Genome-Wide Characterization and Analysis of Expression of the Histone Gene Family in Razor Clam, Sinonovacula constricta

The Chinese razor clam (Sinonovacula constricta), a bivalve species widely distributed in estuaries and mudflats, is often exposed to extreme environmental and microbial stresses. Histones are fundamental components of chromatin and play an important role in innate immunity, as demonstrated by its antimicrobial activities in clams. However, little attention has been paid to histones in bivalves. To fill this gap, we investigated the genomic distribution, structural characteristics, conserved motifs, and phylogenetic relationships of histones in S. constricta. A total of 114 histone genes were detected in the S. constricta genome, which were divided into 25 types in phylogenetic analysis. Among them, partial histones exhibited a tissue-dependent expression pattern, indicating that they may be involved in sustaining the homeostasis of organs/tissues in adult S. constricta. Furthermore, mRNA expression of certain histones changed significantly in S. constricta when infected with Vibrio parahaemolyticus, suggesting that histones play a role in the immune defense of S. constricta. All together, this study on histone genes in S. constricta not only greatly expands our knowledge of histone function in the clam, but also histone evolution in molluscs.

Genes of the month: H3.3 histone genes: H3F3A and H3F3B

Histones constitute the chief protein component of DNA. They help to maintain chromatin structure and regulate gene expression. The long double-stranded DNA molecule winds around histone octamers to form nucleosomes which serve the purpose of compacting DNA within the confines of the nuclear membrane. There are five major types of histones, namely H1/H5, H2, H3 and H4. H3.3 is a subtype of H3 histone and can be encoded either by the H3F3A or H3F3B genes independently. Amino acids such as lysine and arginine found in the histone tails are sites of post-translational modifications (PTMs) such as methylation and acetylation. These PTMs in histones are involved in the regulation of gene expression by chromatin remodelling and by controlling DNA methylation patterns. Mutations in histone genes can affect sites of PTMs causing changes in local and global DNA methylation status. These effects are directly linked to neoplastic transformation by altered gene expression. Recurrent H3.3 histone mutations are increasingly identified in several malignancies and developmental disorders. The following review attempts to shed light on the diseases associated with H3.3 histone mutations.

OMIC-09. MAPPING THE HISTONE MUTATIONAL LANDSCAPE ACROSS ADULT AND PEDIATRIC CANCER GENOMES UNCOVERS NOVEL SOMATIC MUTATIONS IN PEDIATRIC HIGH-GRADE GLIOMAS

There is a growing role for mutations affecting histone linker and histone core-encoding genes across several adult and pediatric cancers. However, the extent to which somatic histone mutations may bridge across different cancers as common tumorigenic events – particularly in the context of pediatric CNS tumors – remains unclear. To address this knowledge gap, we set out to define a comprehensive pan-cancer landscape of somatic histone mutations. We first queried the ICGC PCAWG and TCGA Pan-Cancer Atlas representing >12,500 adult and pediatric cancer patients. We found lymphomas to be most enriched for histone mutations (50–75%) and, in particular, for mutations in linker histones (HIST1H1B-E), yet also in specific core histone genes (eg, HIST2H2BE). Moreover, we observed a significant enrichment of histone mutations in adult high-grade vs low-grade gliomas (10% vs 6%, P<0.05, n=922 patients). Interrogation of whole genome data from 800 pediatric CNS tumor genomes (PBTA/OpenDIPG), identified novel (non-H3K27/non-H3G34) somatic histone mutations in 5–10% of subjects, including pediatric high-grade gliomas (pHGGs) and diffuse midline gliomas (DMGs). We found an overlapping set of histone genes to be recurrently mutated in non-CNS cancers and pediatric CNS tumors alike (eg, HIST1H1B/C/E). Notably, the only pediatric primary CNS lymphoma patient also harbored a histone linker alteration (HIST1H1B), similar to adult non-CNS lymphoma patients. We validated novel somatic histone mutations in DMGs by Sanger sequencing. Ongoing studies include in vitro assessment of the impact of these mutations on cell proliferation, chromatin accessibility, histone spacing, and gene expression. In addition, we will further assess associations with clinical outcome, age, and tumor subtypes. Collectively, oncohistone vulnerabilities were identified and defined as histone gene families recurrently mutated across all cancer types. Our analyses of adult and pediatric cancer genomes have uncovered previously unknown mutations affecting histone linker and core proteins, which may play a yet-undefined role in tumor etiology.

The Adenine/Thymine Deleterious Selection Model for GC Content Evolution at the Third Codon Position of the Histone Genes in Drosophila

The evolution of the GC (guanine cytosine) content at the third codon position of the histone genes (H1, H2A, H2B, H3, H4, H2AvD, H3.3A, H3.3B, and H4r) in 12 or more Drosophila species is reviewed. For explaining the evolution of the GC content at the third codon position of the genes, a model assuming selection with a deleterious effect for adenine/thymine and a size effect is presented. The applicability of the model to whole-genome genes is also discussed.

Mosaic Pattern of H3 K27M-Mutant Protein Expression in a Diffuse Midline Glioma—A Diagnostic Dilemma for the Pathologist

Diffuse midline glioma, H3 K27M-mutant, is a World Health Organization (WHO) grade IV glioma arising in pons, thalamus, and spinal cord. They show mutations resulting in replacement of lysine at position 27 by methionine (K27M) of histone genes, H3F3A, HIST1H3B, and HIST1H3C. The H3 K27M mutant protein is identified in tumor tissue by immunohistochemistry. As these mutations are clonal and homogeneous, the mutant protein is normally identified in all tumor cells. Here we report a case of diffuse midline glioma with mosaic pattern of expression of H3 K27M mutant protein and discuss the diagnostic and therapeutic implications of this unusual pattern.

