4.2.1 Mi-2 functions as a transcriptional repressor at ecdysone induced genes Mi-2 had been previously shown to function as a co-repressor at developmental and proneural genes (Kunert et al., 2009; Murawsky et al., 2001). In contrast, Mi-2 acts as a co-activator to promote full transcriptional activation of heat shock genes (Murawska et al., 2011). The observation that Mi-2 was recruited to several ecdysone dependent genes prompted us to examine its role at the broad and vrille gene. In order to investigate the function of Mi-2, expression of vrille and broad in Mi-2 depleted cells was analysed.
93 Figure 4.9: Expression of broad and vrille in Mi-2 depleted cells. (A) Nuclear extracts from S2 cells treated with dsRNA against GFP, EcR or Mi-2 and incubated with (+) or without (-) 20HE were subjected to Western blot using antibodies indicated on the right. Detection of tubulin served as a loading control. Molecular weight in kDa is depicted on the left. (B) Expression of mRNA of broad and vrille from untreated S2 cells incubated with dsRNA against GFP (white bars), EcR (dark grey bars) or Mi-2 (light grey bars) was determined by RTqPCR.
mRNA levels were calculated relative to the housekeeping gene Rp49 and mRNA levels in cells treated with GFP dsRNA were set to 1. (C) Timecourse of mRNA expression of broad and vrille upon 0, 30, 60, 120, 240 and 360 minutes of 20HE induction in S2 cells treated with dsRNA against GFP (rhombus), EcR (circle) or Mi-2 (square) was determined by RTqPCR. mRNA levels were calculated relative to the housekeeping gene Rp49 and mRNA levels in untreated cells incubated with GFP dsRNA were set to 1. Error bars denote standard deviation of technical triplicates. Standard deviations for broad are not visible in the time course due to the scale of the y-axis. Experiments were performed as biological triplicates and one representative experiment is shown here.
Hence, S2 cells were subjected to dsRNA treatment as described above. In addition, a dsRNA specifically targeting EcR was introduced as a positive control since knockdown of EcR had been shown to efficiently abolish the activation of the ecdysone cascade (Beckstead et al., 2005). The depletion of EcR and Mi-2 was efficient as demonstrated by Western blot (Figure 4.9A, lanes 1-6). Additionally, it was observed that Mi-2 protein level was not affected upon EcR knockdown (lanes 3 and 4).
However, upon Mi-2 depletion, EcR protein levels seemed to be slightly decreased (lanes 5 and 6). Expression analyses demonstrated that knockdown of Mi-2 as well as EcR lead to a 15-fold increase of broad transcript levels in cells that were not treated with 20HE (Figure 4.9B). Also, expression of vrille was fourfold upregulated in EcR and twofold upregulated in Mi-2 dsRNA treated cells. This demonstrated that depletion of Mi-2 and EcR lead to expression of ecdysone-regulated genes that are otherwise less transcribed in untreated S2 cells. Further, expression of broad and vrille was examined
94 at five different time points upon ecdysone induction in dsRNA treated cells (Figure 4.9C). Cells incubated with a dsRNA against GFP showed an eightfold increase of broad expression 60 min after 20HE induction. Expression of broad mRNA further increased with time, reaching about 480-fold increase compared to non-treated cells (t=0’) after six hours. Vrille transcription was sixfold higher after 30 minutes and further increased up to 16-fold after six hours of 20HE treatment. As expected, in cells depleted of EcR, mRNA levels did not show a robust induction after six hours of ecdysone treatment. In contrast, depletion of Mi-2 from S2 cells, led to a significant increase of expression of broad at all measured time points compared to GFP treated samples (300-fold after 2 hours, 720-fold after six hours). Comparable results were found for the vrille gene, where expression in Mi-2 depleted cells was 18-fold upregulated after six hours as compared to a 12-fold increase in vrille mRNA in GFP dsRNA treated cells. From these results, I hypothesised that in untreated cells Mi-2 as well as EcR function as transcriptional repressors at the broad and vrille genes.
Further, I confirmed that EcR is a crucial factor for efficient transcriptional activation upon 20HE treatment. By contrast, Mi-2 appears to retain its function as a co-repressor upon 20HE treatment, as its depletion resulted in superactivation of both genes.
