Cofactors of LIM Domains Associate with Estrogen Receptor α to Regulate the Expression of Noncoding RNA H19 and Corneal Epithelial Progenitor Cell Function

Rachel Herndon Klein,Denise N Stephens,Hsiang Ho,Jefferson K Chen,Michael L Salmans,Winnie Wang,Zhengquan Yu,Bogi Andersen

doi:10.1074/jbc.m115.709386

Abstract

Cofactors of LIM domain proteins, CLIM1 and CLIM2, are widely expressed transcriptional cofactors that are recruited to gene regulatory regions by DNA-binding proteins, including LIM domain transcription factors. In the cornea, epithelium-specific expression of a dominant negative (DN) CLIM under the keratin 14 (K14) promoter causes blistering, wounding, inflammation, epithelial hyperplasia, and neovascularization followed by epithelial thinning and subsequent epidermal-like differentiation of the corneal epithelium. The defects in corneal epithelial differentiation and cell fate determination suggest that CLIM may regulate corneal progenitor cells and the transition to differentiation. Consistent with this notion, the K14-DN-Clim corneal epithelium first exhibits increased proliferation followed by fewer progenitor cells with decreased proliferative potential. In vivo ChIP-sequencing experiments with corneal epithelium show that CLIM binds to and regulates numerous genes involved in cell adhesion and proliferation, including limbally enriched genes. Intriguingly, CLIM associates primarily with non-LIM homeodomain motifs in corneal epithelial cells, including that of estrogen receptor α. Among CLIM targets is the noncoding RNA H19 whose deregulation is associated with Silver-Russell and Beckwith-Wiedemann syndromes. We demonstrate here that H19 negatively regulates corneal epithelial proliferation. In addition to cell cycle regulators, H19 affects the expression of multiple cell adhesion genes. CLIM interacts with estrogen receptor α at the H19 locus, potentially explaining the higher expression of H19 in female than male corneas. Together, our results demonstrate an important role for CLIM in regulating the proliferative potential of corneal epithelial progenitors and identify CLIM downstream target H19 as a regulator of corneal epithelial proliferation and adhesion.

Highlights

CLIMs3 are a family (CLIM1/LDB2 and CLIM2/NLI/LDB1) of ubiquitously expressed cofactors [1,2,3] that form homo- and possibly heterodimers [1, 4] to mediate gene activation
Among the cell proliferation genes down-regulated by keratin 14 (K14)-dominant negative (DN)-Clim in the cornea are cell cycle inhibitors CDKN1A/ p21 and CDKN1B/p27 and noncoding RNA H19, a negative regulator of proliferation, consistent with the hyperproliferation observed in the early postnatal period in DN-CLIM corneas [7]
We identify a previously uncharacterized role for the estrogen receptor in the corneal epithelium both as a cofactor of CLIMs in corneal epithelium and as a potential upstream regulator of H19

Summary

Introduction

CLIMs3 are a family (CLIM1/LDB2 and CLIM2/NLI/LDB1) of ubiquitously expressed cofactors [1,2,3] that form homo- and possibly heterodimers [1, 4] to mediate gene activation. All of the limbal epithelially enriched genes that are affected by DN-CLIM in the mouse cornea are down-regulated, further supporting a role for CLIM as transcriptional activators of a gene program regulating corneal progenitor cells (Fig. 4).

Results

Conclusion