RNA-seq of the medusavirus suggests remodeling of the host nuclear environment at an early infection stage

Nucleo-cytoplasmic large DNA viruses (NCLDVs) undergo a cytoplasmic or nucleo-cytoplasmic cycle, and the latter involves both nuclear and cytoplasmic compartments to proceed viral replication. Medusavirus, a recently isolated NCLDV, has a nucleo-cytoplasmic replication cycle in amoebas during which the host nuclear membrane apparently remains intact, a unique feature among amoeba-infecting giant viruses. The medusavirus genome lacks most transcription genes but encodes a full set of histone genes. To investigate the infection strategy, we performed a time-course RNA-seq experiment. All the viral genes were transcribed and classified into five temporal expression clusters. The immediate early genes (cluster 1, 42 genes) were mostly (83%) of unknown functions, frequently (95%) associated with a palindromic promoter-like motif, and enriched (45%) in putative nuclear-targeting genes. The later genes (clusters 2-5) were assigned to various functional categories. The viral linker histone H1 gene was in cluster 1, whereas the four core histone genes were in cluster 3, suggesting they had distinct roles during the course of the virus infection. The transcriptional profile of the host amoeba, Acanthamoeba castellanii, genes was greatly altered post-infection. Several encystment-related host genes showed increased representation levels at 48 hours post-infection, which is consistent with the previously reported amoeba encystment upon medusavirus infection. Overall, the transcriptional landscape during the course of medusavirus infection suggests that the virus modifies the host nuclear environment immediately after the initiation of infection.

Super resolution light microscopy of the Drosophila Histone Locus Body reveals a core-shell organization associated with expression of replication dependent histone genes

The Histone Locus Body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of replication-dependent (RD) histone mRNAs, which are the only eukaryotic mRNAs lacking a poly-A tail. Many nuclear bodies contain distinct domains, but how internal organization is related to nuclear body function is not fully understood. Here, we demonstrate using structured illumination microscopy that Drosophila HLBs have a “core-shell” organization in which the internal core contains transcriptionally active RD histone genes. The N-terminus of Mxc, which contains a domain required for Mxc oligomerization, HLB assembly, and RD histone gene expression, is enriched in the HLB core. In contrast, the C-terminus of Mxc is enriched in the HLB outer shell as is FLASH, a component of the active U7 snRNP that co-transcriptionally cleaves RD histone pre-mRNA. Consistent with these results, we show biochemically that FLASH binds directly to the Mxc C-terminal region. In the rapid S-M nuclear cycles of syncytial blastoderm Drosophila embryos, the HLB disassembles at mitosis and reassembles the core-shell arrangement as histone gene transcription is activated immediately after mitosis. Thus, the core-shell organization is coupled to zygotic histone gene transcription, revealing a link between HLB internal organization and RD histone gene expression.

Dual RNA 3’-end processing of H2A.X messenger RNA maintains DNA damage repair throughout the cell cycle

Phosphorylated H2A.X is a critical chromatin marker of DNA damage repair (DDR) in higher eukaryotes. However, H2A.X gene expression remains relatively uncharacterised. Replication-dependent (RD) histone genes generate poly(A)- mRNA encoding new histones to package DNA during replication. In contrast, replication-independent (RI) histone genes synthesise poly(A)+ mRNA throughout the cell cycle, translated into histone variants that confer specific epigenetic patterns on chromatin. Remarkably H2AFX, encoding H2A.X, is a hybrid histone gene, generating both poly(A)+ and poly(A)- mRNA isoforms. Here we report that the selective removal of either mRNA isoform reveals different effects in different cell types. In some cells, RD H2A.X poly(A)- mRNA generates sufficient histone for deposition onto DDR associated chromatin. In contrast, cells making predominantly poly(A)+ mRNA require this isoform for de novo H2A.X synthesis, required for efficient DDR. This highlights the importance of differential H2A.X mRNA 3’-end processing in the maintenance of effective DDR.

A region of SLBP outside the mRNA processing domain is essential for deposition of histone mRNA into the Drosophila egg

Replication-dependent histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending in a stemloop instead of a polyA tail, and are normally regulated coordinately with DNA replication. SLBP binds the 3’ end of histone mRNA, and is required for processing and translation. During Drosophila oogenesis, large amounts of histone mRNAs and proteins are deposited in the developing oocyte.The maternally deposited histone mRNA is synthesized in stage 10B oocytes after the nurse cells complete endoreduplication. We report that in WT stage 10B oocytes, the Histone Locus Bodies (HLBs), formed on the histone genes, produce histone mRNAs in the absence of phosphorylation of Mxc, normally required for histone gene expression in S-phase cells. Two mutants of SLBP, one with reduced expression and another with a 10 aa deletion, fail to deposit sufficient histone mRNA in the oocyte, and don't transcribe the histone genes in stage 10B. Mutations in a putative SLBP nuclear localization sequence overlapping the deletion, phenocopy the deletion. We conclude a high concentration of SLBP in the nucleus of stage 10B oocytes is essential for histone gene transcription.

RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells

ABSTRACTIn eukaryotic nuclei, a number of phase-separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post-transcriptional regulation of small nuclear RNA and histone genes. By live-cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC–CB– HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC–HLBs with CBs, and nucleation of PCs near preformed CB– HLBs. Coilin knockout increased the co-localization rate between PCs and HLBs, whereas the number, nucleation timing, and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6-hexanediol revealed that PCs were more liquid-like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs. (187 words)