Deep learning meets histones at the replication fork.

Epigenetic inheritance of heterochromatin requires transfer of parental H3-H4 tetramers to both daughter duplexes during replication. Three recent papers exploit yeast genetics coupled to inheritance assays and AlphaFold2-multimer predictions coupled to biochemistry to reveal that a replisome component (Mrc1/CLASPIN) is an H3-H4 tetramer chaperone important for parental histone transfer to daughters.

RNA quality control factors nucleate Clr4/SUV39H and trigger constitutive heterochromatin assembly.

In eukaryotes, the Suv39 family of proteins tri-methylate lysine 9 of histone H3 (H3K9me) to form constitutive heterochromatin. However, how Suv39 proteins are nucleated at heterochromatin is not fully described. In the fission yeast, current models posit that Argonaute1-associated small RNAs (sRNAs) nucleate the sole H3K9 methyltransferase, Clr4/SUV39H, to centromeres. Here, we show that in the absence of all sRNAs and H3K9me, the Mtl1 and Red1 core (MTREC)/PAXT complex nucleates Clr4/SUV39H at a heterochromatic long noncoding RNA (lncRNA) at which the two H3K9 deacetylases, Sir2 and Clr3, also accumulate by distinct mechanisms. Iterative cycles of H3K9 deacetylation and methylation spread Clr4/SUV39H from the nucleation center in an sRNA-independent manner, generating a basal H3K9me state. This is acted upon by the RNAi machinery to augment and amplify the Clr4/H3K9me signal at centromeres to establish heterochromatin. Overall, our data reveal that lncRNAs and RNA quality control factors can nucleate heterochromatin and function as epigenetic silencers in eukaryotes.

A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance.

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.

Parallel pathways for recruiting effector proteins determine centromere drive and suppression.

Selfish centromere DNA sequences bias their transmission to the egg in female meiosis. Evolutionary theory suggests that centromere proteins evolve to suppress costs of this "centromere drive." In hybrid mouse models with genetically different maternal and paternal centromeres, selfish centromere DNA exploits a kinetochore pathway to recruit microtubule-destabilizing proteins that act as drive effectors. We show that such functional differences are suppressed by a parallel pathway for effector recruitment by heterochromatin, which is similar between centromeres in this system. Disrupting the kinetochore pathway with a divergent allele of CENP-C reduces functional differences between centromeres, whereas disrupting heterochromatin by CENP-B deletion amplifies the differences. Molecular evolution analyses using Murinae genomes identify adaptive evolution in proteins in both pathways. We propose that centromere proteins have recurrently evolved to minimize the kinetochore pathway, which is exploited by selfish DNA, relative to the heterochromatin pathway that equalizes centromeres, while maintaining essential functions.

RNA promotes the formation of spatial compartments in the nucleus.

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

Molecular Mechanisms of Facultative Heterochromatin Formation: An X-Chromosome Perspective.

Facultative heterochromatin (fHC) concerns the developmentally regulated heterochromatinization of different regions of the genome and, in the case of the mammalian X chromosome and imprinted loci, of only one allele of a homologous pair. The formation of fHC participates in the timely repression of genes, by resisting strong trans activators. In this review, we discuss the molecular mechanisms underlying the establishment and maintenance of fHC in mammals using a mouse model. We focus on X-chromosome inactivation (XCI) as a paradigm for fHC but also relate it to genomic imprinting and homeobox (Hox) gene cluster repression. A vital role for noncoding transcription and/or transcripts emerges as the general principle of triggering XCI and canonical imprinting. However, other types of fHC are established through an unknown mechanism, independent of noncoding transcription (Hox clusters and noncanonical imprinting). We also extensively discuss polycomb-group repressive complexes (PRCs), which frequently play a vital role in fHC maintenance.

SnapShot: The Regulatory Genome.

Enhancers switch genes on and off in response to a variety of intrinsic and external cellular signals. They are the cornerstone of gene regulation and the most pervasive constituents of the regulatory genome. Sequence polymorphisms in enhancer DNAs are a major source of population diversity and predilection to disease. To view this SnapShot, open or download the PDF.

Positioning Heterochromatin at the Nuclear Periphery Suppresses Histone Turnover to Promote Epigenetic Inheritance.

