Article

The Histone H3 Acetylase dGcn5 Is a Key Player in Drosophila melanogaster Metamorphosis

Laboratory of Drosophila Genetics and Epigenetics, Department of Developmental Biology, CNRS URA 2578, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Molecular and Cellular Biology (Impact Factor: 4.78). 10/2005; 25(18):8228-38. DOI: 10.1128/MCB.25.18.8228-8238.2005
Source: PubMed

ABSTRACT

Although it has been well established that histone acetyltransferases (HATs) are involved in the modulation of chromatin structure
and gene transcription, there is only little information on their developmental role in higher organisms. Gcn5 was the first
transcription factor with HAT activity identified in eukaryotes. Here we report the isolation and characterization of Drosophila melanogaster dGcn5 mutants. Null dGcn5 alleles block the onset of both oogenesis and metamorphosis, while hypomorphic dGcn5 alleles impair the formation of adult appendages and cuticle. Strikingly, the dramatic loss of acetylation of the K9 and
K14 lysine residues of histone H3 in dGcn5 mutants has no noticeable effect on larval tissues. In contrast, strong cell proliferation defects in imaginal tissues are
observed. In vivo complementation experiments revealed that dGcn5 integrates specific functions in addition to chromosome
binding and acetylation. Surprisingly, a dGcn5 variant protein with a deletion of the bromodomain, which has been shown to
recognize acetylated histones, appears to be fully functional. Our results establish dGcn5 as a major histone H3 acetylase
in Drosophila which plays a key role in the control of specific morphogenetic cascades during developmental transitions.

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    • "The complexes include distinct Ada2 relatives: SAGA contains the Ada2b protein, whereas ATAC contains the Ada2a protein (Kusch et al, 2003; Muratoglu et al, 2003). Gcn5 was found to be essential for in vivo acetylation of larval polytene chromosomes at positions lysine 9 (K9)/K14 of histone H3 and K5/K12 of histone H4 (Carre et al, 2005; Ciurciu et al, 2006). In addition, mutations in the Ada2b gene result in a loss of acetylation of residues H3-K9/K14, whereas mutations in the Ada2a gene only affect acetylation of residues H4-K5/K12 (Qi et al, 2004; Pankotai et al, 2005; Ciurciu et al, 2006). "

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    • "For protein analysis, equal amounts of proteins from total extract, supernatant, or immunoprecipitate samples were boiled in Laemmli buffer and loaded on 12% SDS/PAGE. After transfer onto nitrocellulose membrane and Ponceau staining, membranes were blocked in 5% milk, 1X PBS, 0.1% Tween, and incubated overnight with anti-GFP (1∶2000; Roche), anti-Mbf1 (1∶10000; [28], anti-Ago1 (1∶1000; Abcam ab5070), anti-HA (1∶1000; Roche) or anti-γ-Tubulin (1∶2000; Sigma) antibodies. anti-Dcr-1, anti-Dcr-2 and anti-Ago2 were used at 1∶1000 (a kind gift of Mikiko C. Siomi). "
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    ABSTRACT: Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.
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    • "This concurs with previous reports discussing extensive transcriptional reprogramming at the onset of metamorphosis to induce genes involved in tissue and organ remodeling. For example, HAT activity is mediated by the histone H3 acetylase dGN5, which is a key modulator of chromatin structure and transcription during Drosophila melanogaster metamorphosis [5]. "
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    ABSTRACT: Background Gene expression in eukaryotes is regulated by histone acetylation/deacetylation, an epigenetic process mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) whose opposing activities are tightly regulated. The acetylation of histones by HATs increases DNA accessibility and promotes gene expression, whereas the removal of acetyl groups by HDACs has the opposite effect. Results We explored the role of HDACs and HATs in epigenetic reprogramming during metamorphosis, wounding and infection in the lepidopteran model host Galleria mellonella. We measured the expression of genes encoding components of HATs and HDACs to monitor the transcriptional activity of each enzyme complex and found that both enzymes were upregulated during pupation. Specific HAT inhibitors were able to postpone pupation and to reduce insect survival following wounding, whereas HDAC inhibitors accelerated pupation and increased survival. The administration of HDAC inhibitors modulated the expression of effector genes with key roles in tissue remodeling (matrix metalloproteinase), the regulation of sepsis (inhibitor of metalloproteinases from insects) and host defense (antimicrobial peptides), and simultaneously induced HAT activity, suggesting that histone acetylation is regulated by a feedback mechanism. We also discovered that both the entomopathogenic fungus Metarhizium anisopliae and the human bacterial pathogen Listeria monocytogenes can delay metamorphosis in G. mellonella by skewing the HDAC/HAT balance. Conclusions Our study provides for the first evidence that pathogenic bacteria can interfere with the regulation of HDACs and HATs in insects which appear to manipulate host immunity and development. We conclude that histone acetylation/deacetylation in insects mediates transcriptional reprogramming during metamorphosis and in response to wounding and infection.
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