Structure of the Hir histone chaperone complex.

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.

Regulation of replicative histone RNA metabolism by the histone chaperone ASF1.

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.

DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone network.

A multitude of histone chaperones are required to support histones from their biosynthesis until DNA deposition. They cooperate through the formation of histone co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using exploratory interactomics, we define the interplay between human histone H3-H4 chaperones in the histone chaperone network. We identify previously uncharacterized histone-dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX provides a unique functionality to the histone chaperone network, recruiting histone methyltransferases to promote H3K9me3 catalysis on new histone H3.3-H4 prior to deposition onto DNA. Hereby, DAXX provides a molecular mechanism for de novo H3K9me3 deposition and heterochromatin assembly. Collectively, our findings provide a framework for understanding how cells orchestrate histone supply and employ targeted deposition of modified histones to underpin specialized chromatin states.

Distinct histone H3-H4 binding modes of sNASP reveal the basis for cooperation and competition of histone chaperones.

Chromosomal duplication requires de novo assembly of nucleosomes from newly synthesized histones, and the process involves a dynamic network of interactions between histones and histone chaperones. sNASP and ASF1 are two major histone H3-H4 chaperones found in distinct and common complexes, yet how sNASP binds H3-H4 in the presence and absence of ASF1 remains unclear. Here we show that, in the presence of ASF1, sNASP principally recognizes a partially unfolded Nα region of histone H3, and in the absence of ASF1, an additional sNASP binding site becomes available in the core domain of the H3-H4 complex. Our study also implicates a critical role of the C-terminal tail of H4 in the transfer of H3-H4 between sNASP and ASF1 and the coiled-coil domain of sNASP in nucleosome assembly. These findings provide mechanistic insights into coordinated histone binding and transfer by histone chaperones.

PIF7-mediated epigenetic reprogramming promotes the transcriptional response to shade in Arabidopsis.

For shade-intolerant plants, changes in light quality through competition from neighbors trigger shade avoidance syndrome (SAS): a series of morphological and physiological adaptations that are ultimately detrimental to plant health and crop yield. Phytochrome-interacting factor 7 (PIF7) is a major transcriptional regulator of SAS in Arabidopsis; however, how it regulates gene expression is not fully understood. Here, we show that PIF7 directly interacts with the histone chaperone anti-silencing factor 1 (ASF1). The ASF1-deprived asf1ab mutant showed defective shade-induced hypocotyl elongation. Histone regulator homolog A (HIRA), which mediates deposition of the H3.3 variant into chromatin, is also involved in SAS. RNA/ChIP-sequencing analyses identified the role of ASF1 in the direct regulation of a subset of PIF7 target genes. Furthermore, shade-elicited gene activation is accompanied by H3.3 enrichment, which is mediated by the PIF7-ASF1-HIRA regulatory module. Collectively, our data reveal that PIF7 recruits ASF1-HIRA to increase H3.3 incorporation into chromatin to promote gene transcription, thus enabling plants to effectively respond to environmental shade.

HIRA complex deposition of histone H3.3 is driven by histone tetramerization and histone-DNA binding.

The HIRA histone chaperone complex is comprised of four protein subunits: HIRA, UBN1, CABIN1, and transiently associated ASF1a. All four subunits have been demonstrated to play a role in the deposition of the histone variant H3.3 onto areas of actively transcribed euchromatin in cells. The mechanism by which these subunits function together to drive histone deposition has remained poorly understood. Here we present biochemical and biophysical data supporting a model whereby ASF1a delivers histone H3.3/H4 dimers to the HIRA complex, H3.3/H4 tetramerization drives the association of two HIRA/UBN1 complexes, and the affinity of the histones for DNA drives release of ASF1a and subsequent histone deposition. These findings have implications for understanding how other histone chaperone complexes may mediate histone deposition.

ASF1a Promotes Non-homologous End Joining Repair by Facilitating Phosphorylation of MDC1 by ATM at Double-Strand Breaks.

Double-strand breaks (DSBs) of DNA in eukaryotic cells are predominantly repaired by non-homologous end joining (NHEJ). The histone chaperone anti-silencing factor 1a (ASF1a) interacts with MDC1 and is recruited to sites of DSBs to facilitate the interaction of phospho-ATM with MDC1 and phosphorylation of MDC1, which are required for the recruitment of RNF8/RNF168 histone ubiquitin ligases. Thus, ASF1a deficiency reduces histone ubiquitination at DSBs, decreasing the recruitment of 53BP1, and decreases NHEJ, rendering cells more sensitive to DSBs. This role of ASF1a in DSB repair cannot be provided by the closely related ASF1b and does not require its histone chaperone activity. Homozygous deletion of ASF1A is seen in 10%-15% of certain cancers, suggesting that loss of NHEJ may be selected in some malignancies and that the deletion can be used as a molecular biomarker for cancers susceptible to radiotherapy or to DSB-inducing chemotherapy.

