DNA Methylation and Transcriptome Aberrations Mediated by Erα In

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DNA methylation and transcriptome aberrations PNAS PLUS mediated by ERα in mouse seminal vesicles following developmental DES exposure

Yin Lia, Katherine J. Hamiltona, Tianyuan Wangb, Laurel A. Coonsa, Wendy N. Jeffersona, Ruifang Lic, Yu Wangd, Sara A. Grimmb, J. Tyler Ramseya, Liwen Liue, Kevin E. Gerrishe, Carmen J. Williamsa, Paul A. Wadec, and Kenneth S. Koracha,1

aReproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, ResearchTriangle Park, NC 27709; bIntegrative Bioinformatics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; cThe Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; dPathology Core Facility, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; and eMolecular Genomics Core Facility, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709

Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved March 9, 2018 (received for review November 2, 2017)

Early transient developmental exposure to an endocrine active historically prescribed to pregnant women to prevent miscarriage compound, diethylstilbestrol (DES), a synthetic estrogen, causes (8, 9). Usage was suspended, however, when it was determined that late-stage effects in the reproductive tract of adult mice. Estrogen maternal exposure caused vaginal and breast tumors in young receptor alpha (ERα) plays a role in mediating these developmen- women (10, 11). Several pathologies associated with developmental tal effects. However, the developmental mechanism is not well exposure to DES in humans have been replicated experimentally in known in male tissues. Here, we present genome-wide transcrip- mice (12, 13). A mouse model of neonatal DES exposure was tome and DNA methylation profiling of the seminal vesicles (SVs) widely used to study the effects of EDCs on the reproductive organs during normal development and after DES exposure. ERα mediates with emphasis on critical developmental periods (14). aberrations of the mRNA transcriptome in SVs of adult mice fol- ER-knockout (ERKO) mouse studies demonstrate that ERα lowing neonatal DES exposure. This developmental exposure im- plays a critical role in mediating the toxicological effects of neo- CELL BIOLOGY pacts differential diseases between male (SVs) and female (uterus) natal DES exposure in female and male reproductive organs (15, tissues when mice reach adulthood due to most DES-altered genes 16). Some of the DES toxicity effects result in adult onset atrophy that appear to be tissue specific during mouse development. Cer- of the seminal vesicles (SVs) and aberrant gene expression (15). tain estrogen-responsive gene changes in SVs are cell-type spe- Recently, we reported that neonatal DES exposure induces SV α cific. DNA methylation dynamically changes during development toxicity in adult mice and it is primarily mediated through ER , in the SVs of wild-type (WT) and ERα-knockout (αERKO) mice, which alters gene expression of Svs4 (seminal vesicle secretory which increases both the loss and gain of differentially methylated protein IV) and causes aberrant expression of a uterine protein Ltf regions (DMRs). There are more gains of DMRs in αERKO com- (lactoferrin) (17). DNA methylation is a well-characterized epigenetic modification and is important for gene regulation, tran- pared with WT. Interestingly, the methylation changes between – the two genotypes are in different genomic loci. Additionally, the scriptional silencing, development, and tumorigenesis (18 20). Our expression levels of a subset of DES-altered genes are associated with their DNA methylation status following developmental DES Significance exposure. Taken together, these findings provide an important basis for understanding the molecular and cellular mechanism of Early developmental exposure to endocrine active compounds endocrine-disrupting chemicals (EDCs), such as DES, during devel- causes late-stage effects and alterations in the reproductive opment in the male mouse tissues. This unique evidence contrib- tract of adult mice. Unexpectedly, estrogen receptor alpha utes to our understanding of developmental actions of EDCs in (ERα) plays a pivotal role in mediating these developmental human health. effects. As a model outcome from these developmental effects, we present transcriptome and DNA methylation profiling of estrogen receptor α | transcriptome | DNA methylation | mouse seminal the seminal vesicles (SVs) following neonatal diethylstilbestrol vesicle | neonatal DES exposure (DES) exposure. ERα mediates transcriptome aberrations in SVs of adult mice that impact developmental reprogramming at ormal development and function of reproductive tract or- adulthood. DNA methylation dynamically changes during devel- Ngans are dependent on a highly sensitive and appropriate opment, and methylation is greater in ERα knockout mice com- response to hormone signaling (1). Developmental exposure to pared with wild type. Expression levels of DES-altered genes are estrogenic and antiandrogenic endocrine disrupting chemicals associated with their DNA methylation status. These findings (EDCs) is associated with reproductive dysfunctions in adulthood provide unique evidence for understanding the developmental actions and mechanisms of endocrine-disrupting chemicals in (2). EDCs include natural hormones, synthetic estrogens, phytoes- human health. trogens, plasticizers, and pesticides whose activities are thought to

