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Chromatin structure and DNA accessibility are partly modulated by the incorporation of histone variants. H2A.Z, encoded by the non-essential HTZ1 gene in S. cerevisiae, is an evolutionarily conserved H2A histone variant that is predominantly incorporated at transcription start sites by the SWR1-complex (SWR1-C). While H2A.Z has often been implicated in transcription regulation, htz1Δ mutants exhibit minimal changes in gene expression compared to wild-type. However, given that growth defects of htz1Δ mutants are alleviated by simultaneous deletion of SWR1-C subunits, previous work examining the role of H2A.Z in gene expression regulation may be confounded by deleterious activity caused by SWR1-C when missing its H2A.Z substrate (apo-SWR1-C). Furthermore, as H2A.Z mutants only display significant growth defects in genotoxic stress conditions, a more substantive role for H2A.Z in gene expression may only be uncovered after exposure to cellular stress. To explore this possibility, we generated mRNA transcript profiles for wild-type, htz1Δ, swr1Δ, and htz1Δswr1Δ mutants before and after exposure to hydroxyurea (HU), which induces DNA replication stress. Our data showed that H2A.Z played a more prominent role in gene activation than repression during HU exposure, and its incorporation was important for proper upregulation of several HU-induced genes. We also observed that apo-SWR1-C contributed to gene expression defects in the htz1Δ mutant, particularly for genes involved in phosphate homeostasis regulation. Furthermore, mapping H2A.Z incorporation before and after treatment with HU revealed that decreases in H2A.Z enrichment at transcription start sites was correlated with, but generally not required for, the upregulation of genes during HU exposure. Together this study characterized the regulatory effects of H2A.Z incorporation during the transcriptional response to HU., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Brewis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80's ATPase activity to nucleosome movement. We find two distinct regions of Arp5 binding near the acidic pocket of nucleosomes. One region has an arginine anchor that binds nucleosomes and is vital for INO80 mobilizing nucleosomes. The other region has a hydrophobic/acid patch of Leu and Asp that binds free histone H2A-H2B dimers. These two regions have different roles in remodeling nucleosomes as seen both in vitro and in vivo and the hydrophobic/acidic patch of Arp5 is likely needed for displacing DNA from the H2A-H2B surface and dimer exchange by INO80., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Near the C-terminus of histone H2A in the yeast Saccharomyces cerevisiae, there are 2 serines (S122 and S129) that are targets of phosphorylation. The phosphorylation of serine 129 in response to DNA damage is dependent on the Tel1 and Mec1 kinases. In Schizosaccharomyces pombe and S. cerevisiae, the phosphorylation of serine 122 is dependent on the Bub1 kinase, and S. pombe strains with an alanine mutation of this serine have elevated levels of lagging chromosomes in mitosis. Strains that lack both Tel1 and Mec1 in S. cerevisiae have very elevated rates of nondisjunction. To clarify the functional importance of phosphorylation of serines 122 and 129 in H2A, we measured chromosome loss rates in single-mutant strains and double-mutant combinations. We also examined the interaction of mutations of BUB1, TEL1, and MEC1 in combination with mutations of serines 122 and 129 in H2A. We conclude that the phosphorylation state of S129 has no effect on chromosome disjunction whereas mutations that inactivate Bub1 or a S122A mutation in the histone H2A greatly elevate the rate of chromosome nondisjunction. Based on this analysis, we suggest that Bub1 exerts its primary effect on chromosome disjunction by phosphorylating S122 of histone H2A. However, Tel1, Mec1, and Bub1 are also functionally redundant in a second pathway affecting chromosome disjunction that is at least partially independent of phosphorylation of S122 of H2A., Competing Interests: Conflicts of interest: The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Core histones, synthesized and processed in the cytoplasm, must be chaperoned as they are transported into the nucleus for nucleosome assembly. The importin Kap114 transports H2A-H2B into the yeast nucleus, where RanGTP facilitates histone release. Kap114 and H2A-H2B also bind the histone chaperone Nap1, but how Nap1 and Kap114 cooperate in transport and nucleosome assembly remains unclear. Here, biochemical and structural analyses show that Kap114, H2A-H2B, and a Nap1 dimer (Nap12) associate in the absence and presence of RanGTP to form equimolar complexes. A previous study had shown that RanGTP reduces Kap114's ability to chaperone H2A-H2B, but a new cryo-EM structure of the Nap12•H2A-H2B•Kap114•RanGTP complex explains how both Kap114 and Nap12 interact with H2A-H2B, restoring its chaperoning within the assembly while effectively depositing it into nucleosomes. Together, our results suggest that Kap114 and Nap12 provide a sheltered path that facilitates the transfer of H2A-H2B from Kap114 to Nap12, ultimately directing its specific deposition into nucleosomes., (© 2024 Fung et al.)

