Periodic Reporting for period 1 - TEI (Development of a model system to study the role of chromatin factors during transgenerational epigenetic inheritance (TEI) in C. elegans)
Période du rapport: 2017-05-01 au 2019-04-30
The main purpose of the project was to study the biochemistry of a single locus in the C. elegans genome and discover novel pathways in gene silencing and transgenerational epigenetic inheritance (TEI). So far, we have established a unique technology for the first time in C. elegans and discovered a new pathway that silences selfish elements such as transposons. Unexpectedly, this new pathway functions independent of the germline piRNA pathway and comprises poly-ADP polymerases (PARPs), which are very well studied for DNA damage recognition in humans and mice. Here as a result of our project, we propose a new pathway in C. elegans that uses DNA damage as a signal on transposons for silencing machinery independently of small RNA pathways. As a concept, DNA damage is used as a signal for non-self recognition as well as a defence mechanism in bacterial CRISPR and paramecium DNA elimination mechanisms. Further experiments in this project will detail the mechanistic aspects of this new pathway.
One component of this new pathway is an uncharacterised protein with a catalytically dead PARP (poly-ADP polymerase) domain. This finding is crucial since PARP enzymes are important targets in breast and ovarian cancer patients. Further understanding of PARP enzyme regulation and inhibition in C. elegans might have potential therapeutic applications for patients with breast and ovarian cancer. What is more, a potential role for PARP enzymes has been reported in immunological and neurobiological disorders. Our discovery do not just have a potential for a therapeutic application for cancer, but also an additional understanding in neurobiological and immunological disorders.
One component of this new pathway is an uncharacterised protein with a catalytically dead PARP (poly-ADP polymerase) domain. This finding is crucial since PARP enzymes are important targets in breast and ovarian cancer patients. Further understanding of PARP enzyme regulation and inhibition in C. elegans might have potential therapeutic applications for patients with breast and ovarian cancer. What is more, a potential role for PARP enzymes has been reported in immunological and neurobiological disorders. Our discovery do not just have a potential for a therapeutic application for cancer, but also an additional understanding in neurobiological and immunological disorders.
Transposable elements are segments of non-self DNA that have the ability to copy and move themselves within the host genome and hence create a threat to the genome stability and integrity. To avoid the spreading of these selfish elements, organisms have evolved different defence mechanisms such as small RNAs with the piRNA pathway being one of them in the nematode C. elegans germline. To identify novel proteins involved in the spatial and temporal regulation of the chromatin state and dependent on the piRNA pathway, we have developed a germline specific single locus ChIP system (piChIP) and successfully identified an uncharacterized protein with a catalytically dead PARP (poly-ADP ribosyl polymerase) domain that resembles to the TASOR and MPP8 proteins of the recently discovered HUSH complex in humans.
Interestingly, null mutants f this catalytically dead PARP protein showed synthetic lethality when crossed to a piRNA pathway mutant, hrde-1, at 20C, therefore indicating a parallel pathway. Hence, we started to characterize its role together with catalytically active C. elegans PARP-1 by comparing transcriptome and genome wide binding profiles of wild type and null mutant animals. We found by total RNA sequencing that null mutants desilenced retrotransposons, such as CELE45, to a similar extent, suggesting that these two proteins might function in the same pathway. Additionally, these RNA-seq data sets showed no correlation with those of other known piRNA or nuclear RNAi pathway mutants, demonstrating that they might silence a specific subset of targets. Furthermore, PARP-1 ChIP-seq suggested specific binding on a set of SINE retrotransposons including CELE45 and this enrichment was consistent with PARP-1 ChIP-seq performed in a piRNA pathway mutant.
This new protein with a catalytically dead PARP domain shows domain similarity to the HUSH complex components as well as to the TONSL protein, which interacts with BRCA-1 in humans. Since BRCA-1, together with TONSL, recognize R-loops and single strand DNA breaks as a result of transcription deficiencies and PARP-1 mediates DNA damage repair for single and double strand breaks, we speculate that their specific enrichment on retrotransposons could be transcription-dependent. We are now trying to characterize proteomics analysis in relation to RNA pol II function and DNA damage response on retrotransposons.
Interestingly, null mutants f this catalytically dead PARP protein showed synthetic lethality when crossed to a piRNA pathway mutant, hrde-1, at 20C, therefore indicating a parallel pathway. Hence, we started to characterize its role together with catalytically active C. elegans PARP-1 by comparing transcriptome and genome wide binding profiles of wild type and null mutant animals. We found by total RNA sequencing that null mutants desilenced retrotransposons, such as CELE45, to a similar extent, suggesting that these two proteins might function in the same pathway. Additionally, these RNA-seq data sets showed no correlation with those of other known piRNA or nuclear RNAi pathway mutants, demonstrating that they might silence a specific subset of targets. Furthermore, PARP-1 ChIP-seq suggested specific binding on a set of SINE retrotransposons including CELE45 and this enrichment was consistent with PARP-1 ChIP-seq performed in a piRNA pathway mutant.
This new protein with a catalytically dead PARP domain shows domain similarity to the HUSH complex components as well as to the TONSL protein, which interacts with BRCA-1 in humans. Since BRCA-1, together with TONSL, recognize R-loops and single strand DNA breaks as a result of transcription deficiencies and PARP-1 mediates DNA damage repair for single and double strand breaks, we speculate that their specific enrichment on retrotransposons could be transcription-dependent. We are now trying to characterize proteomics analysis in relation to RNA pol II function and DNA damage response on retrotransposons.
The proposed project used a unique technology for the first time in C. elegans. By using this technology, we identified a new pathway that functions independently of the small RNA pathways. We seek to decipher mechanistic aspects of this unique pathway by biochemistry and genetics. This new pathway comprises of poly-ADP polymerases (PARPs), which are important targets in clinical studies for breast and ovarian cancer. There is also an increasing potential of PARP enzyme regulation for immunological and neurobiological disorders.