Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora. The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate binding, thereby controlling DIM2-mediated DNA methylation. Together, this study uncovers how multiple heterochromatin factors coordinately orchestrate an activity-switching mechanism for region-specific DNA methylation.

Heterochromatin enforces transcriptional gene silencing and can be epigenetically inherited, but the underlying mechanisms remain unclear. Here, we show that histone deacetylation, a conserved feature of heterochromatin domains, blocks SWI/SNF subfamily remodelers involved in chromatin unraveling, thereby stabilizing modified nucleosomes that preserve gene silencing. Histone hyperacetylation, resulting from either the loss of histone deacetylase (HDAC) activity or the direct targeting of a histone acetyltransferase to heterochromatin, permits remodeler access, leading to silencing defects. The requirement for HDAC in heterochromatin silencing can be bypassed by impeding SWI/SNF activity. Highlighting the crucial role of remodelers, merely targeting SWI/SNF to heterochromatin, even in cells with functional HDAC, increases nucleosome turnover, causing defective gene silencing and compromised epigenetic inheritance. This study elucidates a fundamental mechanism whereby histone hypoacetylation, maintained by high HDAC levels in heterochromatic regions, ensures stable gene silencing and epigenetic inheritance, providing insights into genome regulatory mechanisms relevant to human diseases.

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 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.

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.

Chromatin-based epigenetic memory relies on the symmetric distribution of parental histones to newly synthesized daughter DNA strands, aided by histone chaperones within the DNA replication machinery. However, the mechanism of parental histone transfer remains elusive. Here, we reveal that in fission yeast, the replisome protein Mrc1 plays a crucial role in promoting the transfer of parental histone H3-H4 to the lagging strand, ensuring proper heterochromatin inheritance. In addition, Mrc1 facilitates the interaction between Mcm2 and DNA polymerase alpha, two histone-binding proteins critical for parental histone transfer. Furthermore, Mrc1\'s involvement in parental histone transfer and epigenetic inheritance is independent of its known functions in DNA replication checkpoint activation and replisome speed control. Instead, Mrc1 interacts with Mcm2 outside of its histone-binding region, creating a physical barrier to separate parental histone transfer pathways. These findings unveil Mrc1 as a key player within the replisome, coordinating parental histone segregation to regulate epigenetic inheritance.

Epigenetic mechanisms enable cells to develop novel adaptive phenotypes without altering their genetic blueprint. Recent studies show histone modifications, such as heterochromatin-defining H3K9 methylation (H3K9me), can be redistributed to establish adaptive phenotypes. We developed a precision-engineered genetic approach to trigger heterochromatin misregulation on-demand in fission yeast. This enabled us to trace genome-scale RNA and H3K9me changes over time in long-term, continuous cultures. Adaptive H3K9me establishes over remarkably slow timescales relative to the initiating stress. We captured dynamic H3K9me redistribution events which depend on an RNA binding complex MTREC, ultimately leading to cells converging on an optimal adaptive solution. Upon stress removal, cells relax to new transcriptional and chromatin states, establishing memory that is tunable and primed for future adaptive epigenetic responses. Collectively, we identify the slow kinetics of epigenetic adaptation that allow cells to discover and heritably encode novel adaptive solutions, with implications for drug resistance and response to infection.

Repression of facultative heterochromatin is essential for developmental processes in numerous organisms. Methylation of histone H3 lysine 27 (H3K27) by Polycomb repressive complex 2 is a prominent feature of facultative heterochromatin in both fungi and higher eukaryotes. Although this methylation is frequently associated with silencing, the detailed mechanism of repression remains incompletely understood. We utilized a forward genetics approach to identify genes required to maintain silencing at facultative heterochromatin genes in Neurospora crassa and identified three previously uncharacterized genes that are important for silencing: sds3 (NCU01599), rlp1 (RPD3L protein 1; NCU09007), and rlp2 (RPD3L protein 2; NCU02898). We found that SDS3, RLP1, and RLP2 associate with N. crassa homologs of the Saccharomyces cerevisiae Rpd3L complex and are required for repression of a subset of H3K27-methylated genes. Deletion of these genes does not lead to loss of H3K27 methylation but increases acetylation of histone H3 lysine 14 at up-regulated genes, suggesting that RPD3L-driven deacetylation is a factor required for silencing of facultative heterochromatin in N. crassa, and perhaps in other organisms.

Early embryos often have relatively unstructured chromatin that lacks active and inactive domains typical of differentiated cells. In many species, these regulatory domains are established during zygotic genome activation (ZGA). In Drosophila, ZGA occurs after 13 fast, reductive, syncytial nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio grows exponentially. These divisions incorporate maternally-loaded, cytoplasmic pools of histones into chromatin. Previous work found that chromatin incorporation of replication-coupled histone H3 decreases while its variant H3.3 increases in the cell cycles leading up to ZGA. In other cell types, H3.3 is associated with sites of active transcription as well as heterochromatin, suggesting a link between H3.3 incorporation and ZGA. Here, we examine the factors that contribute to H3.3 incorporation at ZGA. We identify a more rapid decrease in the nuclear availability of H3 than H3.3 over the final pre-ZGA cycles. We also observe an N/C ratio-dependent increase in H3.3 incorporation in mutant embryos with non-uniform local N/C ratios. We find that chaperone binding, not gene expression, controls incorporation patterns using H3/H3.3 chimeric proteins at the endogenous H3.3A locus. We test the specificity of the H3.3 chaperone pathways for H3.3 incorporation using Hira (H3.3 chaperone) mutant embryos. Overall, we propose a model in which local N/C ratios and specific chaperone binding regulate differential incorporation of H3.3 during ZGA.

HP1 proteins are essential for establishing and maintaining transcriptionally silent heterochromatin. They dimerize, forming a binding interface to recruit diverse chromatin-associated factors. Although HP1 proteins are known to rapidly evolve, the extent of variation required to achieve functional specialization is unknown. To investigate how changes in amino acid sequence impacts heterochromatin formation, we performed a targeted mutagenesis screen of the S. pombe HP1 homolog, Swi6. Substitutions within an auxiliary surface adjacent to the HP1 dimerization interface produce Swi6 variants with divergent maintenance properties. Remarkably, substitutions at a single amino acid position lead to the persistent gain or loss of epigenetic inheritance. These substitutions increase Swi6 chromatin occupancy in vivo and altered Swi6-protein interactions that reprogram H3K9me maintenance. We show how relatively minor changes in Swi6 amino acid composition in an auxiliary surface can lead to profound changes in epigenetic inheritance providing a redundant mechanism to evolve HP1-effector specificity.

In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin.