Project description:Whole-genome methylomes and total transcriptomes for muscle and liver tissues of Lake Malawi cichlid species characterised in the context of phenotypic diversification.
Project description:Multiple replication abnormalities cause cells lacking BRCA2 to enter mitosis with under-replicated DNA and to activate mitotic DNA synthesis (MiDAS). However, the precise position of these MiDAS sites, as well as their origin, remains unknown. Here we labelled mitotic nascent DNA and performed high-throughput sequencing to identify at high-resolution the sites where MiDAS occurs in the absence of BRCA2. This approach revealed 150 genomic loci affected by MiDAS, which map within regions replicating during early S-phase and are therefore distinct from the aphidicolin-induced common fragile sites. Moreover, these sites largely localise near early firing origins and within genes transcribed in early S, suggesting that they stem from transcription-replication conflicts (TCRs). Inhibiting transcription with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) during early S-phase abrogates MiDAS. Strikingly, MiDAS sites co-localise with genomic loci where R-loops form in unchallenged conditions, suggesting that R-loop accumulation caused by BRCA2 inactivation leads to DNA lesion which are repaired by MiDAS. RAD52 is required in this process, as its abrogation in BRCA2-deficient cells reduces the rate of MiDAS and causes DNA damage accumulation in G1. Furthermore, MiDAS sites triggered by BRCA2 inactivation are hotspots for genomic rearrangement in BRCA2-mutated breast tumours. These results indicate that BRCA2 acts in early S-phase to protect TRC- and R-loop-induced DNA lesions, thereby preventing them from becoming a source of genomic instability and tumorigenesis.
Project description:East African cichlid fishes have radiated in an explosive fashion. The (epi)genetic basis for the abundant phenotypic diversity of these fishes remains largely unknown. As transposable elements (TEs) contribute extensively to genome evolution, we reasoned that TEs may have fuelled cichlid radiations. While TE-derived genetic and epigenetic variability has been associated with phenotypic traits, TE expression and epigenetic silencing remain unexplored in cichlids. Here, we profiled TE expression in African cichlids, and describe dynamic expression patterns during embryogenesis and according to sex. Most TE silencing factors are conserved and expressed in cichlids. We describe an expansion of two truncated Piwil1 genes in Lake Malawi/Nyasa cichlids, encoding a Piwi domain with catalytic potential. To further dissect epigenetic silencing of TEs, we focused on small RNA-driven epigenetic silencing. We detect a small RNA population in gonads consistent with an active Piwi-interacting RNA (piRNA) pathway targeting TEs. We uncover fluid genomic origins of piRNAs in closely related cichlid species. This, along with signatures of positive selection in piRNA pathway factors, points towards fast co-evolution of TEs and the piRNA pathway. Our study is the first step to understand the contribution of ongoing TE-host arms races to the cichlid radiations in Africa.
Project description:DNA double-strand breaks (DSBs) accumulate in mitosis in response to DNA replication stress or deficiency in breast cancer susceptibility type 1 and 2 (BRCA1/2). This is a critical threat to genome stability as the classical DSB repair pathways non-homologous end joining (NHEJ) and homologous recombination (HR) are repressed(1-7). Instead, mitotic repair relies on the processes of microhomology-mediated end joining (MMEJ) or mitotic DNA synthesis (MiDAS), but how these pathways are coordinated and regulated remains unclear. Here, we reveal that the CIP2A-TOPBP1 axis orchestrates DSB repair in mitosis by promoting CDK1-driven phosphorylation of SLX4 at Thr1260, recruiting the SMX complex to promote MiDAS. In addition, the CIP2A-TOPBP1 axis regulates mitotic Polθ recruitment and MMEJ. This dual mechanism ensures coordinated DSBs repair by integrating spatial control of TOPBP1-CIP2A chromatin recruitment with the temporal regulation provided by mitotic kinases, thereby preserving genome stability. These findings provide new insights into DSB repair dynamics in mitosis and offer a mechanistic rationale for the synthetic lethality of CIP2A in BRCA1/2-deficient tumors through the simultaneous impairment of break induced replication (BIR)-like MiDAS and MMEJ, which are genetically redundant pathways to HR.
Project description:This study examines genomic copy-number variation among African cichlids spanning multiple tribes and radiations. We map CNVs and hotspots throughout the Oreochromis niloticus reference genome, categorize gene ontology enrichment within CNV regions, and compare results with sequence-based cichlid phylogenies.
Project description:Deregulation of origin firing and licensing, shortage of deoxyribonucleotides in the cell and interference between transcription and replication are among the causes of DNA replication stress in cancer cells. This leads in various ways (notably, through common fragile sites expression and uncomplete replication of late-replicating regions of the genome during S phase) to the necessity of finishing DNA replication in mitosis by a mechanisms called Mitotic DNA synthesis (MiDAS), which is related to Break-Induced Replication (BIR). Even if it is of primary importance for cancer cells, the molecular mechanism of MiDAS, and generally of BIR, is not yet well understood. Recently, the third subunit of the eukaryotic DNA polymerase delta (POLD3) has been recognized as a key player of BIR, though its role is not yet clear. In this work, using a protocol established in our group to map at high resolution newly replicated DNA at MiDAS sites, we provide new insights into the molecular role of POLD3 in this repair mechanism. In particular, by analyzing MiDAS in mutant HeLa clones lacking the PCNA-interacting domain of POLD3, we demonstrate that the interaction between POLD3 and PCNA is required for coordinating leading and lagging strand synthesis in MiDAS. This work represents an important step forward towards the comprehension of the molecular mechansisms of Mitotic DNA synthesis, the full understanding of which is of primary importance for the possible development of novel cancer therapies targeting BIR-related pathways.
Project description:DNA replication stress is an established driver of cancer-associated chromosomal rearrangements. Replication stress perturbs the duplication of late-replicating loci and activates a mitotic DNA repair pathway (termed MiDAS) for completion of replication. We here investigated RAD51-independent MiDAS.
Project description:East African cichlid fishes have diversified in an explosive fashion, but the (epi)genetic basis of the phenotypic diversity of these fishes remains largely unknown. Although transposable elements (TEs) have been associated with phenotypic variation in cichlids, little is known about their transcriptional activity and epigenetic silencing. Here, we describe dynamic patterns of TE expression in African cichlid gonads and during early development. Orthology inference revealed an expansion of piwil1 genes in Lake Malawi cichlids, likely driven by PiggyBac TEs. The expanded piwil1 copies have signatures of positive selection and retain amino acid residues essential for catalytic activity. Furthermore, the gonads of African cichlids express a Piwi-interacting RNA (piRNA) pathway that target TEs. We define the genomic sites of piRNA production in African cichlids and find divergence in closely related species, in line with fast evolution of piRNA-producing loci. Our findings suggest dynamic co-evolution of TEs and host silencing pathways in the African cichlid radiations. We propose that this co-evolution has contributed to cichlid genomic diversity.