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.

Neurog1 can partially replace Atoh1 to differentiate and maintain hair cells in a disorganized organ of Corti

“…1). We suggest that Neurog1 might activate a second pathway for near normal Pou4f3 expression (Ahmed et al, 2012;Masuda et al, 2012), but fails to fully activate Barhl1 needed for HC maintenance (Fig. 11).…”

“…Elucidating the molecular basis of this critical step of downstream gene activation (Cai et al, 2015;Pan et al, 2012b), in which Atoh1 expression cannot be substituted by Neurog1, could help to transform stem cells more effectively into HCs (Ronaghi et al, 2014;Zine et al, 2014). Atoh1 cooperates with unknown factors to fully express Pou4f3 (Ahmed et al, 2012), which, in turn, cooperates with Atoh1 to maintain HCs (Chen et al, 2015;Hertzano et al, 2004;Masuda et al, 2012;Xiang et al, 2003). In contrast to Pou4f3, Barhl1 expression depends exclusively on Atoh1 (Chellappa et al, 2008).…”

Sensory hair cell development and regeneration: similarities and differences

“…Other transcription factors that are crucial for hair cell development have also proven to be useful for generating new sensory cells (Ahmed et al, 2012;Masuda et al, 2012). For example, Ahmed and colleagues showed that the combined use of Eya1 and Six1 induces a hair cell fate in Sox2-expressing nonsensory cells in embryonic cochlear explants (Ahmed et al, 2012;Masuda et al, 2012).…”

“…For example, Ahmed and colleagues showed that the combined use of Eya1 and Six1 induces a hair cell fate in Sox2-expressing nonsensory cells in embryonic cochlear explants (Ahmed et al, 2012;Masuda et al, 2012). Interestingly, myosin VIIa-positive hair cells can be generated through Atoh1-dependent and -independent pathways, providing support for the existence of putative hair cell reprogramming factors aside from Atoh1.…”

Celastrol enhances Atoh1 expression in inner ear stem cells and promotes their differentiation into functional auditory neuronal-like cells

“…Through collaboration with TCF3/E47, Atoh1 activates E box-dependent transcription and plays critical roles in differentiation of several subsets of neural cells. 8 It was reported that the transcription activity of Atoh1 was readily antagonized by the negative regulator of neurogenesis HES1. 9 Loss-of-function of Atoh1 has been identified in the Goblet cell carcinoid and infratentorial cancer.…”