Figure 1.
Gene models of the Eip74EF and Eip75B genes.
Characteristic transcript variants of the two genes are shown and labeled based on the Drosophila genome annotation by FlyBase [51]. Exons are represented by solid rectangles, promoters are marked by arrows. Arrowtails show the position of amplicons detected in Q-PCR based transcript analysis and ChIP assays. These amplicons fall to the following regions: Eip74EF-RA promoter (RA prom), Eip74EF-RA 5′ transcribed region (intron 1), Eip74EF-RB promoter (RB prom), Eip74EF-RB 5′ transcribed region (intron 5), Eip74EF 3′ transcribed region (exon 8), Eip75B-RC promoter (RC prom), Eip75B-RC 5′ transcribed region (intron 2) and Eip75B-RC 3′ transcribed region (exon 8). The sequences and chromosomal location of primers are in Table S1.
Figure 2.
Transcriptional activity of Eip74EF and Eip75B promoters during the third larval instar.
Transcript levels from the Eip74EF-RA (A), Eip74EF-RB (B) and Eip75B-RC (C) promoters in w1118 larvae are shown. The charts show the average of relative RNA levels in percent of the maximum RNA quantity measured with the given primer pairs, the error bars indicate the standard error.
Figure 3.
Specific histone acetylations show different spatial and temporal patterns on ecdysone induced genes.
Chromatin samples from middle-stage L3 larvae (mid-L3, empty bars), from wandering L3 larvae (w-L3, grey bars) or from late L3 larvae prior pupariation (spev-L3, black bars) were immunoprecipitated with antibodies against (A) histone H3, (B) acetyl-H3K9, (C) acetyl-H3K23, (D) acetyl-H4K8, (E) acetyl-H4K12 or (F) acetyl-H4K16, then quantitated by real-time PCR. In case of histone H3 (A), the amount of precipitated DNA is expressed in the percent of the total input control (TIC). Charts B-F shows the relative quantities of the DNA precipitated by the specific antibodies normalized to the amount of the H3 precipitate, the average ± s.e.m. of three biological replicates are show. (Percentage values do not compare between different antibodies.) The primers detected the following regions: RA-promoter, intron 1, RB-promoter, intron 5 and exon 8 of Eip74EF; RC-promoter, intron 2 and exon 8 of Eip75B, the promoter of Rpl32 and a euchromatic intergenic control region.
Figure 4.
The nej/dCBP protein is responsible for H3K23 specific acetylation in Drosophila.
(A) The level of acetylated H3K23 is unchanged in L3 larvae homo- or hemizygous for loss of function mutant alleles of the gcn5, mof or chm histone acetyltransferase genes, and also in homozygous Ada2a or Ada2b mutants specific for the GCN5 containing ATAC and SAGA complexes, respectively. (B) In nej3 mutant embryos the level of acetyl-H3K23 is significantly reduced compared to the wild-type as quantitated by immunostaining of whole mount embryos. Mean pixel intensity ± s.e.m. are shown, P<0.01. (C) ChIP analysis of chromatin samples from gcn5E333St homozygous null mutant (gcn5) and heterozygous (cont) wandering L3 larvae using acetyl-H3K23 specific antibody detects the presence of the acetyl-H3K23 mark in gcn5 mutants. In engrailed-GAL4 UAS-GFP embryos also carrying either an UAS-dCBP (D) or an UAS-dCBP-FLAD (E) transgene, the UAS transgenes are expressed in the posterior part of every segment, as visualized by GFP fluorescence. Immunostaining with anti-acetyl-H3K23 specific antibody reveals that the level of acetyl-H3K23 is dramatically increased in embryos overexpressing UAS-dCBP (F), while it is unchanged in embryos overexpressing the UAS-dCBP-FLAD construct (G), which is mutated in the acetyltransferase domain of CBP.
Figure 5.
dCBP is required for larval development and ecdysone induced gene expression.
(A) Heat-shock activated one-off expression of a UAS-nejire-RNAi construct in hs-GAL4> UAS-nej-RNAi animals (empty bars) reduces viability compared to non-heat-shocked controls (filled bars). Especially strong effect could be observed if the heat-shock was administered between days 3 and 6 after egg laying. Viability is expressed as the ratio of the number of eclosed hs-GAL4> UAS-nej-RNAi flies and UAS-nej-RNAi non-expressing control siblings. (B) The level of Eip74EF and Eip75B specific transcripts is decreased in nej-RNAi expressing hs-GAL4> nej-RNAi wandering L3 larvae (empty bars) compared to non-expressing control siblings (filled bars). Quantitative PCR analysis was performed with intronic primer pairs specific for the Eip74EF-RA, -RB or Eip75B-RC promoters (E74-RA, E74-RB and E75-RC, respectively), and with primer pairs located in downstream exons detecting mRNA products of the two genes (E74-ex8 and E75-ex8). The chart shows the relative RNA quantities normalized to transcript levels in non-expressing controls ± s.d.