Fig 2: Axial CT scan of the index patients showing both sides. (A,B) Patient #569, arrows in panel (A) indicate the bilateral enlargement of the vestibular aqueduct (EVA), * in panel (B) indicate the bilateral incomplete partition of the cochleae with missing modiolus (IP3), a typical hallmark of POU3F4-related deafness. (C,D) Patient #667, arrow in panel (C) indicates the EVA on the left side, while on the right side the vestibular aqueduct appears normal; * in panel (D) indicate the bilateral IP3.

Fig 3: Slc6a20 and Pou3f4 transcripts and proteins are co-expressed in the mouse cochlea. RT-qPCR on mRNA extracted from whole mouse cochlea, lung, intestine, and kidney and expression of (A) Pou3f4 and (B) Slc6a20 transcripts relative to the housekeeping transcript Actb. n = 5 represents the number of biological replicates. ***p < 0.001, two-tailed, unpaired Student’s t-test. Immunostaining of tissue slices from mouse cochlea with (C–E) an anti-SLC6A20 primary antibody, (F–H) an anti-POU3F4 primary antibody, and (I–K) only the secondary antibody as the negative control. SLC6A20 was clearly localized in the spiral ligament. POU3F4 was more widely distributed in different structures of the cochlea, including the spiral ligament.

Fig 4: POU3F4 regulates the transcription of SLC6A20. (A) HEK293Phoenix cells were transiently transfected with plasmid vectors coding for wild type POU3F4 or the indicated POU3F4 variants. Cells transfected with an empty vector served as negative control. Total RNA was reverse-transcribed and subjected to RT-qPCR. The levels of the SLC6A20 transcript were normalized to those of the housekeeping transcript HPRT1. The SLC6A20 transcript was upregulated in HEK293Phoenix cells overexpressing wild type POU3F4, but not in cells overexpressing the POU3F4 variants. ***p < 0.001, one-way ANOVA with Bonferroni’s post-test. (B,C) RT-qPCR on mRNA extracted from MEFs transfected with a combination of siRNAs designed to silence Pou3f4 or with a scrambled control siRNA (control, CTRL). Expression levels of Pou3f4 and Slc6a20 were normalized to those of the housekeeping transcript Polr2a. Silencing of Pou3f4 led to reduced Slc6a20 expression. n represents the number of biological replicates. **p < 0.01, two-tailed, unpaired Student’s t-test.

Fig 5: Transcriptome analysis (RNAseq) of total RNA extracted from POU3F4-overexpressing HEK293Phoenix cells. The RNAseq analysis was performed on RNA extracted from three independent subcultures of HEK293Phoenix cells transiently transfected with wild type POU3F4, POU3F4 p.C327*, or a control vector encoding solely for the transfection marker EGFP. Volcano plots showing differentially regulated transcripts in wild type POU3F4-transfected cells (A) and POU3F4 p.C327*-transfected cells (C) compared to control cells are shown. Each dot in the plot represents a transcript identified in the analysis. Above the line indicated with a “0” are the transcripts that were upregulated in transfected cells versus the control cells, below the 0 are the downregulated transcripts. Red dots represent transcripts showing a statistically significant upregulation. Heatmap of the top differentially expressed transcripts (DESeq2 results with independent filtering, adj. p-value <0.05, logFC = ± 1) in wild type POU3F4-transfected cells (B) and POU3F4 p.C327*-transfected cells (D) compared to control. (E) Top panel: western blot on total protein extracts from cells overexpressing wild type POU3F4 or POU3F4 p.C327*; control cells were transfected with an empty vector. Bottom panel, the signal of the housekeeping protein GAPDH served as the loading control.