Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7.

David Atac,Kevin Maggi,Samuel Koller,Britta Seebauer,Lucas Mohn,Silke Feil,Wolfgang Berger,Dominik Haenni

doi:10.3390/ijms23031053

Abstract

Basic helix–loop–helix (bHLH) transcription factors are evolutionarily conserved and structurally similar proteins important in development. The temporospatial expression of atonal bHLH transcription factor 7 (ATOH7) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and/or optic nerve abnormalities. Characterization of pathogenic ATOH7 mutations is needed to understand the functions of the conserved bHLH motif. The published ATOH7 in-frame deletion p.(Arg41_Arg48del) removes eight highly conserved amino acids in the basic domain. We functionally characterized the mutant protein by expressing V5-tagged ATOH7 constructs in human embryonic kidney 293T (HEK293T) cells for subsequent protein analyses, including Western blot, cycloheximide chase assays, Förster resonance energy transfer fluorescence lifetime imaging, enzyme-linked immunosorbent assays and dual-luciferase assays. Our results indicate that the in-frame deletion in the basic domain causes mislocalization of the protein, which can be rescued by a putative dimerization partner transcription factor 3 isoform E47 (E47), suggesting synergistic nuclear import. Furthermore, we observed (i) increased proteasomal degradation of the mutant protein, (ii) reduced protein heterodimerization, (iii) decreased DNA-binding and transcriptional activation of a reporter gene, as well as (iv) inhibited E47 activity. Altogether our observations suggest that the DNA-binding basic domain of ATOH7 has additional roles in regulating the nuclear import, dimerization, and protein stability.

Highlights

Basic helix–loop–helix proteins are structurally similar dimerizing transcription factors with a crucial role in development
As previously described by Kondo et al, the homozygous in-frame deletion of 24 bp detected in a three-month-old boy diagnosed with nonsyndromic congenital retinal nonattachment (NCRNA) predicts the removal of eight amino acids (Arg41 to Arg48) within the highly conserved atonal bHLH transcription factor 7 (ATOH7) basic domain [27]
The evolutionary conservation of the basic domain was evaluated by multiple sequence alignment (MSA) of twelve ATOH7 homologs (Figure 1A)

Summary

Introduction

Basic helix–loop–helix (bHLH) proteins are structurally similar dimerizing transcription factors with a crucial role in development. Classification of bHLH transcription factors can be made based on their expression pattern [3]. Class I bHLH transcription factors, known as E proteins, are ubiquitously expressed. The crystal structure of E47 was determined within a DNA-bound dimer involving the bHLH transcription factor neuronal differentiation 1 (NEUROD1; OMIM: * 601724) [4]. Class II bHLH transcription factors exhibit a time-limited and tissue-specific expression pattern and usually require heterodimerization with E proteins in order to exert their function [3]. Class II bHLH transcription factors are involved in the development of various tissues, including muscle, blood, and fly sex determination [5,6,7]

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Discussion

Conclusion