Canonical Histone Research Articles

Abstract 212: Sarcoma-CellMiner: An extensive resource for patient-derived sarcoma cell line epigenetics, genomics and pharmacology

Abstract Sarcomas represent a heterogeneous group of cancers with many histological subtypes. Their prognosis remains poor and treatment is mainly based on palliative chemotherapy at metastatic stage. Tumor genome sequencing failed to identify recurrent somatic drivers while several oncogenic gene fusion-translocations have been identified in specific sarcoma subtypes. Because of the rarity and heterogeneity of sarcomas, only few sarcoma patients are enrolled in clinicals trials according their subtype. Increased translational research according subtypes of sarcoma patients is needed to improve patient management. To characterize the differences between subgroups and build translational hypotheses, we built a novel resource, Sarcoma-Cellminer, which integrates drug sensitivity and genomic data from 112 patient-derived sarcoma cell lines. These data will be available from a web-based tool (https://discover.nci.nih.gov/SclcCellMinerCDB/) derived from our CellMiner cross-database web application (https://discover.nci.nih.gov/cellminercdb). Among the 112 cell lines, 65 are bone sarcomas (including 38 Ewing sarcomas and 22 osteosarcomas), 45 are soft tissue sarcomas (including 21 rhabdomyosarcomas). Transcriptome (RNAseq and microarray), copy number, microRNA, genome-wide methylation, and drug sensitivity data are included and made publicly available. We also generated new genomic data including copy number and methylation (850 k) for 79 cell lines from the NCI in addition to the 42 cell lines from Broad Institute (CCLE) and the 40 cell lines from the MGH-Sanger (GDSC). We created the “SCLC-Global” expression set by regrouping all datasets by Z-score normalization, which enables cross-database analyses of gene expression and molecular pathways. Hierarchical clustering based on expression and methylation data identifies subtypes of sarcomas. Histone genes stand out suggesting that epigenetic regulation of canonical histones is a feature of sarcoma genesis. Sarcoma-CellMiner includes drug sensitivity data for over 500 different drugs tested in the NCI, CCLE and GDSC databases. They show two subgroups of Ewing sarcomas: one sensitive to PARP inhibitors and one resistant. Similarly, profile of response to dasatinib is different when comparing alveolar and embryonal rhabdomyosarcomas. Sarcoma-CellMiner is a powerful tool demonstrating the value of patient-derived cancer cell line databases. It provides hypothesis-driven rationale for using omics, especially transcriptome and epigenetic data to better understand sarcoma heterogeneity and select personalized treatments for clinical trials. Citation Format: Camille Tlemsani, Lorinc Pongor, Javed Khan, Fathi Elloumi, Sudhir Varma, Augustin Luna, Vinodh Rajapakse, Kurt Kohn, Julia Krushkal, Mirit Aladjem, Beverly Teicher, Paul Meltzer, William Reinhold, Christine Heske, Yves Pommier. Sarcoma-CellMiner: An extensive resource for patient-derived sarcoma cell line epigenetics, genomics and pharmacology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 212.

Quantitative proteomics reveals arsenic attenuates stem‐loop binding protein stability via a chaperone complex containing heat shock proteins and ERp44

Arsenic pollution impacts health of millions of people in the world. Inorganic arsenic is a carcinogenic agent in skin and lung cancers. The stem-loop binding protein (SLBP) binds to the stem-loop of the canonical histone mRNA and regulates its metabolism during cell cycle. Our previous work has shown arsenic induces ubiquitin-proteasome dependent degradation of SLBP and contributes to lung cancer. In this study, we established the first comprehensive SLBP interaction network by affinity purification-mass spectrometry (AP-MS) analysis, and further demonstrated arsenic enhanced the association between SLBP and a crucial chaperone complex containing heat shock proteins (HSPs) and ERp44. Strikingly, knockdown of these proteins markedly rescued the protein level of SLBP under arsenic exposure conditions, and abolished the increasing migration capacity of BEAS-2B cells induced by arsenic. Taken together, our study provides a potential new mechanism that a chaperone complex containing HSPs and ERp44 attenuates the stability of SLBP under both normal and arsenic exposure conditions, which could be essential for arsenic-induced high cell migration.

The Role of Histone Variant H3.3 Regulation By Hira in Hematopoiesis

A large proportion of adult hematopoietic stem cells (HSCs) remain quiescent throughout life, rarely undergoing cell division. These quiescent HSCs must undergo global changes in gene expression patterns in order to leave their dormant state and differentiate. Chromatin modifiers that act independent of cell division may therefore be of particular importance in regulating the behavior of quiescent HSCs through alterations in the chromatin landscape.One such chromatin modifier is the histone variant H3.3 chaperone, Hira, which is responsible for incorporating H3.3 independent of DNA replication at poised and transcriptionally active promoters. Despite the fact that this histone variant only differs from the canonical H3 histones by four or five amino acids, its gene-specific association is required for the proper establishment of gene expression and lineage specification in murine embryonic stem cells (mESCs), post-mitotic neurons, and oocytes. Since Hira deposits H3.3 outside of S phase, we hypothesize that Hira plays a critical role in the maintenance of quiescent HSCs through the regulation of H3.3 independent of cell division.HSCs divide rapidly in the fetal liver but become quiescent in adulthood, providing an interesting opportunity to study HSCs that are vastly different in terms of cell cycle properties. In order to study the role of Hira in HSCs, we used a conditional knockout (cKO) mouse model to delete Hira in hematopoietic cells in early development (Vav-iCre; Hirafl/fl). Importantly, we observed severe depletion of adult, but not fetal, HSCs upon loss of Hira. This suggests that Hira is important for the maintenance of quiescent adult HSCs, but not rapidly dividing fetal HSCs, and supports our model of Hira as a regulator of HSC quiescence through the DNA replication-independent incorporation of H3.3. We additionally observed altered differentiation potential, reduced reconstitution capacity, and increased cycling in adult HSCs upon Hira cKO. These results demonstrate that Hira is an essential regulatory factor for the control of adult HSC function.To obtain adult Hira cKO HSCs for further analysis, we also used an inducible model of Hira deletion in the hematopoietic system upon polyinosinic-polycytidylic (pIpC) treatment (Mx1-Cre; Hirafl/fl). We performed RNA-seq analysis on adult Hira cKO HSCs, which showed widespread alterations in gene expression patterns and upregulation of PRC2 target genes. Since Hira is known to recruit polycomb repressive complex 2 (PRC2) to bivalent promoters in mESCs in an H3.3-dependent manner (Banaszynski et. al. Cell 2013), chromatin-immunoprecipitation and sequencing (ChIP-seq) for H3K27me3 will be used to show how loss of Hira affects PRC2 activity in HSCs. We have additionally performed assay for transposase-accessible chromatin and sequencing (ATAC-seq) in order to determine how patterns of DNA accessibility in HSCs are affected by Hira cKO. Altered nucleosome positioning and PRC2 activity will be discussed as potential mechanisms for the role of Hira in regulating the behavior of quiescent HSCs.Our findings reveal a novel paradigm for epigenetic state regulation in adult HSCs. Knowledge gained from this study may inform future research for clinical applications, such as for improved methods of in vitro HSC expansion. Characterizing the importance of H3.3 regulation by Hira in normal HSC activity will therefore provide invaluable information regarding the epigenetic mechanisms underlying hematopoietic homeostasis. DisclosuresNo relevant conflicts of interest to declare.

Histone H4: Genomic identification and function analysis in pearl oyster Pinctada fucata martensii

Histone H4 together with histones H2A, H2B and H3 constitutes the nucleosome and is involved in DNA packaging and regulation of immune response. In this study, we identified 123 H4 genes from nine species by compressive genomic analysis, and H4 gene was contracted with only two members (designed as PmH4-1 and PmH4-2) in the genome of pearl oyster Pinctada fucata martensii. Evolution analysis showed that this contraction happened after the divergence of P. f. martensii and Crassostrea gigas. The full-length cDNA of PmH4-1 is 561 bp, including an open reading frame (ORF) of 312 bp, a 5′-untranslated region (UTR) of 37 bp and a 3′-UTR of 212 bp. The full-length cDNA of PmH4-2 is 556 bp, containing an ORF of 342 bp, a 5′-UTR of 84 bp and a 3′-UTR of 130 bp. Sequence analysis indicated that PmH4-1 was one canonical histone, and PmH4-2 was considered as a variant H4 that showed different structure and have only 55.34% identity with the canonical H4. Tissue expression pattern showed that PmH4-1 was highly expressed in the gonad, and PmH4-2 was mostly expressed in the mantle edge. After lipopolysaccharide stimulation, PmH4-2 was highly induced, and PmH4-1 was slightly upregulated without significance, indicating that PmH4-2 participates in the innate immune responses in pearl oyster P. f. martensii. These results lay the foundation for the study of the H4 family and provide theoretical support for understanding the immune defence mechanism of pearl oyster.

Depletion of Stem‐loop binding protein and polyadenylation of canonical histone mRNAs by nicotine

The use of electronic-cigarettes has increased substantially in recent years among the younger population in particular. The liquid nicotine is the main component of the e-cigarette. Mice exposed to e-cigarette smoke (ECS) generated by heating the liquid nicotine developed lung adenocarcinoma and bladder hyperplasia. This implicated a potential role for ECS/nicotine in cancer development, while the underlying mechanisms remain unclear. Here we report that nicotine reduces the protein level of Stem-loop binding protein (SLBP), a critical factor for the stability, processing and translation of canonical histone pre-mRNAs, in human lung epithelial BEAS-2B cells. It has been known that canonical histone mRNAs typically do not end in a poly(A) tail and that depletion of SLBP results in polyadenylation of canonical histone mRNAs that appeared to be carcinogenic. Indeed, nicotine exposure induced polyadenylation of mRNAs for histones H2A, H2B, H3.1, H3.2, and H4. Interestingly, the inhibitor for nAChRs (nicotinic acetylcholine receptors) reversed nicotine-induced SLBP depletion. Moreover, the depletion of SLBP was attenuated not only by both the inhibitors and siRNAs specific for CDK1/2 and CK2, but also by inhibition of AKT signal pathway. Importantly, nicotine-induced anchorage-independent cell growth was rescued by overexpression of SLBP. These results suggest that activation of nAChR and a series of signal transduction pathways are involved in nicotine-mediated SLBP reduction and they may play an important role in nicotine-induced cell transformation and carcinogenesis.

Combinatorial Histone H3 Modifications Are Dynamically Altered in Distinct Cell Cycle Phases.

The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.

An updated map of Trypanosoma cruzi histone post-translational modifications

In humans and other eukaryotes, histone post-translational modifications (hPTMs) play an essential role in the epigenetic control of gene expression. In trypanosomatid parasites, conversely, gene regulation occurs mainly at the post-transcriptional level. However, our group has recently shown that hPTMs are abundant and varied in Trypanosoma cruzi, the etiological agent of Chagas Disease, signaling for possible conserved epigenetic functions. Here, we applied an optimized mass spectrometry-based proteomic workflow to provide a high-confidence comprehensive map of hPTMs, distributed in all canonical, variant and linker histones of T. cruzi. Our work expands the number of known T. cruzi hPTMs by almost 2-fold, representing the largest dataset of hPTMs available to any trypanosomatid to date, and can be used as a basis for functional studies on the dynamic regulation of chromatin by epigenetic mechanisms and the selection of candidates for the development of epigenetic drugs against trypanosomatids.

A KDM5-Prospero transcriptional axis functions during early neurodevelopment to regulate mushroom body formation.

Mutations in the lysine demethylase 5 (KDM5) family of transcriptional regulators are associated with intellectual disability, yet little is known regarding their spatiotemporal requirements or neurodevelopmental contributions. Utilizing the mushroom body (MB), a major learning and memory center within the Drosophila brain, we demonstrate that KDM5 is required within ganglion mother cells and immature neurons for proper axogenesis. Moreover, the mechanism by which KDM5 functions in this context is independent of its canonical histone demethylase activity. Using in vivo transcriptional and binding analyses, we identify a network of genes directly regulated by KDM5 that are critical modulators of neurodevelopment. We find that KDM5 directly regulates the expression of prospero, a transcription factor that we demonstrate is essential for MB morphogenesis. Prospero functions downstream of KDM5 and binds to approximately half of KDM5-regulated genes. Together, our data provide evidence for a KDM5-Prospero transcriptional axis that is essential for proper MB development.

Kaposi's Sarcoma-Associated Herpesvirus Processivity Factor, ORF59, Binds to Canonical and Linker Histones, and Its Carboxy Terminus Is Dispensable for Viral DNA Synthesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus and the causative agent of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. During lytic reactivation, there is a temporal cascade of viral gene expression that results in the production of new virions. One of the viral factors that is expressed during reactivation is open reading frame 59 (ORF59), the viral DNA polymerase processivity factor. ORF59 plays an essential role for DNA synthesis and is required for the nuclear localization of the viral DNA polymerase (ORF9) to the origin of lytic replication (oriLyt). In addition to its functions in viral DNA synthesis, ORF59 has been shown to interact with chromatin complexes, including histones and cellular methyltransferases. In this study, a series of KSHV BACmids containing 50-amino acid (aa) deletions within ORF59 were generated to determine the interaction domains between ORF59 and histones, as well as to assess the effects on replication fitness as a result of these interactions. These studies show that in the context of infection, ORF59 51 to 100 and 151 to 200 amino acids (aa) are required for interaction with histones, and ORF59 301 to 396 aa are not required for DNA synthesis. Since full-length ORF59 is known to localize to the nucleus, we performed an immunofluorescent assay (IFA) with the ORF59 deletion mutants and showed that all deletions are localized to the nucleus; this includes the ORF59 deletion without the previously identified nuclear localization signal (NLS). These studies further characterize ORF59 and demonstrate its essential role during lytic replication.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the causative agent of potentially fatal malignancies. Lytic replication of KSHV is an essential part of the viral life cycle, allowing for virus dissemination within the infected host and shedding to infect naive hosts. Viral DNA synthesis is a critical step in the production of new infectious virions. One of the proteins that is vital to this process is open reading frame 59 (ORF59), the viral encoded polymerase processivity factor. Previous work has demonstrated that the function of ORF59 is closely connected to its association with other viral and cellular factors. The studies presented here extend that work to include the interaction between ORF59 and histones. This interaction offers an additional level of regulation of the chromatinized viral genome, ultimately influencing DNA synthesis and transcription dynamics.

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac, respectively) and histone 3 lysine 4 (H3K4) methylation, histone modifications common to most active genes. Here we describe a chromatin state featuring unique histone 3 lysine 14 acetylation (H3K14ac) peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defective wing patterning. We find that the SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. Our results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.

Effects of H2A.B incorporation on nucleosome structures and dynamics

The H2A.B histone variant is an epigenetic regulator involved in transcriptional upregulation, DNA synthesis, and splicing that functions by replacing the canonical H2A histone in the nucleosome core particle. Introduction of H2A.B results in less compact nucleosome states with increased DNA unwinding and accessibility at the nucleosomal entry and exit sites. Despite being well characterized experimentally, the molecular mechanisms by which H2A.B incorporation alters nucleosome stability and dynamics remain poorly understood. To study the molecular mechanisms of H2A.B, we have performed a series of conventional and enhanced sampling molecular dynamics simulation of H2A.B- and canonical H2A-containing nucleosomes. Results of conventional simulations show that H2A.B weakens protein-protein and protein-DNA interactions at specific locations throughout the nucleosome. These weakened interactions result in significantly more DNA opening from both the entry and exit sites in enhanced sampling simulations. Furthermore, free energy profiles show that H2A.B-containing nucleosomes have significantly broader free wells and that H2A.B allows for sampling of states with increased DNA breathing, which are shown to be stable on the hundreds of nanoseconds timescale with further conventional simulations. Together, our results show the molecular mechanisms by which H2A.B creates less compacted nucleosome states as a means of increasing genetic accessibility and gene transcription.

Implementing Precision Medicine in Human Frailty through Epigenetic Biomarkers.

