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Treatment with a DNA methyltransferase inhibitor feminizes zebrafish and induces long-term expression changes in the gonads

ABSTRACT: The role of epigenetic modifications such as DNA methylation during vertebrate sexual development is far from being clear. Using the zebrafish model, we tested the effects of one of the most common DNA methyltransferase (dnmt) inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), which is approved for the treatment of acute myeloid leukaemia and is under active investigation for the treatment of solid tumours. Several dose-response experiments were carried out during two periods, including not only the very first days of development (0–6 days post fertilization, dpf), as done in previous studies, but also, and as a novelty, the period of gonadal development (10–30 dpf). Early treatment with 5-aza-dC altered embryonic development, delayed hatching and increased teratology and mortality, as expected. The most striking result, however, was an increase in the number of females, suggesting that alterations induced by 5-aza-dC treatment can affect sexual development as well. Results were confirmed when treatment coincided with gonadal development. In addition, we also found that the adult gonadal transcriptome of 5-aza-dC-exposed females included significant changes in the expression of key reproduction-related genes (e.g., cyp11a1, esr2b and figla), and that several pro-female related pathways such as the Fanconi anemia or the Wnt signaling pathways were downregulated. Furthermore, an overall inhibition of genes implicated in epigenetic regulatory mechanisms (e.g., dnmt1, dicer, cbx4) was also observed. Taken together, our results indicate that treatment with a DNA methylation inhibitor can also alter the sexual development in zebrafish, with permanent alterations of the adult gonadal transcriptome, at least in females. Our results show the importance of DNA methylation for proper control of sexual development, open new avenues for the potential control of sex ratios in fish (aquaculture, population control), and call attention to possibly hidden long-term effects of dnmt therapy when used, for example, in the treatment of prepuberal children affected by some types of cancer.

ORGANISM(S): Danio rerio

PROVIDER: GSE93367 | GEO | 2017/11/30

SECONDARY ACCESSION(S): PRJNA360816

REPOSITORIES: GEO

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Project description:Expression profiling the response to inhibition of DNA methylation and histone deacetylation. Comparison of expression in HepG2 cells treated with 5-aza-dC, Trichostatin A, both, or none (control) to change methylation and acetylation status. Background:DNA methylation and histone deacetylation are epigenetic mechanisms that play major roles in eukaryotic gene regulation. We hypothesize that many genes in the human hepatoma cell line HepG2 are regulated by DNA methylation and histone deacetylation. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) to inhibit DNA methylation with and/or Trichostatin A (TSA) to inhibit histone deacetylation should allow us to identify genes that are regulated epigenetically in hepatoma cells. Results:5-aza-dC had a much larger effect on gene expression in HepG2 cells than did TSA, as measured using Affymetrix® HG-U133A Plus 2.0 microarrays. The expression of 1504 probe sets was affected by 5-aza-dC (at p < 0.01), 535 probe sets by TSA, and 1929 probe sets by the combination of 5-aza-dC and TSA. 5-aza-dC treatment turned on the expression of 211 probe sets that were not detectably expressed in its absence. Expression of imprinted genes regulated by DNA methylation, such as H19 and NNAT, was turned on or greatly increased in response to 5-aza-dC. Genes involved in liver processes such as xenobiotic metabolism (CYP3A4, CYP3A5, and CYP3A7) and steroid biosynthesis (CYP17A1 and CYP19A1), and CCAAT element-binding proteins (CEBPA, CEBPB, and CEBPG) were affected by 5-aza-dC or the combination. Many of the genes that fall within these groups are also expressed in the developing fetal liver. Quantitative real-time RT-PCR assays confirmed selected gene expression changes seen in microarray analyses. Conclusions:Epigenetics play a role in regulating the expression of several genes involved in essential liver processes such as xenobiotic metabolism and steroid biosynthesis in HepG2 cells. Many genes whose expression is normally silenced in these hepatoma cells were re-expressed by 5-aza-dC treatment. Many genes that are expressed in the fetal liver are up-regulated by demethylation, indicating that DNA methylation is a major factor in restricting the expression of fetal genes during liver development. Keywords: comparison of treatments

