The fork protection complex promotes parental histone recycling and epigenetic memory.

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.

Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice.

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.

Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation.

Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.

The Logic of Transgenerational Inheritance: Timescales of Adaptation.

Myriad mechanisms have evolved to adapt to changing environments. Environmental stimuli alter organisms' physiology to create memories of previous environments. Whether these environmental memories can cross the generational barrier has interested scientists for centuries. The logic of passing on information from generation to generation is not well understood. When is it useful to remember ancestral conditions, and when might it be deleterious to continue to respond to a context that may no longer exist? The key might be found in understanding the environmental conditions that trigger long-lasting adaptive responses. We discuss the logic that biological systems may use to remember environmental conditions. Responses spanning different generational timescales employ different molecular machineries and may result from differences in the duration or intensity of the exposure. Understanding the molecular components of multigenerational inheritance and the logic underlying beneficial and maladaptive adaptations is fundamental to understanding how organisms acquire and transmit environmental memories across generations.

A Prion Epigenetic Switch Establishes an Active Chromatin State.

Covalent modifications to histones are essential for development, establishing distinct and functional chromatin domains from a common genetic sequence. Whereas repressed chromatin is robustly inherited, no mechanism that facilitates inheritance of an activated domain has been described. Here, we report that the Set3C histone deacetylase scaffold Snt1 can act as a prion that drives the emergence and transgenerational inheritance of an activated chromatin state. This prion, which we term [ESI+] for expressed sub-telomeric information, is triggered by transient Snt1 phosphorylation upon cell cycle arrest. Once engaged, the prion reshapes the activity of Snt1 and the Set3C complex, recruiting RNA pol II and interfering with Rap1 binding to activate genes in otherwise repressed sub-telomeric domains. This transcriptional state confers broad resistance to environmental stress, including antifungal drugs. Altogether, our results establish a robust means by which a prion can facilitate inheritance of an activated chromatin state to provide adaptive benefit.

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.

Neuronal Small RNAs Control Behavior Transgenerationally.

It is unknown whether the activity of the nervous system can be inherited. In Caenorhabditis elegans nematodes, parental responses can transmit heritable small RNAs that regulate gene expression transgenerationally. In this study, we show that a neuronal process can impact the next generations. Neurons-specific synthesis of RDE-4-dependent small RNAs regulates germline amplified endogenous small interfering RNAs (siRNAs) and germline gene expression for multiple generations. Further, the production of small RNAs in neurons controls the chemotaxis behavior of the progeny for at least three generations via the germline Argonaute HRDE-1. Among the targets of these small RNAs, we identified the conserved gene saeg-2, which is transgenerationally downregulated in the germline. Silencing of saeg-2 following neuronal small RNA biogenesis is required for chemotaxis under stress. Thus, we propose a small-RNA-based mechanism for communication of neuronal processes transgenerationally.

Genome Regulation by Polycomb and Trithorax: 70 Years and Counting.

Polycomb (PcG) and Trithorax (TrxG) group proteins are evolutionarily conserved chromatin-modifying factors originally identified as part of an epigenetic cellular memory system that maintains repressed or active gene expression states. Recently, they have been shown to globally control a plethora of cellular processes. This functional diversity is achieved by their ability to regulate chromatin at multiple levels, ranging from modifying local chromatin structure to orchestrating the three-dimensional organization of the genome. Understanding this system is a fascinating challenge of critical relevance for biology and medicine, since misexpression or mutation of multiple PcG components, as well as of TrxG members of the COMPASS family and of the SWI/SNF complex, is implicated in cancer and other diseases.

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.

Coordination of histone chaperones for parental histone segregation and epigenetic inheritance.

Chromatin-based epigenetic memory relies on the accurate distribution of parental histone H3-H4 tetramers to newly replicated DNA strands. Mcm2, a subunit of the replicative helicase, and Dpb3/4, subunits of DNA polymerase ε, govern parental histone H3-H4 deposition to the lagging and leading strands, respectively. However, their contribution to epigenetic inheritance remains controversial. Here, using fission yeast heterochromatin inheritance systems that eliminate interference from initiation pathways, we show that a Mcm2 histone binding mutation severely disrupts heterochromatin inheritance, while mutations in Dpb3/4 cause only moderate defects. Surprisingly, simultaneous mutations of Mcm2 and Dpb3/4 stabilize heterochromatin inheritance. eSPAN (enrichment and sequencing of protein-associated nascent DNA) analyses confirmed the conservation of Mcm2 and Dpb3/4 functions in parental histone H3-H4 segregation, with their combined absence showing a more symmetric distribution of parental histone H3-H4 than either single mutation alone. Furthermore, the FACT histone chaperone regulates parental histone transfer to both strands and collaborates with Mcm2 and Dpb3/4 to maintain parental histone H3-H4 density and faithful heterochromatin inheritance. These results underscore the importance of both symmetric distribution of parental histones and their density at daughter strands for epigenetic inheritance and unveil distinctive properties of parental histone chaperones during DNA replication.

The Inherent Asymmetry of DNA Replication.

Semiconservative DNA replication has provided an elegant solution to the fundamental problem of how life is able to proliferate in a way that allows cells, organisms, and populations to survive and replicate many times over. Somewhat lost, however, in our admiration for this mechanism is an appreciation for the asymmetries that occur in the process of DNA replication. As we discuss in this review, these asymmetries arise as a consequence of the structure of the DNA molecule and the enzymatic mechanism of DNA synthesis. Increasing evidence suggests that asymmetries in DNA replication are able to play a central role in the processes of adaptation and evolution by shaping the mutagenic landscape of cells. Additionally, in eukaryotes, recent work has demonstrated that the inherent asymmetries in DNA replication may play an important role in the process of chromatin replication. As chromatin plays an essential role in defining cell identity, asymmetries generated during the process of DNA replication may play critical roles in cell fate decisions related to patterning and development.

