TFE2 and GATA3 enhance induction of POU4F3 and myosin VIIa positive cells in nonsensory cochlear epithelium by ATOH1

Abstract

Transcription factors (TFs) can regulate different sets of genes to determine specific cell types by means of combinatorial codes. We previously identified closely-spaced TF binding motifs located 8.2-8.5 kb 5' to the ATG of the murine Pou4f3 gene, a gene required for late hair cell (HC) differentiation and survival. These motifs, 100% conserved among four mammalian species, include a cluster of E-boxes preferred by TCF3/ATOH1 heterodimers as well as motifs for GATA factors and SP1. We hypothesized that these factors might interact to regulate the Pou4f3 gene and possibly induce a HC phenotype in non-sensory cells of the cochlea. Cochlear sensory epithelium explants were prepared from postnatal day 1.5 transgenic mice in which expression of GFP is driven by 8.5 kb of Pou4f3 5' genomic DNA (Pou4f3/GFP). Electroporation was used to transfect cells of the greater epithelial ridge with multiple plasmids encoding human ATOH1 (hATOH1), hTCF3 (also known as E2A or TEF2), hGATA3, and hSP1. hATOH1 or hTCF3 alone induced Pou4f3/GFP cells but hGATA3 and hSP1 did not. hATOH1 but not hTCF3 induced conversion of greater epithelial ridge cells into Pou4f3/GFP and myosin VIIa double-positive cells. Transfection of hATOH1 in combination with hTCF3 or hGATA3 induced 2-3X more Pou4f3/GFP cells, and similarly enhanced Pou4f3/GFP and myosin VIIa double-positive cells, when compared to hATOH1 alone. Triple or quadruple TF combinations were generally not more effective than double TF combinations except in the middle turn, where co-transfection of hATOH1, hE2A, and hGATA3 was more effective than hATOH1 plus either hTCF3 or hGATA3. The results demonstrate that TFs can cooperate in regulation of the Pou4f3 gene and in the induction of at least one other element of a HC phenotype. Our data further indicate that combinations of TFs can be more effective than individual TFs in the inner ear.

Fig. 1. Conserved 5′ TF biinding sites in the Pou4f3 gene

A region that is conserved across four mammalian species is located 8.2-8.5 kb 5′ to the ATG of Pou4f3 in the mouse, where it supports ATOH1 binding (Masuda et al., 2011). Clustered E-boxes (CANNTG) are conserved at this site, including two of a type that is activated by ATOH1 (red boxes) (Klisch et al., 2011). The E-boxes are located in close proximity to conserved binding motifs for GATA and SP1.

Fig. 2. A Pou4f3/GFP transgenic

Neonatal cochlea of a transgenic mouse (Pou4f3/GFP mouse) in which GFP driven by 8.5 kb of 5′ Pou4f3 DNA is expressed only in hair cells (HCs). The scale bar = 200 μm.

Fig. 3. Electroporation paradigm

A postnatal day 1.5 (P1.5) cochlear sensory epithelial explant is placed on a filter membrane and covered with 5 μl of HBSS containing a desired plasmid(s). The gap between the filter membrane and the dish electrode is filled with 500 μl HBSS. Five rectangular electrical pulses (12 V, 30 ms duration, 970 ms interval) are applied through the 2 mm diameter round cover (anode) and dish (cathode) electrodes.

Fig. 4. Typical location of transfected cells in the P1.5 sensory epithelium of the Pou4f3-GFP transgenic mouse

The explant shown was electroporated with 1 πg/πl dsRed-expression vector, and fixed 2 days after transfection. The majority of the transfected cells are located in the greater epithelial ridge (GER) which was medial to the organ of Corti (OC). As in this example, HCs (expressing GFP) were never transfected. Blue fluorescence shows nuclei labeled with DAPI. The scale bar = 200 μm.

Fig. 5. Verification of single and double transfections

A P1.5 wildtype cochlear sensory epithelial explant (in which HCs do not express GFP) co-transfected with eGFP- and dsRed-expression plasmids at 0.5 μg/μl for each plasmid (a, b). Almost all transfected cells (94%) expressed both eGFP and dsRed. The scale bar = 100 μm.

Fig. 6. Co-transfection enhances Pou4f3/GFP induction in the GER by ATOH1

The apical turn of P1.5 explants, 5 days after transfection with transcription factors (TFs). Transfection with 0.5 μg/μl of hATOH1 alone (a) or hTCF3 alone (e) induced Pou4f3/GFP+ cells in the GER, well inside the native HCs (arrow heads) of Pou4f3/GFP mouse sensory epithelium. Transfection of hATOH1 in combination with either hTCF3 (i) or hGATA3 (j) induced more Pou4f3/GFP+ GER cells than hATOH1 alone. Transfection of an empty vector (k) did not induce Pou4f3/GFP+ GER cells. The figure also illustrates the loss of HCs (arrowheads) induced by electroporation. It should be noted that the explants were imaged daily and the position of all HCs were traced, and that no migration of HCs into the GER was ever observed (see Fig. 7) Panels b-d and f-h show a higher magnification of the sample transfected with hATOH1 alone (a) and hTCF3 alone (e) respectively. hATOH1 alone induced myosin VIIa (Myo VIIa) expression, but hTCF3 did not. The scale bar in e = 200 μm. The scale bar in h = 50 μm. A, 0.5 μg/μl of hATOH1 expression plasmid; T, 0.5 μg/μl of hTCF3 expression plasmid; G, 0.5 μg/μl of hGATA3 expression plasmid; P, 0.5 μg/μl of empty plasmid.

