Biology subjects

Karasu, M. E.

Publications and source records attributed to Karasu, M. E..

7 recordsLinked to original sources

bioRxiv2023

CCAR1 promotes DNA repair via an unanticipated role in alternative splicing

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology directed repair (HDR) in the context of [~]18000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi Anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

molecular biology↗

bioRxiv2022

TREX1 restricts CRISPR-Cas9 genome editing in human cells

CRISPR-Cas mediated homology-directed repair (HDR) can flexibly introduce desired mutations at targeted sites in a genome. But achieving high HDR efficiencies is a major hurdle in many cellular contexts. Moreover, cells from patients with mutations in DNA repair factors can exhibit low CRISPR-Cas-mediated HDR, complicating genome editing as a potential treatment. We used genome-wide screening in Fanconi anemia (FA) patient lymphoblastic cell lines to uncover suppressors of CRISPR-Cas mediated HDR. Surprisingly, we found that a single exonuclease called TREX1 is an important determinant of HDR efficiency when single-stranded templates are used as a repair template. TREX1 expression acts as a biomarker for CRISPR-Cas mediated HDR, such that cell lines expressing high levels of TREX1 have poor HDR that can be rescued by TREX1 removal. CRISPR-Cas mediated HDR can also be rescued by using single-stranded DNA templates that are chemically protected in a manner consistent with TREX1s exonucleolytic activity. Overall, our data provide a mechanistic explanation for why some cells are easier to edit than others and suggest a route to increase CRISPR-Cas mediated HDR in TREX-expressing context.

molecular biology↗

bioRxiv2022

Adenine base editing is an efficient approach to restore function in FA patient cells without double-stranded DNA breaks

Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions lead to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from an FA patient was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.

molecular biology↗

bioRxiv2019

Cyclin B3 is dispensable for mouse spermatogenesis

Cyclins, as regulatory partners of cyclin-dependent kinases (CDKs), control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Compared to mitosis, relatively little is known about how cyclin-CDK complexes control meiosis, the specialized cell division that generates gametes for sexual production. Mouse cyclin B3 was previously shown to have expression restricted to the beginning of meiosis, making it a candidate to regulate meiotic events. Indeed, female mice lacking cyclin B3 are sterile because oocytes arrest at the metaphase-to-anaphase transition of meiosis I. However, whether cyclin B3 functions during spermatogenesis was untested. Here, we found that males lacking cyclin B3 are fertile and show no detectable defects in spermatogenesis based on histological analysis of seminiferous tubules. Cytological analysis further showed no detectable defects in homologous chromosome synapsis or meiotic progression, and suggested that recombination is initiated and completed efficiently. Moreover, absence of cyclin B3 did not exacerbate previously described meiotic defects in mutants deficient for cyclin E2, suggesting a lack of redundancy between these cyclins. Thus, unlike in females, cyclin B3 is not essential for meiosis in males despite its prominent meiosis-specific expression.

genetics↗

bioRxiv2019

Ensuring meiotic DNA break formation in the mouse pseudoautosomal region

Sex chromosomes in males share only a diminutive homologous segment, the pseudoautosomal region (PAR), wherein meiotic double-strand breaks (DSBs), pairing, and crossing over must occur for correct segregation. How cells ensure PAR recombination is unknown. Here we delineate cis-and trans-acting factors that control PAR ultrastructure and make the PAR the hottest area of DSB formation in the male mouse genome. Prior to DSB formation, PAR chromosome axes elongate, sister chromatids separate, and DSB-promoting factors hyperaccumulate. These phenomena are linked to mo-2 minisatellite arrays and require ANKRD31 protein. We propose that the repetitive PAR sequence confers unique chromatin and higher order structures crucial for DSB formation, X-Y pairing, and recombination. Our findings establish a mechanistic paradigm of mammalian sex chromosome segregation during spermatogenesis.

molecular biology↗

bioRxiv2018

REC114 partner ANKRD31 controls number, timing and location of meiotic DNA breaks

Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to sex chromosomes pseudoautosomal regions (PAR). They also have delayed/fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures--stochastic on autosomes, nearly absolute on X and Y--cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines direct ANKRD31- REC114 molecular contacts and reveals a surprising pleckstrin homology domain in REC114. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model that ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, promoting efficient and timely DSB formation but possibly also suppressing formation of clustered DSBs.

genetics↗

bioRxiv2018

Cyclin B3 promotes APC/C activation and anaphase I onset in oocyte meiosis

As obligate kinase partners, cyclins control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Meiosis, the cell division that generates gametes for sexual reproduction, poses unique challenges because two rounds of chromosome segregation must be executed without intervening DNA replication. Mammalian cells express numerous, temporally regulated cyclins, but how these proteins collaborate to control meiosis remains poorly understood. Here, we delineate an essential function for mouse cyclin B3 in the first meiotic division of oocytes. Females genetically ablated for cyclin B3 are viable, indicating the protein is dispensable for mitotic divisions, but are sterile. Mutant oocytes appear normal until metaphase I but then display a highly penetrant failure to transition to anaphase I. They arrest with hallmarks of defective APC/C activation, including no separase activity and high MPF, cyclin B1, and securin levels. Partial APC/C activation occurs, however, as exogenously expressed APC/C substrates can be degraded and arrest can be suppressed by inhibiting MPF kinase. Cyclin B3 is itself targeted for degradation by the APC/C. Cyclin B3 forms active kinase complexes with CDK1, and meiotic progression requires cyclin B3-associated kinase activity. Collectively, our findings indicate that cyclin B3 is essential for oocyte meiosis because it fine-tunes APC/C activity as a kinase-activating CDK partner. Cyclin B3 homologs from frog, zebrafish, and fruitfly rescue meiotic progression in cyclin B3-deficient mouse oocytes, indicating conservation of the biochemical properties and possibly cellular functions of this germline-critical cyclin.

cell biology↗