FACT mediates the depletion of macroH2A1.2 to expedite gene transcription.

The histone variant macroH2A is generally linked to transcriptionally inactive chromatin, but how macroH2A regulates chromatin structure and functions in the transcriptional process remains elusive. This study reveals that while the integration of human macroH2A1.2 into nucleosomes does not affect their stability or folding dynamics, it notably hinders the maintenance of facilitates chromatin transcription's (FACT's) function. We show that FACT effectively diminishes the stability of macroH2A1.2-nucleosomes and expedites their depletion subsequent to the initial unfolding process. Furthermore, we identify the residue S139 in macroH2A1.2 as a critical switch to modulate FACT's function in nucleosome maintenance. Genome-wide analyses demonstrate that FACT-mediated depletion of macroH2A-nucleosomes allows the correct localization of macroH2A, while the S139 mutation reshapes macroH2A distribution and influences stimulation-induced transcription and cellular response in macrophages. Our findings provide mechanistic insights into the intricate interplay between macroH2A and FACT at the nucleosome level and elucidate their collective role in transcriptional regulation and immune response of macrophages.

Epigenetic Regulation of DNA Repair Pathway Choice by MacroH2A1 Splice Variants Ensures Genome Stability.

The inactive X chromosome (Xi) is inherently susceptible to genomic aberrations. Replication stress (RS) has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that macroH2A1.2, an RS-protective histone variant enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its structurally distinct and equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics identified a role for macroH2A1.1 in alternative end joining (alt-EJ), which accounts for Xi anaphase defects in the absence of macroH2A1.2. Genomic instability was rescued by simultaneous depletion of macroH2A1.1 or alt-EJ factors, and mice deficient for both macroH2A1 variants harbor no overt female defects. Notably, macroH2A1 splice variant imbalance affected alt-EJ capacity also in tumor cells. Together, these findings identify macroH2A1 splicing as a modulator of genome maintenance that ensures Xi integrity and may, more broadly, predict DNA repair outcome in malignant cells.

DNA Damage Signaling-Induced Cancer Cell Reprogramming as a Driver of Tumor Relapse.

Accumulating evidence supports the role of the DNA damage response (DDR) in the negative regulation of tumorigenesis. Here, we found that DDR signaling poises a series of epigenetic events, resulting in activation of pro-tumorigenic genes but can go as far as reactivation of the pluripotency gene OCT4. Loss of DNA methylation appears to be a key initiating event in DDR-dependent OCT4 locus reactivation although full reactivation required the presence of a driving oncogene, such as Myc and macroH2A downregulation. Using genetic-lineage-tracing experiments and an in situ labeling approach, we show that DDR-induced epigenetic reactivation of OCT4 regulates the resistance to chemotherapy and contributes to tumor relapse both in mouse and primary human cancers. In turn, deletion of OCT4 reverses chemoresistance and delays the relapse. Here, we uncovered an unexpected tumor-promoting role of DDR in cancer cell reprogramming, providing novel therapeutic entry points for cancer intervention strategies.

Replication Stress Shapes a Protective Chromatin Environment across Fragile Genomic Regions.

Recent integrative epigenome analyses highlight the importance of functionally distinct chromatin states for accurate cell function. How these states are established and maintained is a matter of intense investigation. Here, we present evidence for DNA damage as an unexpected means to shape a protective chromatin environment at regions of recurrent replication stress (RS). Upon aberrant fork stalling, DNA damage signaling and concomitant H2AX phosphorylation coordinate the FACT-dependent deposition of macroH2A1.2, a histone variant that promotes DNA repair by homologous recombination (HR). MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from RS-induced DNA damage. Consequently, replicating primary cells steadily accrue macroH2A1.2 at fragile regions, whereas macroH2A1.2 loss in these cells triggers DNA damage signaling-dependent senescence, a hallmark of RS. Altogether, our findings demonstrate that recurrent DNA damage contributes to the chromatin landscape to ensure the epigenomic integrity of dividing cells.

Evolution, structure and function of divergent macroH2A1 splice isoforms.