Fig. 7. TF co-transfection enhances Myo7A induction in the GER by ATOH1

Time course of Pou4f3/GFP expression in the GER of a middle and basal turn explant transfected with hATOH1 plus hTCF3 plus hGATA3, and myosin VIIa expression in the same sample. GER cells showed increasing expression of Pou4f3/GFP from Day 1 to 5, while Pou4f3/GFP+ HCs spread outward and many outer HCs were lost (a-f). Panels g-i show a higher magnification of Pou4f3/GFP, and myosin VIIa expression, for the region indicated in panel f on Day 5. Many Pou4f3/GFP+ GER cells (g) were also positive for myosin VIIa (h, i). While no myosin VIIa+ cells were negative for Pou4f3/GFP, not all Pou4f3/GFP+ GER cells expressed myosin VIIa. Pou4f3/GFP+ cells negative for myosin VIIa tended to show weaker GFP intensity (arrows in g-i indicate some such cells). The scale bar in f = 500 μm. The scale bar in i = 100 μm.

Fig. 8. Quantitative analysis of Pou4f3/GFP induction by TF co-transfection

The numbers of Pou4f3/GFP+ cells observed in the GER following transfection with plasmids encoding TFs, alone or in combination. The numbers of Pou4f3/GFP+ GER cells observed along the entire length of the sensory epithelium (a), in the apical turn (b), in the middle turn (c), or in the basal turn (d) are shown separately. Transfection of hATOH1 alone and hTCF3 alone induced Pou4f3/GFP+ GER cells, but hGATA3 alone did not. Co-transfection of hATOH1 with either hTCF3 or hGATA3 induced more Pou4f3/GFP+ GER cells, and the increase was greater than that induced by hATOH1 alone or hTCF3 alone in the entire cochlea (a) and apical turn (b), although the effect of adding hGATA3 to hATOH1 was not significant statistically in the basal turn, while the triple combination of hATOH1, hTCF3 and hGATA3 was required to increase Pou4f3/GFP+ GER cells significantly over that observed with hATOH1 alone in the middle turn. These indicate that transfection of hATOH1 in combination with either hTCF3 or hGATA3 enhances the hATOH1 effect on 8.5 kb of 5′ Pou4f3 regulatory DNA synergistically. 0.5 A, 0.5 μg/μl of hATOH1 expression plasmid; 1.0 A, 1.0 μg/μl of hATOH1 expression plasmid; T, 0.5 μg/μl of hTCF3 expression plasmid; G, 0.5 μg/μl of hGATA3 expression plasmid; S, 1.0 μg/μl of hSP1 expression plasmid; P, 0.5 μg/μl of empty vector. # or ## indicates a significant difference at p<0.05 or p<0.01 from 0.5 μg/μl of hATOH1 alone by one-way ANOVA with Dunnett’s multiple comparison test. *or ** indicates a significant difference at p<0.05 or p<0.01 from 1.0 μg/μl of hATOH1 alone with the same test.

Fig. 9. Quantitative analysis of Myo7A induction by TF co-transfection

Comparison of the number of Pou4f3/GFP+ GER cells that also expressed myosin VIIa+, observed after transfection with hATOH1 alone or TF combinations. The numbers of Pou4f3/GFP+/myosin VIIa+ GER cells in the entire length of the sensory epithelium (a), in the apical turns (b), in the middle turn (c), and in the basal turn (d) are shown separately. While hATOH1 alone induced Pou4f3/GFP+/myosin VIIa+ GER cells, hTCF3 alone or hGATA3 alone did not. However, co-transfection of each of these TFs with hATOH1 induced more Pou4f3/GFP+/myosin VIIa+ GER cells than hATOH1 alone in the apical turn (a). In the basal turn only hTCF3 enhanced the efeects of hATOH1, while in the middle turn both hTCF3 and hGATA3 were required. The result suggests a synergistic effect among hATOH1, hTCF3, and hGATA3 in the regulation of myosin VIIa expression. Indicators and abbreviations are the same as those in Fig. 8.

Fig. 10. High levels of Pou4f3/GFP induction are required for Myo7A induction

Comparison of normalized GFP intensity between native HCs, and transfected GER cells that expressed Pou4f3/GFP alone or Pou4f3/GFP and myosin VIIa. GFP intensities were measured in the samples transfected with 0.5 μg/μl hATOH1 alone (A), hATOH1 plus hTCF3 (AT), and hATOH1 plus hTCF3 plus hGATA3 (ATG). GER cells that expressed both Pou4f3/GFP and myosin VIIa exhibited significantly greater GFP intensity than cells that expressed only Pou4f3/GFP. While the intensity of Pou4f3/GFP+/myosin VIIa+ GER cells was similar to that of native HCs, there were no differences of GFP intensity of GER cells among the three transfection groups. * indicates p<0.001 with the Mann-Whitney’s U test using Bonferroni correction for multiple comparison. Abbreviations are the same as those of Fig. 8.