be mediated through the estrogen receptors (ERs) (3). ERs, in- Author contributions: Y.L., C.J.W., P.A.W., and K.S.K. designed research; Y.L., K.J.H., cluding ERα and ERβ, are members of a large superfamily of nu- W.N.J., R.L., Y.W., J.T.R., and K.E.G. performed research; Y.L., T.W., L.A.C., S.A.G., L.L., clear receptors and can act as ligand-inducible transcription factors K.E.G., and P.A.W. analyzed data; and Y.L. and K.S.K. wrote the paper. (TFs) (4). The main mechanism involves ER directly bound to The authors declare no conflict of interest. DNA estrogen-response elements (EREs) of target genes to reg- This article is a PNAS Direct Submission. ulate gene expression (5, 6). EDC’s activities have been shown to be Published under the PNAS license. mainly through the ERs to regulate many ER-dependent genes (7). 1To whom correspondence should be addressed. Email: [email protected]. Developmental origins of adult reproductive disease are associ- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ated with EDC exposure in both human and animal studies (2). 1073/pnas.1719010115/-/DCSupplemental. Diethylstilbestrol (DES) is a potent synthetic estrogen which was

www.pnas.org/cgi/doi/10.1073/pnas.1719010115 PNAS Latest Articles | 1of10 Downloaded by guest on September 28, 2021 studies indicated that DES alters the DNA methylation status in histology with the vehicle sample as shown in Fig. 1C.Toexamine certain CpGs of the Svs4 and Ltf gene promoters and the meth- the effects of neonatal DES exposure on the Esr1 (encodes ERα ylation status correlates with the levels of gene expression (21). protein) and Ar [encodes androgen receptor (AR) protein] gene Based on our previous findings, we hypothesized that neonatal expression during development, we performed qPCR analysis in DES exposure alters the transcriptome and DNA methylation both WT and αERKO male mice at ages 3, 5, and 10 wk treated profiles in the mouse SVs and ERα plays a role in these alterations. with either vehicle or DES. We found an increase of Esr1 and a To test our hypothesis, we profiled genome-wide transcriptome lower amount of Ar in the WT SV during development. However, and DNA methylation of the wild-type (WT) and ERα-knockout there was no significant alteration observed in either Esr1 or Ar (αERKO) mouse SVs following neonatal DES exposure. We per- expression following DES exposure (Fig. 1D). As expected, Esr1 was formed similar experiments on uterine samples as a comparison for absent in all αERKO samples. These observations indicate that α tissue specificity. We investigated the changes of DNA methylation ERKO mice exhibit resistance to the developmental effects of DES caused by DES exposure during development in the mouse SVs. exposure on mouse SVs during development (before adulthood). Furthermore, we explored the association of DES-altered tran- α scriptome expression and DNA methylation status in the SVs of ER -Mediated Aberration of the Transcriptome in the SV of Adult adult mice. Mice After Neonatal DES Exposure. To investigate the role of ERα in mediating DES-altered SV gene expression, we profiled Results genome-wide transcriptome of adult mouse (week 10) SVs in the WT and αERKO using RNA-sequencing (RNA-Seq) and micro- αERKO Mice Are Resistant to the Developmental Effects of DES in the array analyses. For RNA-Seq, differentially expressed (DE) genes Mouse SV. Our previous studies showed that neonatal DES ex- ± α were analyzed by examining fold changes with a 1.5-fold cutoff posure reduced SV weight of WT adult mice and ERKO mice (Fig. 2A). As for DES’s effect on the WT SVs, a total of 2,162 DE exhibited resistance to this developmental effect (15). To explore α genes were found (Fig. 2A, comparison 1). In contrast, only 20 DE the role of ER in the mouse SV during development, we col- genes were found in DES-treated αERKO SVs (Fig. 2A, com- α lected the SVs from WT and ERKO mice at 3 wk (before pu- parison 2). As for ERα’s effect, 116 DE genes were found between berty), 5 wk (puberty), and 10 wk (adult) after neonatal DES the WT vehicle and αERKO vehicle SVs (Fig. 2A, comparison 3). exposure (Fig. 1A). In the WT SVs, neonatal DES exposure re- When comparing the WT vehicle to the αERKO DES-treated duced SV weight by over 60% during development compared with SVs, 121 DE genes were found (Fig. 2A, comparison 4). Addi- the vehicle (veh) at all three time points (Fig. 1B). In contrast, this tionally, a total of 2,442 DE genes were identified when comparing reduction in SV weight did not occur in αERKO mice after DES the WT DES and αERKO DES samples and 2,598 genes were exposure (Fig. 1B). We also found that neonatal DES exposure identified in the comparison between WT DES and αERKO veh resulted in significant histological alterations at week 5 in the SV (SI Appendix,Fig.S1A). When we compared the expression of WT males but not in αERKO (Fig. 1C). The histology of WT heatmap of 2,162 (1,678 induced and 484 repressed) DE genes, SV DES samples showed a significant increase in thickness of the DES-altered genes were only in the WT SVs but were not seen in smooth muscle layer. In contrast, αERKO SV DES had similar αERKO SVs (Fig. 2B).

