Search

SMARCAL1, ZRANB3 and HLTF are required for the remodeling of replication forks upon stress to promote genome stability. RAD51, along with the RAD51 paralog complex, were also found to have recombination-independent functions in fork reversal, yet the underlying mechanisms remained unclear. Using reconstituted reactions, we build upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions. SMARCAL1 uniquely anneals RPA-coated ssDNA, which depends on its direct interaction with RPA, but not on ATP. SMARCAL1, along with ZRANB3, but not HLTF efficiently employ ATPase driven translocase activity to rezip RPA-covered bubbled DNA, which was proposed to mimic elements of fork reversal. In contrast, ZRANB3 and HLTF but not SMARCAL1 are efficient in branch migration that occurs downstream in fork remodeling. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), directly stimulate the motor-driven activities of SMARCAL1 and ZRANB3 but not HLTF, and the interplay is underpinned by physical interactions. Our data provide a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal., Nucleic Acids Research, 50 (14), ISSN:1362-4962, ISSN:0301-5610

Breast cancer heterogeneity demands new reliable prognostic markers and therapeutic targets for the personalized management of patients. Over recent years, knowledge on the involvement of different types of kinases in cancer has guided the design of a variety of kinase inhibitors as novel molecularly targeted anti-cancer agents. In a previous high-throughput screening performed by in situ hybridization (ISH) and immunohistochemistry (IHC) on breast cancer tissue microarrays (TMAs), the cyclin-dependent kinase 12 (CDK12) was found to be frequently overexpressed in breast cancer and its overexpression correlated with clinical/pathological parameters of aggressive disease. CDK12 was therefore proposed to be a novel prognostic biomarker in breast cancer. In the present thesis, we extend these preliminary studies and demonstrate, by IHC analysis on TMAs comprising large cohorts of breast cancer patients, that CDK12 overexpression is significantly associated with clinical/pathological parameters of poor prognosis (high tumor grade, high Ki67 proliferative index, positive ERBB2 status) and with a higher risk of disease recurrence and death. We also show, either in human breast tumors or in breast cancer cell lines, that CDK12 overexpression is due to the amplification of the CDK12 gene. Through the use of amenable cell models in diverse in vitro and in vivo assays, we provide evidences that CDK12 is a bona fide oncogene in breast cancer. By genome-wide expression analysis, we show that alterations in CDK12 expression are associated with changes in the transcription and splicing of genes involved in cancer-relevant cellular processes, such as DNA damage response, cell cycle and epithelial-to-mesenchymal transition. In conclusion, we have established that CDK12 is a novel prognostic biomarker in breast cancer and represents a potential novel molecular target for therapeutic intervention in breast cancer.

Pooled genetic screens are powerful tools to study gene function in a high-throughput manner. Typically, sequencing-based screens require cell lysis, which limits the examination of critical phenotypes such as cell morphology, protein subcellular localization, and cell-cell/tissue interactions. In contrast, emerging optical pooled screening methods enable the investigation of these spatial phenotypes in response to targeted CRISPR perturbations. In this study, we report a multi-omic optical pooled CRISPR screening method, which we have named CRISPRmap. Our method combines a novel in situ CRISPR guide identifying barcode readout approach with concurrent multiplexed immunofluorescence and in situ RNA detection. CRISPRmap barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency, while reducing both dependency on third party proprietary sequencing reagents and assay cost. Notably, we conducted a multi-omic base-editing screen in a breast cancer cell line on core DNA damage repair genes involved in the homologous recombination and Fanconi anemia pathways investigating how nucleotide variants in those genes influence DNA damage signaling and cell cycle regulation following treatment with ionizing radiation or DNA damaging agents commonly used for cancer therapy. Approximately a million cells were profiled with our multi-omic approach, providing a comprehensive phenotypic assessment of the functional consequences of the studied variants. CRISPRmap enabled us to pinpoint likely-pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance. Furthermore, our approach effectively distinguished barcodes of a pooled library in tumor tissue, and we coupled it with cell-type and molecular phenotyping by cyclic immunofluorescence. Multi-omic spatial analysis of how CRISPR-perturbed cells respond to various environmental cues in the tissue context offers the potential to significantly expand our understanding of tissue biology in both health and disease., Competing Interests: Declaration of competing interests Columbia University has filed a patent application related to this work for which J.T.G. is an inventor. E.M.C.’s laboratory receives support from Novartis. D.A.L. has served as a consultant for AbbVie, AstraZeneca, and Illumina and is on the Scientific Advisory Board of Mission Bio, Pangea, Alethiomics, and C2i Genomics; D.A.L. has received prior research funding from BMS, 10x Genomics, Ultima Genomics, and Illumina unrelated to the current manuscript.

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication (BIR), evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), unique to ALT cells, are considered potential precursors of C-circles, their generation process remains undefined. Here, we introduce a highly sensitive method to detect single stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear C-rich ssDNA accumulation may indeed precede C-circle formation. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a new model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.