Ada2 proteins are present in many Gcn5-containing complexes. To confirm that Drosophila Ada2b and Gcn5 are able to interact, a GST pull-down assay was performed. Bacterially expressed GST-dGcn5 efficiently binds to in vitro-translated dAda2b protein. Embryonic expression pattern of dAda2b was compared to that of dGcn5 by in situ hybridization. Both dAda2b and dGcn5 are maternally contributed; their transcripts can be detected at the precellular stage. This finding was confirmed by staining ovaries with the dAda2b and dGcn5 probes. Both transcripts are present in nurse cells of stage 10 egg chambers and are dumped in the oocyte when the nurse cells degenerate at late oogenesis (Qi, 2004).
In wild-type embryos undergoing cellularization, no maternal dAda2b mRNA remains, whereas that of dGcn5 persists. In advanced-stage embryos, zygotic expression of both dAda2b and dGcn5 is strongest in the central nervous system (CNS) and is present at lower levels in most other tissues. These results suggest that dAda2b and dGcn5 are found in many of the same cells during embryo development. As expected, in dAda2b homozygous embryos, no dAda2b mRNA could be detected after stage 5, when the maternal message is no longer present. In contrast, the maternal protein persists until late in stage 15, at which point reduced dAda2b protein levels are found in homozygous mutant embryos. In order to investigate the contribution of dAda2b to early embryo development, attempts were made to remove maternal dAda2b in germ line clones. However, dAda2b homozygous germ cells arrest at an early stage of oogenesis, and for this reason it was not possible to assess dAda2b function in early embryo development (Qi, 2004).
The insertion sequences of several P elements that cytologically mapped close to the 69C4 position of Gcn5 on polytene chromosomes were determined, as well as which ones were lethal over the Df(3L)iro-2 deficiency. The 1479/10 P element was found inserted 521 bp upstream of the Gcn5 transcription start site, in the coding sequence of the CG14121 gene. This insertion was used to generate deletions via transposase-mediated P-element excision and the lethal deficiency sex204 was recovered that removes a part of the CG14121 coding sequence, the Gcn5 and CG10686 genes, and the transcription start site of the citron gene. To obtain Gcn5-specific alleles, a screen was performed for EMS-induced Gcn5 mutants by noncomplementation of the sex204 deficiency. Eleven mutants were obtained that failed to complement the sex204 deficiency for viability. Five mutants were rescued by a PL transgenic construct encompassing the Gcn5 and CG14121 genes. Four of these alleles were fully rescued by a UAS-Gcn5 cDNA transgene expressed under the control of the ubiquitous da-GAL4 driver, indicating that these alleles specifically impair Gcn5 function. The remaining allele is a mutation of the CG14121 gene. The Gcn5 mutations were sequenced and it was found that they all lie within the first dGcn5 exon. Two of them are localized in the Pcaf homology domain and result in a cysteine-to-tyrosine substitution (C137T) or a small deletion from tyrosine 280 to phenylalanine 285 (DeltaT280-F285). The two other mutations generate stop codons (Q186st and E333st). Gcn5E333st mutants were outcrossed and then rescued as homozygous by the PL transgene, indicating that they do not bear other irrelevant EMS-induced lethal mutations (Carré, 2005).
To determine the lethal phase associated with the Gcn5 loss of function, the Gcn5E333st allele was maintained over a GFP-expressing balancer chromosome. About 26% of eggs laid by females from this stock did not reach the first larval instar, a number attributable to embryonic lethality from the homozygous balancer chromosome. Nonfluorescent homozygous Gcn5E333st larvae developed similarly to their GFP-expressing fluorescent heterozygous siblings until the end of the third larval instar, despite the absence of detectable Gcn5 protein in mutant tissues at this stage. However, Gcn5E333st mutant larvae did not form their puparium at the normal time and continued to wander for 4 to 5 additional days. They eventually stopped moving but failed to evert spiracles, formed abnormally long prepupae, and died with partial separation of the posterior part of the prepupa from the pupal case. Similar developmental arrest and defects were observed with the heteroallelic null combination Gcn5Q186st/Gcn5E333st (Carré, 2005).
Puparium formation is triggered by a pulse of 20-hydroxyecdysone at the end of the third larval instar. This involves the coordinated induction of a small number of early puff genes, whose products in turn regulate the expression of a larger set of late puff genes. Homozygous Gcn5E333st mutants were taken at various times during their extended wandering stage, and polytene chromosomes from their salivary glands were squashed. A strong reduction was observed of the size of the early puffs 2B, 74EF, and 75B in these animals, indicating a failure of the ecdysone-controlled genetic program in Gcn5 mutants (Carré, 2005).