The yeast SWR1 complex catalyses the exchange of histone H2A-H2B dimers in nucleosomes, with Htz1-H2B dimers 1-3 . Here we used single-molecule analysis to demonstrate two-step double exchange of the two H2A-H2B dimers in a canonical yeast nucleosome with Htz1-H2B dimers, and showed that double exchange can be processive without release of the nucleosome from the SWR1 complex. Further analysis showed that bound nucleosomes flip between two states, with each presenting a different face, and hence histone dimer, to SWR1. The bound dwell time is longer when an H2A-H2B dimer is presented for exchange than when presented with an Htz1-H2B dimer. A hexasome intermediate in the reaction is bound to the SWR1 complex in a single orientation with the 'empty' site presented for dimer insertion. Cryo-electron microscopy analysis revealed different populations of complexes showing nucleosomes caught 'flipping' between different conformations without release, each placing a different dimer into position for exchange, with the Swc2 subunit having a key role in this process. Together, the data reveal a processive mechanism for double dimer exchange that explains how SWR1 can 'proofread' the dimer identities within nucleosomes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The SWR1 chromatin remodeling complex is recruited to +1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the +1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes., Competing Interests: Declaration of interests B.F.P. is an owner of and has a financial interest in Peconic, which uses the ChIP-exo technology (US Patent 20100323361A1) implemented in this study and could potentially benefit from the outcomes of this research., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In chromatin replication, faithful recycling of histones from parental DNA to replicated strands is essential for maintaining epigenetic information across generations. A previous experiment has revealed that disrupting interactions between the N-terminal tail of Mcm2, a subunit in DNA replication machinery, and a histone H3/H4 tetramer perturb the recycling. However, the molecular pathways and the factors that regulate the ratio recycled to each strand and the destination location are yet to be revealed. Here, we performed molecular dynamics simulations of yeast DNA replication machinery, an H3/H4 tetramer, and replicated DNA strands. The simulations demonstrated that histones are recycled via Cdc45-mediated and unmediated pathways without histone chaperones, as our in vitro biochemical assays supported. Also, RPA binding regulated the ratio recycled to each strand, whereas DNA bending by Pol ε modulated the destination location. Together, the simulations provided testable hypotheses, which are vital for elucidating the molecular mechanisms of histone recycling., (© 2024. The Author(s).)

To maintain protein homeostasis in changing nutrient environments, cells must precisely control the amount of their proteins, despite the accompanying changes in cell growth and biosynthetic capacity. As nutrients are major regulators of cell cycle length and progression, a particular challenge arises for the nutrient-dependent regulation of 'cell cycle genes', which are periodically expressed during the cell cycle. One important example are histones, which are needed at a constant histone-to-DNA stoichiometry. Here we show that budding yeast achieves histone homeostasis in different nutrients through a decoupling of transcript and protein abundance. We find that cells downregulate histone transcripts in poor nutrients to avoid toxic histone overexpression, but produce constant amounts of histone proteins through nutrient-specific regulation of translation efficiency. Our findings suggest that this allows cells to balance the need for rapid histone production under fast growth conditions with the tight regulation required to avoid toxic overexpression in poor nutrients., (© 2024. The Author(s).)

Spt5 is a well-conserved factor that manipulates multiple stages of transcription from promoter-proximal pausing (PPP) to termination. Recent studies have revealed an unexpected increase of antisense transcripts near promoters in cells expressing mutant Spt5. Here, we identify Spt5p-restricted intragenic antisense transcripts and their close relationship with sense transcription in yeast. We confirm that Spt5 CTR phosphorylation is also important to retain Spt5's facility to regulate antisense transcription. The genes whose antisense transcription is strongly suppressed by Spt5p share strong endogenous sense transcription and weak antisense transcription, and this pattern is conserved in humans. Mechanistically, we found that Spt5p depletion increased histone acetylation to initiate intragenic antisense transcription by altering chromatin structure. We additionally identified termination factors that appear to be involved in the ability of Spt5p to restrict antisense transcription. By unveiling a new role of Spt5 in finely balancing the bidirectionality of transcription, we demonstrate that Spt5-mediated suppression of DSIF complex regulated-unstable transcripts (DUTs) is essential to sustain the accurate transcription by RNA polymerase II., (© 2024. The Author(s).)