Fig. 1. Experimental designs and changes in SV weight and gene expression during development after neonatal exposure. (A) Timeline for neonatal treatment (corn oil vehicle or DES, 2 μg per day) and collection of tissues. (B) SV weights of male WT and αERKO mice (intact) for the three time points after neonatal treatment on days 1–5 with vehicle (veh) or DES. The mouse number in each group was n = 10– 15. WT-veh was compared at each time point by two-way ANOVA using a Dunnett’s multiple comparison test, ***P < 0.001. (C) Pathology of SVs of male WT and αERKO mice at week 5 (veh or DES- treated samples) stained with hematoxylin and eo- sin (magnification, 10x). (D) The expression levels of Esr1 and Ar genes in SVs of WT and αERKO mice. Total RNA samples were extracted from frozen SV tissues of three individual mice for WT-veh, αERKO- veh, and αERKO-DES, or three pools (n = 5–8) for WT- DES samples. The expression levels were quantified by qPCR and normalized against the 18S housekeeping gene. Data shown represent mean fold change (±SEM) relative to SVs from week 3 WT-veh. *P < 0.05, ****P < 0.0001 by two-way ANOVA using a Tukey’s multiple comparison test.

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Fig. 2. ERα-mediated aberrations of the transcriptome in the SVs of adult mice following developmental DES exposure. (A) Differentially expressed (DE) gene analysis for week 10 SVs RNA-Seq (n = 4 for each group). DE genes from the four comparisons are shown as fold change with a cutoff of >1.5-fold, fragments per kilobase million (FPKM) >1 and q < 0.05. (B) Heatmap depicting 2,162 DE genes from comparison 1 WT-DES vs. WT-veh (1,678 induced and 484 reduced genes). (C) IPA analysis of 1,678 induced or 484 reduced genes.

To understand the biological function underlying the dramatic works associated with metabolic disease and cellular function and gene changes by DES exposure during development in the WT maintenance (Fig. 2C, Right). In addition, we observed a similar SVs, ingenuity pathway analysis (IPA) was performed for both transcriptome with the same samples using GeneChip, a micro- DES-induced and -repressed genes. As for the 1,678 induced array analysis. Around 68% of DE genes overlapped between genes, they appeared to be related to immunological, inflam- RNA-Seq and GeneChip data, suggesting that the two analyses matory, and hematological diseases. There were 750 genes re- were comparable. Using chromosome view analysis, we found that lated to the hepatic system and 585 genes related to lymphoid those DE genes were distributed throughout all chromosomes (SI tissue structure developmental function (Fig. 2C, Left). The top Appendix, Fig. S1B). These findings indicate that ERα mediates upstream regulators were lipopolysaccharide, TNF, IFNG, IL4, aberrations of the transcriptome in the SV of adult mice following and IL1B and the pathway networks were associated with en- DES exposure and many of the DES-altered genes are related to docrine system disorders, cell death, and developmental disor- developmental functions and diseases. ders (Fig. 2C, Left). As for the 484 repressed genes, they appeared to be related to developmental disorders and metabolic disease. Comparison of Altered Genes in the SV and Uterus of Adult Mice After There were 50 genes related to organ morphology and 35 genes Neonatal DES Exposure. DES-induced toxicity in the mouse uterus related to organismal developmental function. Interestingly, 18 has been well documented and many estrogen-response genes DES-repressed genes were related to reproductive system de- have been identified (22, 23). To explore whether DES exposure velopmental function (Fig. 2C, Right). Some major upstream results in similar or tissue-specific gene expression differences, regulators were XBP1, AR, and androgen, through pathway net- we performed RNA-Seq analysis of the uterus of adult mice

