Mechanism of the transcription factor FoxP3 in regulatory T cell development - Proper functions of all immune systems rely on maintenance of immune homeostasis. One family of immune cells that play key roles in immune homeostasis are regulatory T cells (Tregs), a subset of T cells that prevent excessive inflammation and autoimmunity. Tregs differentiate from naïve T cells in a manner that depends on the transcription factor FoxP3. Loss-of-function mutations in FoxP3 lead to fatal multi-organ autoimmune and inflammatory conditions. Despite the importance, the molecular functions and mechanisms of FoxP3 remain poorly understood. We have recently made a significant progress in our structural approach to understand the versatile mode of DNA recognition by FoxP3. We determined the crystal structure of FoxP3 in complex with DNA (Leng et al, Immunity, 2022), breaking the decades-old dogma that FoxP3 forms a domain-swap dimer. More recently, we found that FoxP3 recognizes a new motif composed of TnG repeats by forming a novel multimeric assembly and that this is accompanied by bridging of two DNA molecules (unpublished). Additional preliminary data suggest that this mode of DNA binding also occurs in Treg cells and is important for FoxP3 functions. These findings suggest an exciting new model: unique functions of FoxP3 in Treg development is mediated by its ability to recognize TnG repeats and to form or stabilize DNA loops. We here propose three aims to test and further develop this model. Aim 1 is to determine the cryo-EM structure of the FoxP3 multimers in complex with TnG repeats and to validate the structural mechanism using a combination of biochemistry and cellular immunology. Aim 2 is to investigate functional consequence of such assemblies in T cells using genomic approaches, such as ChIP-seq and HiChIP. Aim 3 is to identify and characterize the direct interacting partners of FoxP3 that mediate its transcriptional function, and to investigate the role of FoxP3 multimerization on its interactome. Together, the proposed research builds upon a strong set of exciting findings from my lab and will have a broad impact on TFs beyond FoxP3. That is, it will help understand how TFs can utilize a multimeric scaffold to recognize new sequence motifs and alter chromatin conformation. Additionally, TnG repeats belong to a group of genetic elements called microsatellites, which are often thought as “junk” or pathogenic. Thus, our work also implicates a novel role for microsatellites in transcriptional regulation and, at the same time, FoxP3 as one of the first exemplary TFs that exploit microsatellites for their functions.