Structural insights into instability of the nucleosome driven by histone variant H3T.

The testis-specific histone variant H3T plays a crucial role in chromatin reorganization during spermatogenesis by destabilizing nucleosomes. However, the structure basis for the nucleosome instability driven by H3T is not fully understand. In this study, we determinate the crystal structure of H3T-H4 in complex with histone chaperone ASF1a at 2.8 Å resolution. Our findings reveal that H3T-H4 binds ASF1a similarly to the conventional H3.1-H4 complex. However, significant structural differences are observed in the H3 α1 helix, the N- and C-terminal region of α2, and N-terminal region of L2. These differences are driven by H3T-specific residues, particularly Val111. Unlike the smaller Ala111 in H3.1, we find that bulkier residue Val111 fits well within the ASF1-H3T-H4 complex, but is difficult to arrange in nucleosome structure. Given that H3.1-Ala111/H3T-Val111 is located at the DNA binding and tetramerization interface of H3-H4, it is likely that Ala111Val substitution will lead to the instability of the corresponding area in nucleosome, contributing to instability of H3T-containing nucleosome. These structural findings may elucidate the role of H3T in chromatin reorganization during spermatogenesis.

Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair.

To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.

Histone chaperone ASF1 acts with RIF1 to promote DNA end joining in BRCA1-deficient cells.

Replication timing regulatory factor 1 (RIF1) acts downstream of p53-binding protein 53BP1 to inhibit the resection of DNA broken ends, which plays critical roles in determining the DNA double-strand break repair pathway choice between nonhomologous end joining and homologous recombination (HR). However, the mechanism by which this choice is made is not yet clear. In this study, we identified that histone chaperone protein ASF1 associates with RIF1 and regulates RIF1-dependent functions in the DNA damage response. Similar to loss of RIF1, we found that loss of ASF1 resulted in resistance to poly (ADP-ribose) polymerase (PARP) inhibition in BRCA1-deficient cells with restored HR and decreased telomere fusion in telomeric repeat-binding protein 2 (TRF2)-depleted cells. Moreover, we showed that these functions of ASF1 are dependent on its interaction with RIF1 but not on its histone chaperone activity. Thus, our study supports a new role for ASF1 in dictating double-strand break repair choice. Considering that the status of 53BP1-RIF1 axis is important in determining the outcome of PARP inhibitor-based therapy in BRCA1- or HR-deficient cancers, the identification of ASF1 function in this critical pathway uncovers an interesting connection between these S-phase events, which may reveal new strategies to overcome PARP inhibitor resistance.

TOF1 and RRM3 reveal a link between gene silencing and the pausing of replication forks.

Eukaryotic DNA replication is accompanied by the disassembly and reassembly of nucleosomes and the transmission of epigenetic marks to the newly assembled chromatids. Several histone chaperones, including CAF-1 and Asf1p, are central to these processes. On the other hand, replication forks pause at numerous positions throughout the genome, but it is not known if and how this pausing affects the reassembly and maintenance of chromatin structures. Here, we applied drug-free gene silencing assays to analyze the genetic interactions between CAC1, ASF1, and two genes that regulate the stability of the paused replisome (TOF1) and the resumption of elongation (RRM3). Our results show that TOF1 and RRM3 differentially interact with CAF-1 and ASF1 and that the deletions of TOF1 and RRM3 lead to reduced silencing and increased frequency of epigenetic conversions at three loci in the genome of S. cerevisiae. Our study adds details to the known activities of CAF-1 and Asf1p and suggests that the pausing of the replication fork can lead to epigenetic instability.

miR-24-3p Regulates Epithelial-Mesenchymal Transition and the Malignant Phenotype of Pancreatic Adenocarcinoma by Regulating ASF1B Expression.

Anti-silencing function protein 1 homolog B (ASF1B) has been implicated in the occurrence and development of cancers. The present work explored the functional role and the expression regulation of ASF1B in pancreatic ductal adenocarcinoma (PDAC). Based on the real-time quantitative PCR (qRT-PCR) and immunohistochemistry (IHC), ASF1B was significantly upregulated in PDAC tissues. High expression of ASF1B was associated with a poor overall survival (OS) and recurrence-free survival (DFS) in the PDAC patients. ASF1B also showed a relatively higher expression in PDAC cells (AsPC-1, PANC-1) when compared with human pancreatic ductal epithelial cells (HPDFe-6). CCK8 and clone formation assay demonstrated that silencing ASF1B impaired the proliferation in PANC-1 and AsPC-1 cells, and Annexin V-PI staining showed an increased level of apoptosis upon ASF1B silencing. ASF1B silencing also suppressed the migration and invasion in PDAC cells, as revealed by Transwell assays. We further showed that miR-24-3p was downregulated in PDAC tissues and cells, which functionally interacted with ASF1B by dual-luciferase reporter assay. miR-24-3p negatively regulated ASF1B expression to modulate the malignant phenotype of PDAC cells. ASF1B shows high expression in PDAC, which promotes the malignancy and EMT process of PDAC cells. miR-24-3p is a negative regulator of ASF1B and is downregulated in PDAC cells. Our data suggest that targeting ASF1B/miR-24-3p axis may serve as an intervention strategy for the management of PDAC.

