Title Transcriptional regulation of RET by Nkx 2 - 1 , Phox 2 b , Sox 10 , andPax

The rearranged during transfection (RET) gene encodes a single-pass receptor whose proper expression and function are essential for the development of enteric nervous system (ENS). Mutations in RET regulatory regions are also associated with Hirschsprung’s disease (HSCR) (aganglionosis of the colon). We have previously showed that two polymorphisms in RET promoter are associated with the increased risk of HSCR. These SNPs overlap with the NK2 homeobox 1 (Nkx2-1) binding motif interrupting the physical interaction of NKX2-1 with the RET promoter and result in reduced RET transcription. In this study, we further delineated Nkx2-1 mediated RET transcription. First, we demonstrated that PHOX2B, like SOX10 and NKX2-1, is expressed in the mature enteric ganglions of human gut. Second, subsequent dualluciferase-reporter studies indicated that Nkx2-1 indeed works co-ordinately with Phox2b and Sox10, but not Pax3, to mediate RET transcription. In addition, identification of Phox2b responsive region in RET promoter further provides solid evidence of the potential functional interaction between Phox2b and RET. In sum, Phox2b and Sox10 act together with Nkx2.1 to modify RET signalling and this interaction may also contribute to HSCR susceptibility.