ABSTRACT: Purpose: In Drosophila, we determined that neural activity-dependent transcription is controlled by CBP, Rpd3, and CoRest-C. To investigate the dynamics of those binding to the chromatin, and the relationship with activity-dependent transcription, we conducted the ChIP-seq assay for the three proteins, and nuclear RNA-seq analysis in mushroom body in Drosophila, either in naive state or after optogenetic activation. Methods: The nuclei of mushroom body were collected by immunoprecipitation-based method (Hirano Y., et al, 2016, Nature Communications, also see extraction protocol below). The chromatin prepared from the collected mushroom body was used for ChIP-seq with antibody for CBP, Rpd3 or CoRest-C. For CBP and Rpd3, antibody was raised against the endogenous proteins. For CoRest-C, which was tagged by myc, anti-myc antibody was used. In nuclear RNA-seq, mRNA was purified from the mushroom body nuclei with oligo dT magnetic beads, and used to prepare library. After deep sequencing in triplicate using illumine Hiseq X, the adaptors were trimmed via Trimommatic, followed by mapping to the Drosophila reference genome, dm6 from UCSC using STAR. The reads with low mapping quality below 8 and the non-primary mapped reads were eliminated. MACS was used for peak calling in ChIP-seq on Strand NGS software, using a default setting except for the following parameters; 10-4 as a P-value cutoff, and 3 as a enrichment factor. The binding sites were determined when the peaks were overlapped in at least 2 out of 3 biological replicates, using the optogenetically activated samples. For RNA-seq, the filtered reads were analyzed with HTSeq-count to obtain numbers of the reads mapped on exons, which was further analyzed on R using DESeq2 Results: Using an optimized data analysis workflow, we mapped about 9-18 million reads for ChIP-seq, and 30-40 million reads for nuclear RNA-seq to Drosophila reference genome, dm6 In ChIP-seq, binding of all proteins was enriched nearby the transcriptional start site (TSS). The binding sites determined were 1,237 for CoRest-C, 2,717 for Rpd3, and 6,684 for CBP. Importantly, among CoRest-C binding sites, overlapping sites of CoRest-C and CBP were 1,038/1,237 (83.9%), those of CoRest-C and Rpd3 were 789/1,237 (63.8%), and those of CoRest-C, Rpd3, and CBP were 704/1,237 (56.9%), suggesting that these three proteins colocalize on the specific genomic regions In nuclear RNA-seq, we found that upregulation of gene expression is robust at 10-min after 5-min optogenetic activation. The genes showing significant difference were 2,702 at this time point, in which 1,582 genes showed increase, and the rest showed decrease in the expression. Among the 1,582 increased genes, 1,055 genes were bound by CBP, supporting the idea that CBP is an important factor in activity-dependent transcription. Furthermore, 338 was bound by CoRest-C, 254 of which showed colocalization with CBP and Rpd3. Conclusions: Our study represents the first detailed analysis of activity-dependent transcription in mushroom body in Drosophila, with biologic replicates, generated by ChIP-seq and RNA-seq technology. We found the rapid response of transcription after optogenetic activation, and its colocalization with CBP, Rpd3 and CoRest-C binding sites. These data strongly support the idea that these three proteins control activity-dependent transcription.
