Coexpression of nuclear receptors and histone methylation modifying genes in the testis: implications for endocrine disruptor modes of action

Abstract

Background: Endocrine disruptor chemicals elicit adverse health effects by perturbing nuclear receptor signalling systems. It has been speculated that these compounds may also perturb epigenetic mechanisms and thus contribute to the early origin of adult onset disease. We hypothesised that histone methylation may be a component of the epigenome that is susceptible to perturbation. We used coexpression analysis of publicly available data to investigate the combinatorial actions of nuclear receptors and genes involved in histone methylation in normal testis and when faced with endocrine disruptor compounds.

Methodology/principal findings: The expression patterns of a set of genes were profiled across testis tissue in human, rat and mouse, plus control and exposed samples from four toxicity experiments in the rat. Our results indicate that histone methylation events are a more general component of nuclear receptor mediated transcriptional regulation in the testis than previously appreciated. Coexpression patterns support the role of a gatekeeper mechanism involving the histone methylation modifiers Kdm1, Prdm2, and Ehmt1 and indicate that this mechanism is a common determinant of transcriptional integrity for genes critical to diverse physiological endpoints relevant to endocrine disruption. Coexpression patterns following exposure to vinclozolin and dibutyl phthalate suggest that coactivity of the demethylase Kdm1 in particular warrants further investigation in relation to endocrine disruptor mode of action.

Conclusions/significance: This study provides proof of concept that a bioinformatics approach that profiles genes related to a specific hypothesis across multiple biological settings can provide powerful insight into coregulatory activity that would be difficult to discern at an individual experiment level or by traditional differential expression analysis methods.

Figure 1. Coexpression patterns in human, mouse and rat testis.

Coexpression between ASCOM genes in rat human and mouse are represented as networks. Green octagons = methyltransferases and purple vee = histone demethylases. Edge labels represent Pearson correlation significant at p<0.05.

Figure 2. Gatekeeper gene coexpression patterns across species.

Coexpression between genes implicated in the gatekeeper mechanism described by Garcia_Bassets et al are represented as networks. Pink ellipses = nuclear receptors, yellow = histone methyltransferases and blue vee = histone demethylase. Edge labels = Pearson correlation significant at p<0.05. These coexpression relationships are reflected in the coexpression patterns between gatekeeper genes and genes relevant to endocrine disruptor phenotypes and the methyltransferase Dnmt1, shown in associated bar graphs for rat, human and mouse Bar lengths represent Pearson correlation, positive values = positive correlation and negative values = inverse correlation, significant at p<0.05. Genes relevant to endocrine disruptor phenotypes: sexual differentiation (Bmp7, Bmp8a, Dmrt1, Dmrt2, Dmrt3, Dmrta1, Fgf9, Jag2, Shh, Sry); embryonic development (Bmp4, Hoxa4, Hoxa10, Hoxa11, Hoxd3, Hoxd8); obesity (Lep, Lepr, Leprot); spermatogenesis (Prm1, Prm2) and steroidogenesis (Star).

Figure 3. Gatekeeper gene coexpression patterns from Experiment 6.

A: Coexpression between genes implicated in the gatekeeper mechanism identified by Garcia- Bassets et al are represented as a network. Pink ellipses = nuclear receptors, yellow and olive green = histone methyltransferases and blue vee = histone demethylase. Edge labels = Pearson correlation significant at p<0.05. 3.B: The coexpression relationships in 3.A are reflected in the coexpression patterns between gatekeeper genes and genes relevant to endocrine disruptor phenotypes: sexual differentiation (Dmrt1, Dmrta1, Fgf9, Shh, Insl3); embryonic development (Hoxa10), steroidogenesis (Cyp17a1, Star);obesity (Leprot) and spermatogenesis (Prm2).

Figure 4. Coexpression between gatekeeper genes and ASCOM component Wdr5.

Coexpression patterns observed in both controls and exposed samples support a role for a gatekeeper mechanism. Given the dynamic nature of histone methylation, perturbation cannot be inferred from differences between control and exposed groups. More in-depth studies are warranted to investigate exposure effects on these mechanisms. A: In control samples from Experiment 2 Kdm1 was observed to have coexpression higher than the study cut-off limits with only three other histone modifiers including the gatekeeper genes Ehmt1 and Prdm2(approaching significance), and no nuclear receptors. In contrast, in samples from the same experiment that are derived from decedents of an F0 vinclozolin exposed dam, Kdm1 showed strong coexpression with Esr2, multiple other nuclear receptors and histone modifiers including the core ASCOM component Wdr5 and the methyltransferase Dnmt1. B: In Experiments 3 and 4 Wdr5 is anti-correlated with Esr1 and has correlated expression with Kdm1. In control samples of Experiment 3 Wdr5 shows coexpression approaching significance with Esr1 (p = 0.06) and the gatekeeper gene Prdm2. In Experiment 4 only one gatekeeper gene Ehmt1 shows coexpression with Wdr5. C: In Experiment 6 Wdr5 showed similar coexpression with Esr2, Ehmt2 and Kdm1 in both control and exposed samples and with additional gatekeeper genes in the exposed group. Pink ellipses = nuclear receptors, green = histone methyltransferase and blue = histone demethylase. Edges represent Pearson correlation; some of these relationships were approaching significance as indicated by the associated p-values.