Developmental reprogramming of myometrial stem cells by endocrine disruptor linking to risk of uterine fibroids

Abstract

Background: The stage, when tissues and organs are growing, is very vulnerable to environmental influences, but it's not clear how exposure during this time causes changes to the epigenome and increases the risk of hormone-related illnesses like uterine fibroids (UFs).

Methods: Developmental reprogramming of myometrial stem cells (MMSCs), the putative origin from which UFs originate, was investigated in vitro and in the Eker rat model by RNA-seq, ChIP-seq, RRBS, gain/loss of function analysis, and luciferase activity assays.

Results: When exposed to the endocrine-disrupting chemical (EDC) diethylstilbestrol during Eker rat development, MMSCs undergo a reprogramming of their estrogen-responsive transcriptome. The reprogrammed genes in MMSCs are known as estrogen-responsive genes (ERGs) and are activated by mixed lineage leukemia protein-1 (MLL1) and DNA hypo-methylation mechanisms. Additionally, we observed a notable elevation in the expression of ERGs in MMSCs from Eker rats exposed to natural steroids after developmental exposure to EDC, thereby augmenting estrogen activity.

Conclusion: Our studies identify epigenetic mechanisms of MLL1/DNA hypo-methylation-mediated MMSC reprogramming. EDC exposure epigenetically targets MMSCs and leads to persistent changes in the expression of a subset of ERGs, imparting a hormonal imprint on the ERGs, resulting in a "hyper-estrogenic" phenotype, and increasing the hormone-dependent risk of UFs.

Keywords: DNA hypo-methylation; Developmental reprogramming; Eker rat; Endocrine-disrupting chemicals; Epigenome; Estrogen signaling; Estrogen-responsive genes; Hormonal imprint; Hyper-estrogenic; Leiomyoma; MLL1 activation; Progenitor cells; TASP1.

Fig. 1

Experimental paradigm. Eker rat pups were exposed to VEH and EDC diethylstilbestrol at postnatal days 10-12. The pups were sacrificed at 5 months of age representing the early adult stage. Myometrial tissues were isolated from the animals and subjected to MMSC isolation using Stro-1/CD44 surface markers. Myometrium from five animals was pooled for each treatment. Multi-omics analyses, including RNA-sequencing, ChIP-sequencing, and RRBS, were performed to determine the global alterations of the transcriptome, histone modification, and DNA methylation, respectively. In addition, targeted bisulfite NGS was performed to examine the DNA methylation within CpG islands of genes

Fig. 2

Developmental EDC-induced reprogramming of rat MMSCs genes. a Five animals from each of the EDC diethylstilbestrol and VEH groups were pooled and subjected to MMSC isolation using a FACS strategy. Pie chart showing the percentage of genes that exhibited changes in RNA expression between EDC-MMSCs and VEH-MMSCs as measured by RNA-seq; the cutoff value is twofold with an FDR < 0.05. b List of the top 20 up-DEGs in EDC- vs. VEH-MMSCs. c List of the top 20 down-DEGs in EDC- vs. VEH-MMSCs. d Over-representation analysis showed that multiple UF-related pathways, including the estrogen response pathways (red color) were affected by early-life EDC exposure. e Relevant diseases affected by developmental EDC diethylstilbestrol exposure were analyzed by Ingenuity Pathway Analysis (IPA) and highlighted in red color

Fig. 3

Transcriptional profiling reveals reprogramming of estrogen-responsive gene. a Pie chart showing the percentage of ERGs that exhibited changes in RNA expression in EDC-MMSCs compared to VEH-MMSCs, as measured by RNA-seq. b List of the top 20 up-ERGs, that showed differential expression as measured by RNA-seq in EDC- vs. VEH-MMSCs. c List of the top 20 down-ERGs, that showed differential expression as measured by RNA-seq in EDC- vs. VEH-MMSCs. d ER-a is upregulated in EDC-MMSCs compared to VEH-MMSCs. Lysates were prepared from EDC-MMSCs and VEH-MMSCs and subjected to Western blot analysis using antibody against ER-a. e Luciferase activities in the presence or absence of estrogen (10 nM) were compared between EDC-MMSCs and VEH-MMSCs. f Alteration of ERG expression in the presence or absence of estrogen in DES-MMSCs and VEH-MMSCs. Student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

Developmental exposure to EDC activates MLL1. a Western blotting with anti-MLL1, -H3K4me3, and -Cd9 antibodies was performed to determine the levels of MLL1C (the activated form of MLL1), H3K4me3 and CD9. Total H3 and β-actin were used as loading controls. Biological replicates (n = 3) were used for quantification. b Confocal imaging showing the staining of H3K4me3 in EDC- vs. VEH-MMSCs and quantitative analysis. Three separate coverslips of cells were used for quantification. Student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

