Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation.

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.

H2A.Z chaperones converge on E2F target genes for melanoma cell proliferation.

High levels of H2A.Z promote melanoma cell proliferation and correlate with poor prognosis. However, the role of the two distinct H2A.Z histone chaperone complexes SRCAP and P400-TIP60 in melanoma remains unclear. Here, we show that individual subunit depletion of SRCAP, P400, and VPS72 (YL1) results in not only the loss of H2A.Z deposition into chromatin but also a reduction of H4 acetylation in melanoma cells. This loss of H4 acetylation is particularly found at the promoters of cell cycle genes directly bound by H2A.Z and its chaperones, suggesting a coordinated regulation between H2A.Z deposition and H4 acetylation to promote their expression. Knockdown of each of the three subunits downregulates E2F1 and its targets, resulting in a cell cycle arrest akin to H2A.Z depletion. However, unlike H2A.Z deficiency, loss of the shared H2A.Z chaperone subunit YL1 induces apoptosis. Furthermore, YL1 is overexpressed in melanoma tissues, and its upregulation is associated with poor patient outcome. Together, these findings provide a rationale for future targeting of H2A.Z chaperones as an epigenetic strategy for melanoma treatment.

HIRA-dependent boundaries between H3 variants shape early replication in mammals.

The lack of a consensus DNA sequence defining replication origins in mammals has led researchers to consider chromatin as a means to specify these regions. However, to date, there is no mechanistic understanding of how this could be achieved and maintained given that nucleosome disruption occurs with each fork passage and with transcription. Here, by genome-wide mapping of the de novo deposition of the histone variants H3.1 and H3.3 in human cells during S phase, we identified how their dual deposition mode ensures a stable marking with H3.3 flanked on both sides by H3.1. These H3.1/H3.3 boundaries correspond to the initiation zones of early origins. Loss of the H3.3 chaperone HIRA leads to the concomitant disruption of H3.1/H3.3 boundaries and initiation zones. We propose that the HIRA-dependent deposition of H3.3 preserves H3.1/H3.3 boundaries by protecting them from H3.1 invasion linked to fork progression, contributing to a chromatin-based definition of early replication zones.

Essential histone chaperones collaborate to regulate transcription and chromatin integrity.

Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.

Transcription-coupled nucleosome assembly.

Eukaryotic transcription occurs on chromatin, where RNA polymerase II encounters nucleosomes during elongation. These nucleosomes must unravel for the DNA to enter the active site. However, in most transcribed genes, nucleosomes remain intact due to transcription-coupled chromatin assembly mechanisms. These mechanisms primarily involve the local reassembly of displaced nucleosomes to prevent (epi)genomic instability and the emergence of cryptic transcription. As a fail-safe mechanism, cells can assemble nucleosomes de novo, particularly in highly transcribed genes, but this may result in the loss of epigenetic information. This review examines transcription-coupled chromatin assembly, with an emphasis on studies in yeast and recent structural studies. These studies shed light on how elongation factors and histone chaperones coordinate to enable nucleosome recycling during transcription.

Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate.

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.

The Histone Chaperones ASF1 and CAF-1 Promote MMS22L-TONSL-Mediated Rad51 Loading onto ssDNA during Homologous Recombination in Human Cells.

The access-repair-restore model for the role of chromatin in DNA repair infers that chromatin is a mere obstacle to DNA repair. However, here we show that blocking chromatin assembly, via knockdown of the histone chaperones ASF1 or CAF-1 or a mutation that prevents ASF1A binding to histones, hinders Rad51 loading onto ssDNA during homologous recombination. This is a consequence of reduced recruitment of the Rad51 loader MMS22L-TONSL to ssDNA, resulting in persistent RPA foci, extensive DNA end resection, persistent activation of the ATR-Chk1 pathway, and cell cycle arrest. In agreement, histones occupy ssDNA during DNA repair in yeast. We also uncovered DNA-PKcs-dependent DNA damage-induced ASF1A phosphorylation, which enhances chromatin assembly, promoting MMS22L-TONSL recruitment and, hence, Rad51 loading. We propose that transient assembly of newly synthesized histones onto ssDNA serves to recruit MMS22L-TONSL to efficiently form the Rad51 nucleofilament for strand invasion, suggesting an active role of chromatin assembly in homologous recombination.

