Investigating the Molecular Basis of PPCD3: Characterization of ZEB1 Regulation of COL4A3 Expression

Abstract

Purpose: To investigate the role of the zinc finger e-box binding homeobox 1 (ZEB1) transcription factor in posterior polymorphous corneal dystrophy 3 by demonstrating its ability to regulate type IV collagen gene transcription via binding to putative E2 box motifs.

Methods: Putative E2 box motifs were identified by in silico analysis within the promoter region of collagen, type IV, alpha3 (COL4A3) and collagen, type IV, alpha4 (COL4A4). To test the ability of ZEB1 to bind to each identified E2 box, electrophoretic mobility shift assays were performed by incubating ZEB1-enriched nuclear extracts with DIG-labeled probes containing one of each of the identified E2 box motifs. Dual-luciferase reporter assays were performed to test the effects of ZEB1 on the luciferase activity of COL4A3 and cadherin 1 (CDH1) promoter constructs, and to determine the effect of a ZEB1 truncating mutation on CDH1 promoter activity.

Results: ZEB1 exhibited binding to six of the nine COL4A3 E2 box probes, whereas no binding was observed for either of the two COL4A4 E2 box probes. ZEB1 overexpression resulted in reduced activity of the COL4A3 promoter construct containing all identified E2 box motifs, whereas a truncating ZEB1 mutation led to the loss of ZEB1-dependent repression of the CDH1 promoter.

Conclusions: COL4A3 gene expression is negatively regulated by ZEB1 binding to E2 box motifs in the COL4A3 promoter region. Therefore, the altered expression of type IV collagens, particularly COL4A3, in the corneal endothelium in individuals with PPCD3 is likely due to reduced transcriptional repression in the setting of a single functional ZEB1 allele.

Figure 1

Schematic depicting the locations of the putative nine E2 box motifs (C4A3, E2A-E2I) and two E2 box motifs (C4A4, E2A and E2B) identified within the 5-kb region upstream of the transcriptional start sites (TSS) for COL4A3 and COL4A4, respectively.

Figure 2

ZEB1 protein from HEK293T nuclear extracts bound to six of the nine identified COL4A3 E2 box motifs. (A) Electrophoretic mobility shift assay performed using HEK293T nuclear extracts as a source of ZEB1 protein and DIG-labeled oligonucleotide probes containing each of the nine identified COL4A3 E2 box motifs. Lane 1: only a single band represented by unbound probes was visible and no complex was formed with the addition of DIG-labeled probes only. Lane 2: protein(s) in the HEK293T nuclear extracts interacted with DIG-labeled E2 box probes causing multiple shifted bands. Lane 3: the addition of unlabeled E2 box probes in 100-fold excess compared with the DIG-labeled E2 box probes resulted in the disappearance of the multiple bands observed in lane 2. Lane 4: the addition of ZEB1 antibody (D80D3; Cell Signaling Technology) to the HEK293T nuclear lysates resulted in the disappearance of the lower shifted band observed with six COL4A3 E2 box probes (ZEB1-shift). Lane 5: the addition of nonspecific rabbit IgG to the nuclear lysates did not cause the disappearance of the lower shifted band. (B) Electrophoretic mobility shift assay blot and corresponding graph show the relative intensities of the ZEB1-shifted bands from each of the nine DIG-labeled COL4A3 E2 box probes.

Figure 3

Effect of ZEB1 overexpression on COL4A3 promoter activity. (A) Schematic diagram of luciferase reporter constructs (not drawn to scale). Black arrows represent E2 box motifs that have been shown to bind ZEB1 in vitro. (B) Relative luciferase activity of CDH1 and COL4A3 promoter constructs was measured when cotransfected into HEK293T cells with an empty vector, pCMV6-Entry, or a ZEB1 cDNA containing construct, pReceiver MO2-ZEB1. Reporter constructs CDH1 P⁻⁶⁰¹ and COL4A3 P⁻⁵⁰⁰⁰, which contain identified E2 box motifs, exhibited significant decreases in luciferase activity when cotransfected into HEK293T cells with ZEB1 compared to being cotransfected with pCMV6-Entry. The reporter construct COL4A3 P⁻³⁰⁷ did not demonstrate a decrease in luciferase activity when cotransfected with ZEB1. **P < 0.05, ****P < 0.0001, n = 6, error bars denote SEM). (C) Anti-ZEB1 (D80D3; Cell Signaling Technology) immunoblotting verified ZEB1 overexpression in each of the HEK293T cell lysates used in the luciferase activity assays cotransfected with pReceiver MO2-ZEB1.

Figure 4

Impact of ZEB1 truncating mutations on CDH1 and COL4A3 promoter activities. (A) The relative luciferase activities of CDH1 and COL4A3 promoter constructs measured when cotransfected into HEK293T cells with either an empty vector (pCMV6-Entry), a ZEB1^WT construct or a ZEB1^R325* mutant construct. Relative to cotransfections with the empty vector, a significant reduction in both the CDH1 and COL4A3 promoter activities were observed when cotransfected with the ZEB1^WT construct, while no significant change in promoter activities was noted when cotransfectioned with the ZEB1^R325* mutant construct. ***P < 0.001, ****P < 0.0001, n = 6, error bars denote SEM. (B) ZEB1^WT (MO2-ZEB1^WT) and ZEB1^R325* (MO2- ZEB1^R325*) expression in the HEK293T cells was confirmed by Western blotting using a ZEB1 antibody (sc-10572; Santa Cruz Biotechnology).