miR-767-3p suppresses melanoma progression by inhibiting ASF1B expression.

BACKGROUND: Melanoma, the type of skin cancer considered as most malignant, and known to be linked with a high incidence as well as high mortality rate. Although the dysregulation of ASF1B and miR-767-3p expression is involved in the progression of various cancers, their biological function in melanoma remains unclear. METHODS: Real-time qPCR was the primary source for determining the levels of ASF1B and miR-767-3p in melanoma. For the validation of association among miR-767-3p and ASF1B, luciferase activity assay was used. Quantification of cell apoptosis, proliferation, migration and viability in melanoma cells were carried out by flow cytometry, BrdU, transwell assays, and CCK-8, respectively. Further evaluation of tumor growth was achieved by xenograft in vivo. RESULTS: Results showed an increased expression of ASF1B while declined expression of miR-767-3p in melanoma. ASF1B knockdown repressed cell migration, viability, proliferation, and tumor growth whereas boosted apoptosis in A375 as well as in A875 melanoma cells. Moreover, miR-767-3p attenuated the migration and proliferation of melanoma cells and encouraged cell apoptosis by reducing ASF1B levels. CONCLUSION: In this study, miR-767-3p was shown to inhibit ASF1B which will attenuate melanoma tumorigenesis, and by this it can be a potential new effective biomarker for the treatment of melanoma.

Involvement of elevated ASF1B in the poor prognosis and tumorigenesis in pancreatic cancer.

Anti-silencing function 1B (ASF1B) has been reported to be associated with the occurrence of many kinds of tumors. However, the biological effect and action mechanism of ASF1B in pancreatic cancer (PC) tumorigenesis remain unclear. The expression and prognosis value of ASF1B in PC were analyzed using GEPIA, GEO, and Kaplan-Meier plotter databases. The diagnostic value of ASF1B in PC was determined by receiver operating characteristic curve. The relationship between ASF1B expression and the clinical feathers in PC was investigated based on TCGA. qRT-PCR and western blot analyses were used to measure ASF1B expression in PC cells. Cell proliferation was evaluated by MTT and EdU assays, and apoptosis was examined by TUNEL and caspase-3 activity assays. Western blot analysis was utilized to detect the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, Bax, Bcl-2, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling proteins. ASF1B was overexpressed in several digestive cancers, including PC. Upregulated ASF1B was correlated with the poor prognosis and clinical features in PC patients. The area under the curve (AUC) value of ASF1B was 0.990. ASF1B was also overexpressed in PC cells. ASF1B silencing inhibited PC cell proliferation, promoted apoptosis, and increased caspase-3 activity, which were accompanied by the reduction of PCNA and cyclin D1 expression and increase of the ratio of Bax/Bcl-2 expression. Additionally, ASF1B silencing suppressed the PI3K/Akt pathway and 740Y-P treatment partially abolished the effects of ASF1B knockdown on PC cells. In conclusion, ASF1B silencing retarded proliferation and promoted apoptosis in PC cells by inactivation of the PI3K/Akt pathway.

Structural insights into histone chaperone Asf1 and its characterization from Plasmodium falciparum.

Asf1 is a highly conserved histone chaperone that regulates tightly coupled nucleosome assembly/disassembly process. We observed that Plasmodium falciparum Asf1 (PfAsf1) is ubiquitously expressed in different stages of the life cycle of the parasite. To gain further insight into its biological activity, we solved the structure of N-terminal histone chaperone domain of PfAsf1 (1-159 amino acids) by X-ray crystallography to a resolution of 2.4 Å. The structure is composed of two beta-sheet to form a beta-sandwich, which resembles an immunoglobulin-like fold. The surface-charge distribution of PfAsf1 is distinct from yAsf1 and hAsf1 although the core-structure shows significant similarity. The crystal-structure indicated that PfAsf1 may exist in a dimeric-state which was further confirmed by solution cross-linking experiment. PfAsf1 was found to specifically interact with Plasmodium histone H3 and H4 and was able to deposit H3/H4 dimer onto DNA-template to form disomes, showing its characteristic histone chaperone activity. We mapped the critical residues of PfAsf1 involved in histone H3/H4 interaction and confirmed by site-directed mutagenesis. Further analysis indicates that histone interacting surface of Asf1 is highly conserved while the dimerization interface is variable. Our results identify the role of PfAsf1 as a mediator of chromatin assembly in Plasmodium falciparum, which is the causative agent of malignant malaria in humans.

