Epigenetic DNA Modifications Are Correlated With B Chromosomes and Sex in the Cichlid Astatotilapia latifasciata

Abstract

Supernumerary B chromosomes are dispensable elements found in several groups of eukaryotes, and their impacts in host organisms are not clear. The cichlid fish Astatotilapia latifasciata presents one or two large metacentric B chromosomes. These elements affect the transcription of several classes of RNAs. Here, we evaluated the epigenetic DNA modification status of B chromosomes using immunocytogenetics and assessed the impact of B chromosome presence on the global contents of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) and the molecular mechanisms underlying these variations. We found that the B chromosome of A. latifasciata has an active pattern of DNA epimarks, and its presence promotes the loss of 5mC in gonads of females with B chromosome (FB+) and promotes the loss of 5hmC in the muscle of males with the B element (MB+). Based on the transcriptional quantification of DNA modification genes (dnmt, tet, and tdg) and their candidate regulators (idh genes, microRNAs, and long non-coding RNAs) and on RNA-protein interaction prediction, we suggest the occurrence of passive demethylation in gonads of FB+ and 5hmC loss by Tet inhibition or by 5hmC oxidation in MB+ muscle. We suggest that these results can also explain the previously reported variations in the transcription levels of several classes of RNA depending on B chromosome presence. The DNA modifications detected here are also influenced by sex. Although the correlation between B chromosomes and sex has been previously reported, it remains unexplained. The B chromosome of A. latifasciata seems to be active and impacts cell physiology in a very complex way, including at the epigenetic level.

Keywords: 5-hydroxymethylcytosine; 5-methylcytosine; DNA demethylation; DNA methylation; microRNA; supernumerary chromosome.

FIGURE 1

Immunocytogenetics in Astatotilapia latifasciata. Representative image of immunostaining of 5mC (A) and 5hmC (B) in the metaphasic chromosomes of one cell. 5mC and 5hmC signals are observed in green and red, respectively. The chromosomes are counterstained in blue with DAPI. For each karyotype chromosome pair, the fluorescent signals of DAPI and 5mC or 5hmC are shown separated and merged.

FIGURE 2

DNA modifications in the presence of B chromosome in A. latifasciata. Global quantification of 5mC and 5hmC in the encephalon, muscle and gonads. Statistically significant p-values determined by t-test (α = 0.05) are indicated by horizontal lines. Bar colors represent the different samples analyzed, which included males without B chromosome (orange, MB-), males with B chromosome (brown, MB+), females without B chromosome (green, FB-), and females with B chromosome (blue, FB+).

FIGURE 3

Relative expression of DNA modification genes in A. latifasciata. Transcription levels of the genes dnmt1, dnmt3a, dnmt3b, tet1, tet2, tet3, and tdg in encephalon measured by RT-qPCR. p, p-values determined by generalized linear model (α = 0.05) followed by Bonferroni’s post hoc test. Bar colors represent the different samples analyzed, which included males without B chromosome (orange, MB-), males with B chromosome (brown, MB+), females without B chromosome (green, FB-), and females with B chromosome (blue, FB+). The box define median and the lines the minimum and maximum values.

FIGURE 4

Relative expression of DNA modification genes in A. latifasciata. Transcription levels of the genes dnmt1, dnmt3a, dnmt3b, tet1, tet2, tet3, and tdg in muscle measured by RT-qPCR. p-values determined by generalized linear model (α = 0.05) followed by Bonferroni’s post hoc test. Bar colors represent the different samples analyzed, which included males without B chromosome (orange, MB-), males with B chromosome (brown, MB+), females without B chromosome (green, FB-), and females with B chromosome (blue, FB+). The box define median and the lines the minimum and maximum values.

FIGURE 5

Relative expression of DNA modification genes in A. latifasciata. Transcription levels of the genes dnmt1, dnmt3a, dnmt3b, tet1, tet2, tet3, and tdg in gonads measured by RT-qPCR. p, p-values determined by generalized linear model (α = 0.05) followed by Bonferroni’s post hoc test. Bar colors represent the different samples analyzed, which included males without B chromosome (orange, MB-), males with B chromosome (brown, MB+), females without B chromosome (green, FB-), and females with B chromosome (blue, FB+). The box define median and the lines the minimum and maximum values.

FIGURE 6

Relative expression of the regulators of DNA modification genes in A. latifasciata. Transcription levels of idh genes in the muscle measured by RT-qPCR (A). Transcription levels of selected candidate miRNAs regulating the DNA modification genes in encephalon (B), muscle (C), and gonads (D), as revealed by RT-qPCR. p, p-values determined by generalized linear model (α = 0.05) followed by Bonferroni’s post hoc test. Bar colors represent the different samples analyzed, which included males without B chromosome (orange, MB-), males with B chromosome (brown, MB+), females without B chromosome (green, FB-), and females with B chromosome (blue, FB+). The box define median and the lines the minimum and maximum values.

FIGURE 7

Comparison of the percentages of 5mC and transcriptional levels of BncRNA per sample in FB- females without B chromosome (blue, FB-) and females with B chromosome (red, FB+). Numbers inside parentheses indicate sample ID. Pearson’s correlation indicates inverse correlation between 5mC levels and BncRNA expression in encephalons (R = –0.3336 and p < 0.05) and ovaries (R = -0.4689 and p < 0.05) of FB+.

FIGURE 8

Hypothesis of causes of 5mC and 5hmC variations with B chromosome presence in A. latifasciata. (A) In individuals without B chromosomes, regular function of Dnmt and Tet is observed. (B) In ovary of FB+, the chromosome transcribes BncRNA, which can inhibit Dnmt1 and promote loss of 5mC due to passive dilution. (C) In muscle of MB+, the B chromosome reduces the inhibition of Tet function by an unidentified mechanism and promotes loss of 5mhC due to passive dilution. (D) Alternatively, in muscle of MB+, the B chromosome can regulate Tet by an unidentified mechanism, which promotes 5hmC oxidation.