Abstract
During tissue development, transcription factors bind regulatory DNA regions called enhancers, often located at great distances from the genes they regulate, to control gene expression. The enhancer landscape during embryonic stem cell differentiation has been well characterized. By contrast, little is known about the shared and unique enhancer regulatory mechanisms in different ectodermally derived epithelial cells. Here we use ChIP sequencing (ChIP-seq) to identify domains enriched for the histone marks histone H3 lysine 4 trimethylation, histone H3 lysine 4 monomethylation, and histone H3 lysine 27 acetylation (H3K4me3, H3K4me1, and H3K27ac) and define, for the first time, the super enhancers and typical enhancers active in primary human corneal epithelial cells. We show that regulatory regions are often shared between cell types of the ectodermal lineage and that corneal epithelial super enhancers are already marked as potential regulatory domains in embryonic stem cells. Kruppel-like factor (KLF) motifs were enriched in corneal epithelial enhancers, consistent with the important roles of KLF4 and KLF5 in promoting corneal epithelial differentiation. We now show that the Kruppel family member KLF7 promotes the corneal progenitor cell state; on many genes, KLF7 antagonized the corneal differentiation-promoting KLF4. Furthermore, we found that two SNPs linked previously to corneal diseases, astigmatism, and Stevens-Johnson syndrome fall within corneal epithelial enhancers and alter their activity by disrupting transcription factor motifs that overlap these SNPs. Taken together, our work defines regulatory enhancers in corneal epithelial cells, highlights global gene-regulatory relationships shared among different epithelial cells, identifies a role for KLF7 as a KLF4 antagonist in corneal epithelial cell differentiation, and explains how two SNPs may contribute to corneal diseases.
Highlights
During tissue development, transcription factors bind distal regulatory regions called enhancers to control and coordinate gene expression in a temporally and spatially specific manner
We have identified potential roles for super enhancers (SEs) and typical enhancers (TEs) in corneal epithelial cell differentiation and characterized the differences in enhancer landscapes in different epithelial cell types, defining the regulatory regions important for cell identity
We have defined a novel role for KLF7 in corneal epithelial cells, where it acts antagonistically to the pro-differentiation factor KLF4
Summary
Both typical enhancers and super enhancers associate with corneal epithelial identity genes. TEs unique to HCE cells were enriched for the retinoic acid receptor ␣ (RXRA) motif, whereas SEs unique to HCE cells were enriched in AP1 and KLF motifs These results indicate that ETS and KLF family members, important regulators of corneal epithelial differentiation, carry out their functions in part by binding to and activating cell type– specific enhancers. Consistent with this possibility, in previously published siRNA data for EHF in corneal epithelial cells, we found that knockdown of EHF caused a small but significant increase in IRS1 gene expression (Fig. 7G) [18] Together, these data suggest that EHF regulates IRS1 through binding to the rs6758183 enhancer and that disruption of the EHF motif causes reduced affinity of EHF for this site, resulting in aberrant IRS1 expression during corneal development
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