ATR checkpoint kinase and CRL1ßTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks.

Many agents used for chemotherapy, such as doxorubicin, interfere with DNA replication, but the effect of this interference on transcription is largely unknown. Here we show that doxorubicin induces the firing of dense clusters of neoreplication origins that lead to clusters of stalled replication forks in gene-rich parts of the genome, particularly on expressed genes. Genes that overlap with these clusters of stalled forks are actively dechromatinized, unwound, and repressed by an ATR-dependent checkpoint pathway. The ATR checkpoint pathway causes a histone chaperone normally associated with the replication fork, ASF1a, to degrade through a CRL1(ßTRCP)-dependent ubiquitination/proteasome pathway, leading to the localized dechromatinization and gene repression. Therefore, a globally active checkpoint pathway interacts with local clusters of stalled forks to specifically repress genes in the vicinity of the stalled forks, providing a new mechanism of action of chemotherapy drugs like doxorubicin. Finally, ASF1a-depleted cancer cells are more sensitive to doxorubicin, suggesting that the 7%-10% of prostate adenocarcinomas and adenoid cystic carcinomas reported to have homozygous deletion or significant underexpression of ASF1a should be tested for high sensitivity to doxorubicin.

Yeast Protein Asf1 Possesses Modulating Activity towards Protein Kinase CK2.

Protein kinase CK2 plays an important role in cell survival and protects regulatory proteins from caspase-mediated degradation during apoptosis. The consensus sequence of proteins phosphorylated by CK2 contains a cluster of acidic amino acids around the phosphorylation site. The poly-acidic sequence in yeast protein Asf1 is similar to the acidic loop in CK2ß, which possesses a regulatory function. We observed that the overexpression of Asf1 in yeast cells influences cell growth. Experiments performed in vitro and in vivo indicate that yeast protein Asf1 inhibits protein kinase CK2. Our data suggest that each CK2 isoform might be regulated in a different way. Deletion of the amino or carboxyl end of Asf1 reveals that the acidic cluster close to the C-terminus is responsible for the activation or inhibition of CK2 activity.

GTP Binding Protein Gtr1 Cooperating with ASF1 Regulates Asexual Development in Stemphylium eturmiunum.

The Gtr1 protein was a member of the RagA subfamily of the Ras-like small GTPase superfamily and involved in phosphate acquisition, ribosome biogenesis and epigenetic control of gene expression in yeast. However, Gtr1 regulation sexual or asexual development in filamentous fungi is barely accepted. In the study, SeGtr1, identified from Stemphylium eturmiunum, could manipulate mycelial growth, nuclear distribution of mycelium and the morphology of conidia in Segtr1 silenced strains compared with its overexpression transformants, while the sexual activity of Segtr1 silenced strains were unchanged. SeASF1, a H3/H4 chaperone, participated in nucleosome assembly/disassembly, DNA replication and transcriptional regulation. Our experiments showed that deletion Seasf1 mutants produced the hyphal fusion and abnormal conidia. Notably, we characterized that Segtr1 was down-regulated in Se∆asf1 mutants and Seasf1 was also down-regulated in SiSegtr1 strains. We further confirmed that SeGtr1 interacted with SeASF1 or SeH4 in vivo and vitro, respectively. Thus, SeGtr1 can cooperate with SeASF1 to modulate asexual development in Stemphylium eturmiunum.

Structural basis for histone H3 recognition by NASP in Arabidopsis.

The structural basis for histone recognition by the histone chaperone nuclear autoantigenic sperm protein (NASP) remains largely unclear. Here, we showed that Arabidopsis thaliana AtNASP is a monomer and displays robust nucleosome assembly activity in vitro. Examining the structure of AtNASP complexed with a histone H3 α3 peptide revealed a binding mode that is conserved in human NASP. AtNASP recognizes the H3 N-terminal region distinct from human NASP. Moreover, AtNASP forms a co-chaperone complex with ANTI-SILENCING FUNCTION 1 (ASF1) by binding to the H3 N-terminal region. Therefore, we deciphered the structure of AtNASP and the basis of the AtNASP-H3 interaction.

Mild dyserythropoiesis and ß-like globin gene expression imbalance due to the loss of histone chaperone ASF1B.

The expression of the human ß-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to ß). The γ- to ß-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (ß-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to ß-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to ß-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.