Periodic Reporting for period 1 - GLoopID (Mechanisms underlying regulation and removal of G-quadruplex/R-loop transcription-replication conflicts)
Reporting period: 2021-09-01 to 2023-08-31
Guanine-rich repetitive DNA sequences, which can form four-stranded structures (G-quadruplex or G4), preferentially form in single stranded DNA (ssDNA) upon transient melting and can also occur in RNA. Transcribed G4s often co-exist with stable DNA:RNA hybrids forming a G4/R-loop, potentially causing deleterious transcription-replication conflicts. G4 motifs are enriched at regulatory loci such as active promoters or enhancers and at telomeres, and they have been implicated in several key biological processes, such as DNA replication, transcription, and telomere homeostasis. Telomeric G4/R-loops are linked to cancers that maintain telomere length not by upregulating telomerase but instead using a recombination-based telomere maintenance mechanism called alternative lengthening of telomeres (ALT). ALT constitutes a pathological scenario where aberrant telomeric chromatin triggers G4/R-loops that drives telomeric replication stress and recombination. ALT cancers are a major cancer type of unmet clinical need: typically mesenchymal in origin, with poor clinical outcomes and account for approximately 10-15% of all tumours. Whereas ALT tumours are strongly associated with mutations in the chromatin remodeller ATRX, inactivation of ATRX alone in culture is insufficient for a cell to become ALT.
• Specific Aim 1:
Determine the proximity proteome composition of site-specific G4/R-loop TRCs (WP1)
• Specific Aim 2:
Characterise the genetic vulnerabilities of G4/R-loop helicase-deficient cells (WP2)
• Specific Aim 3:
Identify the protein domains of G4/R-loop helicases essential for TRC removal (WP3)
• Specific Aim 1:
Determine the proximity proteome composition of site-specific G4/R-loop TRCs (WP1)
• Specific Aim 2:
Characterise the genetic vulnerabilities of G4/R-loop helicase-deficient cells (WP2)
• Specific Aim 3:
Identify the protein domains of G4/R-loop helicases essential for TRC removal (WP3)
This project has provided in-depth mechanistic insights, summarised below, into novel concepts for synthetic lethality in a ATRX deficiency, a cancer relevant setting. In addition, it has provided important new insights into the regulation of ATRX and how cells overcome G4/R-loop induced transcription-replication collisions (TRCs) to safeguard genome stability.
Summary of milestones:
WP1: Determine the proximity proteome composition of site-specific G4/R-loop TRCs.
To achieve site-specific G4/R-loop TRCs, I have exploited a previously reported episomal system in mammalian cells where transcription and replication orientation are controlled. This system utilises unidirectional replication from the Epstein–Barr virus origin and doxycycline-inducible transcription of the insert to locally study TRCs. The miniTurboID biotin ligase enables investigation of the site-specific proteome at the episome. Upon 24 hr of doxycycline and addition of exogenous biotin, cells can biotinylate the proximity proteome. This systematic approach will result in curated dataset with general and telomere-specific G4-protective factors.
M1 (pending): Completion of successful proteomics of site-specific G4/R-loop TRCs.
WP2: Characterise the genetic vulnerabilities of G4/R-loop helicase-deficient cells.
The intrinsic difficult to replicate long tracts of GC-rich repetitive telomeric sequences together with their active transcription has been posed as the basis for constitutive recombination activity at ALT telomeres. ATRX is a G4-helicase and SNF2-like chromatin remodeller recruited to transcriptionally active ectopic telomeric loci, which form G4/R-loops. ATRX forms a multifunctional chromatin remodelling complex with the H3.3 chaperone DAXX and together they suppress spurious transcription and replication stress at heterochromatin. Due to RTEL1 and SETX full knockouts being lethal in eHAP cells, WP2-3 have focused on ATRX. I conducted genome-wide CRISPR/Cas9 knockout screens in isogenic wild type and ATRX knockout cells to identify factors that are essential/reduce the viability of cells lacking ATRX. For that I used a lentiviral two-vector pooled library (Brunello, Addgene), where cells initially express Doxycycline-inducible Cas9 nuclease and the transduction with lentiviral vectors expressing >76,000 sgRNA for 19,114 genes (4 sgRNAs per gene). The screen identified several hits, whose loss is synthetic lethal with the loss of ATRX. Importantly, I also identified factors whose loss confers resistance to specific drug treatments specifically in cells lacking ATRX. In-depth characterisation of selected hits enabled the discovery of novel synthetic genetic interactions of the chromatin remodelling enzyme ATRX, revealing new insights into distinct ATRX functions in global genome and telomere maintenance
M2.1 (completed): Completion of successful CRISPR/Cas9 screens.
