Periodic Reporting for period 4 - PUNCTUATION (Pervasive Upstream Non-Coding Transcription Underpinning Adaptation)
Période du rapport: 2022-08-01 au 2023-07-31
According to the convention in current textbooks, RNA acts as a passive carrier of information between DNA and protein. This view is highly over-simplified: it only applies to protein-coding messenger RNA (mRNA) that represents about 2% of the DNA in our cells. An astounding 80-90% of our genome produces RNA with unclear functional roles, since these do not encode proteins (non-coding RNA). Sequence changes of non-coding DNA reduces cellular health and causes human diseases. A key question of contemporary biology is therefore: how does non-coding DNA affect cellular processes on the molecular level? My proposal addresses this question by focusing on the hypothesis that non-coding DNA can affect biological processes through conversion into non-coding RNA by the processes of transcription. The results of my proposal highlighted the purpose of mysterious non-coding DNA in genomes.
The project identified cellular roles of non-coding RNAs in plants. Beyond the roles of the non-coding genome as RNAs, a key aim of my proposal was the characterization where in genomes non-coding DNA may function through the act of transcription mechanism. Here, we made strong progress to characterize this phenomenon in plants. We utilized a cutting-edge chromatin profiling method to identify where this mechanisms operates during plant growth and development. It is now clear that gene repression by the act of non-coding transcription is frequent in plants. We are now in a much better position to identify and predict gene regulation by this mechanism in genomes. With the conclusion of the action, we are preparing a manuscript outlining this for publication. Since DNA sequence conservation is not a key requirement for this mechanism, our results have a strong potential for societal impact, as they promise to highlight a different kind of conservation that operates in at least parts of the non-coding genome.
The project identified cellular roles of non-coding RNAs in plants. Beyond the roles of the non-coding genome as RNAs, a key aim of my proposal was the characterization where in genomes non-coding DNA may function through the act of transcription mechanism. Here, we made strong progress to characterize this phenomenon in plants. We utilized a cutting-edge chromatin profiling method to identify where this mechanisms operates during plant growth and development. It is now clear that gene repression by the act of non-coding transcription is frequent in plants. We are now in a much better position to identify and predict gene regulation by this mechanism in genomes. With the conclusion of the action, we are preparing a manuscript outlining this for publication. Since DNA sequence conservation is not a key requirement for this mechanism, our results have a strong potential for societal impact, as they promise to highlight a different kind of conservation that operates in at least parts of the non-coding genome.
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.
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.
Our experimental methods detected a high number of genomic regions without prior knowledge of RNA production. To combine the information of our experimental approaches, and useful data from colleagues, we developed a computational tool to generate for data-driven genome annotation files. Here, we combine methods informing on the 5´-end, 3´-end and the middles of RNAs. Then, based on the data, we identify transcripts and RNA isoforms. In a proof-of-concept study on Arabidopsis on budding yeast, we could show that our genome annotations matched experimental data on gene boundaries better than commonly used community annotations. We also identified several thousands of RNAs that were lacking in the annotations. Our release of the genome annotation files promises to impact research in this area.
The molecular characterization of FLAIL offers a strong case-study of a trans-acting lncRNA. We focused on a strong foundation by utilizing several strategies to generate mutations of FLAIL. Mechanistic dissection revealed a cellular mechanism of FLAIL as an accessory spliceosomal RNA that affects alternative splicing of the LACCASE 8 mRNA to promote expression and repress flowering. Genetic analysis of double mutants confirmed that FLAIL and LAC8 act in one pathway to repress flowering.
The genetic dissection of my proposal informed on chromatin-based pathways that shape the molecular decisions to activate or repress TSSs overlaid by another transcription unit. The ChIP-seq data for chromatin modifications relies on peptide antibodies. However, exactly these reagents are connected to the reproducibility crisis in life sciences, where antibodies against the same modifications give different results. To control for the variation of peptide antibodies, we collaborated with Epicypher, and used their K-Met-stat panel designed as internal control reagents in epigenome profiling experiments. We are preparing this manuscript for publication including all data on the K-met-stat panel. Moreover, we performed epigenome profiling using CUT&Tag instead of ChIP-seq. The combination of CUT&Tag and specific antibodies was necessary to improve our input data for the computational identification of tTI in Arabidopsis seedlings. Learning how to identify this mechanism was a key goal of PUNCTUATION. We identified tTI based on improved resolution of TSSs, detection of read-through and overlapping transcription and CUT&Tag data. We estimate that tTI represses mRNA initiation at about 2.000 gene promoters in Arabidopsis seedlings. Excellent chromatin-based flags for RNAPII elongation are H3K4me1 and H3K36me2.
The molecular characterization of FLAIL offers a strong case-study of a trans-acting lncRNA. We focused on a strong foundation by utilizing several strategies to generate mutations of FLAIL. Mechanistic dissection revealed a cellular mechanism of FLAIL as an accessory spliceosomal RNA that affects alternative splicing of the LACCASE 8 mRNA to promote expression and repress flowering. Genetic analysis of double mutants confirmed that FLAIL and LAC8 act in one pathway to repress flowering.
The genetic dissection of my proposal informed on chromatin-based pathways that shape the molecular decisions to activate or repress TSSs overlaid by another transcription unit. The ChIP-seq data for chromatin modifications relies on peptide antibodies. However, exactly these reagents are connected to the reproducibility crisis in life sciences, where antibodies against the same modifications give different results. To control for the variation of peptide antibodies, we collaborated with Epicypher, and used their K-Met-stat panel designed as internal control reagents in epigenome profiling experiments. We are preparing this manuscript for publication including all data on the K-met-stat panel. Moreover, we performed epigenome profiling using CUT&Tag instead of ChIP-seq. The combination of CUT&Tag and specific antibodies was necessary to improve our input data for the computational identification of tTI in Arabidopsis seedlings. Learning how to identify this mechanism was a key goal of PUNCTUATION. We identified tTI based on improved resolution of TSSs, detection of read-through and overlapping transcription and CUT&Tag data. We estimate that tTI represses mRNA initiation at about 2.000 gene promoters in Arabidopsis seedlings. Excellent chromatin-based flags for RNAPII elongation are H3K4me1 and H3K36me2.