Abstract
BACKGROUND: Pediatric high grade gliomas (pHGG) are localized to different neuroanatomic compartments with significant differences in their gene expression profiles. In Histone H3.3 G34V/R mutants, DLX2 homeobox gene expression is increased with a corresponding decrease in myelin transcription factor (MYT1) expression. DLX genes are necessary for tangential migration and differentiation of inhibitory interneurons during CNS development. Neural progenitors derived from DLX1/DLX2 double knockout (DKO) mice transplanted into a wild-type forebrain differentiate into oligodendrocytes, providing support for the DLX homeobox genes in neuronal-glial cell fate decisions. METHODS: Chromatin immunoprecipitation (ChIP) assays using a DLX2 antibody were followed by qPCR. ChIP-reChIP assays were performed using antibodies that recognize specific histone modifications. Electrophoretic mobility shift assays (EMSA) were performed using recombinant DLX2 protein and oligonucleotide probes from ChIP-specified promoter regions. Reporter gene assays provided functional assessment of protein-DNA interactions in vitro. Target gene expression was assessed comparing wild-type (WT) and DLX1/DLX2 DKO tissues. RESULTS: Homeodomain binding sites were localized to the promoters of Olig2, Myt1, Nkx2.2 and others in silico. ChIP assays confirmed promoter occupancy by DLX2. ChIPseq experiments are underway. EMSA studies demonstrated specific DLX2-promoter complexes. Luciferase assays showed repression of Olig2 and Nkx2.2 reporter gene expression, consistent with co-occupancy of H3K27me3 in WT and increased target gene expression in DLX1/DLX2 DKO embryonic tissues. We also demonstrated DLX2 expression in a cohort of gangliogliomas. CONCLUSIONS: Our results support a role for DLX transcription factors in controlling neural progenitor specification by activating GABAergic and inhibiting oligodendroglial cell fates through transcriptional repression of a suite of genes required for oligodendrocyte differentiation. Understanding how pHGG and glioneuronal tumors co-opt these neurodevelopmental programs will lead to novel pharmacologic approaches that promote glioma differentiation.
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