RESUMEN
CENP-A is a centromere-specific histone 3 variant essential for centromere specification. CENP-A partially replaces canonical histone H3 at the centromeres. How the particular CENP-A/H3 ratio at centromeres is precisely maintained is unknown. It also remains unclear how CENP-A is excluded from non-centromeric chromatin. Here, we identify Ccp1, an uncharacterized NAP family protein in fission yeast that antagonizes CENP-A loading at both centromeric and non-centromeric regions. Like the CENP-A loading factor HJURP, Ccp1 interacts with CENP-A and is recruited to centromeres at the end of mitosis in a Mis16-dependent manner. These data indicate that factors with opposing CENP-A loading activities are recruited to centromeres. Furthermore, Ccp1 also cooperates with H2A.Z to evict CENP-A assembled in euchromatin. Structural analyses indicate that Ccp1 forms a homodimer that is required for its anti-CENP-A loading activity. Our study establishes mechanisms for maintenance of CENP-A homeostasis at centromeres and the prevention of ectopic assembly of centromeres.
Asunto(s)
Carboxipeptidasas/genética , Proteínas Portadoras/genética , Proteínas Cromosómicas no Histona/genética , Eucromatina/química , Regulación Fúngica de la Expresión Génica , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Sitios de Unión , Carboxipeptidasas/química , Carboxipeptidasas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Centrómero/química , Centrómero/metabolismo , Centrómero/ultraestructura , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Eucromatina/metabolismo , Eucromatina/ultraestructura , Histonas/química , Histonas/genética , Histonas/metabolismo , Mitosis , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestructura , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Transducción de SeñalRESUMEN
During DNA replication, chromatin is disrupted ahead of the replication fork, and epigenetic information must be restored behind the fork. How epigenetic marks are inherited through DNA replication remains poorly understood. Histone H3 lysine 9 (H3K9) methylation and histone hypoacetylation are conserved hallmarks of heterochromatin. We previously showed that the inheritance of H3K9 methylation during DNA replication depends on the catalytic subunit of DNA polymerase epsilon, Cdc20. Here we show that the histone-fold subunit of Pol epsilon, Dpb4, interacts an uncharacterized small histone-fold protein, SPCC16C4.22, to form a heterodimer in fission yeast. We demonstrate that SPCC16C4.22 is nonessential for viability and corresponds to the true ortholog of Dpb3. We further show that the Dpb3-Dpb4 dimer associates with histone deacetylases, chromatin remodelers, and histones and plays a crucial role in the inheritance of histone hypoacetylation in heterochromatin. We solve the 1.9-Å crystal structure of Dpb3-Dpb4 and reveal that they form the H2A-H2B-like dimer. Disruption of Dpb3-Dpb4 dimerization results in loss of heterochromatin silencing. Our findings reveal a link between histone deacetylation and H3K9 methylation and suggest a mechanism for how two processes are coordinated during replication. We propose that the Dpb3-Dpb4 heterodimer together with Cdc20 serves as a platform for the recruitment of chromatin modifiers and remodelers that mediate heterochromatin assembly during DNA replication, and ensure the faithful inheritance of epigenetic marks in heterochromatin.
Asunto(s)
Proteínas Cdc20/química , ADN Polimerasa II/química , Epigénesis Genética , Heterocromatina/química , Histonas/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/genética , Animales , Sitios de Unión , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Clonación Molecular , Cristalografía por Rayos X , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , Replicación del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Histone lysine methylation is an important epigenetic modification in regulating chromatin structure and gene expression. Histone H3 lysine 4 methylation (H3K4me), which can be in a mono-, di-, or trimethylated state, has been shown to play an important role in gene expression involved in plant developmental control and stress adaptation. However, the resetting mechanism of this epigenetic modification is not yet fully understood. In this work, we identified a JmjC domain-containing protein, JMJ703, as a histone lysine demethylase that specifically reverses all three forms of H3K4me in rice. Loss-of-function mutation of the gene affected stem elongation and plant growth, which may be related to increased expression of cytokinin oxidase genes in the mutant. Analysis of crystal structure of the catalytic core domain (c-JMJ703) of the protein revealed a general structural similarity with mammalian and yeast JMJD2 proteins that are H3K9 and H3K36 demethylases. However, several specific features were observed in the structure of c-JMJ703. Key residues that interact with cofactors Fe(II) and N-oxalylglycine and the methylated H3K4 substrate peptide were identified and were shown to be essential for the demethylase activity in vivo. Several key residues are specifically conserved in known H3K4 demethylases, suggesting that they may be involved in the specificity for H3K4 demethylation.
Asunto(s)
Dominio Catalítico , Histona Demetilasas con Dominio de Jumonji , Oryza , Tallos de la Planta , Aminoácidos , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Metilación , Mutación , Oryza/genética , Oryza/crecimiento & desarrollo , Tallos de la Planta/genética , Tallos de la Planta/crecimiento & desarrollo , Conformación ProteicaRESUMEN
Mammalian TRICs function as K+-permeable cation channels that provide counter ions for Ca2+ handling in intracellular stores. Here we describe the structures of two prokaryotic homologues, archaeal SaTRIC and bacterial CpTRIC, showing that TRIC channels are symmetrical trimers with transmembrane pores through each protomer. Each pore holds a string of water molecules centred at kinked helices in two inverted-repeat triple-helix bundles (THBs). The pores are locked in a closed state by a hydrogen bond network at the C terminus of the THBs, which is lost when the pores assume an open conformation. The transition between the open and close states seems to be mediated by cation binding to conserved residues along the three-fold axis. Electrophysiology and mutagenesis studies show that prokaryotic TRICs have similar functional properties to those of mammalian TRICs and implicate the three-fold axis in the allosteric regulation of the channel.