Myocyte Enhancer Factor 2C and bHLH Proteins Regulate the Expression of Nrl, the bZIP Transcription Factor Essential for Rod Differentiation

Abstract

Abstract: : Purpose: NRL, a bZIP transcription factor, is required for the expression of rod-specific genes and is essential for rod differentiation. The goal of this study is to identify cis-regulatory elements and trans-acting factors that control the rod-specific expression of NRL in developing retina. Methods: Transgenic mice were generated with Nrl-upstream regions tagged to the GFP reporter gene. DNaseI footprinting was carried out for all conserved sequences within the Nrl-5'-upstream region. Gelshift and supershift assays were performed using bovine retinal nuclear extracts and different antibodies. Co-transfections of candidate regulators, myocyte enhancer factor (Mef2C) and bHLH proteins, were performed in cultured cells using luciferase assays. Results: Four highly conserved regions (I-IV) were identified between the 5'-upstream regions of the human and mouse Nrl genes. The most proximal region I contains GC box, TATA-like box, E-box, and other conserved sequences, while the distal enhancer region IV contains Mef2 and E-box-like sites. Transgenic mouse study showed that the distal region IV includes the rod specific enhancer element. Gelshift and supershift assays revealed the binding of Mef2C to the distal region IV. Co-transfection of Mef2C and Mash1 into COS7 cells showed synergistic activation of the luciferase reporter gene that was under the control of Nrl upstream promoter region. NeuroD or Math3 also induce luciferase activity but did not show synergistic effect with Mef2C. Conclusions: Mef2C and Mash1 bind to the distal conserved region and cooperatively transactivate Nrl gene expression. Middle conserved region appears to have a suppressor element. Regulation of Nrl promoter by NeuroD or Math3 is mediated by the proximal conserved region.

Keywords: photoreceptors • retinal development • transcription factors