Hypermethylation of homeobox A10 by in utero diethylstilbestrol exposure: an epigenetic mechanism for altered developmental programming
2009 Study Abstract
Diethylstilbestrol (DES) is a nonsteroidal estrogen that induces developmental anomalies of the female reproductive tract. The homeobox gene HOXA10 controls uterine organogenesis, and its expression is altered after in utero DES exposure.
We hypothesized that an epigenetic mechanism underlies DES-mediated alterations in HOXA10 expression. We analyzed the expression pattern and methylation profile of HOXA10 after DES exposure.
Expression of HOXA10 is increased in human endometrial cells after DES exposure, whereas Hoxa10 expression is repressed and shifted caudally from its normal location in mice exposed in utero. Cytosine guanine dinucleotide methylation frequency in the Hoxa10 intron was higher in DES-exposed offspring compared with controls (P = 0.017). The methylation level of Hoxa10 was also higher in the caudal portion of the uterus after DES exposure at the promoter and intron (P < 0.01). These changes were accompanied by increased expression of DNA methyltransferases 1 and 3b. No changes in methylation were observed after in vitro or adult DES exposure.
DES has a dual mechanism of action as an endocrine disruptor; DES functions as a classical estrogen and directly stimulates HOXA10 expression with short-term exposure, however, in utero exposure results in hypermethylation of the HOXA10 gene and long-term altered HOXA10 expression.
We identify hypermethylation as a novel mechanism of DES-induced altered developmental programming.
Women exposed to DES in utero commonly demonstrate abnormalities of the reproductive tract. The frequency of müllerian abnormalities is related to the gestational age of exposure and the total dose of DES received. DES is widely used as a model for estrogenic endocrine disruption that continues to be a significant health concern. An association between clear cell adenocarcinoma of the vagina in women and in utero DES exposure has been clearly established, yet the mechanism by which DES leads to carcinogenesis is not understood. In addition, DES-mediated endocrine disruption during gestation results in developmental anomalies that persist into adulthood in both human and laboratory animals.
Hox genes comprise a highly evolutionarily conserved family of proteins, and they act as the principal regulators of tissue identity during development. They also play an essential role in defining multiple anterior-posterior axes in the developing embryo. Alterations in Hox gene expression are known to lead to anomalies in tissues that depend on their expression for proper developmental signaling (3). Thus, it is not surprising that the uterine anomalies seen after DES exposure are accompanied by a change in Hox expression. Specifically, the alterations seen in Hox gene expression after exposure to DES in utero result from a posterior or caudal shift from the normal pattern. The decreased levels of expression of Hoxa10 cranially may allow for the increased Hoxa9 expression that is demonstrated after DES exposure. This alteration of Hoxa10 expression by DES could provide a molecular mechanism by which DES produces reproductive tract anomalies.
DNA methylation has regulated a variety of aspects of cellular physiology, including growth and differentiation, through alterations in gene expression. Specifically, HOXA10 methylation has been associated with several human cancers . Yoshida et al. found that down-regulation of HOXA10 expression in endometrial carcinomas is associated with methylation of the HOXA10 promoter. Wu et al. reported that aberrant methylation at HOXA10 may be the cause of its lower expression in the endometrium of women with endometriosis. However, altered methylation in an essential developmental gene resulting in permanent reproductive tract anomalies has not been previously reported, nor has a mechanism responsible for these changes been described.
We investigated the frequency of DNA methylation of Hoxa10 upon in uteroDES exposure, and have shown aberrant methylation in the promoter and intron of Hoxa10 and that this altered methylation also persisted into adulthood. Furthermore, we have also shown increased levels of mRNA expression of two important DNMTs, the enzymes responsible for DNA methylation. We speculate that the mechanism by which DNMT expression is up-regulated by DES is mediated by the estrogen receptor (ER). It has been recently been demonstrated that estrogens can alter the expression of at least one of the DNMTs. Furthermore, alterations in HOX gene expression in the uterus are not seen on ER knockout α-mice.
