Epigenetic modifications DNA methylation in particular

Epigenetic modifications, DNA methylation in particular, regulate key developmental processes including germ cell imprinting and stem cell maintenance/differentiation and play a crucial role in the early periods of embryogenesis (Bartolomei, 2003; Kiefer, 2007; Kondo, 2006; Surani et al., 2007). DNA methylation is also a fundamental aspect of programmed fetal development, determining cell fate, pattern formation, terminal differentiation and maintenance of cellular memory required for developmental stability (Cavalli, 2006; Kafri et al., 1992). Moreover, aberrant epigenetic changes in response to environmental stimuli have been shown to contribute to developmental disorders (Zhao et al., 2007). Several hypotheses involving alcohol (ethanol, EtOH)-induced changes in genetic and epigenetic regulation of glucose transporter as possible molecular mechanisms of FASDs have been recently advanced (Haycock, 2009; Haycock and Ramsay, 2009; Kleiber et al., 2012; Luo, 2009; Ramsay, 2010; Zeisel, 2011). However, the precise mechanisms by which EtOH alters the transcriptional landscape are still largely unknown. In addition, EtOH dose-dependently influences the molecular, cellular, and physiological regulation of adult stem cells which likely contributes to the deleterious consequences of excessive alcohol consumption in adults (Crews et al., 2003; Crews and Nixon, 2003; Nixon et al., 2010; Roitbak et al., 2011). Human stem cells may serve as useful models for delineating the molecular effects of EtOH, especially given the ethical issues of administering alcohol to pregnant women. Alcohol researchers have already taken advantage of these models by observing the consequences of EtOH administration on stem cell differentiation (Garic et al., 2011; Miranda, 2012; Nash et al., 2012; Palmer et al., 2012; Vangipuram and Lyman, 2012). However, genome and methylome-wide studies of EtOH\'s effects on hESCs have not been reported. Here we show that EtOH can induce DNA methylomic changes in hESCs that may have a significant impact on gene regulatory mechanisms potentially involved in stem cell maintenance and differentiation, and by extension, in proper embryo development processes.
Material and methods
Results
Discussion The effect of alcohol on development has been extensively studied in many different animal species (Cudd, 2005). In particular, a single exposure to EtOH during the pre-implantation period was shown to enhance post-implantation fetal death and resorption and to retard normal embryo development (Padmanabhan and Hameed, 1988). In humans, fetal alcohol exposure (FAE) is also correlated with low birth weight, growth, and morphological abnormalities (Day et al., 1989), and in higher rates of spontaneous abortions (Kline et al., 1980; Windham et al., 1997). Numerous other reports have demonstrated genetic, cellular, and biochemical association of alcohol with teratogenesis (Armant and Saunders, 1996; Goodlett and Horn, 2001; Resnicoff et al., 1994; Wozniak et al., 2004). The wide range of physiological and morphological defects associated with FAE suggests that the etiology of FASDs involves a high degree of cellular and molecular heterogeneity. The gastrulation period is considered to be the most sensitive to teratogenic insult, suggesting that differentiating cells might be especially vulnerable to the teratogenic effects of EtOH (Armant and Saunders, 1996). Here we examined the effect of EtOH on the pluripotency of undifferentiated hESCs as a model of early FAE that includes the pre-implantation period. Specifically, we demonstrated that a 24hour low dose (20mM) EtOH treatment significantly reduced the pluripotency (differentiation potential) of hESCs. While to our knowledge no other hESC studies have addressed this issue, somewhat analogous observations were made with rhesus monkey ESCs, albeit at much higher EtOH concentrations and over a course of 4weeks (VandeVoort et al., 2011). We also, for the first time, demonstrated overall increases in DNA methylation in hESCs, defined the genetic and epigenetic molecular landscapes affected by low dose EtOH exposure, and identified genome-wide hotspots that could potentially be vulnerable to FAE. Furthermore, we have identified landscapes of molecular networks that are potentially deregulated by EtOH exposure through DNA methylomic alterations. This is in contrast to the recently reported lack of methylation changes after exposure of hESCs to 20mM EtOH (Krishnamoorthy et al., 2013). Interestingly, their reported selective gene subsets are, for the most part, also unchanged in our study, whereas the much larger set of genes which do show methylation changes in our study are not reported by these authors. Furthermore, numerous studies have linked epigenetic mechanisms as potential regulatory events involved in alcohol teratogenesis (Bielawski et al., 2002; Garro et al., 1991; Haycock, 2009; Kaminen-Ahola et al., 2010). Epigenetic imprinting or genome-wide epigenetic reprogramming has been proposed as a mechanism responsible for alcohol-induced teratogenesis in preimplantation embryos (Haycock, 2009; Haycock and Ramsay, 2009). Interestingly, even paternal or maternal alcohol consumption prior to conception has been shown to result in a wide range of birth defects and fetal abnormalities. It is likely that alcohol-induced epigenetic changes in the gametes or within germ line are responsible for pre-conceptional effects of alcohol (Abel, 2004).