Li et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 28, 2021 following the same neonatal DES exposure as used for the SV tween male and female tissues and this is due to a set of DES-altered study and then compared DES-altered gene profiles between the genes that appears tissue-specific during mouse development. two tissues. In a comparison of WT vehicle and WT DES-treated samples, 425 DE genes were found in the adult uterus (Fig. 3A, Differential Alteration of Estrogen-Responsive Genes During Development Left). When overlapping this gene profile with the 2,162 SV DE in the Mouse SV Following Neonatal DES Exposure. Our previous studies gene profile, we found that 155 DES-altered genes were shared showed that ERα played a role in mediating the aberrant Ltf (an in the two tissues (SI Appendix, Table S2). Interestingly, 15 well- estrogen-response gene) expression in the adult SV following DES known estrogen-response genes were found as listed in Fig. 3A. exposure (17, 21). To investigate the specificity of the effects of Ingenuity pathway analysis showed the differences of the top five DES exposure on estrogen-responsive genes during development in diseases and disorders occurring between SV and uterine genes the SVs, we selected eight well-known estrogen-responsive genes following DES exposure (Fig. 3A, Right). Of note, 111 overlapping found in the mouse uterus (24) from the 155 overlapping genes and genes (out of 155) were related to reproductive system diseases. performed qPCR analysis at the three developmental stages, in- To determine whether DES may have differential effects on cluding week 3 (before puberty), week 5 (puberty), and week 10 shared genes in the adult mouse SV and uterine tissues, we per- (adult). Ltf and C3 are normally expressed in female tissues and formed gene pattern analysis for the 155 overlapping genes. As therefore both basal levels were lower or nondetectable in WT shown in Fig. 3B, six patterns with a varying number of genes were vehicle and αERKO vehicle SVs (Fig. 4A). However, DES strongly identified. Most genes belong to pattern 2 and pattern 3. Pattern induced Ltf and C3 expression in WT SVs compared with vehicle at 2 showed genes that were induced in both SV and uterus and in- all time points, and increasing levels were observed as the mouse clude genes known to be altered following DES, such as Ltf, Ccna2, aged. In contrast, there was no induction observed in the αERKO and Six1. This pattern also showed gene expression that could SVs following DES exposure (Fig. 4A). DES exposure weakly in- contribute to feminization in the male with genes being expressed in duced the expression levels of Six1 and Padi4 from the early de- the SV after DES exposure at a similar level as in the control uterus. velopmental stages (weeks 3 and 5) and levels were significantly Interestingly, in pattern 3, the well-known estrogen-response genes, increased when mice reached adulthood (week 10) (Fig. 4A). Igfbp3 Dcn, Igfbp3, Inhbb,andTgfbi showed differential changes in the SV and Krt15 had a dynamic change during development (Fig. 4A). and uterus, which were induced in the SV but repressed in the uterus Interestingly, Wnt4 and Krt5 had expression pattern changes asso- after DES exposure. These findings suggest that DES exposure ciated with the developmental stage when mice reached adulthood impacts the development of differential pathological changes be- (Fig. 4A). These data suggest that further hormonal regulation at

Fig. 3. Comparison of altered genes in the SVs and uterus of adult mice following neonatal DES exposure. (A) Venn diagram of the overlapping between DES-altered 2,162 SV and 425 uterus DE genes (Left). IPA analysis for the 2,007 SV, 270 uterine, and 155 overlapping DE genes (Right). (B) Gene pattern analysis for 155 overlapping genes using EPIG with the expression value of FPKM. See also SI Appendix, Table S1.

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Fig. 4. Differential alterations of estrogen-response genes during development in the mouse SV following neonatal DES exposure. (A) The qPCR data for developmental changes. Total RNA samples were extracted from SV frozen tissues of three individual mice for WT-veh, αERKO-veh, and αERKO-DES, or three pools (n = 5–8) for WT-DES samples. The expression levels of Ltf, C3, Six1, Padi4, Igfbp, Krt15, Wnt4, and Krt5 were quantified by qPCR and normalized to the 18S housekeeping gene. Data shown represent mean fold change (±SEM) relative to SVs from week 3 WT-veh, ***P < 0.001, ****P < 0.0001 by two-way ANOVA using a Dunnett’s multiple comparison test. (B) The qPCR data for analyzing cell-type specificity in gene changes. Total RNA was isolated from the epithelial and stromal cells of whole SV tissues in WT adult mice using LCM as described in Materials and Methods. The quantification of RNA was shown in SI Appendix, Fig. S2. The expression levels were quantified as above. Data shown represent mean fold change (±SEM) relative to SVs from the epithelial cells (Epi) or stromal cells (Strom) WT-veh, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA using a Dunnett’s multiple comparison test.