4.2.2. Depletion of Iswi does not lead to superactivation of ecdysone dependent genes
To investigate whether the function of Mi-2 is specific for this particular ATP-dependent chromatin remodeler, we depleted a different ATP-dependent chromatin remodeler of the SNF2 family, namely Iswi, from S2 cells. Iswi was previously shown to function as a transcriptional activator of ecdysone regulated genes in Drosophila (Badenhorst et al., 2005). Efficient knockdown of Iswi by RNAi was verified by decreased levels of mRNA in RTqPCR as no antibody for detection of protein levels in Western blot was available (Figure 4.10A). In cells treated with a dsRNA against Iswi, expression of Iswi was 30-fold downregulated compared to GFP treated cells. Interestingly, Iswi expression was fivefold decreased in GFP dsRNA treated cell that were induced with 20HE. Therefore, I hypothesised, that Iswi mRNA expression was negatively influenced by ecdysone.
95 Figure 4.10: Expression of broad and vrille in Iswi depleted cells. (A) Expression of mRNA of Iswi incubated with dsRNA against GFP (white and grey bars) and Iswi (light and dark purple bars) in S2 cells incubated with (grey and dark purple bars) or without (white and light purple bars) 20HE was determined by RTqPCR. mRNA levels were calculated relative to the housekeeping gene Rp49 and mRNA levels in untreated cells incubated with GFP dsRNA were set to 1. (B) Timecourse of mRNA expression of broad and vrille upon 0, 30, 60, 120, 240 and 360 minutes of 20HE induction in S2 cells treated with dsRNA against GFP (rhombus) and Iswi (triangle) was determined by RTqPCR. mRNA levels were calculated relative to the housekeeping gene Rp49 and mRNA levels in untreated cells incubated with GFP dsRNA were set to 1. Error bars denote standard deviation of technical triplicates. Standard deviations for broad are not visible in the time course due to the scale of the y-axis. Experiments were performed as biological triplicates and one representative experiment is shown here.
In uninduced cells (t=0’) broad was twofold upregulated when treated with Iswi dsRNA compared to GFP dsRNA treated cells, whereas no change was detected for vrille expression (Figure 4.10B and data not shown). Upon hormonal stimulation, expression of broad was decreased twofold in Iswi depleted cells as compared to GFP dsRNA after 60 and 120 minutes. This difference in expression levelled off four hours after induction when comparable amounts of mRNA in GFP and Iswi dsRNA treated cells were detected. No significant difference between GFP and Iswi dsRNA treated cells was observed for vrille expression. These experiments demonstrated that Iswi does not appear to function as a transcriptional repressor or activator of broad and vrille.
Further, I hypothesised that Mi-2 functions as a major repressive ATP-dependent chromatin remodeler at the ecdysone induced genes broad and vrille.
4.2.3 Mi-2 regulates transcription of two non-coding RNAs
Mi-2 binding upon 20HE treatment occurred in a genomic region that does not only contain promoter sequences of the vrille gene, but also codes for two non-coding
96 RNAs CR44742 and CR44743. Thus, I proposed that Mi-2 may not only contribute to the regulation of vrille expression but could also influence expression of these ncRNAs.
Both RNAs were expressed to a low extent in S2 cells, but were upregulated about 20-fold upon 20HE treatment (Figure 4.11). Interestingly, changes in expression of both RNAs in EcR and Mi-2 depleted cells were comparable with the findings for the vrille gene (Figure 4.9C). In uninduced cells treated with a dsRNA against Mi-2 CR44742 and CR44743 transcripts were upregulated about fivefold compared to GFP treated cells. In the presence of hormone, noncoding transcript levels were fourfold higher in Mi-2 depleted cells than in GFP dsRNA treated cells. In agreement with the findings above (Figure 4.9B), depletion of EcR resulted in derepression of both ncRNAs in uninduced S2 cells by about twofold. Induction of the ecdysone cascade resulted in an increase in both transcripts in EcR depleted cells, however this transcriptional activation was not as strong as the effect seen in GFP dsRNA treated cells. I concluded that Mi-2 and EcR contributed to the regulation of the two ncRNAs CR44742 and CR44743, in a manner comparable to what was observed for broad and vrille expression.
Figure 4.11: Expression of non-coding RNAs in Mi-2 depleted cells. Expression of CR44743 and CR44742 incubated with dsRNA against GFP (light and dark grey bars), EcR (light and dark green bars) or Mi-2 (light and dark red bars) in untreated (light bars) and six hours 20HE treated (dark bars) S2 cells was determined by RTqPCR. mRNA levels were calculated relative to the housekeeping gene Rp49 and mRNA levels in untreated cells incubated with GFP dsRNA were set to 1.Error bars denote standard deviation of technical triplicates. Experiments were performed as biological triplicates and one representative experiment is shown here.