Periodic Reporting for period 4 - MetDNASecStr (Metabolism of DNA secondary structures and their impact on genome stability)
Período documentado: 2019-11-01 hasta 2021-04-30
DNA replication is an essential process for genome duplication, cell division and ultimately organismal survival that ensures faithful transmission of the genome to progeny. Certain genomic loci represent major obstacles to DNA replication including fragile sites, G-rich tracts and repetitive sequences, such as ribosomal DNA and telomeres. Mammalian telomeres have the propensity to adopt complex DNA secondary structures, including telomere-loops and telomeric G-quadruplex DNA, which are believed to play essential roles in telomere maintenance. However, recent work has established that these structures are also a hindrance to DNA replication and failure to stabilise, repair or restart the replication fork is a potential source of genome instability, the hallmark of many diseases including cancer.
Increasing evidence suggests that telomere secondary structures that are essential for chromosome end protection and appropriate chromosome segregation might also represent a hindrance during DNA replication. Thus cells have developed mechanisms to ensure proper genome duplication that require specific factors to alleviate DNA replication stress, which is causative of genome instability and ultimately tumourigenesis. I am confident that the proposed aims will contribute to an improved understanding of the structure and nature of telomere replication stress in eukaryotes. Ultimately, the program of research will provide a framework for comprehending the contributions of replication stress response factors in general DNA replication and cancer in humans. My following proposal describes three complementary projects that will focus on telomere replication which specifically aim to address the key questions: (1) what are the enzymatic activities that result in fragile telomeres; (2) what is the structure of telomere fragility; (3) what is the the role of Activity-Dependent Neuroprotective Protein (ADNP) in telomere biology and the importance of telomere instability in some neurological disorders including a syndromic form of autism-like disorder.
My research financed with this ERC starting grant has enabled my group to identify a pathway of repair when telomeres are under replication stress (Porreca et al., 2020; eLife), a novel RNA helicase SKIV2L part of hSKI complex involved in MRNA mediated RNA decay pathway that is also responsible for regulating RNA-DNA hybrids at telomeres and suppressing telomere replication stress and DNA damage (Herrera-Moyano et al., 2021, in revision in Cell reports), contribute to the understanding of in-cellulo G-quadruplex dynamics in live cell imaging (Summers et al., 2020, Nature Communications) and help develop new probes (Lewis et al., 2021, Chemistry), contribute to the understanding of senescence pathways when cells are subjected to telomere DNA damage stress (Innes et al., 2021, Genes & Development).
Increasing evidence suggests that telomere secondary structures that are essential for chromosome end protection and appropriate chromosome segregation might also represent a hindrance during DNA replication. Thus cells have developed mechanisms to ensure proper genome duplication that require specific factors to alleviate DNA replication stress, which is causative of genome instability and ultimately tumourigenesis. I am confident that the proposed aims will contribute to an improved understanding of the structure and nature of telomere replication stress in eukaryotes. Ultimately, the program of research will provide a framework for comprehending the contributions of replication stress response factors in general DNA replication and cancer in humans. My following proposal describes three complementary projects that will focus on telomere replication which specifically aim to address the key questions: (1) what are the enzymatic activities that result in fragile telomeres; (2) what is the structure of telomere fragility; (3) what is the the role of Activity-Dependent Neuroprotective Protein (ADNP) in telomere biology and the importance of telomere instability in some neurological disorders including a syndromic form of autism-like disorder.
My research financed with this ERC starting grant has enabled my group to identify a pathway of repair when telomeres are under replication stress (Porreca et al., 2020; eLife), a novel RNA helicase SKIV2L part of hSKI complex involved in MRNA mediated RNA decay pathway that is also responsible for regulating RNA-DNA hybrids at telomeres and suppressing telomere replication stress and DNA damage (Herrera-Moyano et al., 2021, in revision in Cell reports), contribute to the understanding of in-cellulo G-quadruplex dynamics in live cell imaging (Summers et al., 2020, Nature Communications) and help develop new probes (Lewis et al., 2021, Chemistry), contribute to the understanding of senescence pathways when cells are subjected to telomere DNA damage stress (Innes et al., 2021, Genes & Development).
Using the power of Proteomics, we identified the function of TRF1 (Porreca et al., 2020, eLife) and identified novel factors recruited to telomeres. We focused our research on the SKI complex: To tackle the challenge of aberrant or excessive cytoplasmic and nuclear RNA molecules, cells have evolved different RNA decay pathways. Among these, the nonsense-mediated mRNA decay, NMD, surveillance pathway is involved in the degradation and quality-control of mRNAs. Although human NMD has been widely investigated for its cytoplasmic functions, some of its core components have been identified in the nucleus, targeting several essential biological processes including telomere homeostasis. Our data show that the hSKI complex, another NMD component also localises to chromatin and is essential in maintaining telomere physiology. This work is accessible on BioRxiv: Herrera-Moyano et al., 2021 and is in revision in Cell reports.
