CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis

Yang, Seung Chel; Park, Mira; Hong, Kwon-Ho; La, Hyeonwoo; Park, Chanhyeok; Wang, Peike; Li, Gaizhen; Chen, Qionghua; Choi, Youngsok; DeMayo, Francesco J.; Lydon, John P.; Skalnik, David G.; Lim, Hyunjung J.; Hong, Seok-Ho; Park, So Hee; Kim, Yeon Sun; Kim, Hye-Ryun; Song, Haengseok

doi:10.1038/s41467-023-39008-0

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Published: 03 June 2023

CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis

Nature Communications volume 14, Article number: 3220 (2023) Cite this article

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Abstract

Progesterone (P₄) is required for the preparation of the endometrium for a successful pregnancy. P₄ resistance is a leading cause of the pathogenesis of endometrial disorders like endometriosis, often leading to infertility; however, the underlying epigenetic cause remains unclear. Here we demonstrate that CFP1, a regulator of H3K4me3, is required for maintaining epigenetic landscapes of P₄-progesterone receptor (PGR) signaling networks in the mouse uterus. Cfp1^f/f;Pgr-Cre (Cfp1^d/d) mice showed impaired P₄ responses, leading to complete failure of embryo implantation. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 regulates uterine mRNA profiles not only in H3K4me3-dependent but also in H3K4me3-independent manners. CFP1 directly regulates important P₄ response genes, including Gata2, Sox17, and Ihh, which activate smoothened signaling pathway in the uterus. In a mouse model of endometriosis, Cfp1^d/d ectopic lesions showed P₄ resistance, which was rescued by a smoothened agonist. In human endometriosis, CFP1 was significantly downregulated, and expression levels between CFP1 and these P₄ targets are positively related regardless of PGR levels. In brief, our study provides that CFP1 intervenes in the P₄-epigenome-transcriptome networks for uterine receptivity for embryo implantation and the pathogenesis of endometriosis.

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Introduction

CXXC finger protein 1 (CFP1) is an important player in the epigenetic regulation of genes by inducing trimethylation at histone H3 lysine 4 (H3K4me3) with SETD1, a histone methyltransferase¹. H3K4me3 is mainly found in active promoters and turns chromatin into transcriptionally active euchromatin in the transcription start site (TSS) and CpG island (CGI)². When CFP1 binds to unmethylated CpG, it recruits SETD1A/B to trigger H3K4me3 in the promoters of target genes to increase gene expression^1,3. The deletion of CFP1, SETD1A, or SETD1B in mice caused embryonic lethality during or after gastrulation, suggesting the roles of SETD1-CFP1 complexes in early mammalian development^4,5. Furthermore, the conditional deletion of CFP1 highlighted that CFP1-associated H3K4me3 has fundamental roles in various biological processes. CFP1-deficient embryonic stem cells failed to differentiate in vitro because of aberrant H3K4me3 at non-methylated CGI promoters, leading to transcriptional disturbance^2,4,6. CFP1 plays an important role in intrathymic T-cell development and differentiation program of TH17 cells^7,8. Recent studies have also demonstrated that CFP1 is required for epigenetic modification in non-replicative cells, such as germ cells, in mice^9,10,11.

The endometrium is a highly dynamic organ, and its function and cyclicity are mainly regulated by ovarian progesterone (P₄) and estrogen (E₂). Imbalances between P₄ and E₂ may cause various gynecological disorders such as endometriosis, repeated implantation failure, and endometrial cancer¹². P₄ and E₂ activate their nuclear receptors, progesterone receptors (PGRs), and estrogen receptors (ESRs) to control the expression of local factors for uterine functions. PGR-dependent signaling networks for epithelium–stroma interaction are necessary to promote uterine receptivity for embryo implantation and decidualization in the uterus¹³. The fine-tuning of epigenetic modulation is required for phase-specific transcriptional networks during the reproductive cycle, embryo implantation, and subsequent pregnancy^14,15,16. Dynamic changes in histone modifications occur during decidualization^17,18. Silencing and overexpression of EZH2, a histone methyltransferase for H3K27me3, a repressive histone mark, disturb the expression of decidualization markers, such as IGFBP1 and PRL¹⁷. For successful parturition, biphasic modes of H3K27me3 dynamics in decidual stromal cells dictate the regulated gene silencing in the uterine adaptation to pregnancy¹⁹. The acetylation and methylation patterns of H3 and H4 in women with endometriotic lesions are distinct from those of disease-free women²⁰. However, the physiological significance of epigenetic regulatory machineries, such as histone modification during early pregnancy and the pathogenesis of uterine disorders, remains largely unexplored.

