RESUMO
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.
Assuntos
Drosophila melanogaster/genética , Loci Gênicos , Histonas/genética , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Fenótipo , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U7/metabolismo , TransgenesRESUMO
Histones and their posttranslational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have nonhistone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike inferences drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity but not for gene activation. These findings highlight the power of engineering histones to interrogate genome structure and function in animals.
Assuntos
Cromatina/genética , Histonas/metabolismo , Família Multigênica/genética , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Replicação do DNA/genética , Drosophila , Epigênese Genética/genética , Histona-Lisina N-Metiltransferase/metabolismo , MetilaçãoRESUMO
The scaffolding protein Symplekin is part of multiple complexes involved in generating and modifying the 3' end of mRNAs, including cleavage-polyadenylation, histone pre-mRNA processing and cytoplasmic polyadenylation. To study these functions in vivo, we examined the localization of Symplekin during development and generated mutations of the Drosophila Symplekin gene. Mutations in Symplekin that reduce Symplekin protein levels alter the efficiency of both poly A(+) and histone mRNA 3' end formation resulting in lethality or sterility. Histone mRNA synthesis takes place at the histone locus body (HLB) and requires a complex composed of Symplekin and several polyadenylation factors that associates with the U7 snRNP. Symplekin is present in the HLB in the early embryo when Cyclin E/Cdk2 is active and histone genes are expressed and is absent from the HLB in cells that have exited the cell cycle. During oogenesis, Symplekin is preferentially localized to HLBs during S-phase in endoreduplicating follicle cells when histone mRNA is synthesized. After the completion of endoreplication, Symplekin accumulates in the cytoplasm, in addition to the nucleoplasm, and localizes to tricellular junctions of the follicle cell epithelium. This localization depends on the RNA binding protein ypsilon schachtel. CPSF-73 and a number of mRNAs are localized at this same site, suggesting that Symplekin participates in cytoplasmic polyadenylation at tricellular junctions.
Assuntos
Proteínas de Drosophila/biossíntese , Desenvolvimento Embrionário , Histonas/genética , Poliadenilação/genética , Fatores de Poliadenilação e Clivagem de mRNA/biossíntese , Animais , Citoplasma/genética , Drosophila , Proteínas de Drosophila/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Corpos de Inclusão Intranuclear/genética , Corpos de Inclusão Intranuclear/metabolismo , Mutação , Nucleoplasminas/genética , Ribonucleoproteína Nuclear Pequena U7/genética , Fase S/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
UNLABELLED: Whether dietary fiber protects against colorectal cancer is controversial because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumor-suppressive properties in colorectal cancer cell lines. We used gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as a histone deacetylase (HDAC) inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared with normal colonic tissues. SIGNIFICANCE: These results, which link diet and microbiota to a tumor-suppressive metabolite, provide insight into conflicting epidemiologic findings and suggest that probiotic/prebiotic strategies can modulate an endogenous HDAC inhibitor for anticancer chemoprevention without the adverse effects associated with synthetic HDAC inhibitors used in chemotherapy.