We also identified the recruitment of ADNP factor: Activity-Dependent Neuroprotective Protein (ADNP) localise at normal telomeres but noticed a significant increase in ADNP peptide numbers and normalised intensity upon replication stress, which is induced by removal of TRF1 from telomeres, using our conditional knock-out system. This protein is a transcription factor involved in the SWI/SNF remodelling complex, mutated in complex neuro-developmental disorder.
The scientific conclusions of the research financed by this ERC starting Grant has largely been disseminated to the scientific community of researchers working in the fields of Telomere, DNA damage and Repair at various national and International conferences but also when invited in different universities and research centres:
Invited Speaker
• 2020; EMBO Workshop on Telomere Biology delayed to Sep 2022
• 2019; Telomere Network UK meeting (TENUK), Genome Stability Network (GSN)
• 2018; 4th International Congress on Epigenetics & Chromatin; TENUK, University of Newcastle, UK; EMBO Telomere biology in health and human disease, Portugal.
• 2015; EMBO workshop Telomeric chromatin and telomere fragility, Singapore.
Invited seminar
• UMR-INSERM GReD (Fr) Nov 2020;
• Institute of Cancer Research (UK) Oct 2018;
• Laboratory of Molecular Biology (UK) Nov 2018;
• Blaise Pascal University (Fr) Sept 2014.
We also identified the recruitment of ADNP factor: Activity-Dependent Neuroprotective Protein (ADNP) localise at normal telomeres but noticed a significant increase in ADNP peptide numbers and normalised intensity upon replication stress, which is induced by removal of TRF1 from telomeres, using our conditional knock-out system. This protein is a transcription factor involved in the SWI/SNF remodelling complex, mutated in complex neuro-developmental disorder.
The scientific conclusions of the research financed by this ERC starting Grant has largely been disseminated to the scientific community of researchers working in the fields of Telomere, DNA damage and Repair at various national and International conferences but also when invited in different universities and research centres:
Invited Speaker
• 2020; EMBO Workshop on Telomere Biology delayed to Sep 2022
• 2019; Telomere Network UK meeting (TENUK), Genome Stability Network (GSN)
• 2018; 4th International Congress on Epigenetics & Chromatin; TENUK, University of Newcastle, UK; EMBO Telomere biology in health and human disease, Portugal.
• 2015; EMBO workshop Telomeric chromatin and telomere fragility, Singapore.
Invited seminar
• UMR-INSERM GReD (Fr) Nov 2020;
• Institute of Cancer Research (UK) Oct 2018;
• Laboratory of Molecular Biology (UK) Nov 2018;
• Blaise Pascal University (Fr) Sept 2014.
The telomere field and its knowledge have over the recent years extraordinarily evolved from a linear “buffer” sequence to an intricate and complicated chromatin structure where 100+ factors are recruited/regulated in order to elongate, signal, repair, suppress recombination by the bias of DNA secondary structures including T-loop, G-quadruplexes, R-loops and heterochromatin. It is with the ambition to discover novel factors that I started with the optimisation of Dr. Dejardin’s PICh unbiased proteomic approach to compare the telomeric chromatin composition of telomeres during the cell cycle and in different replication stress conditions. In order to establish my niche in the field, we first analysed the consequences of the loss of TRF1 shelterin factor on telomere maintenance. We identified factors and pathways that resemble ALT positive cells and went into proposing a role for mouse TRF1 in averting chromatin remodelling and suppressing break-induced replication (Porreca et al., 2020, eLife). This study is followed-up by a proteomic analysis of human telomeres during the different phases of the cell cycle. We identify the presence of the three components of the hSKI complex (SKIV2L, TTC37 and WDR61), a well characterised cytoplasmic NMD complex at telomeres in G2. We determined the important function of hSKI in regulating RNA-DNA hybrids in order to ensure telomere maintenance and genome stability (Herrera-Moyano et al., 2021; in revision in Cell reports; BioRxiv: https://doi.org/10.1101/2020.05.20.107144). The research focus of the lab on telomere replication and stability will continue to shed light on previously unrecognised function of essential accessory factors that facilitate the various telomere processes during the cell cycle and support pathophysiology understanding when associated to rare syndromes, including the role of ADNP (manuscript in preparation).