During DNA replication, core histones that form nucleosomes on template strands are evicted and associate with newly synthesized strands to reform nucleosomes. Mcm2, a subunit of the Mcm2-7 complex, which is a core component of the replicative helicase, interacts with histones in the amino-terminal region (Mcm2N) and is involved in the parental histone recycling to lagging strands. Herein, the interaction of Mcm2N with histones was biochemically analyzed to reveal the molecular mechanisms underlying histone recycling by Mcm2N. With the addition of Mcm2N, a histone hexamer, comprising an H3-H4 tetramer and an H2A-H2B dimer, was excised from the histone octamer to form a complex with Mcm2N. The histone hexamer, but not H3-H4 tetramer was released from Mcm2N in the presence of Nap1, a histone chaperone. FACT, another histone chaperone, stabilized Mcm2N-histone hexamer complex to protect from Nap1-dependent dissociation. This study indicates cooperative histone transfer via Mcm2N and histone chaperones., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The yeast SWR1 complex catalyzes the exchange of histone H2A/H2B dimers in nucleosomes with Htz1/H2B dimers. We use cryoelectron microscopy to determine the structure of an enzyme-bound hexasome intermediate in the reaction pathway of histone exchange, in which an H2A/H2B dimer has been extracted from a nucleosome prior to the insertion of a dimer comprising Htz1/H2B. The structure reveals a key role for the Swc5 subunit in stabilizing the unwrapping of DNA from the histone core of the hexasome. By engineering a crosslink between an Htz1/H2B dimer and its chaperone protein Chz1, we show that this blocks histone exchange by SWR1 but allows the incoming chaperone-dimer complex to insert into the hexasome. We use this reagent to trap an SWR1/hexasome complex with an incoming Htz1/H2B dimer that shows how the reaction progresses to the next step. Taken together the structures reveal insights into the mechanism of histone exchange by SWR1 complex., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The DNA damage response (DDR) constitutes a vital cellular process that safeguards genome integrity. This biological process involves substantial alterations in chromatin structure, commonly orchestrated by epigenetic enzymes. Here, we show that the epigenetic modifier N-terminal acetyltransferase 4 (Nat4), known to acetylate the alpha-amino group of serine 1 on histones H4 and H2A, is implicated in the response to DNA damage in S. cerevisiae. Initially, we demonstrate that yeast cells lacking Nat4 have an increased sensitivity to DNA damage and accumulate more DNA breaks than wild-type cells. Accordingly, upon DNA damage, NAT4 gene expression is elevated, and the enzyme is specifically recruited at double-strand breaks. Delving deeper into its effects on the DNA damage signaling cascade, nat4-deleted cells exhibit lower levels of the damage-induced modification H2AS129ph (γH2A), accompanied by diminished binding of the checkpoint control protein Rad9 surrounding the double-strand break. Consistently, Mec1 kinase recruitment at double-strand breaks, critical for H2AS129ph deposition and Rad9 retention, is significantly impaired in nat4Δ cells. Consequently, Mec1-dependent phosphorylation of downstream effector kinase Rad53, indicative of DNA damage checkpoint activation, is reduced. Importantly, we found that the effects of Nat4 in regulating the checkpoint signaling cascade are mediated by its N-terminal acetyltransferase activity targeted specifically towards histone H4. Overall, this study points towards a novel functional link between histone N-terminal acetyltransferase Nat4 and the DDR, associating a new histone-modifying activity in the maintenance of genome integrity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Constantinou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The polymerase-associated factor 1 (Paf1) complex (Paf1C) is a conserved protein complex with critical functions during eukaryotic transcription. Previous studies showed that Paf1C is multi-functional, controlling specific aspects of transcription ranging from RNA polymerase II (RNAPII) processivity to histone modifications. However, it is unclear how specific Paf1C subunits directly impact transcription and coupled processes. We have compared conditional depletion to steady-state deletion for each Paf1C subunit to determine the direct and indirect contributions to gene expression in Saccharomyces cerevisiae. Using nascent transcript sequencing, RNAPII profiling, and modeling of transcription elongation dynamics, we have demonstrated direct effects of Paf1C subunits on RNAPII processivity and elongation rate and indirect effects on transcript splicing and repression of antisense transcripts. Further, our results suggest that the direct transcriptional effects of Paf1C cannot be readily assigned to any particular histone modification. This work comprehensively analyzes both the immediate and the extended roles of each Paf1C subunit in transcription elongation and transcript regulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

To maintain the nucleosome organization of transcribed genes, ATP-dependent chromatin remodelers collaborate with histone chaperones. Here, we show that at the 5' ends of yeast genes, RNA polymerase II (RNAPII) generates hexasomes that occur directly adjacent to nucleosomes. The resulting hexasome-nucleosome complexes are then resolved by Chd1. We present two cryoelectron microscopy (cryo-EM) structures of Chd1 bound to a hexasome-nucleosome complex before and after restoration of the missing inner H2A/H2B dimer by FACT. Chd1 uniquely interacts with the complex, positioning its ATPase domain to shift the hexasome away from the nucleosome. In the absence of the inner H2A/H2B dimer, its DNA-binding domain (DBD) packs against the ATPase domain, suggesting an inhibited state. Restoration of the dimer by FACT triggers a rearrangement that displaces the DBD and stimulates Chd1 remodeling. Our results demonstrate how chromatin remodelers interact with a complex nucleosome assembly and suggest how Chd1 and FACT jointly support transcription by RNAPII., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The genome of living cells is constantly challenged by DNA lesions that interfere with cellular processes such as transcription and replication. A manifold of mechanisms act in concert to ensure adequate DNA repair, gene expression, and genome stability. Bulky DNA lesions, such as those induced by UV light or the DNA-damaging agent 4-nitroquinoline oxide, act as transcriptional and replicational roadblocks and thus represent a major threat to cell metabolism. When located on the transcribed strand of active genes, these lesions are handled by transcription-coupled nucleotide excision repair (TC-NER), a yet incompletely understood NER sub-pathway. Here, using a genetic screen in the yeast Saccharomyces cerevisiae, we identified histone variant H2A.Z as an important component to safeguard transcription and DNA integrity following UV irradiation. In the absence of H2A.Z, repair by TC-NER is severely impaired and RNA polymerase II clearance reduced, leading to an increase in double-strand breaks. Thus, H2A.Z is needed for proficient TC-NER and plays a major role in the maintenance of genome stability upon UV irradiation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gaillard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1 Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3 exo- ) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1 Δ and mcm2-3A , do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.

