Histological and transcriptomic effects of 17α-methyltestosterone on zebrafish gonad development.

BACKGROUND: Sex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal via Cyp19a1a aromatase-mediated estrogen production. Androgens and aromatase inhibitors can induce female-to-male sex reversal, however, the mechanisms underlying gonadal masculinisation are poorly understood. We used histological analyses together with RNA sequencing to characterise zebrafish gonadal transcriptomes and investigate the effects of 17α-methyltestosterone on gonadal differentiation. RESULTS: At a morphological level, 17α-methyltestosterone (MT) masculinised gonads and accelerated spermatogenesis, and these changes were paralleled in masculinisation and de-feminisation of gonadal transcriptomes. MT treatment upregulated expression of genes involved in male sex determination and differentiation (amh, dmrt1, gsdf and wt1a) and those involved in 11-oxygenated androgen production (cyp11c1 and hsd11b2). It also repressed expression of ovarian development and folliculogenesis genes (bmp15, gdf9, figla, zp2.1 and zp3b). Furthermore, MT treatment altered epigenetic modification of histones in zebrafish gonads. Contrary to expectations, higher levels of cyp19a1a or foxl2 expression in control ovaries compared to MT-treated testes and control testes were not statistically significant during early gonad development (40 dpf). CONCLUSION: Our study suggests that both androgen production and aromatase inhibition are important for androgen-induced gonadal masculinisation and natural testicular differentiation in zebrafish

Sex differences in DNA methylation and expression in zebrafish brain: a test of an extended ‘male sex drive’ hypothesis

The sex drive hypothesis predicts that stronger selection on male traits has resulted in masculinization of the genome. Here we test whether such masculinizing effects can be detected at the level of the transcriptome and methylome in the adult zebrafish brain. Although methylation is globally similar, we identified 914 specific differentially methylated CpGs (DMCs) between males and females (435 were hypermethylated and 479 were hypomethylated in males compared to females). These DMCs were prevalent in gene body, intergenic regions and CpG island shores. We also discovered 15 distinct CpG clusters with striking sex-specific DNA methylation differences. In contrast, at transcriptome level, more female-biased genes than male-biased genes were expressed, giving little support for the male sex drive hypothesis. Our study provides genome-wide methylome and transcriptome assessment and sheds light on sex-specific epigenetic patterns and in zebrafish for the first time

A Zebrafish Model of Roberts Syndrome Reveals That Esco2 Depletion Interferes with Development by Disrupting the Cell Cycle

The human developmental diseases Cornelia de Lange Syndrome (CdLS) and Roberts Syndrome (RBS) are both caused by mutations in proteins responsible for sister chromatid cohesion. Cohesion is mediated by a multi-subunit complex called cohesin, which is loaded onto chromosomes by NIPBL. Once on chromosomes, cohesin binding is stabilized in S phase upon acetylation by ESCO2. CdLS is caused by heterozygous mutations in NIPBL or cohesin subunits SMC1A and SMC3, and RBS is caused by homozygous mutations in ESCO2. The genetic cause of both CdLS and RBS reside within the chromosome cohesion apparatus, and therefore they are collectively known as “cohesinopathies”. However, the two syndromes have distinct phenotypes, with differences not explained by their shared ontology. In this study, we have used the zebrafish model to distinguish between developmental pathways downstream of cohesin itself, or its acetylase ESCO2. Esco2 depleted zebrafish embryos exhibit features that resemble RBS, including mitotic defects, craniofacial abnormalities and limb truncations. A microarray analysis of Esco2-depleted embryos revealed that different subsets of genes are regulated downstream of Esco2 when compared with cohesin subunit Rad21. Genes downstream of Rad21 showed significant enrichment for transcriptional regulators, while Esco2-regulated genes were more likely to be involved the cell cycle or apoptosis. RNA in situ hybridization showed that runx1, which is spatiotemporally regulated by cohesin, is expressed normally in Esco2-depleted embryos. Furthermore, myca, which is downregulated in rad21 mutants, is upregulated in Esco2-depleted embryos. High levels of cell death contributed to the morphology of Esco2-depleted embryos without affecting specific developmental pathways. We propose that cell proliferation defects and apoptosis could be the primary cause of the features of RBS. Our results show that mutations in different elements of the cohesion apparatus have distinct developmental outcomes, and provide insight into why CdLS and RBS are distinct diseases

Cohesin Mutations in Cancer: Emerging Therapeutic Targets

The cohesin complex is crucial for mediating sister chromatid cohesion and for hierarchal three-dimensional organization of the genome. Mutations in cohesin genes are present in a range of cancers. Extensive research over the last few years has shown that cohesin mutations are key events that contribute to neoplastic transformation. Cohesin is involved in a range of cellular processes; therefore, the impact of cohesin mutations in cancer is complex and can be cell context dependent. Candidate targets with therapeutic potential in cohesin mutant cells are emerging from functional studies. Here, we review emerging targets and pharmacological agents that have therapeutic potential in cohesin mutant cells

Histological and transcriptomic effects of 17α-methyltestosterone on zebrafish gonad development.