The main epigenetic features in aging are: reduced bulk levels of core histones, altered pattern of histone post-translational modifications, changes in the pattern of DNA methylation, replacement of canonical histones with histone variants, and altered expression of non-coding RNA. The identification of epigenetic mechanisms may contribute to the early detection of age-associated subclinical changes or deficits at the molecular and/or cellular level, to predict the development of frailty, or even more interestingly, to improve health trajectories in older adults. Frailty reflects a state of increased vulnerability to stressors as a result of decreased physiologic reserves, and even dysregulation of multiple physiologic systems leading to adverse health outcomes for individuals of the same chronological age. A key approach to overcome the challenges of frailty is the development of biomarkers to improve early diagnostic accuracy and to predict trajectories in older individuals. The identification of epigenetic biomarkers of frailty could provide important support for the clinical diagnosis of frailty, or more specifically, to the evaluation of its associated risks. Interventional studies aimed at delaying the onset of frailty and the functional alterations associated with it, would also undoubtedly benefit from the identification of frailty biomarkers. Specific to the article yet reasonably common within the subject discipline.

DNA Dynamics During SWR1-Catalyzed H2A.Z Histone Exchange

Chromatin is made up of nucleosomes, where each nucleosome core particle consists of ∼147 bp of DNA wrapped around an octamer of histone proteins. A canonical histone octamer contains two H2A-H2B dimers and one H3-H4 tetramer. H2A.Z is a well-conserved histone variant in eukaryotes and is essential for viability in metazoans. It is found at +1 nucleosomes next to transcription start sites, DNA replication origins and sites of DNA damage. H2A.Z is enzymatically deposited by SWR1 through an ATP-dependent reaction known as histone exchange.

Phosphorylation of the Canonical Histone H2A Marks Foci of Damaged DNA in Malaria Parasites.

Plasmodium falciparum parasites proliferate within circulating red blood cells and are responsible for the deadliest form of human malaria. These parasites are exposed to numerous intrinsic and external sources that could cause DNA damage; therefore, they have evolved efficient mechanisms to protect their genome integrity and allow them to proliferate under such conditions. In higher eukaryotes, double-strand breaks rapidly lead to phosphorylation of the core histone variant H2A.X, which marks the site of damaged DNA. We show that in P. falciparum that lacks the H2A.X variant, the canonical P. falciparum H2A (PfH2A) is phosphorylated on serine 121 upon exposure to sources of DNA damage. We further demonstrate that phosphorylated PfH2A is recruited to foci of damaged chromatin shortly after exposure to sources of damage, while the nonphosphorylated PfH2A remains spread throughout the nucleoplasm. In addition, we found that PfH2A phosphorylation is dynamic and that over time, as the parasite activates the repair machinery, this phosphorylation is removed. Finally, we demonstrate that these phosphorylation dynamics could be used to establish a novel and direct DNA repair assay in P. falciparumIMPORTANCEPlasmodium falciparum is the deadliest human parasite that causes malaria when it reaches the bloodstream and begins proliferating inside red blood cells, where the parasites are particularly prone to DNA damage. The molecular mechanisms that allow these pathogens to maintain their genome integrity under such conditions are also the driving force for acquiring genome plasticity that enables them to create antigenic variation and become resistant to essentially all available drugs. However, mechanisms of DNA damage response and repair have not been extensively studied for these parasites. The paper addresses our recent discovery that P. falciparum that lacks the histone variant H2A.X phosphorylates its canonical core histone PfH2A in response to exposure to DNA damage. The process of DNA repair in Plasmodium was mostly studied indirectly. Our findings enabled us to establish a direct DNA repair assay for P. falciparum similar to assays that are widely used in model organisms.

JAZF1, A Novel p400/TIP60/NuA4 Complex Member, Regulates H2A.Z Acetylation at Regulatory Regions.

Histone variants differ in amino acid sequence, expression timing and genomic localization sites from canonical histones and convey unique functions to eukaryotic cells. Their tightly controlled spatial and temporal deposition into specific chromatin regions is accomplished by dedicated chaperone and/or remodeling complexes. While quantitatively identifying the chaperone complexes of many human H2A variants by using mass spectrometry, we also found additional members of the known H2A.Z chaperone complexes p400/TIP60/NuA4 and SRCAP. We discovered JAZF1, a nuclear/nucleolar protein, as a member of a p400 sub-complex containing MBTD1 but excluding ANP32E. Depletion of JAZF1 results in transcriptome changes that affect, among other pathways, ribosome biogenesis. To identify the underlying molecular mechanism contributing to JAZF1’s function in gene regulation, we performed genome-wide ChIP-seq analyses. Interestingly, depletion of JAZF1 leads to reduced H2A.Z acetylation levels at > 1000 regulatory sites without affecting H2A.Z nucleosome positioning. Since JAZF1 associates with the histone acetyltransferase TIP60, whose depletion causes a correlated H2A.Z deacetylation of several JAZF1-targeted enhancer regions, we speculate that JAZF1 acts as chromatin modulator by recruiting TIP60’s enzymatic activity. Altogether, this study uncovers JAZF1 as a member of a TIP60-containing p400 chaperone complex orchestrating H2A.Z acetylation at regulatory regions controlling the expression of genes, many of which are involved in ribosome biogenesis.