How gene activation works in heterochromatin, and how the mechanism might differ from the one used in euchromatin, has been largely unexplored. Previous work has shown that in SIR-regulated heterochromatin of Saccharomyces cerevisiae, gene activation occurs in the absence of covalent histone modifications and other alterations of chromatin commonly associated with transcription.Here we demonstrate that such activation occurs in a substantial fraction of cells, consistent with frequent transcriptional bursting, and this raises the possibility that an alternative activation pathway might be used. We address one such possibility, Pol II CTD phosphorylation, and explore this idea using a natural telomere-linked gene, YFR057w, as a model. Unlike covalent histone modifications, we find that Ser2, Ser5 and Ser7 CTD phosphorylated Pol II is prevalent at the drug-induced heterochromatic gene. Particularly enriched relative to the euchromatic state is Ser2 phosphorylation. Consistent with a functional role for Ser2P, YFR057w is negligibly activated in cells deficient in the Ser2 CTD kinases Ctk1 and Bur1 even though the gene is strongly stimulated when it is placed in a euchromatic context. Collectively, our results are consistent with a critical role for Ser2 CTD phosphorylation in driving Pol II recruitment and transcription of a natural heterochromatic gene - an activity that may supplant the need for histone epigenetic modifications., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: David S. Gross reports financial support was provided by National Institutes of Health. David S. Gross reports financial support was provided by National Science Foundation Division of Molecular and Cellular Biosciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The evolutionarily conserved HIRA/Hir histone chaperone complex and ASF1a/Asf1 co-chaperone cooperate to deposit histone (H3/H4) 2 tetramers on DNA for replication-independent chromatin assembly. The molecular architecture of the HIRA/Hir complex and its mode of histone deposition have remained unknown. Here, we report the cryo-EM structure of the S. cerevisiae Hir complex with Asf1/H3/H4 at 2.9-6.8 Å resolution. We find that the Hir complex forms an arc-shaped dimer with a Hir1/Hir2/Hir3/Hpc2 stoichiometry of 2/4/2/4. The core of the complex containing two Hir1/Hir2/Hir2 trimers and N-terminal segments of Hir3 forms a central cavity containing two copies of Hpc2, with one engaged by Asf1/H3/H4, in a suitable position to accommodate a histone (H3/H4) 2 tetramer, while the C-terminal segments of Hir3 harbor nucleic acid binding activity to wrap DNA around the Hpc2-assisted histone tetramer. The structure suggests a model for how the Hir/Asf1 complex promotes the formation of histone tetramers for their subsequent deposition onto DNA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

H3K4 methylation by Set1-COMPASS (complex of proteins associated with Set1) is a conserved histone modification. Although it is critical for gene regulation, the posttranslational modifications of this complex that affect its function are largely unexplored. This study showed that N-terminal acetylation of Set1-COMPASS proteins by N-terminal acetyltransferases (NATs) can modulate H3K4 methylation patterns. Specifically, deleting NatA substantially decreased global H3K4me3 levels and caused the H3K4me2 peak in the 5' transcribed regions to shift to the promoters. NatA was required for N-terminal acetylation of three subunits of Set1-COMPASS: Shg1, Spp1, and Swd2. Moreover, deleting Shg1 or blocking its N-terminal acetylation via proline mutation of the target residue drastically reduced H3K4 methylation. Thus, NatA-mediated N-terminal acetylation of Shg1 shapes H3K4 methylation patterns. NatB also regulates H3K4 methylation, likely via N-terminal acetylation of the Set1-COMPASS protein Swd1. Thus, N-terminal acetylation of Set1-COMPASS proteins can directly fine-tune the functions of this complex, thereby substantially shaping H3K4 methylation patterns.

Disruptor of telomeric silencing 1 (Dot1p) is an exquisitely conserved histone methyltransferase and is the sole enzyme responsible for H3K79 methylation in the budding yeast Saccharomyces cerevisiae. It has been shown to be highly phosphorylated in vivo; however, the upstream kinases that act on Dot1p are almost entirely unknown in yeast and all other eukaryotes. Here, we used in vitro and in vivo kinase discovery approaches to show that mitogen-activated protein kinase HOG1 (Hog1p) is a bona fide kinase of the Dot1p methyltransferase. In vitro kinase assays showed that Hog1p phosphorylates Dot1p at multiple sites, including at several proline-adjacent sites that are consistent with known Hog1p substrate preferences. The activity of Hog1p was specifically enhanced at these proline-adjacent sites on Dot1p upon Hog1p activation by the osmostress-responsive MAP kinase kinase PBS2 (Pbs2p). Genomic deletion of HOG1 reduced phosphorylation at specific sites on Dot1p in vivo, providing further evidence for Hog1p kinase activity on Dot1p in budding yeast cells. Phenotypic analysis of knockout and phosphosite mutant yeast strains revealed the importance of Hog1p-catalysed phosphorylation of Dot1p for cellular responses to ultraviolet-induced DNA damage. In mammalian systems, this kinase-substrate relationship was found to be conserved: human DOT1L (the ortholog of yeast Dot1p) can be phosphorylated by the proline-directed kinase p38β (also known as MAPK11; the ortholog of yeast Hog1p) at multiple sites in vitro. Taken together, our findings establish Hog1p and p38β as newly identified upstream kinases of the Dot1p/DOT1L H3K79 methyltransferase enzymes in eukaryotes., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Swd2/Cps35 is a common component of the COMPASS H3K4 methyltransferase and CPF transcription termination complex in Saccharomyces cerevisiae. The deletion of SWD2 is lethal, which results from transcription termination defects in snoRNA genes. This study isolated a yeast strain that showed significantly reduced protein level of Swd2 following epitope tagging at its N-terminus (9MYC-SWD2). The reduced level of Swd2 in the 9MYC-SWD2 strain was insufficient for the stability of the Set1 H3K4 methyltransferase, H3K4me3 and snoRNA termination, but the level was enough for viability and growth similar to the wildtype strain. In addition, we presented the genes differentially regulated by the essential protein Swd2 under optimal culture conditions for the first time. The expression of genes known to be decreased in the absence of Set1 and H3K4me3, including NAD biosynthetic process genes and histone genes, was decreased in the 9MYC-SWD2 strain, as expected. However, the effects of Swd2 on the ribosome biogenesis (RiBi) genes were opposite to those of Set1, suggesting that the expression of RiBi genes is regulated by more complex relationship between COMPASS and other Swd2-containing complexes. These data suggest that different concentrations of Swd2 are required for its roles in H3K4me3 and viability and that it may be either contributory or contrary to the transcriptional regulation of Set1/H3K4me3, depending on the gene group., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway., (© 2024. Science China Press.)