The replacement of replication-coupled histones with non-canonical histone variants provides chromatin with additional properties and contributes to the plasticity of the epigenome. MacroH2A histone variants are counterparts of the replication-coupled histone H2A. They are characterized by a unique tripartite structure, consisting of a histone fold, an unstructured linker, and a globular macrodomain. MacroH2A1.1 and macroH2A1.2 are the result of alternative splicing of the MACROH2A1 gene and can have opposing biological functions. Here, we discuss the structural differences between the macrodomains of the two isoforms, resulting in differential ligand binding. We further discuss how this modulates gene regulation by the two isoforms, in cases resulting in opposing role of macroH2A1.1 and macroH2A1.2 in development and differentiation. Finally, we share recent insight in the evolution of macroH2As. Taken together, in this review, we aim to discuss in unprecedented detail distinct properties and functions of the fascinating macroH2A1 splice isoforms.

Histone H2A variants: Diversifying chromatin to ensure genome integrity.

Histone variants represent chromatin components that diversify the structure and function of the genome. The variants of H2A, primarily H2A.X, H2A.Z and macroH2A, are well-established participants in DNA damage response (DDR) pathways, which function to protect the integrity of the genome. Through their deposition, post-translational modifications and unique protein interaction networks, these variants guard DNA from endogenous threats including replication stress and genome fragility as well as from DNA lesions inflicted by exogenous sources. A growing body of work is now providing a clearer picture on the involvement and mechanistic basis of H2A variant contribution to genome integrity. Beyond their well-documented role in gene regulation, we review here how histone H2A variants promote genome stability and how alterations in these pathways contribute to human diseases including cancer.

Histone renegades: Unusual H2A histone variants in plants and animals.

H2A variants are histones that carry out specialized nucleosome function during the eukaryote genome packaging. Most genes encoding H2A histone variants arose in the distant past, and have highly conserved domains and structures. Yet, novel H2A variants have continued to arise throughout the radiation of eukaryotes and disturbed the apparent tranquility of nucleosomes. These species-specific H2A variants contributed to the functional diversification of nucleosomes through changes in both their structure and expression patterns. In this short review, we discuss the evolutionary trajectories of these histone renegades in plants and animal genomes.

Distinct nuclear and cytoplasmic assemblies and interactomes of the mammalian CTLH E3 ligase complex.

The C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit E3 ubiquitin ligase and its cellular functions are poorly characterized. Although some CTLH subunits have been found to localize in both the nucleus and cytoplasm of mammalian cells, differences between the compartment-specific complexes have not been explored. Here, we show that the CTLH complex forms different molecular mass complexes in nuclear and cytoplasmic fractions. Loss of WDR26 severely decreased nuclear CTLH complex subunit levels and impaired higher-order CTLH complex formation, revealing WDR26 as a critical determinant of the nuclear stability of the CTLH complex. Through affinity purification coupled to mass spectrometry of endogenous RanBPM (also called RANBP9), a CTLH complex member, from nuclear and cytoplasmic fractions, we identified over 170 compartment-specific interactors involved in various conserved biological processes, such as ribonucleoprotein biogenesis and chromatin assembly. We validated the nuclear-specific RanBPM interaction with macroH2A1 and the cytoplasm-specific interaction with tankyrase-1/2 (encoded by TNKS and TNKS2). Overall, this study provides critical insights into CTLH complex function and composition in both the cytoplasm and nucleus.

Histone Variant macroH2A1.1 Enhances Nonhomologous End Joining-dependent DNA Double-strand-break Repair and Reprogramming Efficiency of Human iPSCs.

DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.

MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors.

The development of the nervous system requires precise regulation. Any disturbance in the regulation process can lead to neurological developmental diseases, such as autism and schizophrenia. Histone variants are important components of epigenetic regulation. The function and mechanisms of the macroH2A (mH2A) histone variant during brain development are unknown. Here, we show that deletion of the mH2A isoform mH2A1.2 interferes with neural stem cell differentiation in mice. Deletion of mH2A1.2 affects neurodevelopment, enhances neural progenitor cell (NPC) proliferation, and reduces NPC differentiation in the developing mouse brain. mH2A1.2-deficient mice exhibit autism-like behaviors, such as deficits in social behavior and exploratory abilities. We identify NKX2.2 as an important downstream effector gene and show that NKX2.2 expression is reduced after mH2A1.2 deletion and that overexpression of NKX2.2 rescues neuronal abnormalities caused by mH2A1.2 loss. Our study reveals that mH2A1.2 reduces the proliferation of neural progenitors and enhances neuronal differentiation during embryonic neurogenesis and that these effects are at least in part mediated by NKX2.2. These findings provide a basis for studying the relationship between mH2A1.2 and neurological disorders.

