Axenfeld-Rieger syndrome-associated mutants of the transcription factor FOXC1 abnormally regulate NKX2-5 in model zebrafish embryos

Qinxin Zhang,Dong Liang,Yunyun Yue,Luqingqing He,Nan Li,Dongya Jiang,Ping Hu,Qingshun Zhao

doi:10.1074/jbc.ra120.013287

Abstract

FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased, whereas a fourth repressed the expression of NKX2-5 These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.

Highlights

Axenfeld–Rieger syndrome (ARS) is an autosomal dominant disease, mainly characterized by abnormal development of the anterior segment of the eyes
To test whether the promoter containing the presumptive binding sites are directly responsive to mammalian Foxc1, we examined the expression of Nkx2-5 in the rat cardiomyocytes cell line H9c2 overexpressed or knocked down rat Foxc1, respectively
Because FOXC1 directly controls the expression of NKX2-5, we evaluated the functions of the eight FOXC1 mutants (Fig. S3, A and B) identified from ARS patients with congenital heart defects (CHD) [14,15,16,17,18,19,20,21] on regulating the transcriptional activity of NKX2-5 in H9c2 cells to uncover their roles in CHD by overexpressing the same amount of their expression vectors that produced the same level proteins of the FOXC1 mutants in H9c2 cells except for FOXC1(c.210_210delG), which is presumably encoded in the N-terminal part with 70 aa of FOXC1 that would not be recognized by the polyclonal antibody in the western blotting (Fig. S3, B and C)

Summary

Introduction

Axenfeld–Rieger syndrome (ARS) is an autosomal dominant disease, mainly characterized by abnormal development of the anterior segment of the eyes. Embryos carrying knockout alleles of foxc1a, the orthologous gene of mammalian Foxc, die at 9–10 days post-fertilization with severe heart defects, disrupted somites, and smaller eyes [12, 13]. These results reveal that the abnormal phenotypes of the mice and zebrafish carrying Foxc null alleles phenocopy the syndrome of ARS patients with CHD carrying FOXC1 mutants. Together with the facts that Foxc11/2 heterozygous mice and zebrafish have normal heart development [9, 12, 22], it is reasonable to conclude that Foxc or foxc1a knockout model animals should not explain the malfunctional consequences of the human FOXC1 mutants and the roles of the FOXC1 mutants in CHD remains to be answered. R170W) might do it by abnormally repressing the expression of NKX2-5 in the heart of patients

Methods

Results

Conclusion