Abstract
Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes.
Highlights
Medulloblastoma is the most common pediatric brain malignancy
We noted that DNase hypersensitive regions were mostly absent outside of these clustered DHS sites (Fig. S1A) and that the canonical insulator protein, CTCF, bound strongly to regions flanking these clustered DHS sites (,122 kb downstream and,50 kb upstream of Orthodenticle Homeobox 2 (OTX2), grey bars in Fig. S1B), indicating that OTX2 regulatory regions are likely to reside within this range
OTX2 expression associates with accessible chromatin regions and unmethylated promoter DNA at the OTX2 locus, indicating that DNA accessibility limits OTX2 expression to a restricted set of normal and transformed tissues
Summary
Medulloblastoma is the most common pediatric brain malignancy. The substantial comorbidities associated with the standard medulloblastoma treatment of resection, radiation, and chemotherapy necessitate the development of selective therapeutics. OTX2 transcriptionally regulates cell cycle genes [6,7] and the MYC oncogene [5], thereby contributing to medulloblastoma maintenance, and can alter the dynamics of hindbrain progenitor cell migration and proliferation [8] This gene is expressed ubiquitously in Shh-independent medulloblastomas [2,5] and is required for viability by tumors of this subgroup. OTX2 represents an attractive potential drug target in this tumor type; as a ligand-independent transcription factor, inhibiting its function with standard small molecule approaches is challenging With this in mind, we have sought to understand the transcriptional regulation of OTX2 with the goal of identifying strategies of pharmacologically inhibiting its expression
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