Excessive transcription-replication conflicts are a vulnerability of BRCA1-mutant cancers

BRCA1 mutations are associated with increased breast and ovarian cancer risk. BRCA1-mutant tumors are high-grade, recurrent, and often become resistant to standard therapies. Herein, we performed a targeted CRISPR-Cas9 screen and identified MEPCE, a methylphosphate capping enzyme, as a synthetic lethal interactor of BRCA1. Mechanistically, we demonstrate that depletion of MEPCE in a BRCA1-deficient setting led to dysregulated RNA polymerase II (RNAPII) promoter-proximal pausing, R-loop accumulation, and replication stress, contributing to transcription-replication collisions. These collisions compromise genomic integrity resulting in loss of viability of BRCA1-deficient cells. We also extend these findings to another RNAPII-regulating factor, PAF1. This study identifies a new class of synthetic lethal partners of BRCA1 that exploit the RNAPII pausing regulation and highlight the untapped potential of transcription-replication collision-inducing factors as unique potential therapeutic targets for treating cancers associated with BRCA1 mutations

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops

GENOME INTEGRITY AND PROGRAMMED GENOME REARRANGEMENT IN THE CILIATE OXYTRICHA

Ciliates are unicellular eukaryotes that undergo complex programmed genome rearrangements as part of their post-zygotic development and, as such, are long-standing models for studying genome integrity. Oxytricha trifallax has many unique features among ciliate model organisms. It exhibits the greatest number of programmed DNA elimination and translocation events among ciliates, and contains tens of thousands of high copy number nanochromosomes with extremely short telomeres. Similar DNA elimination and translocation events that drive genomic instability and copy number alterations are common occurrences in various types of cancers. The first chapter of this thesis is a comprehensive overview of ciliate programmed genome rearrangements, with a focus on Oxytricha. The second chapter focuses on a common hallmark of genomic instability across eukaryotic genomes, extrachromosomal circular DNAs (eccDNAs). Our work demonstrates that rearrangements in Oxytricha involve the production of copious amounts of eccDNA that originate from repetitive as well as nonrepetitive loci, and are a source of noncoding RNAs. Using an established eccDNA deep-sequencing pipeline, we perform the first genome-wide study of circular DNA in programmed genome rearrangements and identify thousands of eccDNAs in Oxytricha. We also demonstrate eccDNA transcription, suggesting that the circularly eliminated DNA is a source of novel ncRNAs. Our findings yield mechanistic insight into the process of DNA elimination during Oxytricha’s elaborate genome rearrangement. Together with recent findings that implicate a role for eccDNA transcription in humans, our findings also set the stage for a new understanding of extrachromosomal circular DNA, not just as a consequence of genome instability, but as biologically active molecules with important regulatory capacity. The third chapter focuses on another hallmark of genomic instability across eukaryotic genomes, dysfunctional telomeres. Defective telomeres may contribute to senescence and catastrophic chromosomal rearrangements. In this chapter, we uncover a new role for the DNA repair protein complex Ku70/80 at the telomeres in ciliates. This complex has previously been shown to be involved in ciliate programmed genome rearrangements. We show that Ku70/80 is an important component for capping the naturally short telomeres of the ciliate Oxytricha and is required to prevent the loss of the telomeric G-rich tail and telomere-to-telomere fusions. We propose that the Ku heterodimer is an important telomere capping protein that is required for maintaining telomere integrity in Oxytricha likely by preventing the shortening of the G-rich tail

GENOME INTEGRITY AND PROGRAMMED GENOME REARRANGEMENT IN THE CILIATE OXYTRICHA

Ciliates are unicellular eukaryotes that undergo complex programmed genome rearrangements as part of their post-zygotic development and, as such, are long-standing models for studying genome integrity. Oxytricha trifallax has many unique features among ciliate model organisms. It exhibits the greatest number of programmed DNA elimination and translocation events among ciliates, and contains tens of thousands of high copy number nanochromosomes with extremely short telomeres. Similar DNA elimination and translocation events that drive genomic instability and copy number alterations are common occurrences in various types of cancers. The first chapter of this thesis is a comprehensive overview of ciliate programmed genome rearrangements, with a focus on Oxytricha. The second chapter focuses on a common hallmark of genomic instability across eukaryotic genomes, extrachromosomal circular DNAs (eccDNAs). Our work demonstrates that rearrangements in Oxytricha involve the production of copious amounts of eccDNA that originate from repetitive as well as nonrepetitive loci, and are a source of noncoding RNAs. Using an established eccDNA deep-sequencing pipeline, we perform the first genome-wide study of circular DNA in programmed genome rearrangements and identify thousands of eccDNAs in Oxytricha. We also demonstrate eccDNA transcription, suggesting that the circularly eliminated DNA is a source of novel ncRNAs. Our findings yield mechanistic insight into the process of DNA elimination during Oxytricha’s elaborate genome rearrangement. Together with recent findings that implicate a role for eccDNA transcription in humans, our findings also set the stage for a new understanding of extrachromosomal circular DNA, not just as a consequence of genome instability, but as biologically active molecules with important regulatory capacity. The third chapter focuses on another hallmark of genomic instability across eukaryotic genomes, dysfunctional telomeres. Defective telomeres may contribute to senescence and catastrophic chromosomal rearrangements. In this chapter, we uncover a new role for the DNA repair protein complex Ku70/80 at the telomeres in ciliates. This complex has previously been shown to be involved in ciliate programmed genome rearrangements. We show that Ku70/80 is an important component for capping the naturally short telomeres of the ciliate Oxytricha and is required to prevent the loss of the telomeric G-rich tail and telomere-to-telomere fusions. We propose that the Ku heterodimer is an important telomere capping protein that is required for maintaining telomere integrity in Oxytricha likely by preventing the shortening of the G-rich tail

