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The fork protection complex promotes symmetric histone recycling to maintain heterochromatin

ABSTRACT: 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 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. This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus Schizosaccharomyces pombe

PROVIDER: GSE241063 | GEO | 2024/08/01

REPOSITORIES: GEO

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The fork protection complex promotes symmetric histone recycling to maintain heterochromatin [SCAR-seq]

Project description: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 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.

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The fork protection complex promotes symmetric histone recycling to maintain heterochromatin [ChIP-Seq]

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DNA Polymerase Delta Governs Parental Histone Transfer to DNA Replication Lagging Strand

Project description:Chromatin replication is intricately intertwined with DNA replication, and the recycling of parental histones is essential for epigenetic inheritance. The transfer of parental histones to both the DNA replication leading and lagging strands involves two distinct pathways: the leading strand utilizes DNA polymerase ε subunits Dpb3/Dpb4, while the lagging strand is facilitated by the MCM helicase subunit Mcm2. However, the process by which Mcm2, moving along the leading strand, facilitates the transfer of parental histones to the lagging strand remains unclear. Our study reveals that the deletion of Pol32, a non-essential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the replication leading strand. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted by the mutation of Mcm2's histone H3-H4 binding domain. In conclusion, our findings identify the DNA polymerase delta subunit Pol32 as a novel key histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

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Project description:Parental histone recycling is essential for the restoration of chromatin-based epigenetic information during chromatin replication; however, the specific mechanisms underlying the local recycling of parental histones remain poorly understood. Here, we reveal an unexpected role of the Spt16-N domain in histone chaperone FACT during parental histone recycling and transfer in budding yeast. We found that depletion of Spt16 or mutations in the Spt16 middle domain leads to defects in both parental histone recycling and new histone deposition, affecting both the leading and lagging strands of DNA replication forks, highlighting the essential role of the FACT complex in both parental histone recycling and new histone deposition. Surprisingly, Spt16-N deletion results in an apparent defect in parental histone recycling, with a more pronounced defect on the lagging strand than the corresponding leading strand. Mechanistically, the Spt16-N domain acts as a protective barrier, shielding FACT-bound histone H3-H4 and facilitating its interaction with Mcm2, which ensures efficient local parental histone recycling. Collectively, the Spt16-N domain provides a protein–protein interaction module allowing FACT to act as a shuttle chaperone, cooperate with multiple replisome components, which act as co-chaperones, to form a complex involving the shuttle chaperone, histones, and co-chaperones, during parental histone recycling and transfer.

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Project description:During DNA replication parental, nucleosomic histones are segregated almost symmetrically to the two daughter DNA strands enabling mitotic inheritance of histones and their PTMs. In MCM2 histone-binding mutant ESCs (MCM2-2A), histones are recycled asymmetrically to the leading strand. To evaluate if loss of parental histones contributes to the asymmetry, we tracked new and old histones quantitatively by pulse-Triple-SILAC-MS. The quantification showed no change in dilution and incorporation dynamics in MCM2-2A cells, demonstrating that parental histones are not lost at replication forks but re-routed from the lagging to the leading strand.

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The Mcm2-Ctf4-Pola axis facilitates parental histone transfer to lagging strands

Project description:Although essential for epigenetic inheritance, the transfer of parental histone (H3-H4)2 tetramers that contain epigenetic modifications to replicating DNA strands is poorly understood. Here, we show that the Mcm2-Ctf4-Pol axis facilitates the transfer of parental (H3-H4)2 tetramers to lagging strands of DNA replication forks. Mutating the H3-H4 binding domain of Mcm2, a subunit of the CMG (Cdc45-MCM-GINS) replicative helicase that translocates along leading strands, impairs the transfer of parental (H3-H4)2 to lagging strands. Similar effects are observed in Ctf4 and Pol-primase mutants that disrupt the connection of the CMG helicase via Ctf4-Pol to lagging strands. Our results support a model whereby parental (H3-H4)2 displaced from nucleosomes through leading-strand DNA replication are transferred to lagging strands for nucleosome assembly via the CMG-Ctf4-Pol complex.

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Project description:Chromatin-based epigenetic memory relies on the accurate distribution of parental histone tetramers to newly replicated DNA strands, serving as templates for chromatin structure duplication. Mcm2, a subunit of the replicative helicase, and the Dpb3/4, subunits of polymerase epsilon, govern parental histone deposition to the lagging and leading strands, respectively. However, their contribution to epigenetic inheritance remains controversial. Here we show in fission yeast that a Mcm2 histone chaperone mutation severely disrupts heterochromatin inheritance, while Dpb3/4 mutations cause only moderate defects. Surprisingly, simultaneous Mcm2 and Dpb3/4 mutations stabilizes heterochromatin inheritance. eSPAN analyses confirm the conservation of Mcm2 and Dpb3/4 functions in parental histone segregation, with their collective absence reducing segregation bias. Furthermore, the FACT histone chaperone also regulates parental histone transfer independently of strands and collaborates with Mcm2 and Dpb3/4 to maintain parental histone density, ensuring faithful heterochromatin inheritance. These results underscore the importance of precise parental histone segregation to the lagging strand for epigenetic inheritance and unveil unique properties of parental histone chaperones during DNA replication.

2024-04-17 | GSE237770 | GEO

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Project description:Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel relatively large distances from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.

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