Pituitary Adenomas Evade Apoptosis via Noxa Deregulation in Cushing's Disease

Introduction: Sporadic pituitary adenomas are common (>10%) in the general population. Microadenomas (< 1 cm across) can become symptomatic with adrenocorticotropin oversecretion causing Cushing’s disease (CD), leading to high morbidity and mortility. Pathogenic mechanisms in a majority of adenomas including wildtype CD adenomas remain unknown. Mechanistic studies are hindered by a lack of in-vitro models and normal human pituitary gland controls. We hypothesized that comparative analysis of human adenomas with syngeneic normal pituitary glands could reveal pathogenic mechanisms driving CD. Methods: We surgically annotated adenomas and en-route adjacent normal glands in 34 patients. We performed single cell RNAseq and captured 28,724 cells in 6 patients (3 CD and 3 non-CD; 5 pairwise), bulk RNAseq in 7 patients (5 CD and 2 non-CD; 5 pairwise) and DNA methylation analysis in 6 patients (3 CD and 3 non-CD). We confirmed the mechanisms of sporadic CD adenoma pathogenesis in 2 additional syngeneic pairs of human cell lines and using a novel murine normal corticotroph-enriched cell line. Results: Using marker-based classification, we confirmed canonical resident cell classes including corticotrophs in the human pituitary gland. We found SOX2+/S100B+ folliculostellate cells which play a role in pituitary cell replenishment and homeostasis in the normal gland but not in the adenoma compartment. Within the terminally differentiated corticotrophs in the adenoma compartment, we discovered a subset of proliferating cells with upregulation of canonical oncogenes, histone modifiers and DNA repair genes. We also found that consensus dominant genes including PMAIP1 (encoding the pro-apoptotic BH3 only Bcl-2 protein noxa) sharply demarcated adenoma cells from normal gland. The PMAIP1 locus was hypomethylated in CD adenomas compared to non-CD samples. However, despite epigenetic and transcriptional upregulation, we found proteasomal degradation of noxa protein in CD adenomas. Using the selective proteasomal inhibitor bortezomib, we were able to rescue noxa ex-vivo and in-vitro, induce apoptosis, and reduce cell survival of adenoma cell lines. Conclusions: We defined the transcriptionl and DNA methylation landscape of human pituitary adenomas compared to adjacent normal gland tissue. We identified proteasomal-mediated noxa degradation despite PMAIP1 overexpression as an escape mechanism from apoptosis in adenomatous corticotrophs. The proteasomal inhibitor bortezomib rescued noxa and induced apoptosis in adenoma cells, highlighting its potential as a novel therapeutic strategy in CD.

The fungal-specific histone acetyltransferase Rtt109 regulates development, DNA damage response, and virulence in Aspergillus fumigatus.

In eukaryotes, histone acetylation catalyzed by histone acetyltransferase (HAT) has been demonstrated to be critical for various physiological processes. However, the biological functions of HAT and the underlying mechanism by which HAT-regulated processes are involved in fungal development and virulence in the human opportunistic pathogen Aspergillus fumigatus remain largely unexplored. Here, we functionally characterized the roles of Rtt109 in A. fumigatus, an ortholog of Saccharomyces cerevisiae histone acetyltransferase Rtt109. In vivo and in vitro HAT assays revealed that AfRtt109 functions as a canonical histone acetyltransferase, acetylating lysines 9 and 56 of histone H3. Deletion of Afrtt109 leads to severe defects in vegetative growth, conidiation, and causes reduced virulence in the Galleria mellonella model, as well as hypersensitivity to genotoxic agents. Moreover, site-directed mutagenesis revealed that the conserved arginine residues R265 and R306 of Rtt109 are required for the H3K9 and H3K56 acetylation and virulence of A. fumigatus. Unexpectedly, R265E and R306E mutants did not exhibit any detectable phenotypic defects, implying that A. fumigatus Rtt109 regulates fungal development via histone acetylation-independent mechanisms. Together, our results revealed the critical role of fungal-specific HAT Rtt109 in regulating fungal development and virulence, and suggested that it may serve as a unique target for antifungal therapies.

