PP2A inhibitor SET promotes mTORC1 and Bmi1 signaling through Akt activation and maintains the colony-formation ability of cancer cells.

Protein phosphatase 2A (PP2A) is an essential tumor suppressor, with its activity often hindered in cancer cells by endogenous PP2A inhibitory proteins like SE translocation (SET). SET/PP2A axis plays a pivotal role in the colony-formation ability of cancer cells and the stabilization of c-Myc and E2F1 proteins implicated in this process. However, in osteosarcoma cell line HOS, SET knock-down (KD) suppresses the colony-formation ability without affecting c-Myc and E2F1. This study aimed to unravel the molecular mechanism through which SET enhances the colony-formation ability of HOS cells and determine if it is generalized to other cancer cells. Transcriptome analysis unveiled that SET KD suppressed mTORC1 signaling. SET KD inhibited Akt phosphorylation, an upstream kinase for mTORC1. PP2A inhibitor blocked SET KD-mediated decrease in phosphorylation of Akt and a mTORC1 substrate p70S6K. A constitutively active Akt restored decreased colony-formation ability by SET KD, indicating the SET/PP2A/Akt/mTORC1 axis. Additionally, enrichment analysis highlighted that Bmi-1, a polycomb group protein, is affected by SET KD. SET KD decreased Bmi-1 protein by Akt inhibition but not by mTORC1 inhibition, and exogenous Bmi-1 expression rescued the reduced colony formation by SET KD. Four out of eight cancer cell lines exhibited decreased Bmi-1 by SET KD. Further analysis of these cell lines revealed that Myc activity plays a role in SET KD-mediated Bmi-1 degradation. These findings provide new insights into the molecular mechanism of SET-regulated colony-formation ability, which involved Akt-mediated activation of mTORC1/p70S6K and Bmi-1 signaling.

HIRA stabilizes skeletal muscle lineage identity.

The epigenetic mechanisms coordinating the maintenance of adult cellular lineages and the inhibition of alternative cell fates remain poorly understood. Here we show that targeted ablation of the histone chaperone HIRA in myogenic cells leads to extensive transcriptional modifications, consistent with a role in maintaining skeletal muscle cellular identity. We demonstrate that conditional ablation of HIRA in muscle stem cells of adult mice compromises their capacity to regenerate and self-renew, leading to tissue repair failure. Chromatin analysis of Hira-deficient cells show a significant reduction of histone variant H3.3 deposition and H3K27ac modification at regulatory regions of muscle genes. Additionally, we find that genes from alternative lineages are ectopically expressed in Hira-mutant cells via MLL1/MLL2-mediated increase of H3K4me3 mark at silent promoter regions. Therefore, we conclude that HIRA sustains the chromatin landscape governing muscle cell lineage identity via incorporation of H3.3 at muscle gene regulatory regions, while preventing the expression of alternative lineage genes.

Haploinsufficiency of the HIRA gene located in the 22q11 deletion syndrome region is associated with abnormal neurodevelopment and impaired dendritic outgrowth.

The 22q11.2 deletion syndrome (22q11DS) is associated with a wide spectrum of cognitive and psychiatric symptoms. Despite the considerable work performed over the past 20 years, the genetic etiology of the neurodevelopmental phenotype remains speculative. Here, we report de novo heterozygous truncating variants in the HIRA (Histone cell cycle regulation defective, S. Cerevisiae, homolog of, A) gene associated with a neurodevelopmental disorder in two unrelated patients. HIRA is located within the commonly deleted region of the 22q11DS and encodes a histone chaperone that regulates neural progenitor proliferation and neurogenesis, and that belongs to the WD40 Repeat (WDR) protein family involved in brain development and neuronal connectivity. To address the specific impact of HIRA haploinsufficiency in the neurodevelopmental phenotype of 22q11DS, we combined Hira knock-down strategies in developing mouse primary hippocampal neurons, and the direct study of brains from heterozygous Hira+/- mice. Our in vitro analyses revealed that Hira gene is mostly expressed during neuritogenesis and early dendritogenesis stages in mouse total brain and in developing primary hippocampal neurons. Moreover, shRNA knock-down experiments showed that a twofold decrease of endogenous Hira expression level resulted in an impaired dendritic growth and branching in primary developing hippocampal neuronal cultures. In parallel, in vivo analyses demonstrated that Hira+/- mice displayed subtle neuroanatomical defects including a reduced size of the hippocampus, the fornix and the corpus callosum. Our results suggest that HIRA haploinsufficiency would likely contribute to the complex pathophysiology of the neurodevelopmental phenotype of 22q11DS by impairing key processes in neurogenesis and by causing neuroanatomical defects during cerebral development.

FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links.

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.

HIRA deficiency in muscle fibers causes hypertrophy and susceptibility to oxidative stress.