The Histone Chaperone FACT Coordinates H2A.X-Dependent Signaling and Repair of DNA Damage.

Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression.

CHAF1A/B mediate silencing of unintegrated HIV-1 DNAs early in infection.

Early events of the retroviral life cycle are the targets of many host restriction factors that have evolved to prevent establishment of infection. Incoming retroviral DNAs are transcriptionally silenced before integration in most cell types, and efficient viral gene expression occurs only after formation of the provirus. The molecular machinery for silencing unintegrated retroviral DNAs of HIV-1 remains poorly characterized. Here, we identified the histone chaperones CHAF1A and CHAF1B as essential factors for silencing of unintegrated HIV-1 DNAs. Using RNAi-mediated knockdown (KD) of multiple histone chaperones, we found that KD of CHAF1A or CHAF1B resulted in a pronounced increase in expression of incoming viral DNAs. The function of these two proteins in silencing was independent of their interaction partner RBBP4. Viral DNA levels accumulated to significantly higher levels in CHAF1A KD cells over controls, suggesting enhanced stabilization of actively transcribed DNAs. Chromatin immunoprecipitation assays revealed no major changes in histone loading onto viral DNAs in the absence of CHAF1A, but levels of the H3K9 trimethylation silencing mark were reduced. KD of the H3K9me3-binding protein HP1γ accelerated the expression of unintegrated HIV-1 DNAs. While CHAF1A was critical for silencing HIV-1 DNAs, it showed no role in silencing of unintegrated retroviral DNAs of mouse leukemia virus. Our study identifies CHAF1A and CHAF1B as factors involved specifically in silencing of HIV-1 DNAs early in infection. The results suggest that these factors act by noncanonical pathways, distinct from their histone loading activities, to mediate silencing of newly synthesized HIV-1 DNAs.

Single-cell histone chaperones patterns guide intercellular communication of tumor microenvironment that contribute to breast cancer metastases.

BACKGROUND: Histone chaperones (HCs) are crucial for governing genome stability and gene expression in multiple cancers. However, the functioning of HCs in the tumor microenvironment (TME) is still not clearly understood. METHODS: Self-tested single-cell RNA-seq data derived from 6 breast cancer (BC) patients with brain and liver metastases were reanalyzed by nonnegative matrix factorization (NMF) algorithm for 36 HCs. TME subclusters were observed with BC and immunotherapy public cohorts to assess their prognosis and immune response. The biological effect of HSPA8, one of the HCs, was verified by transwell assay and wound-healing assays. RESULTS: Cells including fibroblasts, macrophages, B cells, and T cells, were classified into various subclusters based on marker genes. Additionally, it showed that HCs might be strongly associated with biological and clinical features of BC metastases, along with the pseudotime trajectory of each TME cell type. Besides, the results of bulk-seq analysis revealed that TME cell subclusters mediated by HCs distinguished significant prognostic value for BC patients and were relevant to patients' immunotherapy responses, especially for B cells and macrophages. In particular, CellChat analysis exhibited that HCs-related TME cell subclusters revealed extensive and diverse interactions with malignant cells. Finally, transwell and wound-healing assays exhibited that HSPA8 deficiency inhibited BC cell migration and invasion. CONCLUSIONS: Collectively, our study first dissected HCs-guided intercellular communication of TME that contribute to BC metastases.

How potassium came to be the dominant biological cation: of metabolism, chemiosmosis, and cation selectivity since the beginnings of life.

In the cytoplasm of practically all living cells, potassium is the major cation while sodium dominates in the media (seawater, extracellular fluids). Both prokaryotes and eukaryotes have elaborate mechanisms and spend significant energy to maintain this asymmetric K+ /Na+ distribution. This essay proposes an original line of evidence to explain how bacteria selected potassium at the very beginning of the evolutionary process and why it remains essential for eukaryotes.

The Histone Chaperone Network Is Highly Conserved in Physarum polycephalum.

