Effect of Sin3a deletion on gene expression within FoxP3+ T-regulatory cells

Foxp3 (forkhead box protein 3) functions as the master transcriptional regulator of Treg phenotype. Histone deacetylases 1 and 2 (HDAC1/2) contribute to the regulation of FoxP3 expression via interactions with a myriad of co-regulatory factors. While the nuclear scaffolding protein, Sin3a, has been well established as a co-factor of HDAC1/2, its role within FoxP3+ Tregs has not been. We analyzed the effects of conditional deletion of Sin3a in Foxp3+ Treg cells using three orthogonal approaches. Deletion of Sin3a from FoxP3+ Tregs resulted in the rapid onset of severe and fatal autoimmunity mirroring the phenotype of Scurfy mice. Numbers of Tregs were greatly reduced, residual Tregs had virtually complete loss of suppressive function, and inducible Treg production was blocked. Mice also showed activation of effector T cells, autoantibody production and widespread tissue injury. Mechanistically, Sin3a deletion resulted in decreased transcription of Foxp3 and other Treg signature genes. The reduction of Foxp3 expression was accompanied by the reduction of TET1 and 3 expression and a complete lack of CpG demethylation of the Foxp3 enhancer region CNS2. In addition, Foxp3 protein stability was impaired and fate mapping studies showed conversion of Tregs to ex-Tregs and increased rates of programmed cell death. Thus, Sin3a plays a critical role in the maintenance of Treg identity and function and is essential for the expression and stability of Foxp3. To study the role of Sin3a in T-regulatory cells, Sin3a conditional deletion within FoxP3+ cells was achieved by crossing Sin3a_fl/fl mice with Foxp3_YFPcre. RNA isolated from FACS-sorted FoxP3+ Tregs collected from secondary lymphoid organs of Sin3a-/-FoxP3YFPcre or FoxP3YFPcre controls (equal number of samples from males and females) was used for RNAseq. The differences in gene expression of FACS-sorted FoxP3+ Tregs with or without Sin3a were compared by RNAseq analysis.