Additional file 14 of A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells
Creators: name: Bai, Yu
name: Caussinus, Emmanuel
name: Leo, Stefano
name: Bosshardt, Fritz
name: Myachina, Faina
name: Rot, Gregor
name: Robinson, Mark D.
name: Lehner, Christian F.
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 14: Fig. S7. Evaluation of the role of histone H2Av (His2Av) in transcriptional control of temperature-regulated genes in S2R+ cells. (A) To address the role of His2Av in temperature-dependent regulation of gene expression in S2R+ cells, we added His2Av dsRNA or lacZ dsRNA for control to culture aliquots followed by a shift to the indicated temperatures after four days. Twenty-four hours after the shift, we isolated total RNA for analysis with microarrays. Three replicate experiments were performed. (B) Analysis of transcript levels of His2Av and Act5C (for control) confirmed successful depletion of His2Av transcripts. Bar diagrams display transcript levels (average of three biological replicates and s.d.) at the analyzed temperatures. Average expression at the three temperatures observed after treatment with lacZ dsRNA was set to 100%. (C) Analysis of His2Av depletion by immunoblotting. S2R + _MtnAp-His2Av-mRFP cells (Lidsky et al. 2013) were treated with dsRNA derived from lacZ (for control) and two distinct His2Av amplicons (H2Av_1 and H2Av_2) for three and 5 days as indicated. Cell extracts were analyzed by immunoblotting with anti-mCherry (detecting His2Av-mRFP) and anti-PSTAIR (loading control). The combined treatment with both His2Av dsRNA preparations (H2Av_1 + 2) for 5 days was found to reduce His2Av-mRFP levels to 40% of controls according to quantification of signal intensities and normalization based on anti-PSTAIR signals. (D) Effects of His2Av depletion on the S2R+ cell transcriptome. A volcano plot is displayed with probes associated with signal intensities that were significantly (FDR &lt; 0.05; fold change ≥2) down- (green dots, 320 probes) or upregulated (brown dots, 300 probes) by His2Av depletion in comparison to control (lacZ dsRNA treatment). The comparison based on signals detected at 14 °C is shown. Analogous comparisons at 25 or 30 °C resulted in comparable observations. (E) Effects of His2Av depletion on transcript levels of temperature-regulated genes. Probes associated with a fold change in signal intensity ≥2 in the comparison of 14 and 30 °C in both control experiments (with lacZ dsRNA treatment and without any dsRNA treatment) were selected for further analysis. Plots summarize the signal intensities obtained with these probes, separated into CoolUp and CoolDown probes, after treatment with either lacZ or His2Av dsRNA at the three different temperatures. The comparison revealed at most subtle effects of His2Av depletion on transcript levels of temperature-regulated genes in S2R+ cells, in contrast to results from Arabidopsis thaliana where a reduction in the homolog His2A.Z has been found to result in a dramatic conversion of the expression of genes that are normally temperature-regulated into a constitutive warm-like expression [20]. (F) Fold change correlation analyses for further analysis of the effect of His2Av depletion on temperature-dependent gene regulation. Scatter plots of log2 fold changes for all probes with significant signals are displayed. r = Pearson’s correlation coefficient. As revealed by the left panel, the fold changes observed when comparing expression levels at 14 and 30 °C were very strongly correlated in the two control experiments (with lacZ dsRNA treatment and without any dsRNA treatment). However, there was essentially no correlation between the fold changes resulting from temperature change and those resulting from His2Av depletion at 14 °C (middle panel) or 30 °C (right panel), in contrast to results from similar analyses in Arabidopsis thaliana [20], where reduced loading of the homolog H2A.Z into chromatin (in arp6–10 mutants) results in r = 0.68 for the correlation between transcriptome regulation by temperature and misregulation by reduced H2A.Z.