Endometriosis is a disorder in which endometrial cells grow abnormally outside the uterus. The surgical removal of the ectopic endometrial lesion with hormonal suppression is the current standard of care; however, these therapies have a high incidence of relapse and various side effects^21,22. Endometriosis affects 10%–15% of women of reproductive age and is one of the leading causes of female infertility^21,22,23,24. Leading causes of endometriosis include increased E₂ response, P₄ resistance (decreased P₄ response), and/or abnormal epigenetic regulation^25,26,27,28. Although decreased P₄ response caused by reduced PGR expression is considered a main cause of endometriosis^26,29, the underlying cause of the decreased P₄ response, even with normal P₄ secretion and PGR expression, has not yet been elucidated.

Altered DNA methylation and histone modification on the genes for balanced hormone responses were mainly proposed to affect the endometrial function and the development of endometriosis^9,12. The loss of histone deacetylase 3 (HDAC3) is a result of failures in embryo implantation and decidualization and caused fibrosis in the endometrium, one of the symptoms of human endometriosis with reduced HDAC3 expression²⁵. However, epigenetic causes of how uterine cells grow outside the uterus in patients with endometriosis have not been clearly identified. Using a combination of genetic and pharmacologic tools and various analyses, we demonstrated that CFP1-dependent epigenetic regulation is necessary to maintain uterine transcriptional landscapes for P₄ response for a successful pregnancy and prevent endometriosis with P₄ resistance. Thus, this study provides fundamental insight into understanding the complex interplay between the P₄-PGR signaling pathway and uterine epigenome–transcriptome under physiologic and pathophysiologic conditions, such as endometriosis.

Results

Loss of Cfp1 leads to infertility with multiple failures in oviductal embryo transport, P₄ uterine responses, embryo implantation, and decidualization in mice

CFP1 is expressed in many cells of female reproductive organs, such as the ovary, oviduct, and uterus (Supplementary Fig. 1). During early pregnancy, uterine CFP1 expression gradually increased from day 1 of pregnancy (Day 1) to Day 3. It peaked on Day 4, with the highest expression in the luminal epithelium. However, its expression is not directly affected by the regulation of E₂ and/or P₄ in the mouse uterus (Supplementary Fig. 1). While Cfp1 is deleted in most cell types in the female reproductive tract of adult Cfp1^f/f;Pgr^cre/+ (Cfp1^d/d) mice (Supplementary Fig. 2), they showed normal architectures in the gross morphology and histology of the reproductive tract with regular estrous cycle (Supplementary Fig. 3a–c). Furthermore, serum levels of E₂ and P₄ on Day 4 in Cfp1^d/d mice were comparable to those of Cfp1^f/f mice. We also found that Cfp1^d/d mice ovulate similar numbers of oocytes that can fertilize normally, and the fertilized embryos develop to the blastocyst stage without any aberrations in vitro (Supplementary Fig. 3d–f). However, implantation sites (IS), blue bands along the uterus, were not observed in Cfp1^d/d female mice on Day 5 (Fig. 1a). In addition, Cfp1^d/d uteri did not show decidual responses to artificial stimuli, such as oil (Fig. 1b, c). Accordingly, Cfp1^d/d female mice did not produce any pups (Fig. 1d). Interestingly, all blastocysts were found in the oviduct but not in the uterus of Cfp1^d/d mice on Day 4, whereas they were found in the uterus of Cfp1^f/f mice as expected (Fig. 1e–g).

