RESUMO
The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family.
Assuntos
Proteínas Fúngicas/química , Histona-Lisina N-Metiltransferase/química , Histonas/química , Kluyveromyces/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Humanos , Insetos , Metilação , Proteínas Nucleares/química , Domínios Proteicos , Saccharomyces cerevisiae/química , Alinhamento de Sequência , Especificidade por Substrato , Fatores de Transcrição/químicaRESUMO
DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae, share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication.
Assuntos
Replicação do DNA , DNA Fúngico/biossíntese , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Metiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Fúngico/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Metilação , Metiltransferases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.
Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Replicação do DNA/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , Nucleossomos/genética , RecQ Helicases/genética , Saccharomyces cerevisiae/genética , CoesinasRESUMO
Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While heterochromatin and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. Rather, the gene-protective effect is strictly dependent on Set1's catalytic activity. H3K4 methylation, the Set1 product, antagonizes spreading in two ways: directly inhibiting catalysis by Suv39/Clr4 and locally disrupting nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Nucleossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Fatores de Transcrição/metabolismo , Acetilação , Catálise , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Histonas/metabolismoRESUMO
Various factors differentially recognize trimethylated histone H3 lysine 4 (H3K4me3) near promoters, H3K4me2 just downstream, and promoter-distal H3K4me1 to modulate gene expression. This methylation "gradient" is thought to result from preferential binding of the H3K4 methyltransferase Set1/complex associated with Set1 (COMPASS) to promoter-proximal RNA polymerase II. However, other studies have suggested that location-specific cues allosterically activate Set1. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments show that H3K4 methylation patterns on active genes are not universal or fixed and change in response to both transcription elongation rate and frequency as well as reduced COMPASS activity. Fusing Set1 to RNA polymerase II results in H3K4me2 throughout transcribed regions and similarly extended H3K4me3 on highly transcribed genes. Tethered Set1 still requires histone H2B ubiquitylation for activity. These results show that higher-level methylations reflect not only Set1/COMPASS recruitment but also multiple rounds of transcription. This model provides a simple explanation for non-canonical methylation patterns at some loci or in certain COMPASS mutants.
Assuntos
Histonas/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Metilação , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinação/fisiologiaRESUMO
Embryonic stem cells (ESCs) are defined by their ability to self-renew and the potential to differentiate into all tissues of the developing organism. We previously demonstrated that deleting the catalytic SET domain of the Set1A/complex of proteins associated with SET1 histone methyltransferase (Set1A/COMPASS) in mouse ESCs does not impair their viability or ability to self-renew; however, it leads to defects in differentiation. The precise mechanisms by which Set1A executes these functions remain to be elucidated. In this study, we demonstrate that mice lacking the SET domain of Set1A are embryonic lethal at a stage that is unique from null alleles. To gain insight into Set1A function in regulating pluripotency, we conducted a CRISPR/Cas9-mediated dropout screen and identified the MOZ/MORF (monocytic leukaemia zinc finger protein/monocytic leukaemia zinc finger protein-related factor) and HBO1 (HAT bound to ORC1) acetyltransferase complex member ING5 as a synthetic perturbation to Set1A. The loss of Ing5 in Set1AΔSET mouse ESCs decreases the fitness of these cells, and the simultaneous loss of ING5 and in Set1AΔSET leads to up-regulation of differentiation-associated genes. Taken together, our results point toward Set1A/COMPASS and ING5 as potential coregulators of the self-renewal and differentiation status of ESCs.
