Atoh1, an essential transcription factor in neurogenesis and intestinal and inner ear development: function, regulation, and context dependency

Abstract

Atoh1 (also known as Math1, Hath1, and Cath1 in mouse, human, and chicken, respectively) is a proneural basic helix-loop-helix (bHLH) transcription factor that is required in a variety of developmental contexts. Atoh1 is involved in differentiation of neurons, secretory cells in the gut, and mechanoreceptors including auditory hair cells. Together with the two closely related bHLH genes, Neurog1 and NeuroD1, Atoh1 regulates neurosensory development in the ear as well as neurogenesis in the cerebellum. Atoh1 activity in the cochlea is both necessary and sufficient to drive auditory hair cell differentiation, in keeping with its known role as a regulator of various genes that are markers of terminal differentiation. Atoh1 is known in other fields as an oncogene and a tumor suppressor involved in regulation of cell cycle control and apoptosis. Aberrant Atoh1 activity in adult tissue is implicated in cancer progression, specifically in medullablastoma and adenomatous polyposis carcinoma. We demonstrate through protein sequence comparison that Atoh1 contains conserved phosphorylation sites outside the bHLH domain, which may allow regulation through post-translational modification. With such diverse roles, tight regulation of Atoh1 at both the transcriptional and protein level is essential.

FIG. 1

Gene structure of mouse Atoh1. A A schematic diagram of mouse Atoh1. The Atoh1 open reading frame (ORF), represented by an open box runs from position 1 to position 1053, consisting of a single exon. The two identified Atoh1 enhancer sites, represented by black boxes, are located approximately 3.4 kb 3′ to the ORF. B The two enhancer sites, site (A) and site (B), are separated by 413 bp of non-conserved sequence—the distance between enhancers (A) and (B) might influence enhancer activity. There is little sequence similarity between enhancer (A) and enhancer (B); enhancer (A) contains an E-box motif while enhancer (B) contains a Hairy preferred sequence, an E-box motif and an N box motif.

FIG. 2

Cross-species comparison of the amino acid sequence of Atoh1 and comparison on the bHLH domains of mouse Atoh1, atonal, and mouse Ascl1. A An annotated comparison of the Atoh1 protein sequence in mouse, human, rat, cow, dog, chicken, X. tropicalis, and zebrafish. The mouse Atoh1 protein is 351 amino acids long and consists of three domains: the N terminal domain that is required for protein function but only conserved between mammals; the bHLH domain (green box) which is highly conserved across all eight species; and the C terminus, which contains a serine-rich domain (blue box) extending from amino acid 325–351 and is highly conserved across all eight species. There are several conserved, predicted phosphorylation sites (highlighted in red) annotated with the corresponding predicted kinase. Sequences were obtained from the NCBI database; predicted phosphorylation sites were generated using KinasePhos 2.0. B Comparison of the bHLH domains of mouse Atoh1, Drosophila atonal, and mouse Ascl1 demonstrates that the basic (DNA-binding) domains of Atoh1 and atonal are identical, and Ascl1 shares some common elements. In contrast, the helix–loop–helix domains of Atoh1 and atonal share 60% identity. Given the interchangeable nature of Atoh1 and atonal, these differences may be degenerate in terms of secondary structure and electron cloud topology. Comparison with Ascl1 demonstrates that, while overall similarity to Atoh1 is 46%, the basic domain diverges such that it is likely to select for different E-box motifs. Identical residues are marked in blue and indicated with an exclamation mark (!), residues with similar chemical properties are marked in red (as determined by STRAP chemical similarity alignment labeling), and asterisks mark regions that are somewhat conserved across the three sequences.

FIG. 3

A schematic diagram of the onset of Atoh1 expression in the mouse cochlea. A At E13.5, there is no detectable Atoh1 protein in the cochlea; however, mRNA expression has initiated at the base. Cross-section through the mid-base shows mRNA expression arises in the prosensory epithelia in a broad band spanning the depth of the epithelial layer. B At E14.5, Atoh1 protein is expressed in a single row of cells from the base to the mid-base of the cochlea. Cross-section through the mid-base shows Atoh1 protein is in one cell that will become an inner hair cell (IHC) but mRNA is expressed more broadly. C At E15.5, Atoh1 protein is in four rows of cells running from the base to the mid-base, three rows from the mid-base narrowing to two rows of Atoh1-positive cells towards the apex. Cross-section through the mid-base shows Atoh1 protein is detected in three cells, with Atoh1 mRNA beginning to be restricted to cells closer to the luminal surface. D At E17.5, four rows of cells extend to the mid-base, three rows continue towards the apex that are Atoh1-protein-positive. Cross-section through the mid-base shows Atoh1 protein is found in one IHC and three OHCs. Atoh1 mRNA is now restricted to the HCs. M medial; L lateral; IHC inner hair cell; OHCs outer hair cells. Solid black line indicates level of cross-section.

FIG. 4

A schematic diagram illustrating some of the ways that Atoh1 activity is modulated at the protein level. A Phosphorylation may prevent interaction of Atoh1 with either DNA or with class I bHLH transcription factors such as E47, thereby preventing target gene transcription. B Ids, HLH proteins lacking the DNA binding basic region, may competitively bind E proteins displacing Atoh1. C Atoh1-mediated transcription is dependent on specific cofactors. Atoh1 coexpressed in 3 T3 cells with Tcf4, a class I bHLH transcription factor, results in differentiation of pontine neurons, but if E47 is substituted for Tcf4, Atoh1 does not promote transcription of pontine neuron genes. D Addition of E47 and Atoh1 to HEK293 cells carrying an E-box luciferase reporter results in luminescence; adding the transcription factor Hes1 abolishes luminescence, suggesting that Hes1 competes with Atoh1 in binding E proteins.