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a 'pincer-like' conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition., Competing Interests: AB, NG, PE, EL, ST, CP No competing interests declared, (© 2024, Baier et al.)

Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging strands, respectively. Single Dpb3 deletion (dpb3Δ) or Mcm2 mutation (mcm2-3A), which each disrupts one parental histone transfer pathway, leads to the other's predominance. However, the biological impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3Δ mcm2-3A double mutant did not exhibit the asymmetric parental histone patterns caused by a single dpb3Δ or mcm2-3A mutation, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3Δ, mcm2-3A and dpb3Δ mcm2-3A mutants, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones during DNA replication is essential for maintaining chromatin structure and that lower homologous recombination activity due to parental histone transfer defects is detrimental to cells., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Histone H3K4 tri-methylation (H3K4me3) catalyzed by Set1/COMPASS, is a prominent epigenetic mark found in promoter-proximal regions of actively transcribed genes. H3K4me3 relies on prior monoubiquitination at the histone H2B (H2Bub) by Rad6 and Bre1. Swd2/Cps35, a Set1/COMPASS component, has been proposed as a key player in facilitating H2Bub-dependent H3K4me3. However, a more comprehensive investigation regarding the relationship among Rad6, Swd2, and Set1 is required to further understand the mechanisms and functions of the H3K4 methylation., Results: We investigated the genome-wide occupancy patterns of Rad6, Swd2, and Set1 under various genetic conditions, aiming to clarify the roles of Set1 and Rad6 for occupancy of Swd2. Swd2 peaks appear on both the 5' region and 3' region of genes, which are overlapped with its tightly bound two complexes, Set1 and cleavage and polyadenylation factor (CPF), respectively. In the absence of Rad6/H2Bub, Set1 predominantly localized to the 5' region of genes, while Swd2 lost all the chromatin binding. However, in the absence of Set1, Swd2 occupancy near the 5' region was impaired and rather increased in the 3' region., Conclusions: This study highlights that the catalytic activity of Rad6 is essential for all the ways of Swd2's binding to the transcribed genes and Set1 redistributes the Swd2 to the 5' region for accomplishments of H3K4me3 in the genome-wide level., (© 2024. The Author(s).)

Compact chromatin is closely linked with gene silencing in part by sterically masking access to promoters, inhibiting transcription factor binding and preventing polymerase from efficiently transcribing a gene. However, a broader hypothesis suggests that chromatin compaction can be both a cause and a consequence of the locus histone modification state, with a tight bidirectional interaction underpinning bistable transcriptional states. To rigorously test this hypothesis, we developed a mathematical model for the dynamics of the HMR locus in Saccharomyces cerevisiae , that incorporates activating histone modifications, silencing proteins, and a dynamic, acetylation-dependent, three-dimensional locus size. Chromatin compaction enhances silencer protein binding, which in turn feeds back to remove activating histone modifications, leading to further compaction. The bistable output of the model was in good agreement with prior quantitative data, including switching rates from expressed to silent states (and vice versa), and protein binding/histone modification levels within the locus. We then tested the model by predicting changes in switching rates as the genetic length of the locus was increased, which were then experimentally verified. Such bidirectional feedback between chromatin compaction and the histone modification state may be a widespread and important regulatory mechanism given the hallmarks of many heterochromatic regions: physical chromatin compaction and dimerizing (or multivalent) silencing proteins., Competing Interests: Competing interests statement:The authors declare no competing interest.

The Rpd3S complex plays a pivotal role in facilitating local histone deacetylation in the transcribed regions to suppress intragenic transcription initiation. Here, we present the cryo-electron microscopy structures of the budding yeast Rpd3S complex in both its apo and three nucleosome-bound states at atomic resolutions, revealing the exquisite architecture of Rpd3S to well accommodate a mononucleosome without linker DNA. The Rpd3S core, containing a Sin3 Lobe and two NB modules, is a rigid complex and provides three positive-charged anchors (Sin3_HCR and two Rco1_NIDs) to connect nucleosomal DNA. In three nucleosome-bound states, the Rpd3S core exhibits three distinct orientations relative to the nucleosome, assisting the sector-shaped deacetylase Rpd3 to locate above the SHL5-6, SHL0-1, or SHL2-3, respectively. Our work provides a structural framework that reveals a dynamic working model for the Rpd3S complex to engage diverse deacetylation sites.

