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

• Published in: International journal of molecular sciences Vol. 23; no. 3; p. 1053
• Main Authors: Atac, David, Mohn, Lucas, Feil, Silke, Maggi, Kevin, Haenni, Dominik, Seebauer, Britta, Koller, Samuel, Berger, Wolfgang
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 19.01.2022; MDPI
• Subjects: Abnormalities; Amino acids; Assaying; atonal bHLH transcription factor 7 (ATOH7); Basic Helix-Loop-Helix Transcription Factors - metabolism; basic helix–loop–helix (bHLH); Binding; Cell differentiation; Cycloheximide; cycloheximide (CHX) chase assay; Deoxyribonucleic acid; Dimerization; dimerization-mediated proteasomal degradation; DNA; Energy transfer; Fluorescence; Gene deletion; HEK293 Cells; Humans; Imports; Localization; Microscopy; Mutant Proteins; Mutants; Mutation; Nerve Tissue Proteins - metabolism; Nuclear transport; Optic nerve; Proteasomes; protein dimerization; Protein transport; Proteins; Reporter gene; Retina; Retinal ganglion cells; synergistic nuclear import; Transcription activation; Transcription factors; dimerization-mediated proteasomal degradation; basic helix–loop–helix (bHLH); atonal bHLH transcription factor 7 (ATOH7); retina; cycloheximide (CHX) chase assay; synergistic nuclear import; protein dimerization; fluorescence lifetime imaging-Förster resonance energy transfer (FLIM–FRET); nonsyndromic congenital retinal nonattachment (NCRNA); DNA-binding
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms23031053

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 ( ) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and/or optic nerve abnormalities. Characterization of pathogenic mutations is needed to understand the functions of the conserved bHLH motif. The published 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 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.