Transcriptional and post-transcriptional regulation of human Treg identity and function

About This Grant

ABSTRACT Regulatory T cells (Tregs) are essential for maintaining immune homeostasis by preventing excessive inflammation and autoimmune disease. Treg stability is governed by the lineage-defining transcription factor FOXP3. However, exposure to chronic inflammation or repeated T cell receptor stimulation can cause Tregs to destabilize, resulting in loss of FOXP3 expression and suppressive function. The gene regulatory networks controlling Treg identity and function remain incompletely understood, especially in human T cells. To address this, we conducted CRISPR nuclease screening for trans-regulatory factors that control the maintenance and suppression of FOXP3 expression in human Tregs. Our screen highlighted SATB1, SRF, IRF4, and YBX1 as top regulators for further investigation. This proposal aims to define the multi-level regulatory networks governing Treg stability. Aim 1 focuses on the transcriptional regulation of FOXP3 by elucidating how key transcription factors (TFs) modulate FOXP3 expression and Treg stability. We will define the functional roles of key TFs by mapping their binding sites and assessing their effects on chromatin accessibility. We will employ base editing of key cis-regulatory elements (CREs) that modulate FOXP3 expression to identify critical DNA regions and provide a high-resolution map of TF-CRE interactions in Tregs. Aim 2 investigates post- transcriptional regulation of FOXP3, focusing on YBX1, an RNA-binding protein we identified as a novel regulator of FOXP3 expression. Our preliminary data indicate that YBX1 knockout in human Tregs enhances FOXP3 levels and prevents Treg destabilization under inflammatory conditions. We have also uncovered YBX1-bound transcripts as well as identified pathways regulated by YBX1. We will further characterize broad cellular pathways regulated by YBX1 and assess direct FOXP3 regulation by systematic mutagenesis of YBX1 binding targets in key Treg transcripts. We will perform in vitro and in vivo functional assessments in both aims to test the impacts of modulating key regulatory elements and their targets on Treg lineage stability and function. Together, these studies will provide a comprehensive map of FOXP3 regulation at transcriptional and post-transcriptional levels, and identify strategies to modulate key elements to enhance Treg suppressive function and resistance to destabilization. Our findings will provide fundamental insights into Treg biology and inform the development of more effective Treg-based immunotherapies for autoimmune diseases, transplantation, and inflammatory disorders.