Biology subjects

Sachan, A.

Publications and source records attributed to Sachan, A..

4 recordsLinked to original sources

bioRxiv2026

Interpretable machine learning coupled to gene regulatory networks uncovers subcircuits underlying cell fate decisions

Gene regulatory networks (GRNs) model causal linkages that control cell fate decisions and differentiation transitions. Prioritizing regulatory subnetworks underlying cell state differences is of critical importance, but current methods including those reliant on topological metrics introduce circularity as the metrics prioritizing TFs are computed from the same networks whose assumptions they inherit. Separately, interpretable machine learning methods can identify latent factors (LFs) that discriminate cellular states with formal statistical guarantees but do not model regulatory linkages. Here, we present FOCAL (Factor-Outcome Coupling for Assessment of Linkages), a paradigm to prioritize regulatory subnetworks by coupling state-specific and dynamic GRNs with outcome-supervised LFs learned using interpretable machine learning without reference to network topology. This shifts GRN focus from macroscopic TF nodes to state-specific and dynamic TF-gene linkages. In B and T cells, FOCAL identified GIFs (GRNs coupled to Interpretable latent Factors), prioritized regulatory subnetworks underlying established states as well as transient regulatory episodes preceding them. By coupling LFs learnt from perturbation experiments of lineage-defining TFs, FOCAL identified transcriptional predisposition to alternative fates within progenitor cell populations before overt differentiation. This uncovered a novel NFATC2-IRF8 interplay in activated B cells, that was validated by in-vitro and in-vivo genetic perturbations. The two transcription factors act cooperatively to restrain extrafollicular plasmablast differentiation and promote germinal center B cell fate.

systems biology↗

bioRxiv2025

Rnd3 regulates cell morphodynamics by spatial restriction of cell contraction signaling

Cell migration is enabled by dynamic changes in cell shape, which are controlled by spatio-temporal activity patterns of the Rho GTPases Rac1 and RhoA. Classical models proposed that these activity patterns are generated by mutual inhibition between the front signal Rac1 and the back signal RhoA, leading to opposing gradients that define the direction of cell migration. However, direct measurements of signal crosstalk showed that Rac1 can activate RhoA, which is incompatible with mutual inhibition. Furthermore, opposing Rac1 and RhoA gradients generated by mutual inhibition would need to overlap at least partially in the cell center. In contrast, both Rac and Rho activities were largely absent in the cell center, and both found to be highly localized near the cell edge in the cell periphery. Here, we hypothesized that these spatio-temporal Rho GTPase activity patterns are generated by the mutual inhibition between RhoA and the unconventional Rho family member Rnd3. Using rapid, optogenetic and chemical perturbations, we confirmed this mutual inhibitory crosstalk. However, we found that this crosstalk does not lead to the expected mutually exclusive spatio-temporal patterns of Rnd3 activity and cell retraction in spontaneously migrating, unperturbed cells. Instead, Rnd3 activity was even slightly elevated during cell retraction and was surprisingly depleted during cell protrusion. We discovered that this depletion is caused by the inhibition of Rnd3 by Rac1 activity, and that this newly identified inhibition is much more pronounced compared to the inhibition of Rnd3 by Rho. By combining rapid optogenetic perturbations with pharmacological manipulations, we found that Rac1 inhibition by Rnd3 is mediated by p21-activated kinases (PAKs). Investigations into the function of Rnd3 showed that it is required for the tight spatio-temporal regulation of the cell contraction/retraction signal Rho, and that it prevents ectopic, highly dynamic, spontaneous Rho activity pulses within the whole cell attachment area. Interestingly, protrusion-retraction dynamics were also severely inhibited in the absence of Rnd3, and its overexpression stimulated this process. Taken together, we show that Rac and not Rho acts as the major Rnd3 inhibitor in cells. Furthermore, our findings support a mechanism, in which Rnd3 acts as a global inhibitor of Rho that spatially restricts Rho activity to regions near the edge of migrating cells.

cell biology↗

bioRxiv2025

Cell cycle-coupled transcriptional network orchestrates human B cell fate bifurcation

Bifurcation of activated human B cells into plasmablast (PB) and germinal center precursor (preGC) fates underlies protective and autoimmune antibody responses, yet gene regulatory networks (GRNs) governing the alternative trajectories remain poorly defined. Using temporal single-cell multiomics, we assembled state-specific human B cell GRNs spanning four scales: transcription factor (TF)-to-fate, TF-to-gene, cis-regulatory element (CRE)-to-gene and nucleotide-to-CRE. Applying the framework to in vitro differentiated and tonsil B cells revealed concordant regulatory architectures and impacts of in silico TF perturbations. CRISPR perturbations validated many TF-to-fate and TF-to-gene predictions and revealed a mutually repressive BATF-IRF4/PRDM1 network module. The GRNs were used to predict and interpret effects of autoimmune disease variants, uncovering partitioning of disease risk at distinct TF motifs and B cell states. Predicted functional variants were independently supported by chromatin and expression QTLs and reporter assays. A web application enables exploration of B cell regulatory determinants of vaccine responses and autoimmune diseases.

immunology↗

bioRxiv2023

Crosstalk between Rac and Rho GTPase activity mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles.

Rho GTPase crosstalk is thought to play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigated crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining acute activity perturbation with activity measurements in individual, mammalian cells. As expected for their proposed mutual inhibition, we confirmed that Rho inhibits Rac activity. However, surprisingly, we found that Rac strongly stimulates Rho activity. We hypothesized that this crosstalk might play a role in mediating the tight spatio-temporal coupling between cell protrusions and retractions that are typically observed during mesenchymal cell migration. Using new, improved activity sensors for endogenous Rho GTPases, we find that Rac activation is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. In a screen for potential crosstalk mediators, we find that a subset of the Rho activating Lbc-type GEFs, in particular Arhgef11 and Arhgef12, are enriched at transient cell protrusions and retractions. Furthermore, via an optogenetic approach, we show that these Lbc GEFs are recruited to the plasma membrane by active Rac, suggesting that they might link cell protrusion and retraction by mediating Rac/Rho activity crosstalk. Indeed, depletion of these GEFs impaired cell protrusion-retraction dynamics, which was accompanied by a decrease in migration distance and an increase in migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate effective exploratory cell migration by coordinating the central cell morphogenic processes of cell protrusion and retraction by coupling the activity of the associated small GTPases Rac and Rho.

cell biology↗