**Fig. 1: *Cfp1*^d/d mice suffer from aberrant epithelial cell proliferation and complete failure of embryo implantation and decidualization.**

To examine embryo implantation in the uterus of Cfp1^d/d mice with defective oviductal embryo transport, wildtype blastocysts were transferred to the uteri of Cfp1^f/f and Cfp1^d/d mice on day 4 of pseudopregnancy. Distinct IS were observed in Cfp1^f/f but not in Cfp1^d/d recipients, and unimplanted blastocysts were retrieved from Cfp1^d/d recipients 24 h after embryo transfer (Fig. 1h, i and Table 1), indicating that CFP1-deficient uterine environments do not support embryo implantation. In this aspect, cell proliferation profiles are aberrant in the uterus of Cfp1^d/d mice on Day 4 (Fig. 1j, k), whereas the levels of E₂ and P₄ (Supplementary Fig. 3d) and their receptors (Supplementary Fig. 4) are comparable between Cfp1^f/f and Cfp1^d/d mice. Nevertheless, the uterine epithelium persistently proliferates, and the stroma showed less proliferation potential in the uterus of Cfp1^d/d mice, suggesting that CFP1, as an epigenetic regulator, is required for proper hormone responses in the uterus for a successful pregnancy.

Table 1 Embryo implantation in pseudopregnant Cfp1^f/f and Cfp1^d/d recipients after blastocyst transfer

Full size table

CFP1 regulates uterine mRNA landscapes in both H3K4me3-dependent and H3K4me3-independent manners

To understand the CFP1-dependent epigenetic regulation on transcriptional landscapes in the uterus, we performed chromatin immunoprecipitation sequencing (ChIP-seq) and mRNA sequencing (mRNA-seq) with the uteri of Cfp1^f/f and/or Cfp1^d/d mice on Day 4 (Fig. 2). ChIP-seq with antibodies for CFP1 and H3K4me3 provided evidence that CFP1-binding sites are highly enriched in TSS and CGI, and H3K4me3 in TSS and CGI was generally reduced in the uterus of Cfp1^d/d mouse (Fig. 2a). De novo motif analysis for CFP1 ChIP-seq data showed that CFP1 recognizes the CCGG motifs, including CGG and its reverse complement CCG (Fig. 2b), which are consistent with the CFP1-binding motifs found in humans^30,31,32. CFP1 binding was noticeably enriched in extended gene bodies, including promoters (9%), exons (4%), and introns (38%) in mouse uterus, considering that the mouse genome consists of promoters (2%), exons (2%), introns (20%), and intergenic factors (76%) (Fig. 2c). When the mRNA-seq and H3K4me3 ChIP-seq data in ±2 kilo base pairs (Kbp) of TSS were analyzed together, 40.3% of differentially expressed genes (DEGs) had a reduction in both gene expression and H3K4me3 levels in Cfp1^d/d mice (Fig. 2d) as expected from the known actions of CFP1 on H3K4me3. Generally, the lower gene expression levels are, the lower the H3K4me3 levels in the +2Kbp TSS region are (Supplementary Fig. 5). However, a significant portion of DEGs (18.2%) was made up of genes with decreased expression levels and increased H3K4me3 levels in Cfp1^d/d mouse uterus on Day 4, suggesting that CFP1 could promote gene expression in the uterus in H3K4me3-independent manner.

