RESUMEN
The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.
Asunto(s)
Replicación del ADN , Epigénesis Genética , Histonas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Histonas/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Mutación , Memoria EpigenéticaRESUMEN
Transcription factors can exert opposite effects depending on the chromosomal context. The fission yeast transcription factor Atf1 both activates numerous genes in response to stresses and mediates heterochromatic gene silencing in the mating-type region. Investigating this context dependency, we report here that the establishment of silent heterochromatin in the mating-type region occurs at a reduced rate in the absence of Atf1 binding. Quantitative modeling accounts for the observed establishment profiles by a combinatorial recruitment of histone-modifying enzymes: locally by Atf1 at two binding sites and over the whole region by dynamically appearing heterochromatic nucleosomes, a source of which is the RNAi-dependent cenH element. In the absence of Atf1 binding, the synergy is lost, resulting in a slow rate of heterochromatin formation. The system shows how DNA-binding proteins can influence local nucleosome states and thereby potentiate long-range positive feedback on histone-modification reactions to enable heterochromatin formation over large regions in a context-dependent manner.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Factor de Transcripción Activador 1/genética , Factor de Transcripción Activador 1/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Fosfoproteínas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Methylation of histone H3K9 is a hallmark of epigenetic silencing in eukaryotes. Nucleosome modifications often rely on positive feedback where enzymes are recruited by modified nucleosomes. A combination of local and global feedbacks has been proposed to account for some dynamic properties of heterochromatin, but the range at which the global feedbacks operate and the exact mode of heterochromatin propagation are not known. We investigated these questions in fission yeast. Guided by mathematical modeling, we incrementally increased the size of the mating-type region and profiled heterochromatin establishment over time. We observed exponential decays in the proportion of cells with active reporters, with rates that decreased with domain size. Establishment periods varied from a few generations in wild type to >200 generations in the longest region examined, and highly correlated silencing of two reporters located outside the nucleation center was observed. On a chromatin level, this indicates that individual regions are silenced in sudden bursts. Mathematical modeling accounts for these bursts if heterochromatic nucleosomes facilitate a deacetylation or methylation reaction at long range, in a distance-independent manner. A likely effector of three-dimensional interactions is the evolutionarily conserved Swi6HP1 H3K9me reader, indicating the bursting behavior might be a general mode of heterochromatin propagation.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Heterocromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Genes del Tipo Sexual de los Hongos , Heterocromatina/genética , Modelos Genéticos , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
In fission yeast, the inverted repeats IR-L and IR-R function as boundary elements at the edges of a 20-kb silent heterochromatic domain where nucleosomes are methylated at histone H3K9. Each repeat contains a series of B-box motifs physically associated with the architectural TFIIIC complex and with other factors including the replication regulator Sap1 and the Rix1 complex (RIXC). We demonstrate here the activity of these repeats in heterochromatin formation and maintenance. Deletion of the entire IR-R repeat or, to a lesser degree, deletion of just the B boxes impaired the de novo establishment of the heterochromatic domain. Nucleation proceeded normally at the RNA interference (RNAi)-dependent element cenH but subsequent propagation to the rest of the region occurred at reduced rates in the mutants. Once established, heterochromatin was unstable in the mutants. These defects resulted in bistable populations of cells occupying alternate "on" and "off" epigenetic states. Deleting IR-L in combination with IR-R synergistically tipped the balance toward the derepressed state, revealing a concerted action of the two boundaries at a distance. The nuclear rim protein Amo1 has been proposed to tether the mating-type region and its boundaries to the nuclear envelope, where Amo1 mutants displayed milder phenotypes than boundary mutants. Thus, the boundaries might facilitate heterochromatin propagation and maintenance in ways other than just through Amo1, perhaps by constraining a looped domain through pairing.