Assuntos
Histonas , Células-Tronco Embrionárias Murinas , Animais , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Mutações Sintéticas Letais , Proteínas Supressoras de TumorRESUMO
BACKGROUND: Histone H3K4 tri-methylation (H3K4me3) catalyzed by Set1/COMPASS, is a prominent epigenetic mark found in promoter-proximal regions of actively transcribed genes. H3K4me3 relies on prior monoubiquitination at the histone H2B (H2Bub) by Rad6 and Bre1. Swd2/Cps35, a Set1/COMPASS component, has been proposed as a key player in facilitating H2Bub-dependent H3K4me3. However, a more comprehensive investigation regarding the relationship among Rad6, Swd2, and Set1 is required to further understand the mechanisms and functions of the H3K4 methylation. RESULTS: We investigated the genome-wide occupancy patterns of Rad6, Swd2, and Set1 under various genetic conditions, aiming to clarify the roles of Set1 and Rad6 for occupancy of Swd2. Swd2 peaks appear on both the 5' region and 3' region of genes, which are overlapped with its tightly bound two complexes, Set1 and cleavage and polyadenylation factor (CPF), respectively. In the absence of Rad6/H2Bub, Set1 predominantly localized to the 5' region of genes, while Swd2 lost all the chromatin binding. However, in the absence of Set1, Swd2 occupancy near the 5' region was impaired and rather increased in the 3' region. CONCLUSIONS: This study highlights that the catalytic activity of Rad6 is essential for all the ways of Swd2's binding to the transcribed genes and Set1 redistributes the Swd2 to the 5' region for accomplishments of H3K4me3 in the genome-wide level.
Assuntos
Histona-Lisina N-Metiltransferase , Histonas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Histonas/metabolismo , Histonas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Metilação , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two cis-regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyltransferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions independently of the E1 and E2 cis-regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Cromatina/genética , Células-Tronco Embrionárias/citologia , Deleção de Genes , Histona Metiltransferases , Camundongos , Ligação Proteica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ativação Transcricional/genéticaRESUMO
Of the six members of the COMPASS (complex of proteins associated with Set1) family of histone H3 Lys4 (H3K4) methyltransferases identified in mammals, Set1A has been shown to be essential for early embryonic development and the maintenance of embryonic stem cell (ESC) self-renewal. Like its familial relatives, Set1A possesses a catalytic SET domain responsible for histone H3K4 methylation. Whether H3K4 methylation by Set1A/COMPASS is required for ESC maintenance and during differentiation has not yet been addressed. Here, we generated ESCs harboring the deletion of the SET domain of Set1A (Set1AΔSET); surprisingly, the Set1A SET domain is dispensable for ESC proliferation and self-renewal. The removal of the Set1A SET domain does not diminish bulk H3K4 methylation in ESCs; instead, only a subset of genomic loci exhibited reduction in H3K4me3 in Set1AΔSET cells, suggesting a role for Set1A independent of its catalytic domain in ESC self-renewal. However, Set1AΔSET ESCs are unable to undergo normal differentiation, indicating the importance of Set1A-dependent H3K4 methylation during differentiation. Our data also indicate that during differentiation, Set1A but not Mll2 functions as the H3K4 methylase on bivalent genes and is required for their expression, supporting a model for transcriptional switch between Mll2 and Set1A during the self-renewing-to-differentiation transition. Together, our study implicates a critical role for Set1A catalytic methyltransferase activity in regulating ESC differentiation but not self-renewal and suggests the existence of context-specific H3K4 methylation that regulates transcriptional outputs during ESC pluripotency.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Animais , Proliferação de Células/genética , Células-Tronco Embrionárias/enzimologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , Metilação , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Domínios PR-SET/genéticaRESUMO
Recent studies have shown that a subset of nucleoporins (Nups) can detach from the nuclear pore complex and move into the nuclear interior to regulate transcription. One such dynamic Nup, called Nup98, has been implicated in gene activation in healthy cells and has been shown to drive leukemogenesis when mutated in patients with acute myeloid leukemia (AML). Here we show that in hematopoietic cells, Nup98 binds predominantly to transcription start sites to recruit the Wdr82-Set1A/COMPASS (complex of proteins associated with Set1) complex, which is required for deposition of the histone 3 Lys4 trimethyl (H3K4me3)-activating mark. Depletion of Nup98 or Wdr82 abolishes Set1A recruitment to chromatin and subsequently ablates H3K4me3 at adjacent promoters. Furthermore, expression of a Nup98 fusion protein implicated in aggressive AML causes mislocalization of H3K4me3 at abnormal regions and up-regulation of associated genes. Our findings establish a function of Nup98 in hematopoietic gene activation and provide mechanistic insight into which Nup98 leukemic fusion proteins promote AML.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional , Animais , Células Cultivadas , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Metilação , CamundongosRESUMO
Nuclear pore proteins (Nups) interact with chromosomes to regulate gene expression and chromatin structure. A new study by Franks and colleagues (pp. 2222-2234) provides new mechanistic insight into the molecular basis by which Nup98 promotes gene activation in normal hematopoietic cells and how that process is altered by translocations to cause excess expression of developmental genes in leukemia.