M2.2(completed): Insight obtained in synthetic lethal pathways with ATRX.
WP3: Identify the protein domains of G4/R-loop helicases essential for TRC removal.
I interrogated the importance of known or putative motifs in ATRX and how several point mutations impact on the ability of ATRX to promote DNA replication/telomere instability phenotypes.
M3(completed): Mechanistic establishment of the role of G4/R-loop helicases at TRCs.
WP4: Training in new techniques and transferable skills.
WP5: Dissemination of the project results.
WP6: Science communication.
Summary of milestones:
WP1: Determine the proximity proteome composition of site-specific G4/R-loop TRCs.
To achieve site-specific G4/R-loop TRCs, I have exploited a previously reported episomal system in mammalian cells where transcription and replication orientation are controlled. This system utilises unidirectional replication from the Epstein–Barr virus origin and doxycycline-inducible transcription of the insert to locally study TRCs. The miniTurboID biotin ligase enables investigation of the site-specific proteome at the episome. Upon 24 hr of doxycycline and addition of exogenous biotin, cells can biotinylate the proximity proteome. This systematic approach will result in curated dataset with general and telomere-specific G4-protective factors.
M1 (pending): Completion of successful proteomics of site-specific G4/R-loop TRCs.
WP2: Characterise the genetic vulnerabilities of G4/R-loop helicase-deficient cells.
The intrinsic difficult to replicate long tracts of GC-rich repetitive telomeric sequences together with their active transcription has been posed as the basis for constitutive recombination activity at ALT telomeres. ATRX is a G4-helicase and SNF2-like chromatin remodeller recruited to transcriptionally active ectopic telomeric loci, which form G4/R-loops. ATRX forms a multifunctional chromatin remodelling complex with the H3.3 chaperone DAXX and together they suppress spurious transcription and replication stress at heterochromatin. Due to RTEL1 and SETX full knockouts being lethal in eHAP cells, WP2-3 have focused on ATRX. I conducted genome-wide CRISPR/Cas9 knockout screens in isogenic wild type and ATRX knockout cells to identify factors that are essential/reduce the viability of cells lacking ATRX. For that I used a lentiviral two-vector pooled library (Brunello, Addgene), where cells initially express Doxycycline-inducible Cas9 nuclease and the transduction with lentiviral vectors expressing >76,000 sgRNA for 19,114 genes (4 sgRNAs per gene). The screen identified several hits, whose loss is synthetic lethal with the loss of ATRX. Importantly, I also identified factors whose loss confers resistance to specific drug treatments specifically in cells lacking ATRX. In-depth characterisation of selected hits enabled the discovery of novel synthetic genetic interactions of the chromatin remodelling enzyme ATRX, revealing new insights into distinct ATRX functions in global genome and telomere maintenance
M2.1 (completed): Completion of successful CRISPR/Cas9 screens.
M2.2(completed): Insight obtained in synthetic lethal pathways with ATRX.
WP3: Identify the protein domains of G4/R-loop helicases essential for TRC removal.
I interrogated the importance of known or putative motifs in ATRX and how several point mutations impact on the ability of ATRX to promote DNA replication/telomere instability phenotypes.
M3(completed): Mechanistic establishment of the role of G4/R-loop helicases at TRCs.
WP4: Training in new techniques and transferable skills.
WP5: Dissemination of the project results.
WP6: Science communication.
The impact of the training action has been to complement my previous expertise and reach a position of professional maturity. Apart from the scientific development, I have improved my supervision and mentoring skills by formal training, including the EMBO Laboratory Leadership Course, as well as through transferring my skills and guiding PhD students and mentoring undergraduate students. I have reinforced my dissemination and communication skills through participation in scientific conferences and outreach activities. The results obtained from the project have been disseminated within the scientific community via oral/poster presentations in national and international scientific conferences. The project has resulted in high-quality results that are being compiled in two independent scientific publications. Finally, the research carried out has uncovered novel strategies for therapeutic intervention. My research has generated applicable strategies for targeting ATRX-mutated tumours, thus we have protected the acquired intellectual property through a patent application.