Li et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 28, 2021 the different developmental stages could affect expression levels of enough to change the methylation status at many locations (both estrogen-responsive genes in the SV following DES exposure. loss and gain of methylation), suggesting ERα may be important To examine the cell type specificity in changes of estrogen- for developmentally setting the DNA methylation status in the responsive genes, we used laser capture microdissection (LCM) to SV. In addition, we found more gains of DMRs at week 5 (20,331) isolate the epithelial and stromal cells from whole SV adult tissues with both DES treatment and the absence of ERα compared with (Fig. 4B, Left). The qPCR results showed that DES exposure WT SVs, suggesting ERα may protect certain regions or these are significantly induced the expression levels of Ltf, Six1,andKrt5 sensitive to ERα presence (Fig. 5A,comparison4). genes in epithelial cells, but there was no change in stromal cells. Next, we investigated distribution of DMRs in the genome and C3 gene expression was induced in both cell types in the DES- found that the seven types of genomic regions were comparable treated samples. However, an induction of Igf-1 expression was between weeks 5 and 10 in the WT and αERKO SVs (Fig. 5B). only observed in SV stroma (Fig. 4B, Right). These results indicate In the WT loss of DMRs, there was a developmental switch from that certain gene changes in the mouse SVs following DES ex- a reduction in the introns to an induction in the intergenic re- posure appear to be cell-type specific. gions. However, it was opposite in the WT gains of DMRs (Fig. 5B, Top). In contrast, there was no switch in the mouse genome DNA Methylation Reprogrammed in the Mouse SV After DES Exposure. with either loss or gain of DMRs in the αERKO SVs (Fig. 5B, Based on our previous findings that DNA methylation status in Bottom). The data suggest that a difference in DNA methylation certain CpGs of the Svs4 and Ltf gene promoters associated with pattern may occur during development in the mouse SVs that is thelevelsofgeneexpressionintheadultmouseSVs(21),wehy- governed by the presence of ERα. pothesized that altered DNA methylation may play a role during development in the mouse SVs following DES exposure. To test Comparison of DNA Methylation Changes During Development in the this hypothesis, we performed methyl-CpG binding domain capture WT and αERKO SVs After Neonatal DES Exposure. To determine the sequencing (MBD-Seq) analysis in the SVs of WT and αERKO DNA methylation changes in the two developmental stages, we mice at the two later developmental stages, puberty (week 5) and overlapped the loss or gain of DMRs between week 5 and week adult (week 10). Targeted bisulfite-Seq (TBS-Seq) analysis was used 10 (Fig. 6A). We only found a small percentage of overlapping to validate the MBD-Seq data. A set of well-known estrogen- DMRs in all four comparisons, suggesting that there was a de- responsive genes (24) was selected for testing and the results of velopmental change in DNA methylation in the mouse SV following seven genes are shown in SI Appendix,Fig.S3. Differential DNA DES exposure. In WT, only a small percentage of the overlapping methylation regions (DMRs) were identified for each pair of sam- DMRs in both losses (408) and gains (269) were obtained between ples and summarized in Fig. 5A. We found that DES increased both weeks 5 and 10. In contrast, the overlapping gains of methylation the loss and gain of DNA methylation between week 5 and week DMRs in αERKO (5,264) was far above the number of losses (205), 10 in WT and αERKO SVs. Of note, there were many more gains suggesting that ERα is playing a more significant role in addition to of DMRs in αERKO DES vs. vehicle (16,632) compared with WT methylation than demethylation during development. To investigate DES vs. vehicle (5,236) at week 5, suggesting the absence of ERα whether ERα has a role in DNA methylation reprogramming by may have altered the methylation status in general (Fig. 5A, neonatal DES exposure in the adult SVs, we compared both loss comparisons 1 and 2). To further investigate the role of ERα,we and gain of DMRs for the WT and αERKO SVs at week 10 and compared the loss and gain of DMRs between αERKO and WT found that only a small number of DMRs overlapped between vehicle SVs (Fig. 5A, comparison 3). Lack of ERα alone was the two genotypes (Fig. 6B), suggesting that DNA methylation

Fig. 5. DNA methylation dynamics during development in the mouse SVs following neonatal DES exposure. (A) Differentially methylated regions (DMRs) analysis for week 5 and week 10 SV MBD-Seq using MEDIPS. The numbers of loss or gain of methylation DMRs are shown with the four comparisons. (B) Distribution of DMRs in the mouse genome (mm10). The seven genomic regions are compared between weeks 5 and 10 in WT or αERKO samples.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1719010115 Li et al. Downloaded by guest on September 28, 2021 PNAS PLUS CELL BIOLOGY Fig. 6. Comparison of DMRs and the enriched motifs in the WT and αERKO SVs following developmental DES exposure. (A) Venn diagrams depict the overlap between week 5 and week 10 DMRs with the loss and gain of methylation in four comparisons. (B) Venn diagrams depict the overlap between week 10 WT and αERKO SVs with the loss or gain of methylation DMRs. (C) Venn diagrams depict the overlap of motifs between WT and αERKO DMRs with the loss or gain, or WT loss and αERKO gain of DNA methylation.