Assuntos
Histonas/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Homeodomínio/genética , Leucemia/genética , Metilação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Translocação GenéticaRESUMO
In eukaryotes, methylation of histone H3 at lysine 4 (H3K4me) catalyzed by the complex of proteins associated with Set1 (COMPASS) is crucial for the transcriptional regulation of genes and the development of organisms. In Monascus, the functions of COMPASS in establishing H3K4me remain unclear. This study first identified the conserved COMPASS core subunits MpSet1 and MpSwd3 in Monascus purpureus and confirmed their roles in establishing H3K4me2/3. Loss of MpSet1 and MpSwd3 resulted in slower growth and development and inhibited the formation of cleistothecia, ascospores, and conidia. The loss of these core subunits also decreased the production of extracellular and intracellular Monascus pigments (MPs) by 94.2%, 93.5%, 82.7%, and 82.5%, respectively. In addition, RNA high-throughput sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) showed that the loss of MpSet1 and MpSwd3 altered the expression of 2646 and 2659 genes, respectively, and repressed the transcription of MPs synthesis-related genes. In addition, the ΔMpset1 and ΔMpswd3 strains demonstrated increased sensitivity to cell wall stress with the downregulation of chitin synthase-coding genes. These results indicated that the COMPASS core subunits MpSet1 and MpSwd3 help establish H3K4me2/3 for growth and development, spore formation, and pigment synthesis in Monascus. These core subunits also assist in maintaining cell wall integrity.
Assuntos
Monascus , Monascus/metabolismo , Fermentação , Pigmentos BiológicosRESUMO
Histone H3 methylation on Lys4 (H3K4me) is associated with active gene transcription in all eukaryotes. In Saccharomyces cerevisiae, Set1 is the sole lysine methyltransferase required for mono-, di-, and trimethylation of this site. Although H3K4me3 is linked to gene expression, whether H3K4 methylation regulates other cellular processes, such as mitosis, is less clear. Here we show that both Set1 and H3K4 mutants display a benomyl resistance phenotype that requires components of the spindle assembly checkpoint (SAC), including Bub3 and Mad2. These proteins inhibit Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mutations in Cdc20 that block Mad2 interactions suppress the benomyl resistance of both set1 and H3K4 mutant cells. Furthermore, the HORMA domain in Mad2 directly binds H3, identifying a new histone H3 "reader" motif. Mad2 undergoes a conformational change important for execution of the SAC. We found that the closed (active) conformation of both yeast and human Mad2 is capable of binding methylated H3K4, but, in contrast, the open (inactive) Mad2 conformation limits interaction with methylated H3. Collectively, our data indicate that interactions between Mad2 and H3K4 regulate resolution of the SAC by limiting closed Mad2 availability for Cdc20 inhibition.
Assuntos
Histonas/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Mad2/metabolismo , Benomilo/farmacologia , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Resistência a Medicamentos/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Metilação , Mutação , Ligação Proteica/genética , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/genética , Fuso Acromático/patologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Moduladores de Tubulina/farmacologiaRESUMO
The MLL/KMT2 family enzymes are frequently mutated in human cancers and congenital diseases. They deposit the majority of histone 3 lysine 4 (H3K4) mono-, di-, or tri-methylation in mammals and are tightly associated with gene activation. Structural and biochemical studies in recent years provide in-depth understanding of how the MLL1 and homologous yeast SET1 complexes interact with the nucleosome core particle (NCP) and how their activities for H3K4 methylation are regulated by the conserved core components. Here, we will discuss the recent single molecule cryo-EM studies on the MLL1 and ySET1 complexes bound on the NCP. These studies highlight the dynamic regulation of the MLL/SET1 family lysine methyltransferases with unique features as compared with other histone lysine methyltransferases. These studies provide insights for loci-specific regulation of H3K4 methylation states in cells. The mechanistic studies on the MLL1 complex have already led to the development of the MLL1 inhibitors that show efficacy in acute leukemia and metastatic breast cancers. Future studies on the MLL/SET1 family enzymes will continue to bring to light potential therapeutic opportunities.