**Fig. 2: CFP1 epigenetically regulates uterine transcriptome via H3K4me3 on Day 4.**

Although CFP1 binds to unmethylated CpG and induces H3K4me3 to increase gene expression^1,3, the number of significantly downregulated (3329) and upregulated (2829) genes in Cfp1^d/d mice was comparable (54% vs. 46%) in DEGs from mRNA-seq (Fig. 2e and Supplementary Fig. 6a). However, gene ontology (GO) analyses showed that most GO terms (126/132, 95.5%) with false discovery rate (FDR) of <0.25 were reduced in the uteri of Cfp1^d/d mice on Day 4 (Fig. 2f and Supplementary Fig. 6b). Gene set enrichment analyses (GSEA) showed that various gene sets associated with P₄ response, hedgehog signaling, cancers, ion channel activities, and stromal cell stimulation were significantly downregulated in Cfp1^d/d uterus (Fig. 2g). Interestingly, the Indian hedgehog (IHH)-dependent smoothened signaling pathway (SSP) is a well-known P₄ downstream pathway that inhibits E₂-dependent epithelial proliferation and further stimulates stromal proliferation in the uterus³³. A heatmap, reverse-transcriptase polymerase chain reaction (RT-PCR), and real-time RT-PCR analyses for the SSP gene set validated that IHH-dependent SSP is significantly downregulated in Cfp1^d/d mouse uterus on Day 4 (Fig. 2h, i).

CFP1 epigenetically regulates P₄ responses through the IHH-dependent SSP in mouse uterus

To further investigate the epigenetic actions of CFP1 in the uterus, we tried to identify CFP1 direct target genes by analyzing all sequencing data together. The CFP1 direct target gene candidates should have CFP1-binding site(s) and be downregulated in Cfp1^d/d uteri. We found that the putative direct target genes of CFP1 are regulated not only in H3K4me3-dependent (673 genes) but also inH3K4me3-independent (423 genes) manner (Fig. 3a and Supplementary Data 1–2). Interestingly, the upstream and downstream genes of IHH-dependent SSP were included in the list; Ihh, Gli3, and Gata2 as H3K4me3-dependent and Ptch1, Sox17, and Nr2f2 as H3K4me3-independent target genes (Fig. 3b and Supplementary Data 2). The expression of these genes was reduced with statistical significance in Cfp1^d/d mice (Figs. 2i and 3c). The visualization of sequencing data for Gata2, Ihh, and Sox17 using Integrative Genomics Viewer demonstrated that they all have CFP1 binding sites, and their expression levels were significantly reduced in Cfp1^d/d uteri. However, H3K4me3 levels were decreased in extended gene bodies of Gata2 and Ihh, but not in Sox17 (Fig. 3d). Real-time ChIP PCR reinforced that SETD1 and H3K4me3 were enriched in the promoter regions of Gata2 and Ihh but not in Sox17, whereas CFP1 is enriched in all of them in Cfp1^f/f uterus (Fig. 3e and Supplementary Table 1). In summary, CFP1 works with SETD1 to increase H3K4me3 in Gata2 and Ihh promoters but not for Sox17 promoter for their expression in the uterus.

**Fig. 3: CFP1-dependent epigenetic regulation intervenes in important uterine P₄ responses via H3k4me3-dependent and H3k4me3-independent manners in mice.**

P₄-PGR induces stromal cell proliferation and inhibits epithelial cell proliferation via the activation of SSP downstream of Gata2, Sox17, and Ihh in mouse uterus on Day 4 (Fig. 3b). However, P₄ could not inhibit E₂-dependent epithelial proliferation and facilitate stromal cell proliferation in the uteri of ovariectomized (OVX) Cfp1^d/d mice (Fig. 3f, g) in line with the results on Day 4 (Fig. 1k). Since defective SSP could cause aberrant cell proliferation in Cfp1^d/d uteri, we tried to rescue this phenotype with SAG, a smoothened agonist, in these mice. An intrauterine delivery of SAG successfully rescued aberrant epithelial proliferation in the uteri of OVX Cfp1^d/d mice treated with E₂ and P₄ (Fig. 3f and 3g). Furthermore, SAG significantly restored the decreased expression levels of genes in SSP and its downstream genes, such as Gli1, Gli2, Nr2f2, and Hand2, in Cfp1^d/d uteri to the levels in Cfp1^f/f uteri (Fig. 3h). When our mRNA-seq datasets were compared with other P₄-related transcriptomic data (GSE118264, GSE40661, and GSE178541)^13,34,35, comparative analyses showed that significant numbers of DEGs in Cfp1^d/d uterus overlap with DEGs in other P₄-related datasets (Supplementary Fig. 7). Collectively, these results suggest that CFP1 loss disturbs the epigenetic maintenance of P₄-PGR signaling pathways in the uterus.

