In the last funding period, we used computational approach to detect tTI at promoters of genes. Essentially, here we addressed the questions:
1. What is the evidence for RNA production overlapping the gene promoter?
2. What is the evidence for the repression of transcriptional initiation of the gene promoter?
3. What is the evidence for chromatin signaling of RNAPII elongation at the promoters?
To address 1.) we combined the information from our data that identified both ends of RNA molecules to identify gene promoters that were “invaded” by another transcript on the same strand.
To address 2.), we used data that specifically reveals the 5´-ends of RNAs. We noticed that commonly used genome annotations showed great differences in their annotation of the 5´-ends of RNAs compared to the experimental data identifying 5´-ends of RNAs. To use correct 5´-ends, we thus had to generate genome annotation files that were more accurate than the off-the-shelf annotations that offer a simplified compromise between different tissues and environmental conditions. These efforts resulted in the development of the TranscriptomeReconstructoR pipeline.
To address 3.), our prior analyses of ChIP-seq data suggested that RNAPII elongation in plants is characterized by certain post-translational modification of histone H3 and H4 N-terminal tails, namely mono-methylation at lysine 4 of histone H3 (H3K4me1) and di-methylation at lysine 36 of histone H3 (H3K36me2), perhaps also H3K4me2 and H3K36me3. However, we were unsure how strongly the available ChIP-seq data in Arabidopsis seedlings were confounded by the reproducibility crisis linked to peptide antibodies targeted the same modification. We thus started an industrial collaboration with the epigenomics company Epicypher. We used their K-met-stat panel in combination with cleavage under target and tagmentation (CUT&Tag) method. We compared the specificity of 18 different antibodies covering different the three methylation states an H3K4 and H3K36 as well as H3K27me3. We identified the antibodies with highest on-target specificity, and used these data as the foundation to identify which gene promoters in Arabidopsis seedlings show evidence for chromatin-signatures of RNAPII elongation. We pursued a link to developmental gene regulation by applying our methods to siliques. Indeed, we found that alleviated repression in siliques at many of gene promoters, coinciding with a shifted chromatin profile. We are currently preparing the manuscript addressing 3.) for publication. tTI is a common mechanism to repress mRNA initiation in Arabidopsis. It is connected to chromatin-signaling of RNAPII elongation, reversible throughout development, and linked to the local concentration of transcription factors.
