Periodic Reporting for period 3 - ConflictResolution (Transcription-replication conflicts in disease and development)
Période du rapport: 2023-02-01 au 2024-07-31
Conflicts are a normal part of our social life. After all, it can’t be expected that two people agree on everything at any time. Thus, managing conflicts and finding ways how to avoid and resolve them is an important skill that we need to acquire early-on and continue practicing on an every-day basis in our life. Unexpectedly, conflicts are not only a social phenomenon among human individuals, but they can also arise inside of our body within our cells. More precisely at the molecular level, many molecular machines progress using the same DNA template. "ConflictResolution” investigates how two of these machineries, namely transcription to read the information of genes and DNA replication to duplicate the genome are regulated and coordinated to avoid so-called transcription-replication conflicts (TRCs). In our social environment, mismanagement of conflicts can lead to great harm, and this holds also true for cellular conflicts. TRCs are a potent endogenous source for genetic and epigenetic instability, and can therefore contribute to cancers, aging, developmental and neurological diseases. But how and where do TRCs provoke these instabilities? How frequently do the machineries meet and which fraction of encounters are harmful or get easily resolved?
Unfortunately, these questions are largely unanswered owing to the lack of suitable cellular systems. ConflictResolution deciphers the contribution of collisions to induce pathological transformation of cancer cells as well as physiological changes in embryonic cells.
Unfortunately, these questions are largely unanswered owing to the lack of suitable cellular systems. ConflictResolution deciphers the contribution of collisions to induce pathological transformation of cancer cells as well as physiological changes in embryonic cells.
Aim 1 is focused on characterizing the genetic and epigenetic consequences of collisions on an episomal model locus. In the first 2.5 years of the project, we focused our efforts on subaims 1.2 and 1.3 and achieved the majority of the described goals in the action. We used ChIP-qPCR in synchronized cells to study the fate of the transcription and replication machineries on the mAIRN reporter gene and observed strong promoter-proximal pausing of RNAPII on the reporter gene in S-phase cells. This enrichment of RNAPII coincided with an accumulation of replication factors (MCM2 and PCNA) at the same position, strongly suggesting that the preferred location of the collision site is close to the promoter. Surprisingly, this promoter enrichment was observed independent of HO and CD orientation of the constructs, suggesting that RNAPII promoter escape is an important mechanism to prevent or resolve TRCs. We also mapped chromatin accessibility by limited MNase digestion and observed a strong increase in chromatin accessibility at the mapped promoter TRC site, consistent with the hypothesized “chromatin scar” forming at TRC sites. Finally, we also performed ChIP-qPCR of multiple histone marks along the mAIRN reporter gene including H3K4me3, H3K9me2, H3K36me3 and H3S10ph in synchronized G1-arrested and S-phase-released cells. Most striking results were obtained with H3K4me3 ChIP that showed a specific enrichment under conditions of TRC induction, consistent with recent reports that this mark can mitigate R-loop dependent transcription-replication conflicts. Together, these studies allowed us to characterize in detail the spatial and temporal changes of the local chromatin structure at specific sites of inducible transcription-replication convergence as originally proposed.
Aim 2 is focused on establishing a cellular Split-APEX2 system to map collision sites and identify epigenetic changes in a cancer genome. As proposed, we have tested several combinations of Split-APEX fragments in MCF7 breast cancer cells and established cell lines that allowed us to use proximity labelling to study the proxisome associated with TRCs (Aim 2.1). We performed mass spectrometry on these samples and identified 88 candidate proteins that were specifically enriched over the negative controls (absence of replication or transcription) (Aim 2.2). We validated and functionally characterized one hit of particular interest, the CGG-trinucleotide repeat binding factor 1 (CGGBP1) and showed that this factor functions as a mitigator of DNA secondary structure formation and resulting TRCs. A manuscript summarizing these findings is currently under review in Molecular Cell.
For Aim 2.3 we have performed initial streptavidin pulldown experiments to enrich for genomic fragments associated with TRC sites that resulted in poor yield of DNA fragments that were not sufficient to prepare DNA sequencing libraries. We are currently optimizing different pull-down conditions, use of different biotin substrates and upscaling of the cellular material in order to obtain specific enrichment of TRC-related genomic fragments over the genomic background.
Aim 3 is focused on uncovering the role of transcription-replication interference during zygotic genome activation in early embryonic development. We have successfully implemented the two proposed assays (co-localization of transcripton/replication and RNAPII-PCNA Proximity Ligation assay (TRC-PLA)) in mouse embryonic stem (ES) cells and early 2-cell, mid 2-cell, late 2-cell, 4-cell and 8-cell stage mouse embryos.