These findings suggest that hypermethylation of the Hoxa10 gene could be the mechanism responsible for the DES-altered developmental programming of the Hoxa10 gene and its associated anomalies. It has been previously reported that DES-induced down-regulation of Hoxa10 or Hoxa11 gene expression is not associated with methylation changes at the proximal promoter. However, we did find differences in levels of DNA methylation in both the promoter and the intron in the DES-treated group in the caudal portions of the uterus. It is possible that the changes in DNA methylation are only observed in the regions of the gene that we explored, which were further 5′ than those previously reported. Our methods of MSP and BSP may also have been more sensitive than those previously used.
We found that, spatially, changes in DNA methylation appeared to be confined to the caudal portion of the uterus. This finding was surprising because expression of Hoxa10 was found to be relatively increased in this portion of the uterus, and not decreased, as is typically seen with increased DNA methylation. We suspect that, rather than silencing gene activity altogether, the mechanism of this epigenetic alteration may be mediated through differential methylation of promoter binding sites for transcriptional repressors. For instance, the promoter sequence we investigated contains a binding site for Yin-Yang 1. Yin-Yang 1 is a zinc finger protein that classically functions as a repressor of Hox gene transcription.
At the same time, we found no significant changes in DNA methylation of HOXA10 in DES-treated HESCs or Ishikawa cells, and these changes were not accompanied by decreased HOXA10 expression. In fact, in vitro treatment with DES resulted in elevated levels of HOXA10 gene expression in these cells, similar to a previous report from our laboratory. Thus, the short-term increase in HOXA10 expression upon DES treatment is likely not related to changes in DNA methylation. Instead, this increase in expression is more likely mediated by the direct estrogen-agonist effect of DES on the ER because HOXA10 has been shown previously to both contain an estrogen response element, and to be regulated by estradiol. On the other hand, changes in DNA methylation may take longer to occur, or alternatively, may only occur during a critical developmental window. The absence of changes in DNA methylation after acute DES exposure in the adult uterus further supports this suggestion.
It has been shown previously that ER-α is necessary to obtain the DES phenotype because ER-α knockout mice show none of the anatomical or gene expression changes related to chronic DES exposure in utero. The effect of DES is likely mediated through ER-α, given the loss of response in the knockout mouse.
In conclusion, we have shown that aberrant DNA methylation of the Hoxa10 gene is associated with changes in Hoxa10 expression after in utero DES exposure, This change in gene methylation provides a molecular mechanism through which DES results in altered developmental programming of the reproductive tract. With short-term exposure, DES appears to act as a classical estrogen, stimulating Hox gene expression; however, after longer exposure in utero, DES induces a permanent epigenetic effect on the expression of Hox genes through changes in DNA methylation. The mechanism of these changes in DNA methylation involves DES-mediated up-regulation of DNMTs. It has recently become clear that alterations in DNA methylation are associated with a variety of processes, including control of gene expression, change in chromatin structure, gene imprinting, and accompanying transcriptional silencing of affiliated genes. Thus, methylation-induced epigenetic changes play an important role in development. Because DES is a prototypical xenoestrogen, other EDCs may have similar epigenetic effects. Thus, DNA methylation is a novel mechanism by which endocrine disruptors may regulate developmental programming.
Sources and more information
- Full study (free access) : Hypermethylation of homeobox A10 by in utero diethylstilbestrol exposure: an epigenetic mechanism for altered developmental programming, The Endocrine Society, Volume 150, Issue 7, Pages 3376–3382, 1 July 2009.
- Featured image Immunohistochemical analysis shows that DES exposure alters Hoxa10 expression in 2-wk-old female mice exposed in utero to DES. A, Location of cranial and caudal sections of the mouse uterus. B, Hoxa10 expression in the cranial and caudal sections of the uterus. DES-treated mice showed increased caudal expression and decreased cranial expression of Hoxa10 in the uterus compared with controls credit NCBI PMC2703508/figure/F2.