The nucleosome is composed of histones and DNA. Prior to their deposition on chromatin, histones are shielded by specialized and diverse proteins known as histone chaperones. They escort histones during their entire cellular life and ensure their proper incorporation in chromatin. Physarum polycephalum is a Mycetozoan, a clade located at the crown of the eukaryotic tree. We previously found that histones, which are highly conserved between plants and animals, are also highly conserved in Physarum. However, histone chaperones differ significantly between animal and plant kingdoms, and this thus probed us to further study the conservation of histone chaperones in Physarum and their evolution relative to animal and plants. Most of the known histone chaperones and their functional domains are conserved as well as key residues required for histone and chaperone interactions. Physarum is divergent from yeast, plants and animals, but PpHIRA, PpCABIN1 and PpSPT6 are similar in structure to plant orthologues. PpFACT is closely related to the yeast complex, and the Physarum genome encodes the animal-specific APFL chaperone. Furthermore, we performed RNA sequencing to monitor chaperone expression during the cell cycle and uncovered two distinct patterns during S-phase. In summary, our study demonstrates the conserved role of histone chaperones in handling histones in an early-branching eukaryote.

Disruption of NAP1 genes in Arabidopsis thaliana suppresses the fas1 mutant phenotype, enhances genome stability and changes chromatin compaction.

Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.

Histone variants: dynamic punctuation in transcription.

Eukaryotic gene regulation involves a balance between packaging of the genome into nucleosomes and enabling access to regulatory proteins and RNA polymerase. Nucleosomes are integral components of gene regulation that restrict access to both regulatory sequences and the underlying template. Whereas canonical histones package the newly replicated genome, they can be replaced with histone variants that alter nucleosome structure, stability, dynamics, and, ultimately, DNA accessibility. Here we consider how histone variants and their interacting partners are involved in transcriptional regulation through the creation of unique chromatin states.

Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization.

Nuclear DNA in the male gamete of sexually reproducing animals is organized as sperm chromatin compacted primarily by sperm-specific protamines. Fertilization leads to sperm chromatin remodeling, during which protamines are expelled and replaced by histones. Despite our increased understanding of the factors that mediate nucleosome assembly in the nascent male pronucleus, the machinery for protamine removal remains largely unknown. Here we identify four Drosophila protamine chaperones that mediate the dissociation of protamine-DNA complexes: NAP-1, NLP, and nucleophosmin are previously characterized histone chaperones, and TAP/p32 has no known function in chromatin metabolism. We show that TAP/p32 is required for the removal of Drosophila protamine B in vitro, whereas NAP-1, NLP, and Nph share roles in the removal of protamine A. Embryos from P32-null females show defective formation of the male pronucleus in vivo. TAP/p32, similar to NAP-1, NLP, and Nph, facilitates nucleosome assembly in vitro and is therefore a histone chaperone. Furthermore, mutants of P32, Nlp, and Nph exhibit synthetic-lethal genetic interactions. In summary, we identified factors mediating protamine removal from DNA and reconstituted in a defined system the process of sperm chromatin remodeling that exchanges protamines for histones to form the nucleosome-based chromatin characteristic of somatic cells.

Two Functionally Redundant FK506-Binding Proteins Regulate Multidrug Resistance Gene Expression and Govern Azole Antifungal Resistance.

Increasing resistance to antifungal therapy is an impediment to the effective treatment of fungal infections. Candida glabrata is an opportunistic human fungal pathogen that is inherently less susceptible to cost-effective azole antifungals. Gain-of-function mutations in the Zn-finger pleiotropic drug resistance transcriptional activator-encoding gene CgPDR1 are the most prevalent causes of azole resistance in clinical settings. CgPDR1 is also transcriptionally activated upon azole exposure; however, factors governing CgPDR1 gene expression are not yet fully understood. Here, we have uncovered a novel role for two FK506-binding proteins, CgFpr3 and CgFpr4, in the regulation of the CgPDR1 regulon. We show that CgFpr3 and CgFpr4 possess a peptidyl-prolyl isomerase domain and act redundantly to control CgPDR1 expression, as a Cgfpr3Δ4Δ mutant displayed elevated expression of the CgPDR1 gene along with overexpression of its target genes, CgCDR1, CgCDR2, and CgSNQ2, which code for ATP-binding cassette multidrug transporters. Furthermore, CgFpr3 and CgFpr4 are required for the maintenance of histone H3 and H4 protein levels, and fluconazole exposure leads to elevated H3 and H4 protein levels. Consistent with the role of histone proteins in azole resistance, disruption of genes coding for the histone demethylase CgRph1 and the histone H3K36-specific methyltransferase CgSet2 leads to increased and decreased susceptibility to fluconazole, respectively, with the Cgrph1Δ mutant displaying significantly lower basal expression levels of the CgPDR1 and CgCDR1 genes. These data underscore a hitherto unknown role of histone methylation in modulating the most common azole antifungal resistance mechanism. Altogether, our findings establish a link between CgFpr-mediated histone homeostasis and CgPDR1 gene expression and implicate CgFpr in the virulence of C. glabrata.

