Function- and conservation-based identification of direct regulatory targets of the transcription factor Sox10

Abstract

We describe identification of multiple direct regulatory targets of Sox10, a critical transcription factor for the development of peripheral glia. By combining RNA interference technique and DNA microarray technology, we have identified a set of genes that show a significant down-regulation upon introduction of Sox 10 specific siRNA into schwannoma cells. Potential binding sites for Sox10 conserved across several mammalian species were subsequently identified within genomic regions proximal to these genes. At least 7 such clusters belonging to 4 different genes were shown to be directly interacting with Sox10 by chromatin immunoprecipitation assay. We further confirm the regulation of one of the genes, SOD3, by Sox10 through EMSA and reporter assays. This protocol relies on both transcriptional activation of target genes as well as physical interaction with regulatory elements and thus represents an efficient approach in identifying actively regulated direct targets of transcription factors.