Epigenetic Control of Gonadal Aromatase (cyp19a1) in Temperature-Dependent Sex Determination of Red-Eared Slider Turtles

In the red-eared slider turtle (Trachemys scripta), a species with temperature-dependent sex determination (TSD), the expression of the aromatase gene during gonad development is strictly limited to the female-producing temperature. The underlying mechanism remains unknown. In this study, we identified the upstream 5′-flanking region of the aromatase gene, gonad-specific promoter, and the temperature-dependent DNA methylation signatures during gonad development in the red-eared slider turtle. The 5′-flanking region of the slider aromatase exhibited sequence similarities to the aromatase genes of the American alligator, chicken, quail, and zebra finch. A putative TATA box was located 31 bp upstream of the gonad-specific transcription start site. DNA methylation at the CpG sites between the putative binding sites of the fork head domain factor (FOX) and vertebrate steroidogenic factor 1 (SF1) and adjacent TATA box in the promoter region were significantly lower in embryonic gonads at the female-producing temperature compared the male-producing temperature. A shift from male- to female-, but not from female- to male-, producing temperature changed the level of DNA methylation in gonads. Taken together these results indicate that the temperature, particularly female-producing temperature, allows demethylation at the specific CpG sites of the promoter region which leads the temperature-specific expression of aromatase during gonad development.This work was supported by the National Science Foundation (grant number IOS-1051623). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Integrative BiologyEmail: [email protected]

Total DNA methylation level of the putative aromatase promoter in the gonads.

<p>Total percent DNA methylation during embryonic development at MPT and FPT (A), MPT shifted to FPT (C), and FPT shifted to MPT (D) were examined. (B) The scheme of temperature-shift experiment. Each bar represents twenty to twenty-two clones/stage/temperature from the data of a single pool of 10 gonads. Asterisk (*) is statistically significant (p<0.05) according to the Fisher's exact test. MPT: male-producing temperature. FPT: female-producing temperature.</p

DNA methylation of the putative aromatase promoter at the individual CpG site in the gonads.

<p>(A) DNA methylation status in clones from the gonads at embryonic stage 16, 19, and 21 at MPT and FPT. Each row represents a single clone (1–20∼22 clones/stage/temperature) and a column represents the position of CpG (I – VII). Open circles: unmethylated CpG sites. Closed circles: methylated CpG sites. (B) Percent DNA methylation at the individual CpG site (I – VII) of the putative aromatase promoter at stage 16, 19, and 21 in the slider gonad. Asterisk (*) is statistically significant (p<0.05) according to the Fisher's exact test. MPT: male-producing temperature. FPT: female-producing temperature.</p

Primer sequences to identify the 5′-flanking region of the aromatase gene and gonad-specific transcription start sites in the red-eared slider turtle.

<p>Arom GSP  =  Aromatase gene specific primer.</p

Sequence homology analysis of the 5′-flanking region of the aromatase in the red-eared slider turtle.

<p>The newly identified 4113 bp sequence of the aromatase in the red-eared slider turtle (GenBank accession no. KC554066), consisting of 4066 bp upstream and 47 bp downstream of the translation start codon (ATG), was pair-aligned with the aromatase sequences (upstream bp length from ATG/total bp sequence compared) of the American alligator (3950/4000), chicken (2614/3229), quail (2442/4351), and zebra finch (1356/1401) using the percentage identity plot (PIP) Maker <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063599#pone.0063599-Schwartz1" target="_blank">[24]</a>. GenBank accession numbers are as follows: <i>alligator</i> (AY029233), <i>chicken</i> (D50335), <i>quail</i> (D50336), and <i>zebra finch</i> (AH008871). The additional 5′ flanking region of alligator aromatase was extracted from the assembled genome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063599#pone.0063599-StJohn1" target="_blank">[68]</a>. The horizontal axis represents the base-pair position of the aromatase sequence of the red-eared slider turtle, and the vertical axis represents the percent nucleotide similarity of each aligned segment (i.e., stretch of DNA without any gaps) with the corresponding species.</p

Prediction of transcription factor binding sites (TFBSs) on the 5′-flanking region of aromatase.

<p>TFBSs were predicted using the MatInspector (Genomatix). Colored marks represent the TFBSs with a 5′ to 3′ (above the line) or a 3′ to 5′ (below the line) orientation relative to the transcription start site (TSS). SF1: vertebrate steroidogenic factor 1, ERE: estrogen response elements, FOX: fork head domain factors, DM: DM domain-containing transcription factor, SOX: Sox/SRY sex/testis-determining and related HMG box factors, HEAT: heat shock factors. Horizontal arrow represents TSS (+1). A magnification of gray box is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063599#pone-0063599-g003" target="_blank">Figure 3</a>.</p

DNA methylation of the putative aromatase promoter at the individual CpG site in temperature-shifted gonads.

<p>At embryonic stage 16, eggs are shifted to the opposite temperature, MPT to FPT (A, C) and FPT to MPT (B, D). (A, B) DNA methylation status in clones from the gonads at embryonic stage 19 and 21 after temperature-shift. Each row represents a single clone (1–21∼22 clones/stage/temperature) and a column represents the position of CpG (I – VII). Open circles: unmethylated CpG sites. Closed circles: methylated CpG sites. (C, D) Percent DNA methylation at the individual CpG site (I – VII) of the putative aromatase promoter at stage 19 and 21 in the slider gonad. Asterisk (*) is statistically significant (p<0.05) according to the Fisher's exact test. MPT: male-producing temperature. FPT: female-producing temperature.</p

Putative aromatase promoter (−583 to +170) sequence in the gonads.

<p>The core conserved sequences (four nucleotides) for transcription factor binding sites (TFBSs) and CpG sites (I – VII) are indicated in colored and gray boxes, respectively. The gonad-specific transcription start site (TSS, +1) is indicated with an arrow. The translation initiation codon, ATG, is indicated in bold. The sequences of the Arom Set5 primer pairs used for DNA methylation analysis are underlined. SF1: vertebrate steroidogenic factor 1. ERE: estrogen response elements. FOX: fork head domain factors. DM: DM domain-containing transcription factor. SOX: Sox/SRY sex/testis-determining and related HMG box factors. HEAT: heat shock factors. TATA: TATA box.</p