Nucleosome assembly proceeds through DNA replication-coupled or replication-independent mechanisms. For skeletal myocytes, whose nuclei have permanently exited the cell cycle, replication-independent assembly is the only mode available for chromatin remodeling. For this reason, any nucleosome composition alterations accompanying transcriptional responses to physiological signals must occur through a DNA replication-independent pathway. HIRA is the histone chaperone primarily responsible for replication-independent incorporation of histone variant H3.3 across gene bodies and regulatory regions. Thus, HIRA would be expected to play an important role in epigenetically regulating myocyte gene expression. The objective of this study was to determine the consequence of eliminating HIRA from mouse skeletal myocytes. At 6 weeks of age, myofibers lacking HIRA showed no pathological abnormalities; however, genes involved in transcriptional regulation were downregulated. By 6 months of age, myofibers lacking HIRA exhibited hypertrophy, sarcolemmal perforation and oxidative damage. Genes involved in muscle growth and development were upregulated, but those associated with responses to cellular stresses were downregulated. These data suggest that elimination of HIRA produces a hypertrophic response in skeletal muscle and leaves myofibers susceptible to stress-induced degeneration.

SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin.

Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin.

Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis.

HIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis.

Transcription factor EKLF (KLF1) recruitment of the histone chaperone HIRA is essential for ß-globin gene expression.

The binding of chromatin-associated proteins and incorporation of histone variants correlates with alterations in gene expression. These changes have been particularly well analyzed at the mammalian ß-globin locus, where transcription factors such as erythroid Krüppel-like factor (EKLF), which is also known as Krüppel-like factor 1 (KLF1), play a coordinating role in establishing the proper chromatin structure and inducing high-level expression of adult ß-globin. We had previously shown that EKLF preferentially interacts with histone H3 and that the H3.3 variant is differentially recruited to the ß-globin promoter. We now find that a novel interaction between EKLF and the histone cell cycle regulation defective homolog A (HIRA) histone chaperone accounts for these effects. HIRA is not only critical for ß-globin expression but is also required for activation of the erythropoietic regulators EKLF and GATA binding protein 1 (GATA1). Our results provide a mechanism by which transcription factor-directed recruitment of a generally expressed histone chaperone can lead to tissue-restricted changes in chromatin components, structure, and transcription at specific genomic sites during differentiation.

Histone chaperone Chz1 facilitates the disfavouring property of Spt16 to H2A.Z-containing genes in Saccharomyces cerevisiae.

Htz1 (histone 2A Z1) deposition at promoters is involved in the transcriptional activation of quiescent genes. Chz1 [chaperone for Htz1 (or H2A)-H2B dimer] is an Htz1-H2B-specific chaperone that delivers histone H2A.Z that substitutes for H2A. Spt16 (suppressor of Ty) functions in transcription elongation and also possesses a histone chaperone activity. However, the links among Chz1, Htz1 and Spt16 remain unknown. In the present study, we determined the genomic binding profiling of Htz1, Pol II (RNA polymerase II) and Spt16 using ChIP microarray experiments and sequenced nucleosomal DNA using a next-generation sequencing technique in wild-type and chz1-deletion strains of Saccharomyces cerevisiae. The results of the present study revealed that Spt16 and Pol II are associated, bind at nucleosome-depleted regions, and are positively correlated with the transcription rate. Importantly, Spt16 disfavours the Htz1-bound genes, and this discrimination is impaired upon the deletion of chz1. The negative correlation between the binding profiles of Spt16 and Htz1 at promoters is not an intrinsic repulsion, but is probably due to a requirement for transcription initiation. We showed that chz1 deletion decreases Htz1 binding at promoters and telomeres. Also, in the chz1-deletion mutant, Spt16 binding at ribosomal genes was lost. The results of the present study suggest that the discrimination of Spt16 to Htz1-bound genes is due to the priority of Chz1 over Spt16 in binding to the Htz1-bound genomic regions. Chz1-escorted Htz1 therefore impairs Spt16 binding at chromatin.

HP1-mediated formation of alternative lengthening of telomeres-associated PML bodies requires HIRA but not ASF1a.

Approximately 10% of cancers use recombination-mediated Alternative Lengthening of Telomeres (ALT) instead of telomerase to prevent telomere shortening. A characteristic of cells that utilize ALT is the presence of ALT-associated PML nuclear bodies (APBs) containing (TTAGGG)n DNA, telomere binding proteins, DNA recombination proteins, and heterochromatin protein 1 (HP1). The function of APBs is unknown and it is possible that they are functionally heterogeneous. Most ALT cells lack functional p53, and restoration of the p53/p21 pathway in these cells results in growth arrest/senescence and a substantial increase in the number of large APBs that is dependent on two HP1 isoforms, HP1α and HP1γ. Here we investigated the mechanism of HP1-mediated APB formation, and found that histone chaperones, HIRA and ASF1a, are present in APBs following activation of the p53/p21 pathway in ALT cells. HIRA and ASF1a were also found to colocalize inside PML bodies in normal fibroblasts approaching senescence, providing evidence for the existence of a senescence-associated ASF1a/HIRA complex inside PML bodies, consistent with a role for these proteins in induction of senescence in both normal and ALT cells. Moreover, knockdown of HIRA but not ASF1a significantly reduced p53-mediated induction of large APBs, with a concomitant reduction of large HP1 foci. We conclude that HIRA, in addition to its physical and functional association with ASF1a, plays a unique, ASF1a-independent role, which is required for the localization of HP1 to PML bodies and thus for APB formation.