Transcriptional output of the human FOXP3 locus

CD4+ regulatory T cells (TREGS) are critical for immune tolerance and the transcription factor Forkhead Box P3 (FOXP3) plays a crucial role in their differentiation and function. As for many other human genes, an alternative upstream promoter has been reported for FOXP3. This promoter is active only in TREGS and could have profound implications for the output of the locus and, therefore, for the functionality of the cell. Here, we surveyed the transcriptome of human TREGS to identify FOXP3 transcript isoforms that emanate from the alternative promoter and investigated their function. To this end, we used direct RNA sequencing, western blotting, reanalysis of public mass spectrometry data, transfection of in vitro transcribed RNA, Fluorescence-Activated Cell Sorting, and two distinct RNA smFISH technologies. We identified multiple novel FOXP3 transcriptional products, including one relatively abundant isoform with an extended 5' UTR that we named “longFOXP3”. Together, we show that the transcriptional output of the human FOXP3 locus is far more complex than that of the current annotation, and that nanopore-based methods are required to accurately discriminate between transcript isoforms. Furthermore, our collective observations show that longFOXP3 is not coding and that the activity of the upstream promoter might lead to/correlates with decreased levels of FOXP3. The alternative promoter might interfere with the activity of the canonical promoter and, in this way, fine-tune the levels of FOXP3 in TREGS. Interestingly, the highly cell type-specific activity of the promoter and its potential regulatory role may offer applications as biomarker and/or therapeutic target.