Title: Transcriptional profiles (mRNA-seq) of Drosophila G9aDD1 mutants and control during 0, 6 and 12 hours of paraquat oxidative stress exposure.

Purpose: Investigating the role of Drosophila G9a in oxidative stress responses. Methods: Flies were collected after eclosion and allowed to recover from CO2 exposure for 5 days prior to paraquat exposure. Paraquat (Methyl viologen dichloride hydrate 98 %; Sigma 856177) was mixed into the flyfood at 40 °C to a final concentration of 50 mM. For OS induction, 5-9 day old flies were transferred to paraquat containing food and incubated at 25 °C and 70 % humidity. At each time point, flies were flash frozen in liquid nitrogen followed by vortexing and filtering through a series of sieves to isolate heads from other body parts. 200 fly heads per sample were used for RNA extraction using QIAGEN lipid mini tissue kit. The TruSeq RNA Sample Preparation Kit v2 (Illumina) was used to prepare adapter ligated PCR fragments for sequencing. PCR was used to selectively enrich the fragments containing the adapters. The PCR fragments were validated using Agilent 2200 TapeStation. Single indexed samples were multiplexed and sequenced on an Illumina HiSeq 2000 sequencing system (Illumina) in single-end mode with a read length of 35 bp. Quality of sequenced reads was assessed with FastQC. The RNAseq experiments were conducted on two biological duplicates for each condition. Sequenced reads were aligned with Burrows-Wheeler algorithm (BWA) (Li & Durbin, 2010) to the Drosophila reference genome (BDGP.5, http://www.fruitfly.org/) and per gene read counts were generated with HTSeq count (Anders et al, 2015). 25–30 million reads with high quality alignment were obtained for each sample and used for differential expression analysis. DESeq (Anders & Huber, 2010) was used to obtain library size-normalized read counts and to calculate differential expression of genes in 4 pairwise comparisons: 0 h versus 6 h and 0 h versus 12 h after OS in both G9a mutants and controls (fold change =1.5, adjusted p-value= 0.05, Benjamini-Hochberg). Results: We found 2731 genes to be differential expressed in at least one of the four pairwise comparisons. The largest group of differentially expressed (DE) genes are highly augmented upon OS induction in the G9a mutant (41.7 % of all DE genes). The second largest group of DE genes (23.9 % of all DE genes) were more downregulated in G9a mutant in response to OS. Genes that are over-activated in G9a mutants are predominantly involved in OS response and OS mediated damage, whereas genes that are downregulated in G9a mutants are involved in energy metabolism. Conclusions: Our data suggest that G9a provides an epigenetic mechanism that safeguards an appropriate transcriptional response to OS and preserves immediately available energy, thereby acting as a critical regulatory hub between the transcriptional and physiological responses to oxidative stress. Overall design: fly-head mRNA libraries of 5-9 days old male G9aDD1 mutant and control during 0, 6 and 12 hours of paraquat oxidative stress exposure were sequenced in duplicate on Hi-seq 2000.

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Riahi H, Brekelmans C, Foriel S, Merkling SH, Lyons TA, Itskov PM, Kleefstra T, Ribeiro C, van Rij RP, Kramer JM, Schenck A