Assuntos
Histonas , Proteína de Leucina Linfoide-Mieloide , Animais , Humanos , Histonas/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Lisina/metabolismo , Núcleo Celular/metabolismo , Histona-Lisina N-Metiltransferase/genética , Mamíferos/metabolismoRESUMO
Highly evolutionarily conserved multiprotein complexes termed Complex of Proteins Associated with Set1 (COMPASS) are required for histone 3 lysine 4 (H3K4) methylation. Drosophila Set1, Trx, and Trr form the core subunits of these complexes. We show that flies deficient in any of these three subunits demonstrated high lethality at eclosion (emergence of adult flies from their pupal cases) and significantly shortened lifespans for the adults that did emerge. Silencing Set1, trx, or trr in the heart led to a reduction in H3K4 monomethylation (H3K4me1) and dimethylation (H3K4me2), reflecting their distinct roles in H3K4 methylation. Furthermore, we studied the gene expression patterns regulated by Set1, Trx, and Trr. Each of the COMPASS core subunits controls the methylation of different sets of genes, with many metabolic pathways active early in development and throughout, while muscle and heart differentiation processes were methylated during later stages of development. Taken together, our findings demonstrate the roles of COMPASS series complex core subunits Set1, Trx, and Trr in regulating histone methylation during heart development and, given their implication in congenital heart diseases, inform research on heart disease.
Assuntos
Proteínas de Drosophila , Epigênese Genética , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Coração/crescimento & desenvolvimentoRESUMO
Fungal infections are a major health concern because of limited antifungal drugs and development of drug resistance. Candida can develop azole drug resistance by overexpression of drug efflux pumps or mutating ERG11, the target of azoles. However, the role of epigenetic histone modifications in azole-induced gene expression and drug resistance is poorly understood in Candida glabrata. In this study, we show that Set1 mediates histone H3K4 methylation in C. glabrata. In addition, loss of SET1 and histone H3K4 methylation increases azole susceptibility in both C. glabrata and S. cerevisiae. This increase in azole susceptibility in S. cerevisiae and C. glabrata strains lacking SET1 is due to distinct mechanisms. For S. cerevisiae, loss of SET1 decreased the expression and function of the efflux pump Pdr5, but not ERG11 expression under azole treatment. In contrast, loss of SET1 in C. glabrata does not alter expression or function of efflux pumps. However, RNA sequencing revealed that C. glabrata Set1 is necessary for azole-induced expression of all 12 genes in the late ergosterol biosynthesis pathway, including ERG11 and ERG3. Furthermore, chromatin immunoprecipitation analysis shows histone H3K4 trimethylation increases upon azole-induced ERG gene expression. In addition, high performance liquid chromatography analysis indicated Set1 is necessary for maintaining proper ergosterol levels under azole treatment. Clinical isolates lacking SET1 were also hypersusceptible to azoles which is attributed to reduced ERG11 expression but not defects in drug efflux. Overall, Set1 contributes to azole susceptibility in a species-specific manner by altering the expression and consequently disrupting pathways known for mediating drug resistance.