Cac1 WHD and PIP domains have distinct roles in replisome progression and genomic stability.

Replication-coupled (RC) nucleosome assembly is an essential process in eukaryotic cells to maintain chromatin structure during DNA replication. The deposition of newly-synthesized H3/H4 histones during DNA replication is facilitated by specialized histone chaperones. CAF-1 is an important histone chaperone complex and its main subunit, Cac1p, contains a PIP and WHD domain for interaction with PCNA and the DNA, respectively. While Cac1p subunit was extensively studied in different systems much less is known regarding the importance of the PIP and WHD domains in replication fork progression and genome stability. By exploiting a time-lapse microscopy system for monitoring DNA replication in individual live cells, we examined how mutations in these Cac1p domains affect replication fork progression and post-replication characteristics. Our experiments revealed that mutations in the Cac1p WHD domain, which abolished the CAF-1-DNA interaction, slows down replication fork progression. In contrast, mutations in Cac1p PIP domain, abolishing Cac1p-PCNA interaction, lead to extended late-S/Anaphase duration, elevated number of RPA foci and increased spontaneous mutation rate. Our research shows that Cac1p WHD and PIP domains have distinct roles in high replisome progression and maintaining genome stability during cell cycle progression.

The Histone Chaperone HIRA Is a Positive Regulator of Seed Germination.

Histone chaperones regulate the flow and dynamics of histone variants and ensure their assembly into nucleosomal structures, thereby contributing to the repertoire of histone variants in specialized cells or tissues. To date, not much is known on the distribution of histone variants and their modifications in the dry seed embryo. Here, we bring evidence that genes encoding the replacement histone variant H3.3 are expressed in Arabidopsis dry seeds and that embryo chromatin is characterized by a low H3.1/H3.3 ratio. Loss of HISTONE REGULATOR A (HIRA), a histone chaperone responsible for H3.3 deposition, reduces cellular H3 levels and increases chromatin accessibility in dry seeds. These molecular differences are accompanied by increased seed dormancy in hira-1 mutant seeds. The loss of HIRA negatively affects seed germination even in the absence of HISTONE MONOUBIQUITINATION 1 or TRANSCRIPTION ELONGATION FACTOR II S, known to be required for seed dormancy. Finally, hira-1 mutant seeds show lower germination efficiency when aged under controlled deterioration conditions or when facing unfavorable environmental conditions such as high salinity. Altogether, our results reveal a dependency of dry seed chromatin organization on the replication-independent histone deposition pathway and show that HIRA contributes to modulating seed dormancy and vigor.

Similar yet critically different: the distribution, dynamics and function of histone variants.

Organization of the genetic information into chromatin plays an important role in the regulation of all DNA template-based reactions. The incorporation of different variant versions of the core histones H3, H2A, and H2B, or the linker histone H1 results in nucleosomes with unique properties. Histone variants can differ by only a few amino acids or larger protein domains and their incorporation may directly affect nucleosome stability and higher order chromatin organization or indirectly influence chromatin function through histone variant-specific binding partners. Histone variants employ dedicated histone deposition machinery for their timely and locus-specific incorporation into chromatin. Plants have evolved specific histone variants with unique expression patterns and features. In this review, we discuss our current knowledge on histone variants in Arabidopsis, their mode of deposition, variant-specific post-translational modifications, and genome-wide distribution, as well as their role in defining different chromatin states.

Recognition of ASF1 by Using Hydrocarbon-Constrained Peptides.

Inhibiting the histone H3-ASF1 (anti-silencing function 1) protein-protein interaction (PPI) represents a potential approach for treating numerous cancers. As an α-helix-mediated PPI, constraining the key histone H3 helix (residues 118-135) is a strategy through which chemical probes might be elaborated to test this hypothesis. In this work, variant H3118-135 peptides bearing pentenylglycine residues at the i and i+4 positions were constrained by olefin metathesis. Biophysical analyses revealed that promotion of a bioactive helical conformation depends on the position at which the constraint is introduced, but that the potency of binding towards ASF1 is unaffected by the constraint and instead that enthalpy-entropy compensation occurs.