Origins of DNA replication in eukaryotes.

Errors occurring during DNA replication can result in inaccurate replication, incomplete replication, or re-replication, resulting in genome instability that can lead to diseases such as cancer or disorders such as autism. A great deal of progress has been made toward understanding the entire process of DNA replication in eukaryotes, including the mechanism of initiation and its control. This review focuses on the current understanding of how the origin recognition complex (ORC) contributes to determining the location of replication initiation in the multiple chromosomes within eukaryotic cells, as well as methods for mapping the location and temporal patterning of DNA replication. Origin specification and configuration vary substantially between eukaryotic species and in some cases co-evolved with gene-silencing mechanisms. We discuss the possibility that centromeres and origins of DNA replication were originally derived from a common element and later separated during evolution.

Asymmetric Histone Inheritance: Establishment, Recognition, and Execution.

The discovery of biased histone inheritance in asymmetrically dividing Drosophila melanogaster male germline stem cells demonstrates one means to produce two distinct daughter cells with identical genetic material. This inspired further studies in different systems, which revealed that this phenomenon may be a widespread mechanism to introduce cellular diversity. While the extent of asymmetric histone inheritance could vary among systems, this phenomenon is proposed to occur in three steps: first, establishment of histone asymmetry between sister chromatids during DNA replication; second, recognition of sister chromatids carrying asymmetric histone information during mitosis; and third, execution of this asymmetry in the resulting daughter cells. By compiling the current knowledge from diverse eukaryotic systems, this review comprehensively details and compares known chromatin factors, mitotic machinery components, and cell cycle regulators that may contribute to each of these three steps. Also discussed are potential mechanisms that introduce and regulate variable histone inheritance modes and how these different modes may contribute to cell fate decisions in multicellular organisms.

A composite DNA element that functions as a maintainer required for epigenetic inheritance of heterochromatin.

Epigenetic inheritance of heterochromatin requires DNA-sequence-independent propagation mechanisms, coupling to RNAi, or input from DNA sequence, but how DNA contributes to inheritance is not understood. Here, we identify a DNA element (termed "maintainer") that is sufficient for epigenetic inheritance of pre-existing histone H3 lysine 9 methylation (H3K9me) and heterochromatin in Schizosaccharomyces pombe but cannot establish de novo gene silencing in wild-type cells. This maintainer is a composite DNA element with binding sites for the Atf1/Pcr1 and Deb1 transcription factors and the origin recognition complex (ORC), located within a 130-bp region, and can be converted to a silencer in cells with lower rates of H3K9me turnover, suggesting that it participates in recruiting the H3K9 methyltransferase Clr4/Suv39h. These results suggest that, in the absence of RNAi, histone H3K9me is only heritable when it can collaborate with maintainer-associated DNA-binding proteins that help recruit the enzyme responsible for its epigenetic deposition.

A Family of Argonaute-Interacting Proteins Gates Nuclear RNAi.

Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.

Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance.

Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.

Minimal requirements for the epigenetic inheritance of engineered silent chromatin domains.

Mechanisms enabling genetically identical cells to differentially regulate gene expression are complex and central to organismal development and evolution. While gene silencing pathways involving DNA sequence-specific recruitment of histone-modifying enzymes are prevalent in nature, examples of sequence-independent heritable gene silencing are scarce. Studies of the fission yeast Schizosaccharomyces pombe indicate that sequence-independent propagation of heterochromatin can occur but requires numerous multisubunit protein complexes and their diverse activities. Such complexity has so far precluded a coherent articulation of the minimal requirements for heritable gene silencing by conventional in vitro reconstitution approaches. Here, we take an unconventional approach to defining these requirements by engineering sequence-independent silent chromatin inheritance in budding yeast Saccharomyces cerevisiae cells. The mechanism conferring memory upon these cells is remarkably simple and requires only two proteins, one that recognizes histone H3 lysine 9 methylation (H3K9me) and catalyzes the deacetylation of histone H4 lysine 16 (H4K16), and another that recognizes deacetylated H4K16 and catalyzes H3K9me. Together, these bilingual "read-write" proteins form an interdependent positive feedback loop that is sufficient for the transmission of DNA sequence-independent silent information over multiple generations.

DNA polymerase delta governs parental histone transfer to DNA replication lagging strand.

Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase ε subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. 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 through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase δ subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

Protein-Based Inheritance: Epigenetics beyond the Chromosome.

Epigenetics refers to changes in phenotype that are not rooted in DNA sequence. This phenomenon has largely been studied in the context of chromatin modification. Yet many epigenetic traits are instead linked to self-perpetuating changes in the individual or collective activity of proteins. Most such proteins are prions (e.g., [PSI+], [URE3], [SWI+], [MOT3+], [MPH1+], [LSB+], and [GAR+]), which have the capacity to adopt at least one conformation that self-templates over long biological timescales. This allows them to serve as protein-based epigenetic elements that are readily broadcast through mitosis and meiosis. In some circumstances, self-templating can fuel disease, but it also permits access to multiple activity states from the same polypeptide and transmission of that information across generations. Ensuing phenotypic changes allow genetically identical cells to express diverse and frequently adaptive phenotypes. Although long thought to be rare, protein-based epigenetic inheritance has now been uncovered in all domains of life.