BACKGROUND: Sex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal via Cyp19a1a aromatase-mediated estrogen production. Androgens and aromatase inhibitors can induce female-to-male sex reversal, however, the mechanisms underlying gonadal masculinisation are poorly understood. We used histological analyses together with RNA sequencing to characterise zebrafish gonadal transcriptomes and investigate the effects of 17α-methyltestosterone on gonadal differentiation. RESULTS: At a morphological level, 17α-methyltestosterone (MT) masculinised gonads and accelerated spermatogenesis, and these changes were paralleled in masculinisation and de-feminisation of gonadal transcriptomes. MT treatment upregulated expression of genes involved in male sex determination and differentiation (amh, dmrt1, gsdf and wt1a) and those involved in 11-oxygenated androgen production (cyp11c1 and hsd11b2). It also repressed expression of ovarian development and folliculogenesis genes (bmp15, gdf9, figla, zp2.1 and zp3b). Furthermore, MT treatment altered epigenetic modification of histones in zebrafish gonads. Contrary to expectations, higher levels of cyp19a1a or foxl2 expression in control ovaries compared to MT-treated testes and control testes were not statistically significant during early gonad development (40 dpf). CONCLUSION: Our study suggests that both androgen production and aromatase inhibition are important for androgen-induced gonadal masculinisation and natural testicular differentiation in zebrafish

Cohesin is required for activation of MYC by estradiol.

Cohesin is best known as a multi-subunit protein complex that holds together replicated sister chromatids from S phase until G2. Cohesin also has an important role in the regulation of gene expression. We previously demonstrated that the cohesin complex positively regulates expression of the oncogene MYC. Cell proliferation driven by MYC contributes to many cancers, including breast cancer. The MYC oncogene is estrogen-responsive and a transcriptional target of estrogen receptor alpha (ERα). Estrogen-induced cohesin binding sites coincide with ERα binding at the MYC locus, raising the possibility that cohesin and ERα combine actions to regulate MYC transcription. The objective of this study was to investigate a putative role for cohesin in estrogen induction of MYC expression. We found that siRNA-targeted depletion of a cohesin subunit, RAD21, decreased MYC expression in ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231) breast cancer cell lines. In addition, RAD21 depletion blocked estradiol-mediated activation of MYC in ER-positive cell lines, and decreased ERα binding to estrogen response elements (EREs) upstream of MYC, without affecting total ERα levels. Treatment of MCF7 cells with estradiol caused enrichment of RAD21 binding at upstream enhancers and at the P2 promoter of MYC. Enriched binding at all sites, except the P2 promoter, was dependent on ERα. Since RAD21 depletion did not affect transcription driven by an exogenous reporter construct containing a naked ERE, chromatin-based mechanisms are likely to be involved in cohesin-dependent MYC transcription. This study demonstrates that ERα activation of MYC can be modulated by cohesin. Together, these results demonstrate a novel role for cohesin in estrogen-mediated regulation of MYC and the first evidence that cohesin plays a role in ERα binding

Depletion of RAD21 positively regulates <i>MYC</i> expression in both ER-positive and ER-negative breast cancer cells.

<p><b>A)</b><b><i>RAD21</i></b><b> silencing in T47D cells.</b> RAD21 protein levels were reduced 48 hours after transfection with 10 nM RAD21 siRNA as described in B. <b>B)</b><b>Estradiol-activation of </b><b><i>MYC</i></b><b> is RAD21-dependent in T47D cells</b>. T47D cells were transfected with Control (CON) or RAD21 (RAD) siRNA for 48 hours and then treated with vehicle (V) or estradiol (E) for 6 hours. <i>MYC</i> transcript levels are shown relative to Control siRNA + V treated cells. The ** symbols indicate a significant (p<0.01) reduction in <i>MYC</i> expression in RAD21-depleted cells relative to Control siRNA transfected cells, and between estradiol treated Control cells and estradiol treated RAD21-depleted T47D cells. <b>C)</b><b>RAD21 silencing in MDA-MB-231 cells.</b> RAD21 protein levels were reduced 48 hours after siRNA transfection with 10 nM RAD21 siRNA. <b>D) RAD21 positively regulates </b><b><i>MYC</i></b><b> in ER-negative MDA-MB-231 cells.</b> Relative levels of <i>MYC</i> mRNA in Control (CON) and RAD21 siRNA transfected MDA-MB-231 cells were determined by qRT-PCR. All results are representative or the mean +/− SEM of three independent experiments. The symbol ** indicates a significant (p<0.01) difference in <i>MYC</i> transcript levels between Control siRNA and RAD21 siRNA transfected cells MDA-MB-231 cells.</p

RAD21 binding is enriched by estradiol treatment at <i>MYC</i> regulatory sites within the 8q24 region.

<p>MCF7 cells were fixed following treatment with vehicle (V) or estradiol (E) for 45 minutes. RAD21 binding was analyzed by ChIP. Data are presented as fold enrichment relative to input chromatin and normalized against a negative site (NEG) where no binding was observed. The bar graph represents the mean +/− SEM of three independent experiments. The symbol * indicates a significant increase (p<0.05) in RAD21 binding between vehicle and estradiol treated cells. A schematic of primer locations is shown below the histogram. ChIP primer sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049160#pone.0049160.s004" target="_blank">Table S1</a>. A scale diagram of primer positions relative to the <i>MYC</i> gene and promoters is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049160#pone.0049160.s003" target="_blank">Figure S3</a>.</p

RAD21 binding to <i>MYC</i> regulatory elements varies between breast cancer cell lines.

<p>RAD21 binding in MCF7, T47D and MDA-MB-231 cell lines was analyzed by ChIP. RAD21 binding is shown as fold enrichment relative to input chromatin and normalized against a negative site (NEG) where no binding was observed. The bar graph represents the mean +/− SEM of three independent experiments. The symbols * and ** indicate significant differences (p<0.05 and p<0.01 respectively) in RAD21 binding relative to the MCF7 cell line. A schematic of primer locations is shown below the histogram. ChIP primer sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049160#pone.0049160.s004" target="_blank">Table S1</a>. A scale diagram of primer positions relative to the <i>MYC</i> gene and promoters is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049160#pone.0049160.s003" target="_blank">Figure S3</a>.</p