 variants are contrasted by their strong evolutionary conservation, variability in their potential PTMs, and increased reports of their disparate functions, sub-nuclear localizations and unique expression patterns in different cell types. The commonly accepted notion that histone H1 functions solely in chromatin compaction and transcription repression is now being challenged by work from multiple groups. These studies highlight histone H1 variants as underappreciated facets of chromatin dynamics that function independently in various chromatin-based processes. In this review, we present notable findings involving the individual somatic H1 variants of which there are seven, underscoring their particular contributions to distinctly significant chromatin-related processes.
Assuntos
Cromatina/metabolismo , Variação Genética , Histonas/metabolismo , Animais , Sequência Conservada , Regulação da Expressão Gênica , Histonas/genética , Humanos , Relação Estrutura-AtividadeRESUMO
Ant societies show a division of labor in which a queen is in charge of reproduction while nonreproductive workers maintain the colony. In Harpegnathos saltator, workers retain reproductive ability, inhibited by the queen pheromones. Following the queen loss, the colony undergoes social unrest with an antennal dueling tournament. Most workers quickly abandon the tournament while a few workers continue the dueling for months and become gamergates (pseudoqueens). However, the temporal dynamics of the social behavior and molecular mechanisms underlining the caste transition and social dominance remain unclear. By tracking behaviors, we show that the gamergate fate is accurately determined 3 d after initiation of the tournament. To identify genetic factors responsible for this commitment, we compared transcriptomes of different tissues between dueling and nondueling workers. We found that juvenile hormone is globally repressed, whereas ecdysone biosynthesis in the ovary is increased in gamergates. We show that molecular changes in the brain serve as earliest caste predictors compared with other tissues. Thus, behavioral and molecular data indicate that despite the prolonged social upheaval, the gamergate fate is rapidly established, suggesting a robust re-establishment of social structure.
Assuntos
Formigas , Comportamento Animal , Animais , Feminino , Formigas/genética , Comportamento Animal/fisiologia , Ovário/metabolismo , Reprodução/genética , TranscriptomaRESUMO
Gene expression programmes conferring cellular identity are achieved through the organization of chromatin structures that either facilitate or impede transcription. Among the key determinants of chromatin organization are the histone modifications that correlate with a given transcriptional status and chromatin state. Until recently, the details for the segregation of nucleosomes on DNA replication and their implications in re-establishing heritable chromatin domains remained unclear. Here, we review recent findings detailing the local segregation of parental nucleosomes and highlight important advances as to how histone methyltransferases associated with the establishment of repressive chromatin domains facilitate epigenetic inheritance.
Assuntos
Linhagem da Célula , Montagem e Desmontagem da Cromatina , Replicação do DNA , Epigênese Genética , Padrões de Herança , Nucleossomos/metabolismo , Humanos , Nucleossomos/genética , PaisRESUMO
Mononucleosomes, the basic building blocks of chromatin, contain two copies of each core histone. The associated posttranslational modifications regulate essential chromatin-dependent processes, yet whether each histone copy is identically modified in vivo is unclear. We demonstrate that nucleosomes in embryonic stem cells, fibroblasts, and cancer cells exist in both symmetrically and asymmetrically modified populations for histone H3 lysine 27 di/trimethylation (H3K27me2/3) and H4K20me1. Further, we obtained direct physical evidence for bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Bivalency at target genes was resolved upon differentiation of ES cells. Polycomb repressive complex 2-mediated methylation of H3K27 was inhibited when nucleosomes contain symmetrically, but not asymmetrically, placed H3K4me3 or H3K36me3. These findings uncover a potential mechanism for the incorporation of bivalent features into nucleosomes and demonstrate how asymmetry might set the stage to diversify functional nucleosome states.
