Xin Xie

 Supervisor: Alex Betz

Project Title: Domain-dependent transcription program of Foxp3 in CD4+ T cells and domain-dependent interactions of Foxp3 with other transcription regulators

Regulatory T (Treg) cells are a subset of CD4+ T cells, which play essential modulatory effects in immune tolerance and homeostasis. Forkhead transcription factor Foxp3 is identified as the master regulator in Treg development and function. Ectopic expression of foxp3 in naïve CD4+ T cells confers Treg phenotype and immunosuppressive functions. Foxp3 have been shown to act as both transcription activator and repressor in global gene regulation, and Foxp3 has been reported to interact with more than 300 proteins in purified Foxp3 complex.  Structurally, 4 domains have been well defined in Foxp3 protein: proline-rich domain, Zinc Finger domain, Coil-coil domain and Forkhead-DNA binding domain. My project aims to dissect the roles of different Foxp3 domains in the transcription regulation and its interactions with other transcription regulators. I transduced primary CD4+ T cells with virus carrying Foxp3 mutants and RNA sequencing was performed to analyze the global gene expression profiles. Even though Forkhead domain is reported to interact with NFAT and be involved in regulation of certain genes including IL2, our RNA-seq data showed the deletion of Forkhead domain caused the loss of Foxp3-like regulation only in a small subset of Foxp3 regulated genes.  However, the deletion of proline-rich domain led to the disruption of Foxp3-like regulation in the majority of Foxp3-regulated genes. To show the importance of the proline-rich domain in the Foxp3’s interaction with other transcription factors, I overexpressed WT type Foxp3, proline-rich domain deleted Foxp3 and Forkhead deleted ones together with other transcription factors including HDAC1, 2, 3, Tip60 and Eos, which has been shown to interact with Foxp3. Co-immunoprecipitation showed that the deletion of proline-rich domain either abrogated or impaired their interactions, while Forkhead deletion had no significant effects.  The data suggest that Foxp3 may plays a more important role in its transcription program as a scaffolding protein through its proline-rich domain.  I am planning to use tagged-Foxp3 and its mutants to pull-down the whole protein complex to show the importance of proline-rich domain in foxp3’s interaction with other proteins.