Using the 2C::tbGFP reporter cell line (Aim 3.1 i), we found that mouse ES cells show a poor coordination of transcription and replication processes and high TRC-PLA levels, a molecular feature of these pluripotent cells that is lost after cellular dedifferentiation to 2-cell like cells. We have also modulated TRC levels by knocking down candidate factors such as FACT, INO80, SMARCAD1, CHD1, RECQL5 and p97 (Aim 3.1 ii). Our assays scored positive for the siRNAs targeting INO80, RECQL5 and p97 which showed a higher overlap of transcription/replication and more TRC-PLA foci, consistent with our hypothesis.
We have obtained initial results of TRC levels in early mouse embryos prior (early 2-cell stage), during (mid and late 2-cell stage) and after (4-cell and 8-cell stage) major zygotic genome activation (ZGA) in early mouse embryos. Consistent with the onset of transcription, we observe a major deregulation of transcription-replication coordination during ZGA using the pulse-chase-pulse approach (Aim 3.2 i) and TRC-PLA assay (Aim 3.2 ii). We are currently optimizing the microinjection conditions of siRNAs against INO80, RECQL5 and p97 in mouse embryos to test the significance and follow the developmental outcome of these cells with altered TRC levels (Aim 3.2 iii).
Aim 2 is focused on establishing a cellular Split-APEX2 system to map collision sites and identify epigenetic changes in a cancer genome. As proposed, we have tested several combinations of Split-APEX fragments in MCF7 breast cancer cells and established cell lines that allowed us to use proximity labelling to study the proxisome associated with TRCs (Aim 2.1). We performed mass spectrometry on these samples and identified 88 candidate proteins that were specifically enriched over the negative controls (absence of replication or transcription) (Aim 2.2). We validated and functionally characterized one hit of particular interest, the CGG-trinucleotide repeat binding factor 1 (CGGBP1) and showed that this factor functions as a mitigator of DNA secondary structure formation and resulting TRCs. A manuscript summarizing these findings is currently under review in Molecular Cell.
For Aim 2.3 we have performed initial streptavidin pulldown experiments to enrich for genomic fragments associated with TRC sites that resulted in poor yield of DNA fragments that were not sufficient to prepare DNA sequencing libraries. We are currently optimizing different pull-down conditions, use of different biotin substrates and upscaling of the cellular material in order to obtain specific enrichment of TRC-related genomic fragments over the genomic background.
Aim 3 is focused on uncovering the role of transcription-replication interference during zygotic genome activation in early embryonic development. We have successfully implemented the two proposed assays (co-localization of transcripton/replication and RNAPII-PCNA Proximity Ligation assay (TRC-PLA)) in mouse embryonic stem (ES) cells and early 2-cell, mid 2-cell, late 2-cell, 4-cell and 8-cell stage mouse embryos.
Using the 2C::tbGFP reporter cell line (Aim 3.1 i), we found that mouse ES cells show a poor coordination of transcription and replication processes and high TRC-PLA levels, a molecular feature of these pluripotent cells that is lost after cellular dedifferentiation to 2-cell like cells. We have also modulated TRC levels by knocking down candidate factors such as FACT, INO80, SMARCAD1, CHD1, RECQL5 and p97 (Aim 3.1 ii). Our assays scored positive for the siRNAs targeting INO80, RECQL5 and p97 which showed a higher overlap of transcription/replication and more TRC-PLA foci, consistent with our hypothesis.
We have obtained initial results of TRC levels in early mouse embryos prior (early 2-cell stage), during (mid and late 2-cell stage) and after (4-cell and 8-cell stage) major zygotic genome activation (ZGA) in early mouse embryos. Consistent with the onset of transcription, we observe a major deregulation of transcription-replication coordination during ZGA using the pulse-chase-pulse approach (Aim 3.2 i) and TRC-PLA assay (Aim 3.2 ii). We are currently optimizing the microinjection conditions of siRNAs against INO80, RECQL5 and p97 in mouse embryos to test the significance and follow the developmental outcome of these cells with altered TRC levels (Aim 3.2 iii).
As part of this project, we assessed the contribution of transcription-replication interference during early embryonic development. Upon fertilization, embryos undergo massive changes in gene expression and global chromatin structure. We found that TRC levels are strongly elevated in this particular cell cycle, thus placing collisions as a potential mechanism to contribute to epigenetic reprogramming during development. In contrast to current views that TRCs are undesirable, harmful events, this finding uncovers a new physiological role of TRCs in cell differentiation and therefore provide a significant breakthrough in the field of TRC biology.