Figure 1.
(A) Interspecies comparisons of ATOH8 gene loci. In the plot, the horizontal axis represents the sequence of the base genome, and the amplitude represents the degree of sequence conservation. With the hATOH8 gene locus as a standard reference sequence, the ATOH8 gene locus is divided into several regions, the exons (blue for protein coding regions and light cyan for UTRs) and the introns (pink). In vertebrates, ATOH8 consists of three exons (exon1, exon2 and exon3b shown in blue color) and an exon3a is predicted to encode a novel C-terminus of hATOH8 isoform 2. (B) A potential DNA loop in primate ATOH8 gene loci. One fragment is located in the intron between exon3a and exon3b as a potential loop acceptor (LA, marked with yellow); and the second one is located within the exon3b as a loop donor (LD, marked with red). The pink color marks introns, cyan the UTRs and blue the exons. The sketch of potential DNA loop derived from human ATOH8 is presented in the lower part of the figure.
Figure 2.
Bayesian phylogenetic tree of ATOH8 gene.
The gene tree was built on the basis of complete coding DNA sequence alignment (2713 bp including gaps) of 20 species. The fruit fly NET gene was used as outgroup. Only branches with a posterior probability of at least 0.75 are shown. Branch support indicates the posterior probabilities and Maximum Likelihood Bootstrap.
Figure 3.
Bayesian phylogenetic tree based on the conserved 82 amino acid alignment of the bHLH domain region.
Branches with a posterior probability below 0.75 were collapsed. Support values on the branches resemble the posterior probability values, the PhyML bootstrap, and the RaxML bootstrap values, respectively.
Figure 4.
Comparative analysis of 18 vertebrate ATOH8 proteins.
The identical amino acids are labeled with black background. Sequences more than 80% conserved are highlighted with carmine. Sequences more than 50% conserved are highlighted with cyan. Dots represent the missing amino acids. The bHLH domain, proline-rich and serine-rich regions are defined with bi-directional arrowheads. The bHLH domain was defined by comparison of the ATOH8 sequence (UniProtKB/Swiss-Prot: Q96SQ7, ATOH8_HUMAN) to data from protein database according to the calculation in protein database and protein structure prediction (http://www.compbio.dundee.ac.uk/www-jpred/). The two commonly missing regions in zebrafish, frog and chicken in contrast to mammalian ATOH8 are indicated by blue.
Figure 5.
CpG islands and TATA elements in the upstream region of ATOH8 start codon.
In mammals, the upstream region of ATOH8 coding region is characteristic of CpG islands, of which the human ATOH8 has the longest CpG islands. No TATA elements (green diamonds) are found. In chicken, both CpG islands and TATA elements are found in ATOH8 upstream region. In frog and zebrafish, there are no CpG islands but TATA elements present in the upstream region. From chicken, frog to zebrafish, the number of TATA elements increases.
Figure 6.
Fragments upstream of human ATOH8 could drive gene expression in the chicken embryo.
(A) Diagram of constructed plasmids. 944 nt (1), 442 nt (2) and 9 nt (3) fragments (light cyan) upstream of the hATOH8 translation start codon, together with hATOH8 coding sequence (pink) are integrated into pIRES2-EGFP vector by replacement of its CMV promoter, respectively. The cActin-beta-RFP plasmid (4) is used as an additional indicator for effective transfection. (B) Exogenous gene expression after transfection of these plasmids into chicken embryos. Both 944 nt (1) and 442 nt (2) fragments could drive the expression of their downstream genes shown by, EGFP (green), and hATOH8 as shown here after in situ hybridization. In contrast, the embryo co-transfected with the control plasmid and cActin-beta-RFP plasmid shows no expression of EGFP or hATOH8 (3+4).
Figure 7.
Fish and chromosome painting show the host chromosome of ATOH8 gene in primates.
(A) Demonstration of host chromosomes of ATOH8 in metaphase cells. In human, ATOH8 (green) and WCP2 (red) staining signals are located on the same two chromosomes. ATOH8-positive signal is present on the short arm of WCP2-painted chromosomes. In other primates, ATOH8 signals are positive in two chromosomes, while WCP2 probe marked four chromosomes, out of which two chromosomes are ATOH8 positive. (B) The territories of WCP2-marked chromosomes in interphase cells. In human, WCP2-positive chromosomes (green) have two territories in the nucleus, while four separate territories are clearly visible in non-human primates.