Biology subjects

Sokirniy, I.

Publications and source records attributed to Sokirniy, I..

3 recordsLinked to original sources

bioRxiv2024

A side-by-side comparison of variant function measurements using deep mutational scanning and base editing

Variant annotation is a crucial objective in mammalian functional genomics. Deep Mutational Scanning (DMS) is a well-established method for annotating human gene variants, but CRISPR base editing (BE) is emerging as an alternative. However, questions remain about how well high-throughput base editing measurements can annotate variant function and the extent of downstream experimental validation required. This study presents the first direct comparison of DMS and BE in the same lab and cell line. Results indicate that focusing on the most likely edits and highest efficiency sgRNAs enhances the agreement between a "gold standard" DMS dataset and a BE screen. A simple filter for sgRNAs making single edits in their window could sufficiently annotate a large proportion of variants directly from sgRNA sequencing of large pools. When multi-edit guides are unavoidable, directly measuring the variants created in the pool, rather than sgRNA abundance, can recover high-quality variant annotation measurements in multiplexed pools. Taken together, our data show a surprising degree of correlation between base editor data and gold standard deep mutational scanning.

genomics↗

bioRxiv2023

MicroRNA-375 is induced during astrocyte-to-neuron reprogramming and promotes survival of reprogrammed neurons when overexpressed

While astrocyte-to-neuron (AtN) reprogramming holds great promise in regenerative medicine, the molecular mechanisms that govern this unique biological process remain elusive. MicroRNAs (miRNAs), as post-transcriptional regulators of gene expression, play crucial roles during development and under various pathological conditions. To understand the function of miRNAs during AtN reprogramming process, we performed RNA-seq of both mRNAs and miRNAs on human astrocyte (HA) cultures upon NeuroD1 overexpression. Bioinformatics analyses showed that NeuroD1 not only activates essential neuronal genes to initiate reprogramming process but also induces miRNA changes in HA. Among the upregulated miRNAs, we identified miR-375 and its targets, neuronal ELAVL genes (nELAVLs), which encode a family of RNA-binding proteins and are also upregulated by NeuroD1. We further showed that manipulating miR-375 level regulates nELAVLs expression during NeuroD1-mediated reprogramming. Interestingly, miR-375/nELAVLs are also induced by reprogramming factors Neurog2 and ASCL1 in HA suggesting a conserved function to neuronal reprogramming, and by NeuroD1 in the mouse astrocyte culture and spinal cord. Functionally, we showed that miR-375 overexpression improves NeuroD1-mediated reprogramming efficiency by promoting cell survival at early stages in HA even in cultures treated with the chemotherapy drug Cisplatin. Moreover, miR-375 overexpression doesnt appear to compromise maturation of the reprogrammed neurons in long term HA cultures. Lastly, overexpression of miR-375-refractory ELAVL4 induces apoptosis and reverses the cell survival-promoting effect of miR-375 during AtN reprogramming. Together, we demonstrate a neuro-protective role of miR-375 during NeuroD1-mediated AtN reprogramming and suggest a strategy of combinatory overexpression of NeuroD1 and miR-375 for improving neuronal reprogramming efficiency.

neuroscience↗

bioRxiv2018

USP7 cooperates with NOTCH1 to drive the oncogenic transcriptionalprogram in T cell leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Treatments1-6 show high response rates but have debilitating effects and carry risk of relapse5,7,8. Previous work implicated NOTCH1 and other oncogenes1,2,9-20. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Here, we demonstrate that ubiquitin-specific protease 7 (USP7)21-32 controls leukemia growth by stabilizing the levels of the NOTCH1 and JMJD3 demethylase. USP7 is overexpressed T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 through deubiquitination. USP7 is bound to oncogenic targets and controls gene expression through H2B ubiquitination and H3K27me3 changes via stabilization of NOTCH1 and JMJD3. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and USP7 inhibition alters associated gene activity. These results provide a new model for deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. USP7 inhibition33 significantly blocked T-ALL cell growth in vitro and in vivo. Our studies also show that USP7 is upregulated in the aggressive high-risk cases of T-ALL and suggest that USP7 expression might be a prognostic marker in ALL and its inhibition could be a therapeutic tool against aggressive leukemia.

cancer biology↗