Heteroallelic Gcn5C137T/Gcn5E333st and Gcn5E333st/Gcn5DeltaT280-F285 mutant larvae form their puparium normally, indicating that the Gcn5C137T and Gcn5DeltaT280-F285 variant proteins retain partial function. However, these mutants fail to elongate their metathoracic legs correctly, have rough eyes, and display a partial to complete absence of abdominal cuticle deposition. Both heteroallelic combinations give rise to rare adult escapers with misshapen wings, rough eyes, and crooked and twisted metathoracic legs. These escapers died a few hours after eclosion. Altogether, these data point to an essential function of Gcn5 at the onset of and during metamorphosis (Carré, 2005).
Since an important stock of Gcn5 protein is detected in oocytes and presyncytial embryos, this maternal contribution of Gcn5 may be sufficient to allow embryonic and larval development. To generate embryos lacking a maternal contribution, advantage was taken of the absence of expression of the pUAST-derived construct UAS-Gcn5 in the female germ line. UAS-Gcn5/+; Gcn5E333st/sex204 da-GAL4 rescued adults were generated, and oogenesis in females was found to be arrested at stages 5 and 6. This effect was due to the lack of UAS-Gcn5 expression in the germ line, since control females rescued by the Gcn5 genomic construct (PL/+; Gcn5E333st/sex204) were fertile. With Gcn5 being required for oogenesis, its contribution to embryonic development was not examined (Carré, 2005).
Imaginal discs from homozygous Gcn5E333st mutant third-instar larvae are misshapen and severely reduced in size, suggesting that a Gcn5 loss of function impairs the proliferation of imaginal cells during larval instars. In a previous report, use was made of the inverted-repeat transgenic construct UAS-IR[Gcn5] to target RNA interference (RNAi) against Gcn5 (Roignant, 2003). To further analyze the role of Gcn5 in the cell proliferation of imaginal tissues, a UAS-IR[gcn5] transgenic line was crossed with en-GAL4 UAS-GFP individuals expressing the GAL4 activator. The en-GAL4 driver induced both GFP expression and specific dGcn5 depletion in the posterior (P) compartments of imaginal discs of third-larval-instar progeny. In a control experiment, silencing of the unrelated CBP protein was not observed. The Gcn5-depleted P compartments often had a reduced size and appeared flatter than anterior compartments, suggesting that cell proliferation is slowed down in these compartments. Although Gcn5 RNAi in the P compartments of imaginal discs induced strong lethality during late pupal development, few animals survived until the adult stage. The posterior part of their wings was reduced in size and displayed abnormal veins and cross veins. In the most dramatic cases, a bubble indicative of abnormal adhesion between the dorsal and ventral wing epithelial sheets was observed. The cell density was not significantly changed in the silenced compartment, indicating that the size reduction is due to a reduction in the cell number. A vg-GAL4 driver triggered Gcn5 RNAi in the wing margin and induced a strong reduction of this structure in silenced adults, while the induction of Gcn5 RNAi using a dll-GAL4 driver led to a reduction in the size of the distal part of the tarsus and wings. Finally, the induction of Gcn5 RNAi by an esg-GAL4 driver, which is strongly expressed in imaginal histoblasts, resulted in lethality of pharate adults, with a partial to complete absence of the abdominal cuticle. Collectively, these data strongly suggested that Gcn5 RNAi limits cell proliferation (Carré, 2005).
Surprisingly, however, a greater proportion of cells in S phase as well as a significantly larger number of cells undergoing mitosis was observed in the P compartments of wing discs silenced by UAS-IR[Gcn5] upon activation by en-GAL4. Notably, a high level of apoptosis was detected in imaginal discs from third-instar Gcn5 mutant larvae as well as in response to Gcn5 RNAi triggered by either en-GAL4 or ptc-GAL4. Together, these data suggest that the net effect of the Gcn5 loss of function on the compartment size results from a deregulation of the cell cycle coupled to cell death (Carré, 2005).
The p55 family WD40 repeat-containing histone chaperone proteins are components of several chromatin regulatory complexes (such as PRC2, NURF and CAF-1) and interact with histone H4, yet their functional relevance in vivo is unclear. This study used Drosophila as a genetic model to dissect the function of p55/Caf1 during development. p55 was found essential for Drosophila development and is required for cell proliferation and viability. However, the data further demonstrate that histone H3K27 di-/tri-methylation and PRC2-mediated gene silencing still occur normally when p55 is missing. p55 is also implicated in bridging chromatin regulatory complexes to the chromatin by binding to histone H4, but a transgene of p55 whose binding pocket is disrupted could still functionally substitute the wild-type p55 for the survival. These studies suggest that p55 is not crucial for PRC2-mediated gene silencing in vivo, and the vital function of p55 is probably not dependent on its interaction with histone H4 (Wen, 2012).
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date revised: 20 March 2012
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