Assuntos
Células-Tronco Embrionárias/metabolismo , Código das Histonas , Histonas/metabolismo , Nucleossomos/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Linhagem Celular , Fibroblastos/metabolismo , Células HeLa , Histonas/química , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-TraducionalRESUMO
Heterochromatin serves important functions, protecting genome integrity and stabilizing gene expression programs. Although the Suv39h methyltransferases (KMTs) are known to ensure pericentric H3K9me3 methylation, the mechanisms that initiate and maintain mammalian heterochromatin organization remain elusive. We developed a biochemical assay and used in vivo analyses in mouse embryonic fibroblasts to identify Prdm3 and Prdm16 as redundant H3K9me1-specific KMTs that direct cytoplasmic H3K9me1 methylation. The H3K9me1 is converted in the nucleus to H3K9me3 by the Suv39h enzymes to reinforce heterochromatin. Simultaneous depletion of Prdm3 and Prdm16 abrogates H3K9me1 methylation, prevents Suv39h-dependent H3K9me3 trimethylation, and derepresses major satellite transcription. Most strikingly, DNA-FISH and electron microscopy reveal that combined impairment of Prdm3 and Prdm16 results in disintegration of heterochromatic foci and disruption of the nuclear lamina. Our data identify Prdm3 and Prdm16 as H3K9me1 methyltransferases and expose a functional framework in which anchoring to the nuclear periphery helps maintain the integrity of mammalian heterochromatin.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Heterocromatina , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Lâmina Nuclear/metabolismo , Proto-Oncogenes , Fatores de Transcrição/genéticaRESUMO
Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. Some loops are cell-type specific. Here we asked whether CTCF loops are established by a universal or locus-specific mechanism. Investigating the molecular determinants of CTCF clustering, we found that CTCF self-association in vitro is RNase sensitive and that an internal RNA-binding region (RBRi) mediates CTCF clustering and RNA interaction in vivo. Strikingly, deleting the RBRi impairs about half of all chromatin loops in mESCs and causes deregulation of gene expression. Disrupted loop formation correlates with diminished clustering and chromatin binding of RBRi mutant CTCF, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least two classes: RBRi-independent and RBRi-dependent loops. We speculate that evidence for RBRi-dependent loops may provide a molecular mechanism for establishing cell-specific CTCF loops, potentially regulated by RNA(s) or other RBRi-interacting partners.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/genética , Linhagem Celular , Cromatina/química , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-AtividadeRESUMO
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
As the process that silences gene expression ensues during development, the stage is set for the activity of Polycomb-repressive complex 2 (PRC2) to maintain these repressed gene profiles. PRC2 catalyzes a specific histone posttranslational modification (hPTM) that fosters chromatin compaction. PRC2 also facilitates the inheritance of this hPTM through its self-contained "write and read" activities, key to preserving cellular identity during cell division. As these changes in gene expression occur without changes in DNA sequence and are inherited, the process is epigenetic in scope. Mutants of mammalian PRC2 or of its histone substrate contribute to the cancer process and other diseases, and research into these aberrant pathways is yielding viable candidates for therapeutic targeting. The effectiveness of PRC2 hinges on its being recruited to the proper chromatin sites; however, resolving the determinants to this process in the mammalian case was not straightforward and thus piqued the interest of many in the field. Here, we chronicle the latest advances toward exposing mammalian PRC2 and its high maintenance.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Animais , Cromatina/metabolismo , Humanos , Mutação , Neoplasias/genética , Neoplasias/fisiopatologia , Transporte Proteico , Pesquisa/tendênciasRESUMO
The histone methyltransferase activity of PRC2 is central to the formation of H3K27me3-decorated facultative heterochromatin and gene silencing. In addition, PRC2 has been shown to automethylate its core subunits, EZH1/EZH2 and SUZ12. Here, we identify the lysine residues at which EZH1/EZH2 are automethylated with EZH2-K510 and EZH2-K514 being the major such sites in vivo. Automethylated EZH2/PRC2 exhibits a higher level of histone methyltransferase activity and is required for attaining proper cellular levels of H3K27me3. While occurring independently of PRC2 recruitment to chromatin, automethylation promotes PRC2 accessibility to the histone H3 tail. Intriguingly, EZH2 automethylation is significantly reduced in diffuse intrinsic pontine glioma (DIPG) cells that carry a lysine-to-methionine substitution in histone H3 (H3K27M), but not in cells that carry either EZH2 or EED mutants that abrogate PRC2 allosteric activation, indicating that H3K27M impairs the intrinsic activity of PRC2. Our study demonstrates a PRC2 self-regulatory mechanism through its EZH1/2-mediated automethylation activity.
