Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7
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 (<i>ATOH7</i>) directs the differentiation of retinal ganglion cells and mutati...
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| Format: | Article |
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MDPI AG
2022-01-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/3/1053 |
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| author | David Atac Lucas Mohn Silke Feil Kevin Maggi Dominik Haenni Britta Seebauer Samuel Koller Wolfgang Berger |
| author_facet | David Atac Lucas Mohn Silke Feil Kevin Maggi Dominik Haenni Britta Seebauer Samuel Koller Wolfgang Berger |
| author_sort | David Atac |
| collection | DOAJ |
| description | 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 (<i>ATOH7</i>) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and/or optic nerve abnormalities. Characterization of pathogenic <i>ATOH7</i> mutations is needed to understand the functions of the conserved bHLH motif. The published <i>ATOH7</i> 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 <i>ATOH7</i> 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. |
| first_indexed | 2024-03-09T23:50:38Z |
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| id | doaj.art-69a4175b9d494a97992f9d85c8daf3f9 |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T23:50:38Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-69a4175b9d494a97992f9d85c8daf3f92023-11-23T16:34:11ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-01-01233105310.3390/ijms23031053Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7David Atac0Lucas Mohn1Silke Feil2Kevin Maggi3Dominik Haenni4Britta Seebauer5Samuel Koller6Wolfgang Berger7Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, SwitzerlandInstitute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, SwitzerlandInstitute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, SwitzerlandInstitute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, SwitzerlandCenter for Microscopy and Image Analysis, University of Zurich, 8057 Zurich, SwitzerlandInstitute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, SwitzerlandInstitute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, SwitzerlandInstitute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, SwitzerlandBasic 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 (<i>ATOH7</i>) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and/or optic nerve abnormalities. Characterization of pathogenic <i>ATOH7</i> mutations is needed to understand the functions of the conserved bHLH motif. The published <i>ATOH7</i> 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 <i>ATOH7</i> 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.https://www.mdpi.com/1422-0067/23/3/1053atonal bHLH transcription factor 7 (<i>ATOH7</i>)basic helix–loop–helix (bHLH)cycloheximide (CHX) chase assayprotein dimerizationsynergistic nuclear importdimerization-mediated proteasomal degradation |
| spellingShingle | David Atac Lucas Mohn Silke Feil Kevin Maggi Dominik Haenni Britta Seebauer Samuel Koller Wolfgang Berger Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7 International Journal of Molecular Sciences atonal bHLH transcription factor 7 (<i>ATOH7</i>) basic helix–loop–helix (bHLH) cycloheximide (CHX) chase assay protein dimerization synergistic nuclear import dimerization-mediated proteasomal degradation |
| title | Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7 |
| title_full | Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7 |
| title_fullStr | Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7 |
| title_full_unstemmed | Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7 |
| title_short | Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7 |
| title_sort | functional characterization of an in frame deletion in the basic domain of the retinal transcription factor atoh7 |
| topic | atonal bHLH transcription factor 7 (<i>ATOH7</i>) basic helix–loop–helix (bHLH) cycloheximide (CHX) chase assay protein dimerization synergistic nuclear import dimerization-mediated proteasomal degradation |
| url | https://www.mdpi.com/1422-0067/23/3/1053 |
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