Identification of Sox2 cis-regulatory region specific for cochlear epithelium

Abstract

Sox2 is one of the most important transcription factors in neurosensory fate determination in the otic epithelium. Irx3 and Irx5 genes are TALE homeodomain transcription factors that express in the developing otocyst. Interestingly, Sox2 is ectopically expressed in Irx3-/-Irx5-/- mutant, suggesting a negative regulatory role of Irx3 and Irx5 on Sox2 expression. The cis-acting regulatory regions of Sox2 are complex and their functions vary in a tissue specific manner. To identify cochlear epithelium specific cis-regulatory region of Sox2, we perform bioinformatics analysis and in vitro assays. Analysis of Hi-C data in multiple tissues reveals a Sox2 downstream distal region, which has a strong interaction with Sox2 promoter within the same topologically associating domain (TAD). Cis-regulatory elements are enriched within the same TAD. The identity of these cis-regulatory elements is further characterized by ChIP-seq of active histone marks H3K27ac and H3K4me1. Analysis of FANTOM5 (Functional Annotation of the Mammalian Genome) data reveals these cis-regulatory elements are enriched with transcriptional and enhancer activity. We perform chromatin conformation capture (3C) assay in developing cochlea, which shows the interaction between these cis-regulatory regions and Sox2 promoter. The cochlear epithelium specific Sox2 cis-regulatory regions are also enriched with Irx3 and Irx5 binding. The identification of cochlear epithelium specific cis-regulatory regions provides insights into the regulation of Sox2.