Using Zinc-finger technology to study transcriptional regulation of ThPOK identifies potential cis-acting elements and transcription factors' binding sites (P1460)

Abstract

Abstract T cell maturation involves key branch points at which cells choose between commitment to different lineages, i.e. gd versus ab T cell lineages and CD4+ helper versus CD8+ killer lineages. We focussed on the characterization of a unique line of spontaneous mutant mice, HD-/- mice, which are blocked in the generation of CD4+ helper T cells. Eventually, we discovered of the Zn-finger transcription factor Th-POK. We and others further established that the transcription factor ThPOK is necessary and sufficient to trigger adoption of the CD4 lymphocyte fate. We start to investigate the regulation of ThPOK expression and its subsequent control of CD4+T cell commitment. We identified six separate DHS sites, of which two coincide with putative promoters and the others could represent potential enhancers and silencers. Functional analysis of these putative regulatory elements using a series of reporter transgenics revealed that the DHS site A functions as a lineage specific silencer (referred to as DRE, distal regulatory element).To further dissect the contribution of these DHS sites to the regulation of ThPOK expression, we employed the technology of Zinc finger nucleases (ZFN) mediated genome mutation. Analyzing these mutant mice lines indicates that these both these TF binding motifs work in concert to regulate ThPOK expression, which further affect the determination of thymocytes fate.