Assuntos
Glioma/enzimologia , Glioma/genética , Histonas/metabolismo , Criança , Ativação Enzimática , Inativação Gênica , Histonas/genética , Humanos , Lisina/metabolismo , Metilação , Complexo Repressor Polycomb 2/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Eusocial insects live in societies in which distinct family members serve specific roles in maintaining the colony and advancing the reproductive ability of a few select individuals. Given the genetic similarity of all colony members, the diversity of morphologies and behaviors is surprising. Social communication relies on pheromones and olfaction, as shown by mutants of orco, the universal odorant receptor coreceptor, and through electrophysiological analysis of neuronal responses to pheromones. Additionally, neurohormonal factors and epigenetic regulators play a key role in caste-specific behavior, such as foraging and caste switching. These studies start to allow an understanding of the molecular mechanisms underlying social behavior and provide a technological foundation for future studies of eusocial insects. In this review, we highlight recent findings in eusocial insects that advance our understanding of genetic and epigenetic regulations of social behavior and provide perspectives on future studies using cutting-edge technologies.
Assuntos
Comportamento Animal/fisiologia , Epigênese Genética/genética , Insetos/genética , Comportamento Social , Animais , Epigênese Genética/fisiologia , Insetos/fisiologia , Neurônios/metabolismo , Feromônios/genética , Receptores Odorantes/genética , Olfato/genéticaRESUMO
The human FACT (facilitates chromatin transcription) complex, composed of two subunits SPT16 (Suppressor of Ty 16) and SSRP1 (Structure-specific recognition protein-1), plays essential roles in nucleosome remodeling. However, the molecular mechanism of FACT reorganizing the nucleosome still remains elusive. In this study, we demonstrate that FACT displays dual functions in destabilizing the nucleosome and maintaining the original histones and nucleosome integrity at the single-nucleosome level. We found that the subunit SSRP1 is responsible for maintenance of nucleosome integrity by holding the H3/H4 tetramer on DNA and promoting the deposition of the H2A/H2B dimer onto the nucleosome. In contrast, the large subunit SPT16 destabilizes the nucleosome structure by displacing the H2A/H2B dimers. Our findings provide mechanistic insights by which the two subunits of FACT coordinate with each other to fulfill its functions and suggest that FACT may play essential roles in preserving the original histones with epigenetic identity during transcription or DNA replication.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Nucleossomos/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/genética , Ligação Proteica , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genéticaRESUMO
PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin. Here we report a stepwise feedback mechanism entailing key residues within distinctive interfacing motifs of EZH2 or EED that are found to be mutated in cancers and/or Weaver syndrome. PRC2 harboring these EZH2 or EED mutants manifested little activity in vivo but, unexpectedly, exhibited similar chromatin association as wild-type PRC2, indicating an uncoupling of PRC2 activity and recruitment. With genetic and chemical tools, we demonstrated that targeting allosteric activation overrode the gain-of-function effect of EZH2Y646X oncogenic mutations. These results revealed critical implications for the regulation and biology of PRC2 and a vulnerability in tackling PRC2-addicted cancers.
Assuntos
Regulação Alostérica/fisiologia , Cromatina/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Anormalidades Múltiplas/metabolismo , Linhagem Celular Tumoral , Hipotireoidismo Congênito/metabolismo , Anormalidades Craniofaciais/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Deformidades Congênitas da Mão/metabolismo , Histonas/metabolismo , Humanos , Neoplasias/metabolismoRESUMO
The maintenance of gene expression patterns during metazoan development is achieved, in part, by the actions of polycomb repressive complex 2 (PRC2). PRC2 catalyzes mono-, di-, and trimethylation of histone H3 at lysine 27 (H3K27), with H3K27me2/3 being strongly associated with silenced genes. We demonstrate that EZH1 and EZH2, the two mutually exclusive catalytic subunits of PRC2, are differentially activated by various mechanisms. Whereas both PRC2-EZH1 and PRC2-EZH2 are able to catalyze mono- and dimethylation, only PRC2-EZH2 is strongly activated by allosteric modulators and specific chromatin substrates to catalyze trimethylation of H3K27 in mouse embryonic stem cells (mESCs). However, we also show that a PRC2-associated protein, AEBP2, can stimulate the activity of both complexes through a mechanism independent of and additive to allosteric activation. These results have strong implications regarding the cellular requirements for and the accompanying adjustments in PRC2 activity, given the differential expression of EZH1 and EZH2 upon cellular differentiation.