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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.

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