Novel Foxp3 Regulators discovered by a genome-wide CRISPR Screen

Abstract

Abstract Forkhead box P3 (Foxp3) is an indispensable transcription factor that controls the development of regulatory T cells (Tregs) and programs their suppressive functions. Understanding the mechanisms that regulate Foxp3 expression can potentially lead to the development of therapeutics to manipulate Tregs for treating autoimmune diseases or enhancing cancer immunotherapy. Although a large body of works has been devoted to unraveling the molecular mechanisms controlling Foxp3 expression, a systematic approach is still needed to understand the regulation of Foxp3. In this study, we developed pooled retroviral CRISPR libraries and performed an efficient genome-wide knockout screen in mouse primary T cells to identify the regulators of Foxp3. The outcomes of this screen not only confirmed a number of known Foxp3 regulators but also revealed many novel factors that control Foxp3 expression. Gene ontology analysis showed that positive regulators of Foxp3 are highly enriched with genes involved in chromatin modification, including subunits in the SWI/SNF and SAGA complexes, indicating an underappreciated role for chromatin organization in Treg development and maintenance. Among the negative regulators of Foxp3, we identified a set of genes involved in mRNA processing and methylation, suggesting that transcriptional and post-transcriptional mechanisms actively mediate Foxp3 turnover in Tregs. In summary, our study generated a comprehensive picture of genes and pathways controlling Foxp3 expression and uncovered multiple novel regulators that could be exploited to manipulate Tregs. The genetic tool and strategy used in our study can also be applied to screen for factors determining other traits in mouse primary T cells.