Genetic basis of thermal nociceptive sensitivity and brain weight in a BALB/c reduced complexity cross.

Thermal nociception involves the transmission of temperature-related noxious information from the periphery to the CNS and is a heritable trait that could predict transition to persistent pain. Rodent forward genetics complement human studies by controlling genetic complexity and environmental factors, analysis of end point tissue, and validation of variants on appropriate genetic backgrounds. Reduced complexity crosses between nearly identical inbred substrains with robust trait differences can greatly facilitate unbiased discovery of novel genes and variants. We found BALB/cByJ mice showed enhanced sensitivity on the 53.5°C hot plate and mechanical stimulation in the von Frey test compared to BALB/cJ mice and replicated decreased gross brain weight in BALB/cByJ versus BALB/cJ. We then identified a quantitative trait locus (QTL) on chromosome 13 for hot plate sensitivity (LOD = 10.7; p < 0.001; peak = 56 Mb) and a QTL for brain weight on chromosome 5 (LOD = 8.7; p < 0.001). Expression QTL mapping of brain tissues identified H2afy (56.07 Mb) as the top transcript with the strongest association at the hot plate locus (FDR = 0.0002) and spliceome analysis identified differential exon usage within H2afy associated with the same locus. Whole brain proteomics further supported decreased H2AFY expression could underlie enhanced hot plate sensitivity, and identified ACADS as a candidate for reduced brain weight. To summarize, a BALB/c reduced complexity cross combined with multiple-omics approaches facilitated identification of candidate genes underlying thermal nociception and brain weight. These substrains provide a powerful, reciprocal platform for future validation of candidate variants.

Systemic depletion of histone macroH2A1.1 boosts hippocampal synaptic plasticity and social behavior in mice.

Gene expression and epigenetic processes in several brain regions regulate physiological processes such as cognitive functions and social behavior. MacroH2A1.1 is a ubiquitous variant of histone H2A that regulates cell stemness and differentiation in various organs. Whether macroH2A1.1 has a modulatory role in emotional behavior is unknown. Here, we employed macroH2A1.1 knock-out (-/- ) mice to perform a comprehensive battery of behavioral tests, and an assessment of hippocampal synaptic plasticity (long-term potentiation) accompanied by whole hippocampus RNA sequencing. MacroH2A1.1-/- mice exhibit a stunningly enhancement both of sociability and of active stress-coping behavior, reflected by the increased social behavior in social activity tests and higher mobility time in the forced swim test, respectively. They also display an increased hippocampal synaptic plasticity, accompanied by significant neurotransmission transcriptional networks changes. These results suggest that systemic depletion of histone macroH2A1.1 supports an epigenetic control necessary for hippocampal function and social behavior.

The diagnostic and prognostic value of H2AFY in hepatocellular carcinoma.

BACKGROUND: The potential correlation between H2AFY (also known as MacroH2A1) and the clinical characteristics of hepatocellular carcinoma (HCC) patients was analysed through gene expression profiles and clinical data in The Cancer Genome Atlas (TCGA) database, and the diagnostic and prognostic value of H2AFY in HCC was discussed. METHODS: The gene expression data of HCC and the corresponding clinical characteristics of HCC patients were downloaded from the TCGA database. The differences in H2AFY in normal liver tissues and HCC were analysed. The relationship between H2AFY and clinical characteristics was analysed by Wilcoxon signed-rank test, logistic regression and Kruskal-Wallis test. The Kaplan-Meier method and the Cox regression method were used to analyse the relationship between overall survival and clinical characteristics of the patients. An ROC curve was used to predict the diagnostic value of H2AFY in HCC. Gene set enrichment analysis (GSEA) was used to analyse the pathway enrichment of H2AFY. RESULT: Compared with normal liver tissues, H2AFY was significantly highly expressed in HCC. H2AFY was positively correlated with the age, clinical stage, G stage (grade) and T stage (tumor stage) of liver cancer patients. Higher H2AFY expression predicted a poor prognosis in HCC patients. Cox regression analysis suggested that H2AFY was an independent risk factor for the prognosis of HCC patients. The ROC curve suggested that H2AFY had certain diagnostic value in HCC. GSEA suggested that H2AFY was correlated with lipid metabolism and a variety of tumour pathways. CONCLUSION: Our study showed that H2AFY was significantly overexpressed in HCC. H2AFY may be a potential diagnostic and prognostic marker for HCC, and high expression of H2AFY predicts a poor prognosis in patients with HCC.