Histone Variant H3.3 Mutations in Defining the Chromatin Function in Mammals.

The systematic mutation of histone 3 (H3) genes in model organisms has proven to be a valuable tool to distinguish the functional role of histone residues. No system exists in mammalian cells to directly manipulate canonical histone H3 due to a large number of clustered and multi-loci histone genes. Over the years, oncogenic histone mutations in a subset of H3 have been identified in humans, and have advanced our understanding of the function of histone residues in health and disease. The oncogenic mutations are often found in one allele of the histone variant H3.3 genes, but they prompt severe changes in the epigenetic landscape of cells, and contribute to cancer development. Therefore, mutation approaches using H3.3 genes could be relevant to the determination of the functional role of histone residues in mammalian development without the replacement of canonical H3 genes. In this review, we describe the key findings from the H3 mutation studies in model organisms wherein the genetic replacement of canonical H3 is possible. We then turn our attention to H3.3 mutations in human cancers, and discuss H3.3 substitutions in the N-terminus, which were generated in order to explore the specific residue or associated post-translational modification.

TBIO-08. BASE-RESOLUTION METHYLOMES OF GLIOMAS BEARING HISTONE H3.3 MUTATIONS REVEAL A G34 MUTANT-SPECIFIC SIGNATURE SHARED WITH BONE TUMORS

BACKGROUNDTwo recurrent mutations, K27M and G34R/V, in H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation was prevalent in bone tumors. In contrast to K27 mutation, it remains elusive how G34 mutations affect the epigenome. Here we performed whole-genome bisulfite sequencing of four G34R-mutated gliomas and the G34V-mutated glioma cell line KNS-42. Similarly, we analyzed seven and three gliomas harboring K27M and no mutations in H3F3A, respectively. These data were compared with those on bone tumors.RESULTSG34R-mutated gliomas exhibited lower global methylation levels, similar CpG island (CGI) methylation levels, and compromised hypermethylation of telomere-proximal CGIs compared with those bearing K27M and no mutations. Hypermethylated regions specific to G34R-mutated gliomas were enriched for CGIs, including those of OLIG1, OLIG2, and canonical histone genes in the HIST1 cluster. These CGIs were hypermethylated in osteosarcomas with, but not without, the G34W mutation. In KNS-42 cells, CGIs with G34V-mutated histone H3.3 exhibited higher methylation levels than those with wild-type histone H3.3. This effect was also observed in the G34R-mutated glioma samples.CONCLUSIONSGliomas bearing G34R/V mutations display characteristic methylomic alterations, some of which are shared by osteosarcomas with the G34W mutation. Deposition of G34 variants may lead to elevated methylation of otherwise hypomethylated, histone H3.3-bearing CGIs.

Primate-specific histone variants.

Canonical histones (H2A, H2B, H3, and H4) are present in all eukaryotes where they package genomic DNA and participate in numerous cellular processes, such as transcription regulation and DNA repair. In addition to the canonical histones, there are many histone variants, which have different amino acid sequences, possess tissue-specific expression profiles, and function distinctly from the canonical counterparts. A number of histone variants, including both core histones (H2A/H2B/H3/H4) and linker histones (H1/H5), have been identified to date. Htz1 (H2A.Z) and CENP-A (CenH3) are present from yeasts to mammals, and H3.3 is present from Tetrahymena to humans. In addition to the prevalent variants, others like H3.4 (H3t), H2A.Bbd, and TH2B, as well as several H1 variants, are found to be specific to mammals. Among them, H2BFWT, H3.5, H3.X, H3.Y, and H4G are unique to primates (or Hominidae). In this review, we focus on localization and function of primate- or hominidae-specific histone variants.