dynamically changes in different loci of the mouse genome be- Currently, our group has reported that functionality of ERα in tween the WT and αERKO SVs. the binding sites of target genes is restricted to EREs, which vary To explore the molecular mechanisms that differentiate DNA from the consensus palindromic element by one or two nucleo- methylation patterns in WT and αERKO SVs, we performed TF tides (24). To identify ERα ERE-mediated genes in the adult motif analysis for the loss and gain of DMRs. The results in- mouse SV after developmental DES exposure, we searched for dicated 64 enriched motifs in WT losses, 40 in αERKO losses, consensus ERE sites (ERE, GGTCAnnnTGACC or 1-nt muta- 52 in WT gains, and 64 in αERKO gains (Fig. 6C). When tion) in DES-altered genes that contained DMRs within a dis- overlapping the motifs of WT and αERKO, we found that there tance of ±100 kb of the TSS. After integrating the three datasets, were more gain motifs shared than losses in the two genotypes. including DE genes, DMRs, and EREs (SI Appendix, Fig. S4, Interestingly, there were over 70% overlapping motifs in WT Left), we found 29 DES-induced genes with a loss in methylation losses and αERKO gains (Fig. 6C). These results suggest that and 25 DES-repressed genes with a gain in methylation that α contained EREs (SI Appendix, Fig. S4, Right). To determine lack of ER may cause more methylation, resulting in gene si- α lencing in the adult SVs after DES exposure. whether ER had direct interactions with the ERE binding site after DES exposure, as an example, we selected Epha2 (a DES- The Correlation of DES-Altered Gene Expression and DNA Methylation induced and loss-of-methylation gene) which contained an ERE site in the promoter region for further analysis (SI Appendix, Fig. Status in the Adult Mouse SV. To further understand the re- S5). MBD-Seq data showed that DES caused the loss of DNA lationship between gene expression and DNA methylation, we methylation at week 10 in the two regions of Epha2 and this mapped the week 10 MBD-Seq peaks to the location of DES- ± status was correlated with the gene expression by RNA-Seq and altered genes in the mouse genome within 100 kb of the qPCR analyses. Using ChIP-qPCR analysis, ERα and Pol II transcriptional start site (TSS). When the 1,678 DES-induced enrichments were significantly increased in an Epha2-ERE re- genes were checked against the 11,271 loss-of-methylation gion in DES samples, which was expected. These results indicate DMRs, 855 DES-induced genes were associated with 1,582 that DNA methylation status, as exemplified with Epha2, cor- DMR losses (Fig. 7A, Left). When the 484 DES-repressed genes relates with gene expression and ERα can directly bind to an were checked against 19,381 gain of DMRs, 365 DES-repressed ERE of Epha2 in the SVs of adult mice, which is made accessible genes were associated with 1,170 DMR gains (Fig. 7A, Right). after neonatal DES exposure. These findings demonstrate that DNA methylation status is as- To further explore the molecular mechanisms that differenti- sociated with the expression levels of a subset of altered genes in ate possible DES-altered gene expression via DNA methylation, the SVs of adult mice following neonatal DES exposure. we performed the TF motif analysis for a subset of gene-mapped

Li et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 28, 2021 Fig. 7. Correlation of gene expression and DNA methylation status in the adult mouse SVs following developmental DES exposure. (A) Data integration of DES-altered DE genes and DMRs which are mapped to the closest gene TSS ± 100 kb. (B) The top enriched motifs in 1,582 loss or 1,170 gain of methylation DMRs. See also SI Appendix, Tables S2–S4.(C) UCSC Genome Browser screen shots represent tracks of RNA-Seq, MBD-Seq, and TBS-Seq analysis for DES induced with the loss DMR gene (Mmp8) and DES repressed with the gain DMR gene (Shisa4).

loss and gain of DMRs. The results induced 25 enriched motifs Discussion from the known motif analysis, 30 motifs from the de novo In this study, we examined the developmental effects related to motif analysis in the loss of DMRs (SI Appendix,TablesS3and toxicity in the mouse SVs following synthetic estrogen DES expo- S4), four motifs from the known motif analysis, and nine motifs sure. Our data uncovered that the associated tissue effects appear from the de novo motif analysis in the 1,170 gain-of-methylation linked to alterations in the mRNA transcriptome and DNA DMRs (SI Appendix, Table S5). HRE, E2A, FOXP1, MafA, methylation profile in the mouse SVs following neonatal DES ex- CRX, Foxo1, STAT6, and Smad3 motifs were enriched in the posure. Following a series of analyses, we concluded that ERα- losses (Fig. 7B, Left) and HIF-1a, MYB, GATA3, Nrf2, Nkx2, mediated aberrations of the transcriptome and many of the altered Mycn, IRF4, and TFAP2A motifs were enriched in the gains genes were related to and enriched for developmental functions (Fig. 7B, Right). Interestingly, AR half-site motifs were identi- and potential diseases in the adult SVs when male mice were ex- fied in both the gain and loss lists. This suggests that differential posed to DES in the early stages of development. Interestingly, we androgen signaling could occur from the DES treatment in WT found that a set of estrogen-responsive uterine genes were also SVs and play a significant role in altered gene expression related aberrantly expressed in the SV of adult male mice following DES to the toxicity. In addition, associations between expression and exposure. DNA methylation was reprogrammed dynamically during DNA methylation status for the two genes, Mmp8 (DES-induced development in the mouse SVs following DES exposure. The and loss methylation) and Shisa4 (DES-repressed and gain changes occurred in different loci of the mouse genome between methylation) are shown in Fig. 7C. Using TBS-Seq, we con- the WT and αERKO SVs. The expression levels of about 60% of firmed the loss-of-methylation status of Mmp8 in the two regions DES-altered genes were associated with the corresponding DNA from the two MBD peaks (7 CpGs in chr9: 7,560,386–7,560,591; methylation status. 4 CpGs in chr9: 7,567,566–7,567,676) (Fig. 7C, Left) and the Exposure to EDCs during development can alter susceptibility gain-of-methylation status of Shisa4 in a region from a MBD to adult diseases later in life, including many diseases related to peak (16 CpGs in chr1: 135,372,809–135,373,184) (Fig. 7C, the male and female reproductive tracts affecting fertility (25– Right). Genes that have an association between expression and 27). The toxicity of EDCs, such as DES or genistein exposure in DNA methylation status are listed in Fig. 7C, Bottom. These female mice, has been well documented as a major contributor to results suggest that DNA methylation correlates with and regu- uterine cancer when mice reached adulthood (23, 28–30). In lates both induced and reduced gene expression with differen- agreement, we showed that cancer was the number one disease tially enriched motifs in the adult SV following DES exposure. association following DES exposurefromtheuterinegeneprofiling

