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.

ALS-linked FUS mutants affect the localization of U7 snRNP and replication-dependent histone gene expression in human cells

Genes encoding replication-dependent histones lack introns, and the mRNAs produced are a unique class of RNA polymerase II transcripts in eukaryotic cells that do not end in a polyadenylated tail. Mature mRNAs are thus formed by a single endonucleolytic cleavage that releases the pre-mRNA from the DNA and is the only processing event necessary. U7 snRNP is one of the key factors that determines the cleavage site within the 3ʹUTR of replication-dependent histone pre-mRNAs. We have previously showed that the FUS protein interacts with U7 snRNA/snRNP and regulates the expression of histone genes by stimulating transcription and 3ʹ end maturation. Mutations in the FUS gene first identified in patients with amyotrophic lateral sclerosis (ALS) lead to the accumulation of the FUS protein in cytoplasmic inclusions. Here, we report that mutations in FUS lead to disruption of the transcriptional activity of FUS and mislocalization of U7 snRNA/snRNP in cytoplasmic aggregates in cellular models and primary neurons. As a consequence, decreased transcriptional efficiency and aberrant 3ʹ end processing of histone pre-mRNAs were observed. This study highlights for the first time the deregulation of replication-dependent histone gene expression and its involvement in ALS.

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.

Single-sequence probes reveal chromosomal locations of tandemly repetitive genes in scleractinian coral Acropora pruinosa: a potential tool for karyotyping

The short and similar sized chromosomes of Acropora pose a challenge for karyotyping. Conventional methods, such as staining of heterochromatic regions, provide unclear banding patterns that hamper identification of such chromosomes. In this study, we used short single-sequence probes for tandemly repetitive 5S ribosomal RNA (rRNA) and core histone genes to identify specific chromosomes of Acropora pruinosa. Both the probes produced intense signals in fluorescence in situ hybridization, which distinguished chromosome pairs. The locus of the core histone gene was on chromosome 8, whereas that of 5S rRNA gene was on chromosome 5. The sequence of the 5S rRNA probe was composed largely of U1 and U2 spliceosomal small nuclear RNA (snRNA) genes and their interspacers, flanked by short sequences of the 5S rRNA gene. This is the first report of a tandemly repetitive linkage of snRNA and 5S rRNA genes in Cnidaria. Based on the constructed tentative karyogram and whole genome hybridization, the longest chromosome pair (chromosome 1) was heteromorphic. The probes also hybridized effectively with chromosomes of other Acropora species and population, revealing an additional core histone gene locus. We demonstrated the applicability of short-sequence probes as chromosomal markers with potential for use across populations and species of Acropora.

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 Profiling Reveals a Common Mechanism of Histone Gene Downregulation and Complementary Effects for Radioprotectants in Response to Ionizing Radiation

High-dose ionizing radiation (IR) alters the expression levels of non-coding RNAs (ncRNAs). However, the roles of ncRNAs and mRNAs in mediating radiation protection by radioprotectants remain unknown. Microarrays were used to determine microRNA (miRNA), long ncRNA (lncRNA), and mRNA expression profiles in the bone marrow of irradiated mice pretreated with amifostine, CBLB502, and nilestriol. Differentially expressed mRNAs were functionally annotated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Some histone cluster genes were validated by real-time PCR, and the effects of radioprotectant combinations were monitored by survival analysis. We found that these radioprotectants increased the induction of lncRNAs and mRNAs. miRNA, lncRNA, and mRNA expression patterns were similar with amifostine and CBLB502, but not nilestriol. The radioprotectants exhibited mostly opposite effects against IR-induced miRNAs, lncRNAs, and mRNAs while inducing a common histone gene downregulation following IR, mainly via nucleosome assembly and related signaling pathways. Notably, the effects of nilestriol significantly complemented those of amisfostine or CBLB502; low-dose drug combinations resulted in better radioprotective effects in pretreated mice. Thus, we present histone gene downregulation by radioprotectants, together with the biological functions of miRNA, lncRNA, and mRNA, to explain the mechanism underlying radioprotection.

Structural Analysis of the SANT/Myb Domain of FLASH and YARP Proteins and Their Complex with the C-Terminal Fragment of NPAT by NMR Spectroscopy and Computer Simulations

FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein (YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH–NPAT and YARP–NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and in silico modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple α-helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single α-helix that makes multiple contacts with α-helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. In silico modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.

Drosophila histone locus body assembly and function involves multiple interactions

By using a histone gene replacement platform in Drosophila, we show that interactions among multiple factors contribute to HLB formation, and that the large number of genes at the endogenous histone locus sequesters available factors from attenuated transgenic histone gene arrays, thereby preventing HLB formation and histone gene expression from these arrays.