EDC exposure disrupted the epigenome in MMSCs. a Pie chart showing the number of ERGs with peaks. b Integration of H3K4me3 with RNA expression of ERGs by EDC exposure. c EDC-regulated ERGs with H3K4me3 peaks. d Histograms from Integrative Genomics Viewer showing H3K4me3 occupancy at Esr-1, Cxcl12, Cd9, Mpped2, and Tgm2 (left panel). For each gene, the upper and the lower browser images display an expanded view of a selected region of the H3K4me3 peak distributions in VEH-MMSCs (blue track) and EDC-MMSCs (red track). Middle panel: directed ChIP-sequencing validation of H3K4me3 target genes by ChIP-qPCR; right panel; RNA-sequencing validation by q-PCR. Student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 6

Knockdown of Tasp1 reversed the reprogrammed ERGs induced by EDC exposure. a Western blot analysis was conducted to determine the role of TASP1 in MLL1-mediated epigenetic pathways after knock-down of Tasp1 expression using shRNA lentiviral (pLKD-TASP1) plasmids in EDC-MMSCs. The levels of expression of TASP1, H3K4me3, and CD9 proteins were measured by Western blot analysis using antibodies against TASP1, H3K4me3 and CD9, respectively (left panel). Biological replicates (n = 3) were used for quantification. Quantitative analysis was performed using Image J (right panel). b the upregulation of the expression of 6 reprogrammed ERGs (Esr-1, Pgr, Cxcl12, Ar, Cd9, and Tgm2) induced by EDC exposure was reversed by Tasp1 knock-down in EDC-MMSCs. RNA expression of ERGs was measured by qPCR in EDC-MMSCs infected with either scrambled lentivirus or one of three different individual Tasp1 knock-down lentiviruses. *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t-test

Fig. 7

Developmental EDC exposure alters the methylome and reprograms ERGs via DNA promoter methylation. a Western blot analysis was performed to measure the protein levels of DNMT3A in VEH-MMSCs and EDC-MMSCs. b Heatmap of genes with DNA methylation in VEH- and EDC-MMSCs. c The methylation tracks of Esr1 determined by targeted bisulfite NGS, which covered 14 CpG sites located in its CpG island. d Percentage of EDC-regulated genes with status of H3K4me3 and DNA methylation. e Number of genes with status of H3K4me3 and DNA methylation. f Integrated genome viewer (IGV) plots and integration of multi-omic analysis. Peaks with H3K4me3 enrichment, DNA methylation, and RNA expression in EDC-MMSCs are indicated in red, while peaks with H3K4me3 enrichment, DNA methylation, and RNA expression in VEH-MMSCs are indicated in blue. Student’s t-test, ***p < 0.001

Fig. 8

Specific reprogramming of ERGs in DES-MMSCs, which impacts DMC. a Bar plots showing the differential expression of ERGs, including Bcl11b, Cd9, Cxcl12, and Tgm2, between DES-MMSCs and DES-DMCs. b The correlation between H3K4me3 status and compared expressions of reprogrammed ERGs between MMSCs and DMCs using VEH-MMSC as a reference. The p-value shows the significant difference in gene expression between Stro-1/CD44 double-positive and double-negative cells. The genes with light blue background indicate that two comparisons of the gene expression (Stro-1/CD44 double-positive vs. double-negative cells, or Stro-1⁺/CD44⁺ DES vs. VEH) going in the opposite direction. The genes with white background indicate that two comparisons go in the same direction. c Diagram of experimental design. Serum-free conditioned medium (CM) was prepared from EDC-MMSCs and VEH-MMSCs. DMCs from rat adult myometrium were grown in the CM from EDC-MMSCs and VEH-MMSCs for 2 days. d MTT assays were performed to measure DMC proliferation in the presence of CM from either EDC-MMSCs or VEH-MMSCs. e qPCR was performed to determine the effect of CM from EDC-MMSCs and VEH-MMSCs on the expression of β-catenin and β-catenin-regulated genes (Angpt2, Med12l, and Pitx2) in DMCs. *p < 0.05; **p < 0.01, ****p < 0.0001

Fig. 9

Model for developmental reprogramming of the epigenome in MMSCs. Environmental risk factors, including EDC exposure, disrupt the epigenome via histone modification and DNA methylation, thereby leading to the conversion of MMSCs into UF-initiating cells, and eventually giving rise to the formation of UFs