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1719010115 Li et al. Downloaded by guest on September 28, 2021 analysis in this study. We also provided the natural developmental unmethylated by DES exposure in the adult mouse SVs. These PNAS PLUS transcriptome expression in the mouse SVs following neonatal DES results suggest that DES exposure alters the DNA methylation exposure, which is a unique transcriptome profile in this mouse re- pattern with a sex and/or strain specificity during development productive organ. The observed phenotype difference between WT- that impacts different CpG loci. veh and WT-DES SV during development could be due to upstream DNA methylation was traditionally known as a regulator in regulators such as cytokines (TNF, IL4, and IL1B), kinases (EGFR, gene silencing, but recent studies have shown a more complex IKBKB, JAK, and MAPKs), and many transcription regulators that picture. More recently a study showed that DNA methylation can become activated. Many of these regulate cell death, differentiation, also have different outcomes, including activation of transcription and proliferation, which are vital in the growth of cells and tissue. (40). The direct relationship between DNA methylation and gene The repression of organ and tissue morphology genes such as Ggt1, expression remains unclear (41). The association between DNA Lep,andNr4A3, and feminization protein GPRC6A are critical methylation and gene expression has been proposed to be either for cell adhesion (31). More interestingly, the 111 overlapping active or passive, and can depend on the context in which they DES-altered genes (out of 155) between uterus and SVs are re- occur in the genome (42). Here we found that the expression lated to reproductive tract diseases. These findings provide unique levels of genes, around 50% of DES-induced and 75% of DES- mechanistic information to understand the differential toxicities in repressed genes, were correlated with their DNA methylation males and females following EDC exposure as shown with DES. status. These findings suggest that DNA methylation is involved in The Ltf gene, which is highly expressed in the female reproduc- both DES-induced and -repressed gene expression in the adult tive tract, was used as a marker for the early hormonal response in mouse SVs after neonatal DES exposure. Evidence has indicated the developing mouse uterus (32, 33). Normally, Ltf is not expressed that transcription factors can interact with methylated DNA se- in adult mouse SVs (17). However, neonatal DES exposure sig- quences (40). In our analysis, we also found the sets of DES-induced nificantly induces the basal levels of this gene’s expression and al- genes with a gain of DMRs or repressed genes with the loss of tered programming in the adult mouse SVs (15, 17). We DMRs (SI Appendix,Fig.S6A). Further analysis of those DMRs demonstrated that neonatal DES exposure induced the Ltf gene and genes will be needed for understanding the role of DNA expression continually in the SVs from week 3 to week 10. How- methylation in gene regulation. Additionally, gene silencing ever, it did not occur in the adult mouse uterus, indicating a dif- may have a major impact that inhibits a required gene expres- ferential tissue selective reprogramming of this locus from the sion during development in the mouse SVs following neonatal developmental DES exposure. The Wnt family proteins as secreted DES exposure. Studies looking at natural DNA methylation ligands can act through many receptors to stimulate signaling variations in human populations have shown that genetic vari-

