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.

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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

10.1101/2020.04.16.030577 ◽

2020 ◽

Author(s):

Jennifer Potter-Birriel ◽

Graydon B. Gonsalvez ◽

William F. Marzluff

Keyword(s):

Embryonic Cell ◽

Histone Mrna ◽

Stem Loop ◽

Cell Cycles ◽

Histone Mrnas ◽

Histone Locus Bodies ◽

Cellular Mrnas ◽

Histone Locus ◽

Mrna Binding

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.

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Drosophila Histone Locus Body assembly and function involves multiple interactions

10.1101/2020.03.16.994483 ◽

2020 ◽

Author(s):

Kaitlin P. Koreski ◽

Leila E. Rieder ◽

Lyndsey M. McLain ◽

William F. Marzluff ◽

Robert J. Duronio

Keyword(s):

Gene Expression ◽

Repeat Unit ◽

Gene Array ◽

Homozygous Deletion ◽

Histone Gene ◽

Histone Genes ◽

Histone Mrna ◽

Histone Gene Expression ◽

Histone Locus Body ◽

Histone Locus

AbstractThe 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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e20-10-0645 ◽

2021 ◽

pp. mbc.E20-10-0645

Author(s):

James P. Kemp ◽

Xiao-Cui Yang ◽

Zbigniew Dominski ◽

William F. Marzluff ◽

Robert J. Duronio

Keyword(s):

Gene Expression ◽

Gene Transcription ◽

Nuclear Body ◽

Histone Gene ◽

Core Shell ◽

Histone Genes ◽

The Core ◽

Histone Gene Expression ◽

Histone Locus ◽

Histone Gene Transcription

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.

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CDK-Regulated Phase Separation Seeded by Histone Genes Ensures Precise Growth and Function of Histone Locus Bodies

Developmental Cell ◽

10.1016/j.devcel.2020.06.003 ◽

2020 ◽

Vol 54 (3) ◽

pp. 379-394.e6 ◽

Cited By ~ 5

Author(s):

Woonyung Hur ◽

James P. Kemp ◽

Marco Tarzia ◽

Victoria E. Deneke ◽

William F. Marzluff ◽

...

Keyword(s):

Phase Separation ◽

Histone Genes ◽

Histone Locus Bodies ◽

And Function ◽

Histone Locus

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Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis

The Journal of Cell Biology ◽

10.1083/jcb.201504043 ◽

2016 ◽

Vol 213 (5) ◽

pp. 557-570 ◽

Cited By ~ 39

Author(s):

Deirdre C. Tatomer ◽

Esteban Terzo ◽

Kaitlin P. Curry ◽

Harmony Salzler ◽

Ivan Sabath ◽

...

Keyword(s):

Messenger Rna ◽

Mrna Processing ◽

Histone Mrna ◽

Histone Mrnas ◽

Assembly Factor ◽

U7 Snrnp ◽

Histone Locus Body ◽

Histone Locus ◽

Processing Factors

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination.

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Effect of adenovirus infection on expression of human histone genes.

Molecular and Cellular Biology ◽

10.1128/mcb.4.7.1363 ◽

1984 ◽

Vol 4 (7) ◽

pp. 1363-1371 ◽

Cited By ~ 20

Author(s):

S J Flint ◽

M A Plumb ◽

U C Yang ◽

G S Stein ◽

J L Stein

Keyword(s):

Gene Expression ◽

Dna Synthesis ◽

Histone Gene ◽

Adenovirus Infection ◽

Histone Genes ◽

Histone Mrna ◽

Intact Cells ◽

Mrna Species ◽

Histone Gene Expression ◽

Infected Cells

The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA synthesis, observed when DNA replication is inhibited by a variety of drugs, is not maintained after adenovirus infection.

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CDK-regulated phase separation seeded by histone genes ensures precise growth and function of Histone Locus Bodies

10.1101/789933 ◽

2019 ◽

Cited By ~ 2

Author(s):

Woonyung Hur ◽

Marco Tarzia ◽

Victoria E. Deneke ◽

Esteban A. Terzo ◽

Robert J. Duronio ◽

...

Keyword(s):

Phase Separation ◽

Gene Activation ◽

Nuclear Body ◽

Histone Mrnas ◽

Histone Locus Bodies ◽

Sex Combs ◽

Zygotic Gene ◽

Zygotic Gene Activation ◽

And Function ◽

Histone Locus

SummaryMany membrane-less organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The Histone Locus Body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. Here, we show that Drosophila HLBs form through phase separation of the scaffold protein multi-sex combs (Mxc). The size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which catalyzes phase separation, and the nuclear concentration of Mxc, which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Our experiments thus identify a mechanism linking nuclear body growth and size with gene expression.

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

10.1101/2020.12.25.424380 ◽

2020 ◽

Author(s):

Takashi Imada ◽

Takeshi Shimi ◽

Ai Kaiho ◽

Yasushi Saeki ◽

Hiroshi Kimura

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Clusters ◽

Nuclear Bodies ◽

Histone Genes ◽

Small Nuclear Rna ◽

Pol Ii ◽

Histone Locus Bodies ◽

Dynamic Structures ◽

Histone Locus

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)

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Rapid reversible changes in the rate of histone gene transcription and histone mRNA levels in mouse myeloma cells.

Molecular and Cellular Biology ◽

10.1128/mcb.4.2.351 ◽

1984 ◽

Vol 4 (2) ◽

pp. 351-357 ◽

Cited By ~ 42

Author(s):

R A Graves ◽

W F Marzluff

Keyword(s):

Protein Synthesis ◽

Dna Synthesis ◽

Gene Transcription ◽

Histone Gene ◽

Mrna Levels ◽

Histone Mrna ◽

Myeloma Cells ◽

Histone Mrnas ◽

Histone Gene Transcription ◽

Mouse Myeloma

The levels of histone mRNAs are reduced 90 to 95% after treatment of mouse myeloma cells with inhibitors of DNA synthesis which disrupt deoxynucleotide metabolism. In contrast, novobiocin, which inhibits DNA synthesis but does not alter deoxynucleotide metabolism, did not alter histone mRNA levels. Upon reversing the inhibition by fluorodeoxyuridine by feeding with thymidine, histone mRNA levels are restored to control levels within 40 to 60 min. The rate of histone gene transcription is reduced 75 to 80% within 10 min after treatment with fluorodeoxyuridine and increased to control levels within 10 min after refeeding with thymidine. Inhibition of protein synthesis with cycloheximide or puromycin in cells which had been treated with fluorodeoxyuridine resulted in an increase of histone mRNA levels. This was partly due to an increase in the rate of transcription. The data indicate that both transcription and mRNA degradation are linked to deoxynucleotide metabolism. Continued protein synthesis is necessary for maintaining the inhibition of histone gene transcription.

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