MEDU-50. TRANSCRIPTIONAL REGULATION OF THE HOMEOBOX GENES CRX AND OTX2 - IMPLICATIONS FOR MEDULLOBLASTOMA GROUP 3

BACKGROUND: Generation of disease-specific models and development of new treatments for non-WNT/non-SHH medulloblastomas (MB) is an urgent need. Group 3 MB is characterized by amplifications of MYC and/or OTX2. Furthermore, transcriptional profiling of group 3 MB shows dysregulation of GABAergic and/or photoreceptor gene expression. The distalless/DLX homeobox genes are required for regulation of GABAergic interneuron differentiation and migration. Furthermore, in the retinas of the Dlx1/Dlx2 double knockout (DKO) mouse, which die at birth, there is marked upregulation of the cone rod homeobox, Crx, as well as the related orthodenticle homeobox gene, Otx2. Moreover, OTX2 activates Crx expression. Otx2 is expressed in three domains of the developing central nervous system (CNS): at the hindbrain/midbrain boundary, the forebrain and retina. METHODS/RESULTS: Using chromatin immunoprecipitation (ChIP) of wild-type mouse embryonic retinas and a specific antibody to DLX2, we have identified regions within regulatory elements of the Crx and Otx2 promoters, respectively. DLX2 protein:Crx/Otx2 DNA binding specificity was confirmed by mobility shift assays, including supershifts using the DLX2 antibody, as well as through site-directed mutagenesis of candidate homeodomain DNA binding motifs. Reporter gene assays demonstrated repression of Crx and/or Otx2 expression when co-transfected with Dlx2 expression constructs in vitro. De-repression of CRX and/or OTX2 expression was confirmed in the Dlx1/Dlx2 DKO in vivo. In addition, crossing of a Crx lacZ reporter mouse with the Dlx1/Dlx2 DKO showed increased lacZ expression in the retinas of the reporter mouse with concurrent loss of of Dlx1 and Dlx2 function. CONCLUSIONS: DLX2 directly binds to both Crx and Otx2 promoters in the embryonic retina and represses expression of Crx and Otx2. We are exploring other upstream transcriptional regulators of OTX2 in the developing CNS towards the development of a group 3 MB mouse model for testing novel therapeutic approaches for children with an intermediate to poor prognosis.