Wiest and K
Wiest and K. whereby differential TCR signals are molecularly interpreted to promote or antagonize ThPOK expression, and thereby CD4 versus CD8 lineage fates remains unknown. Here we show, using reverse genetic and molecular methods that an autonomous, position-independent TCR-sensing switch is usually embedded within the ThPOK locus. Further, using an in vivo mutagenesis approach, we demonstrate that differential TCR signals are interpreted during lineage commitment by relative binding of EGR, NFAT and Ebox factors to this bistable switch. Collectively our study reveals the central molecular mechanism whereby TCR signaling influences differential lineage choice. Ultimately, these findings may provide an important new tool for skewing T cell fate to treat malignancy and autoimmune diseases. silencer, SilThPOK, which is located 3?kb upstream of the distal promoter8,9. Germline deletion of the SilThPOK in mice causes promiscuous expression of ThPOK and diverts all thymocytes towards CD4 lineage, demonstrating that this SilThPOK is essential for repression of transcription in cells that would normally adopt the CD8 lineage. Our understanding of how the SilThPOK is usually regulated, however, remains rudimentary. Deletion of 2 Runx consensus binding motifs severely impairs silencing function8,9, and mice lacking Runx1 and Runx3 or the obligate Runx-binding partner Cbfb, exhibit loss of the T-cytotoxic lineage. Interestingly, while constitutive expression of ThPOK causes redirection of class I-restricted SERPINA3 thymocytes to the CD4 lineage, overexpression of Runx3 is not sufficient to redirect MHC II-restricted thymocytes to the CD8?+?lineage10. Furthermore, Runx factors are bound to the SilThPOK at all stages of thymic development, indicating that differential binding by Runx factors is not responsible for differential silencer function in class I- versus II-restricted thymocytes. Hence, the molecular basis for how differential TCR signals regulate ThPOK expression in class I- versus class II-restricted thymocytes remains to be decided. Regulation of the gene during thymic development somewhat parallels that of transcription in SP CD8 but not SP CD4 thymocytes11C13, and which contains functionally crucial Runx-binding sites14,15. However, no evidence has emerged to date that transcription and the SilCD4, in particular, are regulated by TCR signals. Indeed, this would seem intuitively unlikely given that is usually transcribed in both unsignaled DP thymocytes and strongly signaled SP CD4 thymocytes. As layed out above, the molecular genetic mechanisms by which TCR-dependent regulation of transcription is usually controlled remain unknown. Here, we seek to resolve this critical issue using an in vivo gene targeting approach. First, through reciprocal swapping of the SilThPOK with the SilCD4, we Dexmedetomidine HCl provide molecular genetic proof that TCR signals directly target Dexmedetomidine HCl the SilThPOK. Second, using precise in vivo gene editing we identify an autonomous, position-independent TCR-sensing switch within the SilThPOK that controls expression in Dexmedetomidine HCl developing thymocytes. Collectively, our study defines the central molecular genetic mechanism whereby TCR signaling influences lineage choice via regulation of expression. Results In vivo silencer swap discloses the autonomous and position-independent function of SilThPOK While we previously showed that strong TCR signals induce transcription in thymocytes8, the molecular mechanisms that connect TCR signals with induction are unknown. We reasoned that TCR signals may induce expression either by (1) activation of positive regulatory elements (enhancers/promoters), or (2) inactivation of the SilThPOK silencer (Supplementary Fig.?1). To genetically test whether the SilThPOK encodes the autonomous and locus-independent capacity to sense differences in MHC class I- versus class II-restricted TCR signaling, we generated SilThPOK swap mice, in which the SilThPOK is usually inserted into the gene in place of its own SilCD4 silencer element (CD4ThPOKsil mice). We used this approach because SilCD4 and SilThPOK share important functional characteristics, i.e., both are active in developing.