TRANSCRIPTIONAL REGULATION OF OLIGODENDROGLIAL CELL FATE BY DLX HOMEOBOX GENES: POTENTIAL RELEVANCE TO HISTONE H3.3 MUTATIONS IN PEDIATRIC HIGH GRADE GLIOMA

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Abstract

BACKGROUND: The discovery of variant histone H3.3 somatic driver mutations in pediatric high grade gliomas (pHGG) has provided important insights into their pathogenesis. The K27M and G34V/R mutants are usually localized to different neuroanatomic compartments (deep midline/brainstem or superficial/cortical, respectively) and have significant differences in their gene expression profiles. For example, there is a marked increase in the expression of the homeobox gene DLX2 and a corresponding decrease in the expression of the myelin transcription factor, MYT1. When neural progenitors that are mutant for both DLX1 and DLX2 are transplanted into a wild-type background in the developing mouse forebrain, they differentiate into cells of the oligodendrocyte lineage, supporting a role for the DLX homeobox genes in neuronal-glial cell fate decisions. METHODS: Bioinformatics approaches were used to identify homeodomain consensus TAAT/ATTA tetranucleotide DNA binding motifs in regulatory regions of genes required for oligodendrocyte differentiation, Modified chromatin immunoprecipitation (ChIP) assays using a DLX2 antibody were followed by qPCR. ChIP-reChIP assays were performed using antibodies that recognize specific histone modifications at active or repressed regulatory regions of chromatin. 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 mutant forebrain and retina. RESULTS: Homeodomain binding sites were localized to the promoters of several genes, including Olig2, Myt1 and Nkx2.2 in silico. ChIP assays confirmed promoter occupancy by DLX2 in several regions of these gene promoters. ChIPseq experiments are in progress. EMSA studies demonstrated specific DLX2: promoter complexes. Of interest, 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 the DLX1/DLX2 double KO embryonic tissues. CONCLUSIONS: DLX homeobox genes are necessary for tangential migration and differentiation of inhibitory interneurons in the developing CNS. Collectively, our results support a role for these 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 co-opt these neurodevelopmental programs will lead to novel pharmacologic approaches that promote glioma differentiation. SECONDARY CATEGORY: Pediatrics.

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