The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs) 1,2 . However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin., (© 2024. The Author(s).)

We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomycete Sordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase of Neurospora crassa that performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation, and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in Δrtt109 and Δasf1 strains, accompanied by a loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of rtt109 and asf1 are sensitive to the DNA damaging agent methyl methanesulfonate, but not hydroxyurea. In contrast, chk2 mutants are fertile and resistant to methyl methanesulfonate, but not hydroxyurea. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification, and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Histone acetylation is a predominant active chromatin mark deposited by histone acetyltransferases (HATs) that transfer the acetyl group from acetyl coenzyme A (acetyl-CoA) to lysine ε-amino groups in histones. GENERAL CONTROL NON-REPRESSED PROTEIN 5 (GCN5) is one of the best-characterized HATs and functions in association with several adaptor proteins such as ADA2 within multiprotein HAT complexes. ADA2-GCN5 interaction increases GCN5 binding to acetyl-CoA and stimulates its HAT activity. It remains unclear whether the HAT activity of GCN5 (which acetylates not only histones but also cellular proteins) is regulated by acetyl-CoA levels, which vary greatly in cells under different metabolic and nutrition conditions. Here we show that the ADA2 protein itself is acetylated by GCN5 in rice cells. Lysine acetylation exposes ADA2 to a specific E3 ubiquitin ligase and reduces its protein stability. In rice plants, ADA2 protein accumulation reversely parallels its lysine acetylation and acetyl-CoA levels, both of which are dynamically regulated under varying growth conditions. Stress-induced ADA2 accumulation could stimulate GCN5 HAT activity to compensate for the reduced acetyl-CoA levels for histone acetylation. These results indicate that ADA2 lysine acetylation that senses cellular acetyl-CoA variations is a mechanism to regulate HAT activity and histone acetylation homeostasis in plants under changing environments., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

DNA lesions trigger DNA damage checkpoint (DDC) signaling which arrests cell cycle progression and promotes DNA damage repair. In Saccharomyces cerevisiae , phosphorylation of histone H2A (γH2A, equivalent to γH2AX in mammals) is an early chromatin mark induced by DNA damage that is recognized by a group of DDC and DNA repair factors. We find that γH2A negatively regulates the G2/M checkpoint in response to the genotoxin camptothecin, which is a DNA topoisomerase I poison. γH2A also suppresses DDC signaling induced by the DNA alkylating agent methyl methanesulfonate. These results differ from prior findings, which demonstrate positive or no roles of γH2A in DDC in response to other DNA damaging agents such as phleomycin and ionizing radiation, which suggest that γH2A has DNA damage-specific effects on DDC signaling. We also find evidence supporting the notion that γH2A regulates DDC signaling by mediating the competitive recruitment of the DDC mediator Rad9 and the DNA repair factor Rtt107 to DNA lesions. We propose that γH2A/γH2AX serves to create a dynamic balance between DDC and DNA repair that is influenced by the nature of DNA damage.

In S phase, duplicating and assembling the whole genome into chromatin requires upregulation of replicative histone gene expression. Here, we explored how histone chaperones control histone production in human cells to ensure a proper link with chromatin assembly. Depletion of the ASF1 chaperone specifically decreases the pool of replicative histones both at the protein and RNA levels. The decrease in their overall expression, revealed by total RNA sequencing (RNA-seq), contrasted with the increase in nascent/newly synthesized RNAs observed by 4sU-labeled RNA-seq. Further inspection of replicative histone RNAs showed a 3' end processing defect with an increase of pre-mRNAs/unprocessed transcripts likely targeted to degradation. Collectively, these data argue for a production defect of replicative histone RNAs in ASF1-depleted cells. We discuss how this regulation of replicative histone RNA metabolism by ASF1 as a "chaperone checkpoint" fine-tunes the histone dosage to avoid unbalanced situations deleterious for cell survival., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Mislocalization of overexpressed CENP-A (Cse4 in budding yeast, Cnp1 in fission yeast, CID in flies) contributes to chromosomal instability (CIN) in yeasts, flies, and human cells. Mislocalization of CENP-A is observed in many cancers and this correlates with poor prognosis. Structural mechanisms that contribute to mislocalization of CENP-A are poorly defined. Here, we show that interaction of histone H4 with Cse4 facilitates an in vivo conformational change in Cse4 promoting its mislocalization in budding yeast. We determined that Cse4 Y193A mutant exhibits reduced sumoylation, mislocalization, interaction with histone H4, and lethality in psh1Δ and cdc48-3 strains; all these phenotypes are suppressed by increased gene dosage of histone H4. We developed a new in vivo approach, antibody accessibility (AA) assay, to examine the conformation of Cse4. AA assay showed that wild-type Cse4 with histone H4 is in an 'open' state, while Cse4 Y193A predominantly exhibits a 'closed' state. Increased gene dosage of histone H4 contributes to a shift of Cse4 Y193A to an 'open' state with enhanced sumoylation and mislocalization. We provide molecular insights into how Cse4-H4 interaction changes the conformational state of Cse4 in vivo. These studies advance our understanding for mechanisms that promote mislocalization of CENP-A in human cancers., (Published by Oxford University Press on behalf of Nucleic Acids Research 2023.)

Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 ( prp45 (1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1 , and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2 , VPS75 , or HRB1 , the most affected cases with transcription-related function. Intron removal from SRB2 , but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45 (1-169) and htz1 Δ was detectable even in cells with SRB2 intron deleted ( srb2 Δi). The less truncated variant, prp45 (1-330), had a synthetic growth defect with htz1 Δ at 16°C, which also persisted in the srb2 Δi background. Moreover, htz1 Δ enhanced prp45 (1-330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1 , respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45 (1-169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45 (1-169), to the chromatin environment.

Yeast COMPASS (complex of proteins associated with Set1) and human MLL (mixed-lineage leukemia) complexes are histone H3 lysine 4 methyltransferases with critical roles in gene regulation and embryonic development. Both complexes share a conserved C-terminal SET domain, responsible for catalyzing histone H3 K4 methylation on nucleosomes. Notably, their catalytic activity toward nucleosomes is enhanced and optimized with assembly of auxiliary subunits. In this review, we aim to illustrate the recent X-ray and cryo-EM structures of yeast COMPASS and human MLL1 core complexes bound to either unmodified nucleosome core particle (NCP) or H2B mono-ubiquitinated NCP (H2Bub.NCP). We further delineate how each auxiliary component of the complex contributes to the NCP and ubiquitin recognition to maximize the methyltransferase activity., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast), marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful; however, co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast; however, this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell cycle progression and kinetochore function in wild-type (WT) cells. Suppressors of CENP-A toxicity included gene deletions of subunits of 3 conserved chromatin remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation., Competing Interests: Conflicts of interest J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; and a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC, and serves or served on the scientific advisory board of the following: LogoMix Inc.; Modern Meadow Inc.; ROME Therapeutics, Inc.; Sample6 Inc.; Sangamo, Inc.; Tessera Therapeutics Inc., and the Wyss Institute. G.O. and M.A.B.H. declare no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Proper maintenance of epigenetic information after replication is dependent on the rapid assembly and maturation of chromatin. Chromatin Assembly Complex 1 (CAF-1) is a conserved histone chaperone that deposits (H3-H4) 2 tetramers as part of the replication-dependent chromatin assembly process. Loss of CAF-1 leads to a delay in chromatin maturation, albeit with minimal impact on steady-state chromatin structure. However, the mechanisms by which CAF-1 mediates the deposition of (H3-H4) 2 tetramers and the phenotypic consequences of CAF-1-associated assembly defects are not well understood. We used nascent chromatin occupancy profiling to track the spatiotemporal kinetics of chromatin maturation in both wild-type (WT) and CAF-1 mutant yeast cells. Our results show that loss of CAF-1 leads to a heterogeneous rate of nucleosome assembly, with some nucleosomes maturing at near WT kinetics and others showing significantly slower maturation kinetics. The slow-to-mature nucleosomes are enriched in intergenic and poorly transcribed regions, suggesting that transcription-dependent assembly mechanisms can reset the slow-to-mature nucleosomes following replication. Nucleosomes with slow maturation kinetics are also associated with poly(dA:dT) sequences, which implies that CAF-1 deposits histones in a manner that counteracts resistance from the inflexible DNA sequence, promoting the formation of histone octamers as well as ordered nucleosome arrays. In addition, we show that the delay in chromatin maturation is accompanied by a transient and S-phase-specific loss of gene silencing and transcriptional regulation, revealing that the DNA replication program can directly shape the chromatin landscape and modulate gene expression through the process of chromatin maturation., (© 2023 Chen et al.; Published by Cold Spring Harbor Laboratory Press.)

Histones and transcription factors (TFs) are two important DNA-binding proteins that interact, compete, and together regulate transcriptional processes in response to diverse internal and external stimuli. Condition-specific depletion of histones in Saccharomyces cerevisiae using a galactose-inducible H3 promoter provides a suitable framework for examining transcriptional alteration resulting from reduced nucleosome content. However, the effect on DNA binding activities of TFs is yet to be fully explored. In this work, we combine ChIP-seq of H3 with RNA-seq to elucidate the genome-scale relationships between H3 occupancy patterns and transcriptional dynamics before and after global H3 depletion. ChIP-seq of Rap1 is also conducted in the H3-depletion and control treatments, to investigate the interplay between this master regulator TF and nucleosomal H3, and to explore the impact on diverse transcriptional responses of different groups of target genes and functions. Ultimately, we propose a working model and testable hypotheses regarding the impact of global and local H3 depletion on transcriptional changes. We also demonstrate different potential modes of interaction between Rap1 and H3, which sheds light on the potential multifunctional regulatory capabilities of Rap1 and potentially other pioneer factors., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘In the past 5 years, S.A.T. has consulted or been a member of scientific advisory boards at Roche, Genentech, Biogen, GlaxoSmithKline, Qiagen and ForeSite Labs and is an equity holder of Transition Bio. The remaining authors declare no competing interests’., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Acetylation of histones is a key post-translational modification that guides gene expression regulation. In yeast, the class I histone deacetylase containing Rpd3S complex plays a critical role in the suppression of spurious transcription by removing histone acetylation from actively transcribed genes. The S. cerevisiae Rpd3S complex has five subunits (Rpd3, Sin3, Rco1, Eaf3, and Ume1) but its subunit stoichiometry and how the complex engages nucleosomes to achieve substrate specificity remains elusive. Here we report the cryo-EM structure of the complete Rpd3S complex bound to a nucleosome. Sin3 and two copies of subunits Rco1 and Eaf3 encircle the deacetylase subunit Rpd3 and coordinate the positioning of Ume1. The Rpd3S complex binds both trimethylated H3 tails at position lysine 36 and makes multiple additional contacts with the nucleosomal DNA and the H2A-H2B acidic patch. Direct regulation via the Sin3 subunit coordinates binding of the acetylated histone substrate to achieve substrate specificity., (© 2023. The Author(s).)