pathways. These proteins have been linked to oncogenesis and ations influence DNA methylation levels in different tissue/cell CELL BIOLOGY developmental processes in the female reproductive tract (34–36). types (43, 44). Additionally, epigenetic regulation of gene ex- Here, we report that the Wnt4 gene is highly expressed in the early pression and the association of specific histone marks with SV developmental stage and has reduced expression immediately promotor sequence classes are fine tuned in a cell type-specific following puberty. Interestingly, this gene was only induced in the manner (45). To have a better understanding of the epigenetic SVs of WT adult mice and not the αERKO. These findings suggest mechanism of DES-induced toxicity in the mouse SVs, a that estrogen-responsive genes regulated by ERα action, such as Ltf genome-wide analysis of DNA methylation in a single cell type and Wnt4, contribute to hormonal regulation during development or analysis of histone marks should be performed in future in a tissue-specific manner; therefore, alterations in their expression studies. after neonatal DES exposure could result in feminization of the SV. Epigenetic modifications, including DNA methylation and his- Conclusions tone modification, play important roles in regulating cellular dif- Our findings demonstrate that neonatal DES exposure causes ferentiation events. In mammalian cells, DNA methylation occurs toxicity in the mouse SV from early developmental stages through at cytosine, predominantly in CpG dinucleotide contexts (37, 38). α ’ adulthood in mice and many developmental genes are altered. ER Here, we identified the DMR s response to DES in the WT and appeared to play a role in mediating the aberrant transcriptome αERKO SVs and found that there were more DNA methylation α expression induced by DES. Interestingly, we found that a set changes (gain or loss) in the ERKO than in the WT SVs after of estrogen-responsive uterine genes were also now aberrantly neonatal DES exposure. Neonatal DES exposure increases the α expressed in the SV of adult male mice following DES exposure and level of testosterone in the adult ERKO compared with WT mice certain gene changes appeared to be cell-type specific. DES expo- (17). The dynamic DNA methylation changes suggest that there is a sure reprogrammed DNA methylation dynamically during devel- possibility of other hormones, such as testosterone, involved in the opment in the mouse SV. These observations were seen in different DNA methylation changes. Interestingly, when we mapped the α α loci of the mouse genome when considering both WT and ERKO week 10 ERKO gained DMRs to the genes within 10 kb, we SVs. Furthermore, DNA methylation status associated with the discovered that about 25% of DES-induced genes (423 of 1,678 ∼ α expression levels of 60% of DES-altered genes in the mouse SV. genes) in the WT SVs overlapped with ERKO gained DMR genes These findings provide unique evidence for understanding molecu- (SI Appendix,Fig.S6). This could be an explanation as to why some α lar and epigenetic mechanistic consequences in human health fol- of the genes were silenced in the ERKO due to the DNA meth- lowing developmental EDC exposure. ylation status. EDCs act primarily through nuclear receptors, including ER and AR (27). It is expected that different disorders are Materials and Methods seen in males and females as a result of EDC effects that mimic Animal and Neonatal Treatments. All animal studies were conducted in ac- estrogens and/or antagonize androgens (27). In agreement, we found cordance with the NIH Guide for the Care and Use of Laboratory Animals that AR and androgen as the upstream regulators were inhibited in (47). The full protocol can be found in SI Appendix, Materials and Methods. the SVs following DES exposure. A balance between ER and AR regulation and signaling during development could impact the effect DNA Extraction, MBD-Seq, Mapping, and Analysis. Genomic DNA samples were of EDCs in male reproductive organs. extracted from pooled frozen tissues of each group using a Tissue Blood Kit An original DNA methylation study indicated that prenatal DES (Qiagen) according to the manufacturer’s protocol. Pooled genomic DNA samples exposure caused demethylation of three specific CpG sites from a were sonicated with Biorupter (Diogenode) and methylated DNAs were captured methylated status in the Ltf gene promoter in 3-wk-old CD-1 mouse with his-tagged recombinant MBD2b along with its binding partner MBD3L1 uteri (39). We found that the same three CpG sites that were using the MethyCollector Ultra Kit (cat. no. 55005, Active Motif). The full protocol unmethylated in the adult mouse SVs by neonatal DES exposure can be found in SI Appendix, Materials and Methods. were not changed in the 3-wk-old C57BL/6 mouse SVs, which remained in the methylated status (21). In this study, we TBS-Seq, Mapping, and Analysis. Bisulfite conversion sequencing PCR primers identified a CpG region in the intron of the Ltf gene that was were designed using the software program EpiDesigner (www.epidesigner.

Li et al. PNAS Latest Articles | 9of10 Downloaded by guest on September 28, 2021 com/). Bisulfite conversion sequencing PCR was performed using the EZ DNA ACKNOWLEDGMENTS. We thank the members of the K.S.K. laboratory for Methylation-Gold Kit (Zymo Research). The full protocol can be found in SI discussions; Drs. Mitch Eddy and Harriet Kinyamu for critical review of the Appendix, Materials and Methods. manuscript; the National Institute of Environmental Health Sciences (NIEHS)/ NIH Comparative Medicine Branch for supporting the animal study; the Pa- The materials and methods for H&E staining; RNA extraction and qPCR; thology Core facility for the LCM study; the Epigenetic Core facility for pro- microarray (GeneChip) and data analysis; RNA-Seq, mapping, and analysis; viding MBD-Seq and TBS-Seq;, and the NIH Intramural Sequencing Center for laser capture microdissection (LCM); ChIP-qPCR analysis; pattern analysis; motif providing RNA-Seq data. Research funding was provided by the Intramural analysis; ERE motifs contained within peaks; and Statistical analysis can be Research Division of the NIEHS through 1ZIAES70065 (to K.S.K.) and found in SI Appendix, Materials and Methods. 1ZIAES102985 (to C.J.W.).

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