Replicating chromatin in the nucleus: A histone variant perspective.

In eukaryotes, chromatin and DNA replication are intimately linked, whereby chromatin impacts DNA replication control while genome duplication involves recovery of chromatin organisation. Here, we review recent advances in this area using a histone variant lens. We highlight how nucleosomal features interplay with origin definition and how the order of origin firing links with chromatin states in early mammalian development. We next discuss histone recycling and de novo deposition at the fork to finally open on the post-replicative recovery of the chromatin landscape to promote maintenance of cell identity.

Disordered regions and folded modules in CAF-1 promote histone deposition in Schizosaccharomyces pombe.

Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the Saccharomyces cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.

Mapping histone variant genomic distribution: Exploiting SNAP-tag labeling to follow the dynamics of incorporation of H3 variants.

In the eukaryotic cell nucleus, in addition to the genomic information, chromatin organization provides an additional set of information which is more versatile and associates with distinct cell identities. In particular, the marking of the nucleosomes by a choice of specific histone variants can potentially confer distinct functional properties critical for genome function and stability. To understand how this unique marking operates we need to access to the genomic distribution of each variant. A general approach based on ChIP-Seq, relies on the specific isolation of DNA bound to the variant of interest, usually using cross-linked material and specific antibodies. The availability of reliable specific antibodies recognizing with high affinity crosslinked antigen represents a limitation. Here, we describe an experimental approach exploiting a tag fused to the protein of interest. The chose protein is a histone variant and we use native conditions for the selective capture of the histone variant in a nucleosome. Most importantly, we describe how to use a particular labeling system, with a SNAP tag enabling to specifically capture nucleosomes comprising newly synthesized histones. This method allows to follow the newly deposited histone variant at various times thereby offering a unique opportunity to evaluate the dynamics of histone deposition genome wide. We describe the method here for H3 variant, but it can be adapted to any histone variant with the appropriate fused tag to address genome wide a turn-over associated to the biological context of interest.

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.

The cell-cycle choreography of H3 variants shapes the genome.

Histone variants provide versatility in the basic unit of chromatin, helping to define dynamic landscapes and cell fates. Maintaining genome integrity is paramount for the cell, and it is intimately linked with chromatin dynamics, assembly, and disassembly during DNA transactions such as replication, repair, recombination, and transcription. In this review, we focus on the family of H3 variants and their dynamics in space and time during the cell cycle. We review the distinct H3 variants' specific features along with their escort partners, the histone chaperones, compiled across different species to discuss their distinct importance considering evolution. We place H3 dynamics at different times during the cell cycle with the possible consequences for genome stability. Finally, we examine how their mutation and alteration impact disease. The emerging picture stresses key parameters in H3 dynamics to reflect on how when they are perturbed, they become a source of stress for genome integrity.

Transcription-coupled H3.3 recycling: A link with chromatin states.

Histone variant H3.3 is incorporated into chromatin throughout the cell cycle and even in non-cycling cells. This histone variant marks actively transcribed chromatin regions with high nucleosome turnover, as well as silent pericentric and telomeric repetitive regions. In the past few years, significant progress has been made in our understanding of mechanisms involved in the transcription-coupled deposition of H3.3. Here we review how, during transcription, new H3.3 deposition intermingles with the fate of the old H3.3 variant and its recycling. First, we describe pathways enabling the incorporation of newly synthesized vs old H3.3 histones in the context of transcription. We then review the current knowledge concerning differences between these two H3.3 populations, focusing on their PTMs composition. Finally, we discuss the implications of H3.3 recycling for the maintenance of the transcriptional state and underline the emerging importance of H3.3 as a potent epigenetic regulator for both maintaining and switching a transcriptional state.

CENP-A: A Histone H3 Variant with Key Roles in Centromere Architecture in Healthy and Diseased States.

Centromeres are key architectural components of chromosomes. Here, we examine their construction, maintenance, and functionality. Focusing on the mammalian centromere- specific histone H3 variant, CENP-A, we highlight its coevolution with both centromeric DNA and its chaperone, HJURP. We then consider CENP-A de novo deposition and the importance of centromeric DNA recently uncovered with the added value from new ultra-long-read sequencing. We next review how to ensure the maintenance of CENP-A at the centromere throughout the cell cycle. Finally, we discuss the impact of disrupting CENP-A regulation on cancer and cell fate.

HIRA Supports Hepatitis B Virus Minichromosome Establishment and Transcriptional Activity in Infected Hepatocytes.