Epigenetic Regulation of Myogenesis: Focus on the Histone Variants.

Skeletal muscle development and regeneration rely on the successive activation of specific transcription factors that engage cellular fate, promote commitment, and drive differentiation. Emerging evidence demonstrates that epigenetic regulation of gene expression is crucial for the maintenance of the cell differentiation status upon division and, therefore, to preserve a specific cellular identity. This depends in part on the regulation of chromatin structure and its level of condensation. Chromatin architecture undergoes remodeling through changes in nucleosome composition, such as alterations in histone post-translational modifications or exchange in the type of histone variants. The mechanisms that link histone post-translational modifications and transcriptional regulation have been extensively evaluated in the context of cell fate and differentiation, whereas histone variants have attracted less attention in the field. In this review, we discuss the studies that have provided insights into the role of histone variants in the regulation of myogenic gene expression, myoblast differentiation, and maintenance of muscle cell identity.

MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms.

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP-ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1-dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform-specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.

A Lipidomic Signature Complements Stemness Features Acquisition in Liver Cancer Cells.

Lipid catabolism and anabolism changes play a role in stemness acquisition by cancer cells, and cancer stem cells (CSCs) are particularly dependent on the activity of the enzymes involved in these processes. Lipidomic changes could play a role in CSCs' ability to cause disease relapse and chemoresistance. The exploration of lipid composition and metabolism changes in CSCs in the context of hepatocellular cancer (HCC) is still incomplete and their lipidomic scenario continues to be elusive. We aimed to evaluate through high-throughput mass spectrometry (MS)-based lipidomics the levels of the members of the six major classes of sphingolipids and phospholipids in two HCC cell lines (HepG2 and Huh-7) silenced for the expression of histone variant macroH2A1 (favoring stemness acquisition), or silenced for the expression of focal adhesion tyrosine kinase (FAK) (hindering aggressiveness and stemness). Transcriptomic changes were evaluated by RNA sequencing as well. We found definite lipidomic and transcriptomic changes in the HCC lines upon knockdown (KD) of macroH2A1 or FAK, in line with the acquisition or loss of stemness features. In particular, macroH2A1 KD increased total sphingomyelin (SM) levels and decreased total lysophosphatidylcholine (LPC) levels, while FAK KD decreased total phosphatidylcholine (PC) levels. In conclusion, in HCC cell lines knocked down for specific signaling/epigenetic processes driving opposite stemness potential, we defined a lipidomic signature that hallmarks hepatic CSCs to be exploited for therapeutic strategies.

Histone variant MacroH2A1 is downregulated in prostate cancer and influences malignant cell phenotype.

BACKGROUND: Prostate cancer (PCa), a major cause of cancer-related morbidity and mortality worldwide and mostly asymptomatic at earliest stages, is characterized by disruption of genetic and epigenetic balance. A better understanding of how those mechanisms orchestrate disease might improve diagnostic and prognostic tools, allowing for improvements in treatment efficacy. Replacement of canonical histones, an epigenetic mechanism, is highly conserved among species and altered expression of histones variants (e.g., MacroH2A1) has been associated with tumorigenesis. H2AFY gene encodes two isoforms of H2A histone variant MacroH2A1: MacroH2A1.1 and MacroH2A1.2. Specifically, MacroH2A1.1 isoform inhibits cell proliferation and promotes cellular differentiation. Because the contribution of this histone variant to carcinogenesis has been reported in several cancer types, but not for PCa, we aimed to investigate the contribution of MacroH2A1 for prostate carcinogenesis. METHODS: MacroH2A1, MacroH2A1.1 and MacroH2A1.2 isoforms and the corresponding splicing regulators transcript levels were evaluated by RT-qPCR, in a tissue cohort composed by PCa, prostatic intraepithelial neoplasia (PIN) and normal prostate cases. Knockdown for MacroH2A1 and MacroH2A1.1 was performed through lentiviral transduction in DU145 cells, and MacroH2A1.1 overexpression was achieved in LNCaP cells by plasmid transfection, followed by functional assays. Biological and/or experimental replicates were performed when necessary, and specific statistical tests were applied to perform data analysis. RESULTS: MacroH2A1.1 transcript levels were downregulated in PIN and primary PCa compared to normal prostate tissues. The same was found for QKI, a MacroH2A1.1's splicing regulator. Moreover, lower MacroH2A1.1 and QKI expression levels associated with less differentiated tumors (Gleason score ≥ 7). Interestingly, MacroH2A1.1, but more impressively DDX17 (AUC = 0.93; p < 0.0001) and QKI (AUC = 0.94; p < 0.0001), accurately discriminated cancerous from noncancerous prostate tissues. Furthermore, in PCa cell lines, total MacroH2A1 knockdown augmented malignant features, whereas MacroH2A1.1 overexpression impressively attenuated the malignant phenotype. CONCLUSIONS: Overall, our data, derived from primary PCa tissues and cell lines, anticipate a tumor suppressive role for MacroH2A1, particularly for the MacroH2A1.1 isoform, in prostate carcinogenesis.

Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts.

Induction of pluripotency in differentiated cells through the exogenous expression of the transcription factors Oct4, Sox2, Klf4 and cellular Myc involves reprogramming at the epigenetic level. Histones and their metabolism governed by histone chaperones constitute an important regulator of epigenetic control. We hypothesized that histone chaperones facilitate or inhibit the course of reprogramming. For the first time, we report here that the downregulation of histone chaperone Aprataxin PNK-like factor (APLF) promotes reprogramming by augmenting the expression of E-cadherin (Cdh1), which is implicated in the mesenchymal-to-epithelial transition (MET) involved in the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Downregulation of APLF in MEFs expedites the loss of the repressive MacroH2A.1 (encoded by H2afy) histone variant from the Cdh1 promoter and enhances the incorporation of active histone H3me2K4 marks at the promoters of the pluripotency genes Nanog and Klf4, thereby accelerating the process of cellular reprogramming and increasing the efficiency of iPSC generation. We demonstrate a new histone chaperone (APLF)-MET-histone modification cohort that functions in the induction of pluripotency in fibroblasts. This regulatory axis might provide new mechanistic insights into perspectives of epigenetic regulation involved in cancer metastasis.

Xist repression shows time-dependent effects on the reprogramming of female somatic cells to induced pluripotent stem cells.

Although the reactivation of silenced X chromosomes has been observed as part of the process of reprogramming female somatic cells into induced pluripotent stem cells (iPSCs), it remains unknown whether repression of the X-inactive specific transcript (Xist) can greatly enhance female iPSC induction similar to that observed in somatic cell nuclear transfer studies. In this study, we discovered that the repression of Xist plays opposite roles in the early and late phases of female iPSCs induction. Our results demonstrate that the downregulation of Xist by an isopropyl ß-d-1-thiogalactopyranoside (IPTG)-inducible short hairpin RNA (shRNA) system can greatly impair the mesenchymal-to-epithelial transition (MET) in the early phase of iPSC induction but can significantly promote the transition of pre-iPSCs to iPSCs in the late phase. Furthermore, we demonstrate that although the knockdown of Xist did not affect the H3K27me3 modification on the X chromosome, macroH2A was released from the inactivated X chromosome (Xi). This enables the X chromosome silencing to be a reversible event. Moreover, we demonstrate that the supplementation of vitamin C (Vc) can augment and stabilize the reversible X chromosome by preventing the relocalization of macroH2A to the Xi. Therefore, our study reveals an opposite role of Xist repression in the early and late stages of reprogramming female somatic cells to pluripotency and demonstrates that the release of macroH2A by Xist repression enables the transition from pre-iPSCs to iPSCs.

macroH2A1 drives nucleosome dephasing and genome instability in histone humanized yeast.

In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones, providing additional layers of structural and epigenetic regulation. Here, we systematically replace individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. We show that variants H2A.J, TsH2B, and H3.5 complement their respective replicative counterparts. However, macroH2A1 fails to complement, and its overexpression is toxic in yeast, negatively interacting with yeast's native histones and kinetochore genes. To isolate yeast with macroH2A1 chromatin, we uncouple the effects of its macro and histone fold domains, revealing that both domains suffice to override native nucleosome positioning. Furthermore, both uncoupled constructs of macroH2A1 exhibit lower nucleosome occupancy, decreased short-range chromatin interactions (<20 kb), disrupted centromeric clustering, and increased chromosome instability. Our observations demonstrate that lack of a canonical histone H2A dramatically alters chromatin organization in yeast, leading to genome instability and substantial fitness defects.