Successful mitosis depends on the timely establishment of correct chromosomal attachments to microtubules. The kinetochore, a modular multiprotein complex, mediates this connection by recognizing specialized chromatin containing a histone H3 variant called Cse4 in budding yeast and CENP-A in vertebrates. Structural features of the kinetochore that enable discrimination between Cse4/CENP-A and H3 have been identified in several species. How and when these contribute to centromere recognition and how they relate to the overall structure of the inner kinetochore are unsettled questions. More generally, this molecular recognition ensures that only one kinetochore is built on each chromatid and that this happens at the right place on the chromatin fiber. We have determined the crystal structure of a Cse4 peptide bound to the essential inner kinetochore Okp1-Ame1 heterodimer from budding yeast. The structure and related experiments show in detail an essential point of Cse4 contact and provide information about the arrangement of the inner kinetochore., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Crosstalk between histone modifications represents a fundamental epigenetic mechanism in gene regulation. During the transcription elongation process, the histone deacetylase complex Rpd3S is recruited to H3K36-methylated nucleosomes to suppress cryptic transcription initiation. However, how subunits of Rpd3S are assembled and coordinated to recognize nucleosomal substrates and exert their deacetylation function remains unclear. Here we report the structure of Saccharomyces cerevisiae Rpd3S deacetylase bound to H3K36me3-modified nucleosome at 3.1 Å resolution. It shows that Sin3 and Rco1 subunits orchestrate the assembly of the complex and mediate its contact with nucleosome at multiple sites, with the Sin3-DNA interface as a pivotal anchor. The PHD1 domain of Rco1 recognizes the unmodified H3K4 and places the following H3 tail toward the active site of Rpd3, while the chromodomain of Eaf3 subunit recognizes the H3K36me3 mark and contacts both nucleosomal and linker DNA. The second copy of Eaf3-Rco1 is involved in neighboring nucleosome binding. Our work unravels the structural basis of chromatin targeting and deacetylation by the Rpd3S complex., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae , a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target., (© 2023 Gavriil et al.)

Parental histone recycling is vital for maintaining chromatin-based epigenetic information during replication, yet its underlying mechanisms remain unclear. Here, we uncover an unexpected role of histone chaperone FACT and its N-terminus of the Spt16 subunit during parental histone recycling and transfer in budding yeast. Depletion of Spt16 and mutations at its middle domain that impair histone binding compromise parental histone recycling on both the leading and lagging strands of DNA replication forks. Intriguingly, deletion of the Spt16-N domain impairs parental histone recycling, with a more pronounced defect observed on the lagging strand. Mechanistically, the Spt16-N domain interacts with the replicative helicase MCM2-7 and facilitates the formation of a ternary complex involving FACT, histone H3/H4 and Mcm2 histone binding domain, critical for the recycling and transfer of parental histones to lagging strands. Lack of the Spt16-N domain weakens the FACT-MCM interaction and reduces parental histone recycling. We propose that the Spt16-N domain acts as a protein-protein interaction module, enabling FACT to function as a shuttle chaperone in collaboration with Mcm2 and potentially other replisome components for efficient local parental histone recycling and inheritance., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Monoubiquitination of histone H2B-K120/123 plays several roles in regulating transcription, DNA replication and the DNA damage response. The structure of a nucleosome in complex with the dimeric RING E3 ligase Bre1 reveals that one RING domain binds to the nucleosome acidic patch, where it can position the E2 ubiquitin conjugating enzyme Rad6, while the other RING domain contacts the DNA. Comparisons with H2A-specific E3 ligases suggest a general mechanism of tuning histone specificity via the non-E2-binding RING domain., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Loss of transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3) contributes to shorter lifespans in eukaryotes. However, the molecular mechanism of the decline of H3K36me3 during aging remains poorly understood. Here, we report that the degradation of the methyltransferase Set2 is the cause of decreased H3K36me3 levels during chronological aging in budding yeast. We show that Set2 protein degradation during cellular senescence and chronological aging is mainly mediated by the ubiquitin-conjugating E2 enzyme Ubc3 and the E3 ligase Bre1. Lack of Bre1 or abolishment of the ubiquitination stabilizes Set2 protein, sustains H3K36me3 levels at the aging-related gene loci, and upregulates their gene expression, thus leading to extended chronological lifespan. We further illustrate that Gcn5-mediated Set2 acetylation is a prerequisite for Bre1-catalyzed Set2 polyubiquitination and proteolysis during aging. We propose that two sequential post-translational modifications regulate Set2 homeostasis, suggesting a potential strategy to target the Gcn5-Bre1-Set2 axis for intervention of longevity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)