Assuntos
Azóis , Proteínas de Saccharomyces cerevisiae , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Azóis/metabolismo , Azóis/farmacologia , Candida glabrata/genética , Candida glabrata/metabolismo , Farmacorresistência Fúngica/genética , Ergosterol/metabolismo , Regulação Fúngica da Expressão Gênica , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/farmacologia , Histonas/genética , Histonas/metabolismo , Testes de Sensibilidade Microbiana , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Motivos de Aminoácidos , Sítios de Ligação , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Conformação ProteicaRESUMO
Mouse embryonic stem cell (ESC) is a prototype of pluripotent stem cell that undergoes endless self-renewal in culture without losing the pluripotency, the ability to differentiate to all somatic lineages. The self-renewal of ESC relies on a gene expression program, epigenetic state, and cellular metabolism specific to ESC. In this review, we will present the evidence to exemplify how gene regulation, chromatin methylation, and threonine catabolism are specialized to boost ESC self-renewal. It is evident that a feedforward regulatory circuitry forms at the interfaces between the transcriptional, epigenetic and metabolic control to consolidate the pluripotency of ESC.
Assuntos
Autorrenovação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Autorrenovação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Epigênese Genética , Redes Reguladoras de Genes , Histonas/metabolismo , Humanos , Redes e Vias Metabólicas , Metilação , Transdução de Sinais , Treonina/metabolismo , Transcrição GênicaRESUMO
PURPOSE: Although tamoxifen remains the frontline treatment for ERα-positive breast cancers, resistance to this drug limits its clinical efficacy. Most tamoxifen-resistant patients retain ERα expression which may support growth and progression of breast cancers. Therefore, we investigated epigenetic regulation of ERα that may provide a rationale for targeting ERα in these patients. METHODS: Expression levels of the mixed-lineage leukemia (MLL) family of proteins in tamoxifen-resistant breast cancer cells and publicly available breast cancer patient data sets were analyzed. Histone methylation levels in ERα promoter regions were assessed using chromatin immunoprecipitation. Expression levels of ERα and its target gene were analyzed using western blotting and real-time qPCR. Cell-cycle was analyzed by flow cytometry. RESULTS: The expression of MLL3 and SET-domain-containing 1A (SET1A) were increased in tamoxifen-resistant breast cancers. An MLL3 chromatin immunoprecipitation-sequencing data analysis and chromatin immunoprecipitation experiments for MLL3 and SET1A suggested that these proteins bound to enhancer or intron regions of the ESR1 gene and regulated histone H3K4 methylation status. Depletion of MLL3 or SET1A downregulated the expression level of ERα and inhibited the growth of tamoxifen-resistant breast cancer cells. Additional treatment with fulvestrant resulted in a synergistic reduction of ERα levels and the growth of the cells. CONCLUSIONS: The enhanced expression of MLL3 and SET1A in tamoxifen-resistant breast cancer cells supported the ERα-dependent growth of these cells by increasing ERα expression. Our results suggest that targeting these histone methyltransferases might provide an attractive strategy to overcome endocrine resistance.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Histona Metiltransferases/metabolismo , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Biomarcadores Tumorais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metilação , RNA Interferente Pequeno/genética , Tamoxifeno/uso terapêuticoRESUMO
Homologous recombination is essential for chromosome segregation during meiosis I. Meiotic recombination is initiated by the introduction of double-strand breaks (DSBs) at specific genomic locations called hotspots, which are catalyzed by Spo11 and its partners. DSB hotspots during meiosis are marked with Set1-mediated histone H3K4 methylation. The Spo11 partner complex, Rec114-Mer2-Mei4, essential for the DSB formation, localizes to the chromosome axes. For efficient DSB formation, a hotspot with histone H3K4 methylation on the chromatin loops is tethered to the chromosome axis through the H3K4 methylation reader protein, Spp1, on the axes, which interacts with Mer2. In this study, we found genetic interaction of mutants in a histone modification protein complex called PAF1C with the REC114 and MER2 in the DSB formation in budding yeast Saccharomyces cerevisiae. Namely, the paf1c mutations rtf1 and cdc73 showed synthetic defects in meiotic DSB formation only when combined with a wild-type-like tagged allele of either the REC114 or MER2. The synthetic defect of the tagged REC114 allele in the DSB formation was seen also with the set1, but not with spp1 deletion. These results suggest a novel role of histone modification machinery in DSB formation during meiosis, which is independent of Spp1-mediated loop-axis tethering.