Biology subjects

Choudhary, J.

Publications and source records attributed to Choudhary, J..

23 records · Page 2Linked to original sources

bioRxiv2020

Dissecting mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. Here, we generate isogenic CRISPR-Cas9 knockouts ({Delta}) of 43 genes in human induced pluripotent stem cells, culture them in the absence of added DNA damage, and perform wholegenome sequencing of 173 daughter subclones. {Delta}OGG1, {Delta}UNG, {Delta}EXO1, {Delta}RNF168, {Delta}MLH1, {Delta}MSH2, {Delta}MSH6, {Delta}PMS1, and {Delta}PMS2 produce marked mutational signatures indicative of being critical mitigators of endogenous DNA changes. Detailed analyses reveal that 8-oxo-dG removal by different repair proteins is sequence-context-specific while uracil clearance is sequencecontext-independent. Signatures of mismatch repair (MMR) deficiency show components of C>A transversions due to oxidative damage, T>C and C>T transitions due to differential misincorporation by replicative polymerases, and T>A transversions for which we propose a reverse template slippage model. {Delta}MLH1, {Delta}MSH6, and {Delta}MSH2 signatures are similar to each other but distinct from {Delta}PMS2. We validate these gene-specificities in cells from patients with Constitutive Mismatch Repair Deficiency Syndrome. Based on these experimental insights, we develop a classifier, MMRDetect, for improved clinical detection of MMR-deficient tumors.

genomics↗

bioRxiv2019

Systematic re-annotation of 191 genes associated with early-onset epilepsy unmasks de novo variants linked to Dravet syndrome in novel SCN1A exons

The early infantile epileptic encephalopathies (EIEE) are a group of rare, severe neurodevelopmental disorders, where even the most thorough sequencing studies leave 60-65% of patients without a molecular diagnosis. Here, we explore the incompleteness of transcript models used for exome and genome analysis as one potential explanation for lack of current diagnoses. Therefore, we have updated the GENCODE gene annotation for 191 epilepsy-associated genes, using human brain-derived transcriptomic libraries and other data to build 3,550 novel putative transcript models. The extended transcriptional footprint of these genes allowed for 294 intronic or intergenic variants, found in human mutation databases, to be reclassified as exonic, while a further 70 intronic variants were reclassified as splice-site proximal. Using SCN1A as a case study due to its close phenotype/genotype correlation with Dravet syndrome, we screened 122 people with Dravet syndrome, or a similar phenotype, with a panel of novel exon sequences representing eight established genes and identified two de novo SCN1A variants that now, through improved gene annotation can be ascribed to residing among novel exons. These two (from 122 screened patients, 1.6%) new molecular diagnoses carry significant clinical implications. Furthermore, we identified a previously-classified SCN1A intronic Dravet-associated variant that now lies within a deeply conserved novel exon. Our findings illustrate the potential gains of thorough gene annotation in improving diagnostic yields for genetic disorders. We would expect to find new molecular diagnoses in our 191 genes that were originally suspected by clinicians for patients, with a negative diagnosis.

genomics↗

bioRxiv2019

The midbody interactome reveals new unexpected roles for PP1 phosphatases in cytokinesis

The midbody is an organelle assembled at the intercellular bridge that connects the two daughter cells at the end of mitosis. It is composed of a multitude of proteins, organized in a precise and stereotyped pattern, that control the final separation of the daughter cells and prevent incorrect genome segregation. Furthermore, recent evidence indicates that the midbody is involved in many other important processes, including cell fate, pluripotency, apical-basal polarity, tissue organization, and cilium and lumen formation. Understanding the regulation and interactions of midbody proteins is therefore essential to unravel how this organelle executes its multiple functions. Here, we report the first experimentally-based characterization of the intricate midbody protein-protein interaction network (interactome), which identifies a plethora of novel interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome already revealed that PP1{beta}-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex, a key cytokinesis regulator. This de-phosphorylation antagonizes Aurora B kinase in order to modify the functions of centralspindlin and its interactions in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins.

cell biology↗

bioRxiv2017

Regulation of somatosensory mechanotransduction by Annexin A6

Sensory neuron mechanically-activated slowly adapting currents have been linked to noxious mechanosensation. We identified a Conotoxin, Noxious Mechanosensation Blocker -1, that blocks such currents selectively and inhibits mechanical pain Using an active biotinylated form of the toxin we identified 67 binding proteins in sensory neurons and sensory neuron-derived cell lines using mass spectrometry. Annexin A6 was the most frequently identified binding protein. Annexin A6 knockout mice showed an enhanced sensitivity to mechanical stimuli. An increase in rapidly adapting currents was observed in sensory neurons alongside a decrease in slowly adapting currents. Conversely, overexpression of Annexin A6 in sensory neurons inhibited rapidly adapting currents and augmented slowly adapting currents. Furthermore, co-expression of Annexin A6 with Piezo2 led to an inhibition of Piezo-mediated rapidly adapting currents. AAV-mediated overexpression of Annexin A6 in sensory neurons attenuated mechanical pain in a mouse model of osteoarthritis. These data demonstrate a modulatory role for Annexin A6 in somatosensory mechanotransduction.

neuroscience↗

bioRxiv2017

Sub-minute phosphoregulation of cell-cycle systems during Plasmodium gamete formation revealed by a high-resolution time course

Malaria parasites are protists of the genus Plasmodium, whose transmission to mosquitoes is initiated by the production of gametes. Male gametogenesis is an extremely rapid process that is tightly controlled to produce eight flagellated microgametes from a single haploid gametocyte within 10 minutes after ingestion by a mosquito. Regulation of the cell cycle is poorly understood in divergent eukaryotes like Plasmodium, where the highly synchronous response of gametocytes to defined chemical and physical stimuli from the mosquito has proved to be a powerful model to identify specific phosphorylation events critical for cell-cycle progression. To reveal the wider network of phosphorylation signalling in a systematic and unbiased manner, we have measured a high-resolution time course of the phosphoproteome of P. berghei gametocytes during the first minute of gametogenesis. The data show an extremely broad response in which distinct cell-cycle events such as initiation of DNA replication and mitosis are rapidly induced and simultaneously regulated. We identify several protein kinases and phosphatases that are likely central in the gametogenesis signalling pathway and validate our analysis by investigating the phosphoproteomes of mutants in two of them, CDPK4 and SRPK1. We show these protein kinases to have distinct influences over the phosphorylation of similar downstream targets that are consistent with their distinct cellular functions, which is revealed by a detailed phenotypic analysis of an SRPK1 mutant. Together, the results show that key cell-cycle systems in Plasmodium undergo simultaneous and rapid phosphoregulation. We demonstrate how a highly resolved time-course of dynamic phosphorylation events can generate deep insights into the unusual cell biology of a divergent eukaryote, which serves as a model for an important group of human pathogens.

microbiology↗