Project description:Gonad is a bipotential tissue that depending on the genetic and environmental inputs its final phenotype will be determined. Gonadal development is a very complex process because from an undifferentiated gonad the future testis or ovary will be formed. To better understand the complexity of the gonad plasticity throughout development we explored the transcriptomic landscape of gonads from undifferentiated to juvenile fish. For this study we have used turbot (Scophthalmus maximus) that presents one of the most remarkable sexual dimorphism in cultured fish species. With the aim of identifying gene patterns for each gonad developmental stages a microarray species-specific enriched by reproduction- and immune-related genes was assessed. Further genes with sex-differential mRNA expression were mapped in the sex- and growth-QTL (quantitative trait loci) markers. Data revealed that gonadal transcriptome prevail in front of fish biometry as it was possible to classify gonads according to sexual development status by using gene expression profiles rather than their age or size. The same classification was observed with a selection of a subset of reproduction-related genes showing the importance of gene profiles for each of the developing gonadal stage. Overall microarray data showed the importance of the level of expression (intensity) rather than the number of genes found in each development period. Larger numbers of differentially expressed genes (DEG) were found in the differentiating gonads when compared to ovaries and much smaller number resulted when compared to testis. However, more number of DEG was found in testis when compared to ovaries. The earliest presence of cyp19a1a, the key female sex differentiation gene, was found at 90 dpf together with its transcription factor foxl2 showing at the same time a positive correlation with other genes related to extracellular matrix (ecm2), immune system (irf5) or methylation (sall1). The presence of male-related genes like sox9 was evidenced at early developmental stages (70 and 90 dpf) while expression of dmrt3 started not earlier than 150 dpf, coinciding when larger number of male-related genes were found. All developmental stages presented genes related to immune system and epigenetic mechanisms revealing their importance during gonadal development. Sex-QTLs showed larger amount of sex-differential transcripts in the linkage group 6 and 21 both in males and females but transcripts mapped near growth-QTLs were only found in females. Here it is presented the first assessment of the expression profiles observed in the gonads at different developmental stages by identifying genes responsible for male or female traits. It has been identified gene clusters for each developmental stages and gene network interactions for each sexual phenotype. The expression of known male and female-related genes were precisely described throughout gonadal development as well as the expression of other genes with an unknown reproduction function which have been identified for the first time during sexual development process. In addition, it was possible to localize some of these genes on the sex- and growth-QTLs. The data presented here can help to better understand the reproduction system in cultured fish species and improved selection breeding programs to increase their productivity.

Project description:Expression profiling the response to inhibition of DNA methylation and histone deacetylation. Comparison of expression in HepG2 cells treated with 5-aza-dC, Trichostatin A, both, or none (control) to change methylation and acetylation status. Backgroubd: DNA methylation and histone deacetylation are epigenetic mechanisms that play major roles in eukaryotic gene regulation. We hypothesize that many genes in the human hepatoma cell line HepG2 are regulated by DNA methylation and histone deacetylation. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) to inhibit DNA methylation with and/or Trichostatin A (TSA) to inhibit histone deacetylation should allow us to identify genes that are regulated epigenetically in hepatoma cells. RESULTS: 5-aza-dC had a much larger effect on gene expression in HepG2 cells than did TSA, as measured using Affymetrix HG-U133 Plus 2.0 microarrays. The expression of 1504 probe sets was affected by 5-aza-dC (at p < 0.01), 535 probe sets by TSA, and 1929 probe sets by the combination of 5-aza-dC and TSA. 5-aza-dC treatment turned on the expression of 211 probe sets that were not detectably expressed in its absence. Expression of imprinted genes regulated by DNA methylation, such as H19 and NNAT, was turned on or greatly increased in response to 5-aza-dC. Genes involved in liver processes such as xenobiotic metabolism (CYP3A4, CYP3A5, and CYP3A7) and steroid biosynthesis (CYP17A1 and CYP19A1), and genes encoding CCAAT element-binding proteins (C/EBPalpha, C/EBPbeta, and C/EBPgamma) were affected by 5-aza-dC or the combination. Many of the genes that fall within these groups are also expressed in the developing fetal liver and adult liver. Quantitative real-time RT-PCR assays confirmed selected gene expression changes seen in microarray analyses. CONCLUSION: Epigenetics play a role in regulating the expression of several genes involved in essential liver processes such as xenobiotic metabolism and steroid biosynthesis in HepG2 cells. Many genes whose expression is normally silenced in these hepatoma cells were re-expressed by 5-aza-dC treatment. DNA methylation may be a factor in restricting the expression of fetal genes during liver development and in shutting down expression in hepatoma cells

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Treatment with a DNA methyltransferase inhibitor feminizes zebrafish and induces long-term expression changes in the gonads

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