Biology subjects

Sobti, S.

Publications and source records attributed to Sobti, S..

3 recordsLinked to original sources

bioRxiv2026

Systematic identification of chromatin organizers as tuners of intratumoral heterogeneity

Intratumoral transcriptional heterogeneity (ITH) enables tumor cells to explore diverse phenotypic states, fueling therapeutic resistance and metastatic progression. Yet systematic approaches for identifying non-genetic regulators of this phenotype have been lacking. Here, we develop a multi-scale screening framework that integrates bulk patient transcriptomics, functional in vivo genetic screening, and single-cell transcriptomic and epigenomic profiling to nominate chromatin organizers that modulate ITH in breast cancer. From an initial set of 41 candidates nominated in silico, we identify RNF8 and MIS18A as factors whose perturbation produces coordinated shifts in transcriptomic variability and chromatin accessibility dispersion at the single-cell level, without altering mean expression or genomic copy number. MIS18A acts through a centromere-independent, DNA-methylation-associated pathway. Modulating RNF8 or MIS18A expression tunes chemotherapeutic sensitivity and metastatic potential in vivo: knockdown sensitizes cells and reduces metastatic burden, whereas overexpression confers resistance and promotes progression. Higher expression of both genes predicts shorter patient survival. These findings identify RNF8 and MIS18A as chromatin-level stochastic tuners of transcriptional variability in cancer and provide a generalizable framework for discovering regulators of non-genetic tumor heterogeneity.

cancer biology↗

bioRxiv2025

Decoy-seq unlocks scalable genetic screening for regulatory small noncoding RNAs

Small noncoding RNAs (smRNAs) play critical roles in regulating various cellular processes, including development, stress response, and disease pathogenesis. However, functional characterization of smRNAs remains limited by the scale and simplicity of phenotypic readouts. Recently, single-cell perturbation screening methods, which link CRISPR-mediated genetic perturbations to rich transcriptomic profiling, have emerged as foundational and scalable approaches for understanding gene functions, mapping regulatory networks, and revealing genetic interactions. However, a comparable approach for probing the regulatory consequences of smRNA perturbations is lacking. Here, we present Decoy-seq as an extension of this approach for high-content, single-cell perturbation screening of smRNAs. This method leverages U6-driven tough decoys (TuD), which form stable duplexes with their target smRNAs, for inhibition in the cell. Lentiviral-encoded TuDs are compatible with conventional single-cell RNA-sequencing (scRNA-seq) technologies, allowing joint identification of the smRNA perturbation in each cell and its associated transcriptomic profile. We applied Decoy-seq to 336 microRNAs (miRNAs) and 196 tRNA-derived fragments (tRFs) in a human breast cancer cell line, demonstrating its ability to uncover complex regulatory pathways and novel functions of these smRNAs. Notably, we show that tRFs influence mRNA polyadenylation and regulate key cancer-associated processes, such as cell cycle progression and proliferation. Therefore, Decoy-seq provides a powerful framework for exploring the functional roles of smRNAs in normal physiology and disease, and holds promise for accelerating future discoveries.

systems biology↗

bioRxiv2020

Functional tagging of endogenous proteins and rapid selection of cell pools (Rapid generation of endogenously tagged piwi in ovarian somatic sheath cells.)

The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We present a concise workflow to establish modified cells, characterize the edited allele and probe the function of the tagged protein.

cell biology↗