BACKGROUND & AIMS: Upon hepatitis B virus (HBV) infection, partially double-stranded viral DNA converts into a covalently closed circular chromatinized episomal structure (cccDNA). This form represents the long-lived genomic reservoir responsible for viral persistence in the infected liver. Although the involvement of host cell DNA damage response in cccDNA formation has been established, this work investigated the yet-to-be-identified histone dynamics on cccDNA during early phases of infection in human hepatocytes. METHODS: Detailed studies of host chromatin-associated factors were performed in cell culture models of natural infection (ie, Na+-taurocholate cotransporting polypeptide (NTCP)-overexpressing HepG2 cells, HepG2hNTCP) and primary human hepatocytes infected with HBV, by cccDNA-specific chromatin immunoprecipitation and loss-of-function experiments during early kinetics of viral minichromosome establishment and onset of viral transcription. RESULTS: Our results show that cccDNA formation requires the deposition of the histone variant H3.3 via the histone regulator A (HIRA)-dependent pathway. This occurs simultaneously with repair of the cccDNA precursor and independently from de novo viral protein expression. Moreover, H3.3 in its S31 phosphorylated form appears to be the preferential H3 variant found on transcriptionally active cccDNA in infected cultured cells and human livers. HIRA depletion after cccDNA pool establishment showed that HIRA recruitment is required for viral transcription and RNA production. CONCLUSIONS: Altogether, we show a crucial role for HIRA in the interplay between HBV genome and host cellular machinery to ensure the formation and active transcription of the viral minichromosome in infected hepatocytes.

CD8+T cell responsiveness to anti-PD-1 is epigenetically regulated by Suv39h1 in melanomas.

Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an "epigenetic checkpoint" for tumor immunity.

CENP-A Regulation and Cancer.

In mammals, CENP-A, a histone H3 variant found in the centromeric chromatin, is critical for faithful chromosome segregation and genome integrity maintenance through cell divisions. Specifically, it has dual functions, enabling to define epigenetically the centromere position and providing the foundation for building up the kinetochore. Regulation of its dynamics of synthesis and deposition ensures to propagate proper centromeres on each chromosome across mitosis and meiosis. However, CENP-A overexpression is a feature identified in many cancers. Importantly, high levels of CENP-A lead to its mislocalization outside the centromere. Recent studies in mammals have begun to uncover how CENP-A overexpression can affect genome integrity, reprogram cell fate and impact 3D nuclear organization in cancer. Here, we summarize the mechanisms that orchestrate CENP-A regulation. Then we review how, beyond its centromeric function, CENP-A overexpression is linked to cancer state in mammalian cells, with a focus on the perturbations that ensue at the level of chromatin organization. Finally, we review the clinical interest for CENP-A in cancer 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.

H3-H4 histone chaperones and cancer.

Histone chaperones are key regulators of chromatin structure and function. Their frequent mis-regulation in various cancers can impact tumor initiation and progression. Here, we focus on H3-H4 histone chaperones to highlight recent studies concerning their roles in several cancers thereby expanding on previous reports illustrating their functions as tumor-promoting and/or as useful biomarkers for clinical applications. In particular, we discuss how imperfect compensation between H3-H4 histone chaperones favors tumor progression by stimulating the Epithelial mesenchymal transition (EMT) or the Alternative lengthening of telomeres (ALT) pathway. Finally, we present initial studies pointing towards therapies that target H3-H4 histone chaperones for cancer treatment.

CENP-A Subnuclear Localization Pattern as Marker Predicting Curability by Chemoradiation Therapy for Locally Advanced Head and Neck Cancer Patients.

Effective biomarkers predictive of the response to treatments are key for precision medicine. This study identifies the staining pattern of the centromeric histone 3 variant, CENP-A, as a predictive biomarker of locoregional disease curability by chemoradiation therapy. We compared by imaging the subnuclear distribution of CENP-A in normal and tumoral tissues, and in a retrospective study in biopsies of 62 locally advanced head and neck squamous cell carcinoma (HNSCC) patients treated by chemoradiation therapy. We looked for predictive factors of locoregional disease control and patient's survival, including CENP-A patterns, Ki67, HPV status and anisokaryosis. In different normal tissues, we reproducibly found a CENP-A subnuclear pattern characterized by CENP-A clusters both localized at the nuclear periphery and regularly spaced. In corresponding tumors, both features are lost. In locally advanced HNSCC, a specific CENP-A pattern identified in pretreatment biopsies predicts definitive locoregional disease control after chemoradiation treatment in 96% (24/25) of patients (OR = 17.6 CI 95% [2.6; 362.8], p = 0.002), independently of anisokaryosis, Ki67 labeling or HPV status. The characteristics of the subnuclear pattern of CENP-A in cell nuclei revealed by immunohistochemistry could provide an easy to use a reliable marker of disease curability by chemoradiation therapy in locally advanced HNSCC patients.

PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer.

Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers. SIGNIFICANCE: This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2888/F1.large.jpg.