Decoding Immune Disease: Linking non-coding variants to gene regulation in primary CD4+ T cells
EMBARGOED UNTIL 2026-03-04
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Date
2025
Publication Type
Doctoral Thesis
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yes
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EMBARGOED UNTIL 2026-03-04
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Abstract
CD4+ T cells are central to regulated and dysregulated immune responses. Over the past decades, thousands of disease-associated variants have been identified. Many of these variants are linked to immune diseases and colocalize with cis-regulatory elements (CREs) like enhancers, that become active during T cell activation. Although these variants build the genetic basis of immune diseases, the downstream regulatory networks they influence remain largely uncharacterized. In this work, we present the first perturbation-based map of non-coding genome function in primary cells and offer insight into the mechanisms through which genetic variation drives immune disease.
We optimized a CRISPRi system for genome-scale screening in primary CD4+ T cells with scRNA-seq readout, overcoming challenges such as limited culturing times and low lentiviral transduction efficiency. We identified over 1,000 disease-relevant putative CREs, overlapping with over 4,700 immune- associated variants across 14 diseases. Using a multipronged strategy, we predicted target genes for each CRE to prioritize genes for readout in targeted perturb-seq (TAP-seq). By perturbing the CREs in over two million primary CD4+ T cells, we identified 827 CRE-target gene interactions. After stringent filtering, 121 of these were classified as direct, cisregulatory links. Analyzing the links allowed us to characterize enhancer-gene interactions and, for the first time, enabled evaluation of enhancer-target prediction tools beyond K562 cells. Furthermore, we identified novel interactions that suggest previously unrecognized gene functions with clinical relevance. We leveraged the identified links to construct a gene regulatory network, revealing key regulatory hubs that might point to common mechanisms between immune-diseases.
Our approach, which integrates systematic CRE prioritization with high-throughput perturbations and targeted sequencing readouts, is highly sensitive in detecting relevant CRE-gene interactions and is well-suited for adaptation to other primary cell types. The CRE-gene interactions identified here could inform therapeutic strategies, potentially guiding the development of targeted interventions for immune-related diseases.
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Examiner: Platt, Randall
Examiner : Steinmetz, Lars
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ETH Zurich
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Subject
gene regulation; Immune function; Single cell analysis; Functional genomics; CRISPR interference screen; CD4 T cells; Gene Regulatory Network; scRNA-seq
Organisational unit
09580 - Platt, Randall / Platt, Randall