Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Exp Zool B Mol Dev Evol
2023 Oct 13; doi: 10.1002/jez.b.23222.
Show Gene links
Show Anatomy links
Using Xenopus to discover new candidate genes involved in BOR and other congenital hearing loss syndromes.
Neal SJ
,
Rajasekaran A
,
Jusić N
,
Taylor L
,
Read M
,
Alfandari D
,
Pignoni F
,
Moody SA
.
???displayArticle.abstract???
Hearing in infants is essential for brain development, acquisition of verbal language skills, and development of social interactions. Therefore, it is important to diagnose hearing loss soon after birth so that interventions can be provided as early as possible. Most newborns in the United States are screened for hearing deficits and commercially available next-generation sequencing hearing loss panels often can identify the causative gene, which may also identify congenital defects in other organs. One of the most prevalent autosomal dominant congenital hearing loss syndromes is branchio-oto-renal syndrome (BOR), which also presents with defects in craniofacial structures and the kidney. Currently, mutations in three genes, SIX1, SIX5, and EYA1, are known to be causative in about half of the BOR patients that have been tested. To uncover new candidate genes that could be added to congenital hearing loss genetic screens, we have combined the power of Drosophila mutants and protein biochemical assays with the embryological advantages of Xenopus, a key aquatic animal model with a high level of genomic similarity to human, to identify potential Six1 transcriptional targets and interacting proteins that play a role during otic development. We review our transcriptomic, yeast 2-hybrid, and proteomic approaches that have revealed a large number of new candidates. We also discuss how we have begun to identify how Six1 and co-factors interact to direct developmental events necessary for normal otic development.
Figure 1
(a) The amino acid sequence of the human and Xenopus Six Domain (SD, in the dark blue letters) and N-terminal residues of the Homeodomain (HD, in red letters) are identical. The known BOR-causing human variants are noted in teal using the human SIX1 amino acid numbers; seven are in the SD and three are in the HD. The Cwe variant is marked in magenta. The bait used for the Hybrigenics Y2H screen included the entire SD plus the entire HD extending to amino acid 190. (b) Schematic of the SIX5 protein showing the SD, HD, and C-terminal activation domains. The relative positions of the mutations found in BOR patients (Hoskins et al., 2007) are indicated. BOR, branchio-oto-renal; Y2H, yeast two-hybrid.
Figure 2
Candidate Six1 co-factors. (a) Schematic of the domains present in human and Xenopus Sobp proteins. Sobp contains a C-terminal nuclear localization signal (NLS), two FCS-type Zinc finger domains (ZF1 and ZF2), and a proline-rich region of 245 amino acids. Box 1 includes 20 amino acids that are identical in Xenopus and human proteins. Box 2 includes one of the FCS-type ZF domains. Box 3 is a 25 amino acid domain identical in Xenopus and human proteins. The amino acid locations of the known mouse and human SOBP mutations are indicated by red arrows. Each mutation introduces a stop codon (X) that truncates the protein (Birk et al., 2010; Chen et al., 2008). Box 2 and Box 3 were used in a BLAST search that identified Zmym2 and Zmym4 (Neilson et al., 2010). (b) Schematic of the domains present in human and Xenopus Zmym4 proteins. Like Sobp, it contains several FCS-type ZF domains and a C-terminal NLS. A presumed DNA-binding domain is located N-terminal to the ZFs. The yellow box indicates a DUF3504 domain. Regions of homology to Sobp Box 2 and Box 3 are indicated. (c) Wholemount in situ hybridization images of wild-type embryos indicating the expression domains of transcripts encoding Sobp, Zmym2, Zmym4, 2G4 and Mcrs1 (from Neilson et al., 2010). Red arrows indicate the otic vesicle, which gives rise to the inner ear. Arrows indicate the branchial arches, which give rise to elements of the middle ear (asterisks) and cranial cartilages. e, eye; Sobp, sine oculis binding protein.
Figure 3
Overall view of the mass spectrometry (MS) experiments. Tagged Six1 constructs were transfected into HEK293T cells. Proteins were harvested, the expression level of Six1 was evaluated by Western blot (WB), and proteins associated with Six1 immunoprecipitated using either the anti-Flag-tag antibody (left side) or streptavidin beads (right side) for the Six1 Bio-ID2 transfections. All purified proteins were digested with trypsin and peptides were separated and identified by LC/MS/MS. Protein analysis was performed using two different criteria based on the number of unique high-confidence peptides (either three peptides or a one minimum of one peptide). None of the previously identified partners of Six1 were found on the list of the three unique peptides but were found in the list using one minimum peptide. The list of putative candidates was compared to genes expressed in Xenopus placodes (81 proteins in Table 4) and the common proteins were further analyzed using a String network analysis (Figure 4). HRP, horseradish peroxidase; IP, immunoprecipitation; LC/MS/MS, liquid chromatography with tandem mass spectrometry.
Figure 4
Proteins identified by LC/MS/MS and present in Xenopus cranial placodes. (a) Putative interactions were analyzed using the String website (https://version-11-5.string-db.org/cgi/network?networkId=b3V8l02dS8mr). Proteins with known physical or functional interactions are linked. (b) Proteins with roles in otic development, hearing loss, nuclear localization, or transcriptional regulation are color-coded. LC/MS/MS, liquid chromatography with tandem mass spectrometry.
Figure 5
Y2H analysis of Six1 (SD + HD) bait fragments (Six1.L, residues 1–194), harboring clinical and designer substitutions within the SD, with prey fragments of Eya1 (Eya1.L, residues 316–587), Tle4 (Tle4.S, residues 1–138), and Drosophila (D)-Sobp (Sobp-PA, residues 210–354). Interactions were assessed via mating of transformed haploid yeast strains (Y2H Gold “bait” + Y187 “prey”) and assessing diploid cell growth across a gradient of 3-AT concentrations (0.1–10 mM) on media lacking His, Ade, Leu, and Trp. Semiquantitative interaction scores are color-coded as wild-type level (magenta), affected interaction (from light to medium green), or no detectable interaction (dark green). They were inferred from the maximum concentration of 3-AT that supported growth, relative to wild-type controls, for each interaction pair. The approximate positions of the six SD helices are indicated in gray. HD, homeodomain; SD, Six domain; Y2H, yeast two-hybrid.
Figure 6
Alanine scanning mutagenesis reveals potential cofactor binding sites. (a) Alanine-scanning mutagenesis (X > A, A > F) of the Six1 SD was performed and SD + HD fragments were analyzed for their ability to interact with co-factors in Y2H assays; methods and scoring are as in Figure 5. The approximate positions of the six SD helices are indicated in gray. (b) Structural model of the Six1 SD (magenta) and HD (yellow) domains derived from a co-crystal structure with Eya2 (PDB: 4egc). (c–e) Y2H results from (a) were superimposed on the structure (b) to highlight regions (green) where aa substitutions disrupt co-factor interaction. (c) Eya1; (d) Tle4; (e) D-Sobp. (c′) Docking of Eya2 along the green surface of Six1. 3D renderings were generated using Chimera (v1.15; UCSF Resource for Biocomputing, Visualization, and Informatics). 3D, three dimensional; HD, homeodomain; PDB, Protein Data Bank; SD, Six domain.
