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.

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)

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.

A region of Drosophila SLBP distinct from the histone pre-mRNA binding and processing domains is essential for deposition of histone mRNA in the oocyte

ABSTRACTDuring Drosophila oogenesis, large amounts of histone mRNA and proteins are deposited in the developing oocyte. These are sufficient for the first 14 embryonic cell cycles and provide the developing embryo with sufficient histone proteins until the zygotic histone genes are activated. The maternally deposited histone mRNA is synthesized in stage 10b of oogenesis after completion of endoreduplication of the nurse cells. Histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending instead in a conserved stemloop instead of a polyA tail. The Stem-loop binding protein (SLBP) binds the 3’ end of histone mRNA and is essential for both the biosynthesis and translation of histone mRNA. We report that a 10 aa region in SLBP, which is not required for processing in vitro, is essential for transcription of histone mRNA in the stage 10b oocyte. In stage 10b the Histone Locus Bodies (HLBs) produce histone mRNAs in the absence of phosphorylation of Mxc, normally required for histone gene expression in S-phase cells. Mutants expressing this SLBP develop normally, produce small amounts of polyadenylated histone mRNA throughout development, but little histone mRNA in stage 10b resulting in death of the embryos in the first hr of development.

Drosophila Histone Locus Body assembly and function involves multiple interactions

The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and concentrates factors required for RD histone mRNA biosynthesis. The D. melanogaster genome has a single locus comprised of ∼100 copies of a tandemly arrayed repeat unit containing one copy of each of the 5 RD histone genes. To determine sequence elements required for D. melanogaster HLB formation and histone gene expression, we used transgenic gene arrays containing 12 copies of the histone repeat unit that functionally complement loss of the ∼200 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were replaced with H2a-H2b promoters does not form an HLB or express high levels of RD histone mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is active in HLB assembly and RD histone gene expression in the absence of the endogenous RD histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array contains all known factors present in the wild type HLB including CLAMP, which normally binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein and/or protein-DNA interactions contribute to HLB formation, and that the large number of endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic arrays, thereby preventing HLB formation and gene expression.

Loss of Histone Locus Bodies in the Mature Hemocytes of Larval Lymph Gland Result in Hyperplasia of the Tissue in mxc Mutants of Drosophila

Mutations in the multi sex combs (mxc) gene in Drosophila results in malignant hyperplasia in larval hematopoietic tissues, called lymph glands (LG). mxc encodes a component of the histone locus body (HLB) that is essential for cell cycle-dependent transcription and processing of histone mRNAs. The mammalian nuclear protein ataxia-telangiectasia (NPAT) gene, encoded by the responsible gene for ataxia telangiectasia, is a functional Mxc orthologue. However, their roles in tumorigenesis are unclear. Genetic analyses of the mxc mutants and larvae having LG-specific depletion revealed that a reduced activity of the gene resulted in the hyperplasia, which is caused by hyper-proliferation of immature LG cells. The depletion of mxc in mature hemocytes of the LG resulted in the hyperplasia. Furthermore, the inhibition of HLB formation was required for LG hyperplasia. In the mutant larvae, the total mRNA levels of the five canonical histones decreased, and abnormal forms of polyadenylated histone mRNAs, detected rarely in normal larvae, were generated. The ectopic expression of the polyadenylated mRNAs was sufficient for the reproduction of the hyperplasia. The loss of HLB function, especially 3′-end processing of histone mRNAs, is critical for malignant LG hyperplasia in this leukemia model in Drosophila. We propose that mxc is involved in the activation to induce adenosine deaminase-related growth factor A (Adgf-A), which suppresses immature cell proliferation in LG.