In eukaryotes, heterochromatin is generally located at the nuclear periphery. This study investigates the biological significance of perinuclear positioning for heterochromatin maintenance and gene silencing. We identify the nuclear rim protein Amo1NUPL2 as a factor required for the propagation of heterochromatin at endogenous and ectopic sites in the fission yeast genome. Amo1 associates with the Rix1PELP1-containing RNA processing complex RIXC and with the histone chaperone complex FACT. RIXC, which binds to heterochromatin protein Swi6HP1 across silenced chromosomal domains and to surrounding boundary elements, connects heterochromatin with Amo1 at the nuclear periphery. In turn, the Amo1-enriched subdomain is critical for Swi6 association with FACT that precludes histone turnover to promote gene silencing and preserve epigenetic stability of heterochromatin. In addition to uncovering conserved factors required for perinuclear positioning of heterochromatin, these analyses elucidate a mechanism by which a peripheral subdomain enforces stable gene repression and maintains heterochromatin in a heritable manner.

Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage.

Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.

Condensed Chromatin Behaves like a Solid on the Mesoscale In Vitro and in Living Cells.

The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.

A Heterochromatin-Specific RNA Export Pathway Facilitates piRNA Production.

PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, we uncover an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.

SMCHD1 Merges Chromosome Compartments and Assists Formation of Super-Structures on the Inactive X.

Mammalian chromosomes are partitioned into A/B compartments and topologically associated domains (TADs). The inactive X (Xi) chromosome, however, adopts a distinct conformation without evident compartments or TADs. Here, through exploration of an architectural protein, structural-maintenance-of-chromosomes hinge domain containing 1 (SMCHD1), we probe how the Xi is reconfigured during X chromosome inactivation. A/B compartments are first fused into "S1" and "S2" compartments, coinciding with Xist spreading into gene-rich domains. SMCHD1 then binds S1/S2 compartments and merges them to create a compartment-less architecture. Contrary to current views, TADs remain on the Xi but in an attenuated state. Ablating SMCHD1 results in a persistent S1/S2 organization and strengthening of TADs. Furthermore, loss of SMCHD1 causes regional defects in Xist spreading and erosion of heterochromatic silencing. We present a stepwise model for Xi folding, where SMCHD1 attenuates a hidden layer of Xi architecture to facilitate Xist spreading.

Rescue of Fragile X Syndrome Neurons by DNA Methylation Editing of the FMR1 Gene.

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.

N6-methyladenine DNA Modification in Glioblastoma.

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.

Heterochromatin: Hiding from the remodeling machines.

In this issue of Molecular Cell, Sahu et al.1 find that shielding heterochromatin from SWI/SNF chromatin remodelers is essential to maintain and epigenetically propagate pre-existing heterochromatin domains, whereas SWI/SNF action protects facultative heterochromatic regions from premature silencing.

Mrc1 regulates parental histone segregation and heterochromatin inheritance.

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.

Nucleosome remodeler exclusion by histone deacetylation enforces heterochromatic silencing and epigenetic inheritance.

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.

Heterochromatic 3D genome organization is directed by HP1a- and H3K9-dependent and independent mechanisms.

Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wild-type tissues is the segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a⋅H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, the self-association of pericentromeric regions is largely preserved despite the loss of H3K9 methylation and HP1a occupancy. Thus, the HP1a⋅H3K9 interaction contributes to but does not solely drive the segregation of euchromatin and heterochromatin inside the nucleus.

Regularly spaced tyrosines in EBF1 mediate BRG1 recruitment and formation of nuclear subdiffractive clusters.

B lineage priming by pioneer transcription factor EBF1 requires the function of an intrinsically disordered region (IDR). Here, we examine the role of regularly spaced tyrosines in the IDR as potential determinants of IDR function and activity of EBF1. We found that four Y > A mutations in EBF1 reduced the formation of condensates in vitro and subdiffractive clusters in vivo. Notably, Y > A mutant EBF1 was inefficient in promoting B cell differentiation and showed impaired chromatin binding, recruitment of BRG1, and activation of specific target genes. Thus, regularly spaced tyrosines in the IDR contribute to the biophysical and functional properties of EBF1.

ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline.

H3K9 trimethylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that in male meiosis, ATF7IP2 amasses on autosomal and X-pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X-pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global up-regulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.