Programmed Genome Rearrangements in the Ciliate Oxytricha

The ciliate Oxytricha is a microbial eukaryote with two genomes, one of which experiences extensive genome remodeling during development. Each round of conjugation initiates a cascade of events that construct a transcriptionally active somatic genome from a scrambled germline genome, with considerable help from both long and small noncoding RNAs. This process of genome remodeling entails massive DNA deletion and reshuffling of remaining DNA segments to form functional genes from their interrupted and scrambled germline precursors. The use of Oxytricha as a model system provides an opportunity to study an exaggerated form of programmed genome rearrangement. Furthermore, studying the mechanisms that maintain nuclear dimorphism and mediate genome rearrangement has demonstrated a surprising plasticity and diversity of non-coding RNA pathways, with new roles that go beyond conventional gene silencing. Another aspect of ciliate genetics is their unorthodox patterns of RNA-mediated, epigenetic inheritance, that rival Mendelian inheritance. This review takes the reader through the key experiments in a model eukaryote that led to fundamental discoveries in RNA biology and pushes the biological limits of DNA processing

Programmed Genome Rearrangements in the Ciliate Oxytricha

The ciliate Oxytricha is a microbial eukaryote with two genomes, one of which experiences extensive genome remodeling during development. Each round of conjugation initiates a cascade of events that construct a transcriptionally active somatic genome from a scrambled germline genome, with considerable help from both long and small noncoding RNAs. This process of genome remodeling entails massive DNA deletion and reshuffling of remaining DNA segments to form functional genes from their interrupted and scrambled germline precursors. The use of Oxytricha as a model system provides an opportunity to study an exaggerated form of programmed genome rearrangement. Furthermore, studying the mechanisms that maintain nuclear dimorphism and mediate genome rearrangement has demonstrated a surprising plasticity and diversity of non-coding RNA pathways, with new roles that go beyond conventional gene silencing. Another aspect of ciliate genetics is their unorthodox patterns of RNA-mediated, epigenetic inheritance, that rival Mendelian inheritance. This review takes the reader through the key experiments in a model eukaryote that led to fundamental discoveries in RNA biology and pushes the biological limits of DNA processing

Programmed Chromosome Deletion in the Ciliate Oxytricha trifallax

The ciliate Oxytricha trifallax contains two nuclei: a germline micronucleus and a somatic macronucleus. These two nuclei diverge significantly in genomic structure. The micronucleus contains approximately 100 chromosomes of megabase scale, while the macronucleus contains 16,000 gene-sized, high ploidy “nanochromosomes.” During its sexual cycle, a copy of the zygotic germline micronucleus develops into a somatic macronucleus via DNA excision and rearrangement. The rearrangement process is guided by multiple RNA-based pathways that program the epigenetic inheritance of sequences in the parental macronucleus of the subsequent generation. Here, we show that the introduction of synthetic DNA molecules homologous to a complete native nanochromosome during the rearrangement process results in either loss or heavy copy number reduction of the targeted nanochromosome in the macronucleus of the subsequent generation. This phenomenon was tested on a variety of nanochromosomes with different micronuclear structures, with deletions resulting in all cases. Deletion of the targeted nanochromosome results in the loss of expression of the targeted genes, including gene knockout phenotypes that were phenocopied using alternative knockdown approaches. Further investigation of the chromosome deletion showed that, although the full length nanochromosome was lost, remnants of the targeted chromosome remain. We were also able to detect the presence of telomeres on these remnants. The chromosome deletions and remnants are epigenetically inherited when backcrossed to wild type strains, suggesting that an undiscovered mechanism programs DNA elimination and cytoplasmically transfers to both daughter cells during conjugation. Programmed deletion of targeted chromosomes provides a novel approach to investigate genome rearrangement and expands the available strategies for gene knockout in Oxytricha trifallax

Exploration of the Nuclear Proteomes in the Ciliate <i>Oxytricha trifallax</i>

Nuclear dimorphism is a fundamental feature of ciliated protozoa, which have separate somatic and germline genomes in two distinct organelles within a single cell. The transcriptionally active somatic genome, contained within the physically larger macronucleus, is both structurally and functionally different from the silent germline genome housed in the smaller micronucleus. This difference in genome architecture is particularly exaggerated in Oxytricha trifallax, in which the somatic genome comprises tens of thousands of gene-sized nanochromosomes maintained at a high and variable ploidy, while the germline has a diploid set of megabase-scale chromosomes. To examine the compositional differences between the nuclear structures housing the genomes, we performed a proteomic survey of both types of nuclei and of macronuclear histones using quantitative mass spectrometry. We note distinct differences between the somatic and germline nuclei, with many functional proteins being highly enriched in one of the two nuclei. To validate our conclusions and the efficacy of nuclear separation, we used protein localization through a combination of transformations and immunofluorescence. We also note that the macronuclear histones strikingly display only activating marks, consistent with the conclusion that the macronucleus is the hub of transcription. These observations suggest that the compartmentalization of different genome features into separate structures has been accompanied by a similar specialization of nuclear components that maintain and facilitate the functions of the genomes specific to each nucleus

Nucleolar RNA polymerase II drives ribosome biogenesis

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli . Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease