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Developmental Exposure to DES Alters Uterine Gene Expression That Maybe Associated with Neoplasia Later in Life

ABSTRACT: Previously, we described a mouse model where the well-known reproductive carcinogen, diethylstilbestrol (DES), caused uterine adenocarcinoma following neonatal treatment. Tumor incidence was dose-dependent reaching >90% by 18 mo. following 1000 µg/kg/day of DES. These tumors followed the initiation/promotion model of hormonal carcinogenesis with developmental exposure as the initiator, and exposure to ovarian hormones at puberty as the promoter. To identify molecular pathways involved in DES-initiation events, uterine gene expression profiles were examined in prepubertal mice exposed to DES (1, 10 or 1000 µg/kg/day) on days 1-5 and compared to age-matched controls. Of more than 20,000 transcripts, approximately 3% were differentially expressed in at least one DES treatment group compared to controls; several transcripts demonstrated dose-responsiveness. Assessment of gene ontology annotation revealed alterations in genes associated with cell growth, differentiation, and adhesion. When expression profiles were compared to published studies of uteri from 5 day old DES-treated mice, or adult mice treated with 17β estradiol, similarities were seen suggesting persistent differential expression of estrogen responsive genes following developmental DES exposure. Moreover, several significantly altered genes have been identified in human uterine adenocarcinomas. Four altered genes [Lactotransferrin (Ltf), Transforming growth factor beta inducible (Tgfβ1), Cyclin D1 (Ccnd1), and Secreted frizzled-related protein 4 (Sfrp4)], selected for real time RT-PCR analysis, correlated well with the directionality of the microarray data. These data suggest altered gene expression profiles observed two weeks after treatment ceased, were imprinted at the time of developmental exposure and maybe related to the initiation events resulting in carcinogenesis. Keywords: dose reponse

ORGANISM(S): Mus musculus

PROVIDER: GSE5825 | GEO | 2007/06/14

SECONDARY ACCESSION(S): PRJNA97213

REPOSITORIES: GEO

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Project description:Previously, we described a mouse model where the well-known reproductive carcinogen, diethylstilbestrol (DES), caused uterine adenocarcinoma following neonatal treatment. Tumor incidence was dose-dependent reaching >90% by 18 mo. following 1000 µg/kg/day of DES. These tumors followed the initiation/promotion model of hormonal carcinogenesis with developmental exposure as the initiator, and exposure to ovarian hormones at puberty as the promoter. To identify molecular pathways involved in DES-initiation events, uterine gene expression profiles were examined in prepubertal mice exposed to DES (1, 10 or 1000 µg/kg/day) on days 1-5 and compared to age-matched controls. Of more than 20,000 transcripts, approximately 3% were differentially expressed in at least one DES treatment group compared to controls; several transcripts demonstrated dose-responsiveness. Assessment of gene ontology annotation revealed alterations in genes associated with cell growth, differentiation, and adhesion. When expression profiles were compared to published studies of uteri from 5 day old DES-treated mice, or adult mice treated with 17β estradiol, similarities were seen suggesting persistent differential expression of estrogen responsive genes following developmental DES exposure. Moreover, several significantly altered genes have been identified in human uterine adenocarcinomas. Four altered genes [Lactotransferrin (Ltf), Transforming growth factor beta inducible (Tgfβ1), Cyclin D1 (Ccnd1), and Secreted frizzled-related protein 4 (Sfrp4)], selected for real time RT-PCR analysis, correlated well with the directionality of the microarray data. These data suggest altered gene expression profiles observed two weeks after treatment ceased, were imprinted at the time of developmental exposure and maybe related to the initiation events resulting in carcinogenesis. Experiment Overall Design: There were 3 DES doses (1, 10 and 1000 ug/kg/day) administered to neonates on days 1-5. RNA was pooled for 10 mice, with each dose having its own matching control (corn oil). Dye-flipped hybridizations were performed for each paired comparison.

Project description:Neonatal exposure to diethylstilbestrol (DES) results in abnormal reproductive tract morphology, female infertility and uterine cancer in mice. This exposure also causes altered gene expression in the female reproductive tract that persists into adulthood. To further explore on a genome-wide basis how neonatal estrogen exposure results in permanent epigenetic changes, we performed RNA-seq and ChIP-seq analyses of DES-treated and control mice. CD-1 mice were treated on postnatal days (PND) 1-5 with DES (2 µg/pup/day) or corn oil as a control; uterine tissues were collected on PND5. RNA-seq analysis resulted in 4,498 differentially expressed genes (>1.5 fold difference; RPKM >1; FDR <0.05). ChIP-seq was performed on uterine samples using H3K4me3, H3K27me3, H3K27acetyl and H3K4me1 as the precipitating antibodies. The most significant findings at the TSS were increased H3K4me3 at up-regulated genes (22%), increased H3K27ac at up-regulated genes (55%) and differential H3K27ac at down-regulated genes (33%). The most striking finding was differential association of H3K27ac at presumed enhancer regions; 3,012 out of 4498 altered genes (67%) had a differential H3K27ac peak associated with them and 2,962 out of 4,498 (66%) had differential H3K4me1. These differences were generally coordinated with gene expression with increased gene expression having more H3K27ac associated although a small subset of genes showed the opposite. To further investigate the mechanism of differential H3K27ac binding in enhancer regions near estrogen regulated genes, we overlapped these enhancers to Esr1 ChIP seq data; a large percentage of them overlap with Esr1 peaks (39%). We also performed motif analysis where ERE was enriched in the differential H3K27ac peaks. We further investigated Esr1’s role in differential H3K27ac enhancer binding by using a conditional uterine deletion of Esr1 by crossing Esr1 floxed mice with PgR-cre and then treating with vehicle or DES and collecting uteri on day 5. We performed a microarray and pattern analysis and determined there were 4,073 out of 4,617 altered genes (88%) protected by conditional uterine deletion of Esr1. We also performed H3K27ac ChIP seq on these samples and found a large number of these protected genes have a differential H3K27ac enhancer. Several Fox genes were up-regulated following DES treatment and Fox binding motifs were found to be enriched in both H3K27ac and H3K4me1 differential enhancer peaks. To further study the impact of Foxa2 or Foxo1, we conditionally deleted each of these using PgR-cre. There were far fewer genes impacted by deletion of either Foxa2 or Foxo1 suggesting these Fox genes are not responsible for DES induced gene expression and these two lines were not followed further. Taken together, these data suggest estrogen regulates gene expression in the neonatal mouse uterus by H3K27ac association at Esr1 binding sites near estrogen regulated genes. Changes in the epigenome during the time of treatment may contribute to the permanent alterations in gene expression observed in aged DES treated mice.

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Developmental Exposure to DES Alters Uterine Gene Expression That Maybe Associated with Neoplasia Later in Life

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