Biology subjects

Guruvayurappan, K.

Publications and source records attributed to Guruvayurappan, K..

4 recordsLinked to original sources

bioRxiv2024

Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states

Cell atlas projects have nominated recurrent transcriptional states as drivers of biological processes and disease, but their origins, regulation, and properties remain unclear. To enable complementary functional studies, we developed a scalable approach for recapitulating cell states in vitro using CRISPR activation (CRISPRa) Perturb-seq. Aided by a novel multiplexing method, we activated 1,836 transcription factors in two cell types. Measuring 21,958 perturbations showed that CRISPRa activated targets within physiological ranges, that epigenetic features predicted activatable genes, and that the protospacer seed region drove an off-target effect. Perturbations recapitulated in vivo fibroblast states, including universal and inflammatory states, and identified KLF4 and KLF5 as key regulators of the universal state. Inducing the universal state suppressed disease-associated states, highlighting its therapeutic potential. Our findings cement CRISPRa as a tool for perturbing differentiated cells and indicate that in vivo states can be elicited via perturbation, enabling studies of clinically relevant states ex vivo.

systems biology↗

bioRxiv2024

A pluripotent stem cell platform for in vitro systems genetics studies of mouse development

The directed differentiation of pluripotent stem cells (PSCs) from panels of genetically diverse individuals is emerging as a powerful experimental system for characterizing the impact of natural genetic variation on developing cell types and tissues. Here, we establish new PSC lines and experimental approaches for modeling embryonic development in a genetically diverse, outbred mouse stock (Diversity Outbred mice). We show that a range of inbred and outbred PSC lines can be stably maintained in the primed pluripotent state (epiblast stem cells -- EpiSCs) and establish the contribution of genetic variation to phenotypic differences in gene regulation and directed differentiation. Using pooled in vitro fertilization, we generate and characterize a genetic reference panel of Diversity Outbred PSCs (n = 230). Finally, we demonstrate the feasibility of pooled culture of Diversity Outbred EpiSCs as "cell villages", which can facilitate the differentiation of large numbers of EpiSC lines for forward genetic screens. These data can complement and inform similar efforts within the stem cell biology and human genetics communities to model the impact of natural genetic variation on phenotypic variation and disease-risk.

developmental biology↗

bioRxiv2023

Genome-wide analysis of CRISPR perturbations indicates that enhancers act multiplicatively and without epistatic-like interactions

A single gene may have multiple enhancers, but how they work in concert to regulate transcription is poorly understood. To analyze enhancer interactions throughout the genome, we developed a generalized linear modeling framework, GLiMMIRS, for interrogating enhancer effects from single-cell CRISPR experiments. We applied GLiMMIRS to a published dataset and tested for interactions between 46,166 enhancer pairs and corresponding genes, including 264 high-confidence enhancer pairs. We found that enhancer effects combine multiplicatively but with limited evidence for further interactions. Only 31 enhancer pairs exhibited significant interactions (FDR < 0.1), of which none came from the high confidence subset and 20 were driven by outlier expression values. Additional analyses of a second CRISPR dataset and in silico enhancer perturbations with Enformer both support a multiplicative model of enhancer effects without interactions. Altogether, our results indicate that enhancer interactions are uncommon or have small effects that are difficult to detect. HighlightsO_LIAnalysis of a large single-cell CRISPRi screen finds limited evidence for synergistic or redundant interactions between enhancers C_LIO_LIThe collective action of multiple enhancers on a common target gene follows a multiplicative model of activity C_LIO_LIA new statistical framework for simulating and modeling data from single-cell CRISPRi screens C_LI

bioinformatics↗

bioRxiv2022

Deletion mapping of regulatory elements for GATA3 reveals a distal T helper 2 cell enhancer involved in allergic diseases

The GATA3 gene is essential for T cell differentiation and is surrounded by risk variants for immune traits. Interpretation of these variants is challenging because the regulatory landscape of GATA3 is complex with dozens of potential enhancers spread across a large topological associating domain (TAD) and gene expression quantitative trait locus (eQTL) studies provide limited evidence for variant function. Here, we perform a tiling deletion screen in Jurkat T cells to identify 23 candidate regulatory elements. Using small deletions in primary T helper 2 (Th2) cells, we validate the function of five of these elements, two of which contain risk variants for asthma and allergic diseases. We fine-map genome-wide association study (GWAS) signals in a distal regulatory element, 1 Mb downstream, to identify 14 candidate causal variants. Small deletions spanning candidate rs725861 decrease GATA3 expression in Th2 cells suggesting a causal mechanism for this variant in allergic diseases. Our study demonstrates the power of integrating GWAS signals with deletion mapping and identifies critical regulatory sequences for GATA3.

genomics↗