Promoter CpG methylation of Hox-a10 and Hox-a11 in mouse uterus not altered upon neonatal diethylstilbestrol exposure

Mouse abdominal B-like Hoxa genes are expressed and functionally required i n the developing reproductive tracts. Mice lacking either Hoxa-10 or Hoxa-1 1, two of the AbdB Hoxa genes, exhibit abnormal uterine development similar to that induced by in utero diethylstilbestrol (DES) exposure. Indeed, ute rine Hoxa-10 and Hoxa-11 expression is potently repressed by perinatal DES exposure, providing a potential molecular mechanism for DES-induced reprodu ctive tract malformations. We have shown previously that DES can permanentl y alter uterine lactoferrin gene expression through modulation of the lacto ferrin promoter methylation pattern. Here we ask whether a similar mechanis m also functions to deregulate uterine Hoxa-10 or Hoxa-11 expression during neonatal DES exposure. We mapped the Hoxa-10 promoter by cloning a 1.485 k b DNA fragment 5' of the Hoxa-10 exon1a. A 5' rapid amplification of cDNA e nds (RACE) experiment revealed a transcription start site for the a 10-1 tr anscript. Functional analysis of the proximal 200-bp sequences demonstrated significant promoter activity, confirming the location of the Hoxa-10 prom oter. Moreover, methylation assays performed on eight CpGs in Hoxa-10 and 1 9 CpGs in Hoxa-11 proximal promoters demonstrated that all these CpGs were highly unmethylated in both control and DES-dosed mice from postnatal day 5 to day 30. Significant methylation around Hoxa-10 and Hoxa-11 promoters wa s only observed in DES-induced uterine carcinomas in 18-mo-old mice. Our re sults suggest that DES-induced downregulations of Hoxa10 or Hoxa-11 gene ex pression are not associated with methylation changes in their proximal prom oters and that gene imprinting by developmental DES exposure may be a gene- specific phenomenon. Published 2001 Wiley-Liss, Inc.