CFP1 is required for P₄ function to inhibit the growth of ectopic endometriotic lesions in mice

Endometriosis can occur possibly via a decreased P₄ response, and some patients with endometriosis show P₄ resistance even with normal PGR expression^36,37,38,39. To further evaluate CFP1 function for uterine P₄ responses, we established a mouse model of endometriosis with some modifications from previous reports^25,26. When P₄ was given with E₂, the size of ectopic lesions was significantly smaller than with E₂ alone, although the number of ectopic lesions was not different (Supplementary Fig. 8a, b). Ectopic lesions were mainly observed on organs with highly developed blood vessels, such as the small intestine, kidney, uterus, and fat pad (Supplementary Fig. 8c). When small pieces of Cfp1^f/f and Cfp1^d/d uterus as endometriotic lesions were transplanted to wildtype recipients (Fig. 4a), P₄ effectively suppressed the E₂-induced growth of Cfp1^f/f but not Cfp1^d/d ectopic lesions even if the number of ectopic lesions was not affected by Cfp1 genotypes (Fig. 4b–e). The mRNA expression of Gata2, Sox17, and Ihh was also significantly downregulated in Cfp1^d/d ectopic uterine lesions (Fig. 4f). When SAG was administered to rescue P₄ resistance and/or insensitivity in Cfp1^d/d ectopic uterine lesions, P₄ suppressed the size of Cfp1^d/d ectopic lesions in the endometriosis model (Fig. 4b–e), suggesting that CFP1 is required for proper P₄ responses to suppress ectopic growth of uterine tissues in mice.

**Fig. 4: CFP1 is required for the counteraction of P₄ on E₂-dependent uterine growth in a mouse model of human endometriosis.**

Downregulation of the epigenetic factor CFP1 may be associated with endometriosis in humans

To further evaluate the potential actions of CFP1 on the pathogenesis of endometriosis in humans, we analyzed datasets (GSE51981) of the endometria of patients with endometriosis (endometriosis group, n = 77) and healthy women (control group, n = 71) from a previous study⁴⁰. While no correlation exists between the expression levels of CFP1 and PGR mRNAs, GATA2, SOX17, and IHH mRNA expression levels were positively correlated with that of CFP1 (Fig. 5a) and PGR mRNA (Supplementary Fig. 9a) regardless of endometrial pathologic conditions. We further analyzed a subset of GSE51981, i.e., endometria of women with mild and severe endometriosis in the mid-secretory phase where P₄ is dominant (Fig. 5b). In patients with mild endometriosis, the expression profiles of PGR, CFP1, and P₄ target genes are not different from those of the control group. However, there was a statistically significant reduction in their expression levels in patients with severe endometriosis (Supplementary Fig. 9b). Among patients with severe endometriosis, some had a comparable level of PGR but a low level of CFP1 (circled red, Fig. 5b). In these patients, the expression patterns of GATA2, SOX17, and IHH mRNAs were significantly decreased. It suggests that aberrantly reduced expression of CFP1 may be associated with endometriosis via abnormal P₄ response in patients with normal PGR expression. We also performed immunohistochemistry and real-time RT-PCR for these genes in endometrial samples from patients with severe endometriosis and disease-free control women in the secretory phase. The eutopic and ectopic endometria from patients with endometriosis showed decreased expression levels of CFP1, GATA2, SOX17, and IHH mRNAs compared with the endometrium from the control group (Fig. 5c, d), suggesting that reduced CFP1 expression may disturb epigenetic landscapes that mediate P₄-PGR signaling pathways in human endometrium, often leading to endometriosis.

**Fig. 5: Concomitant reduction of *CFP1* and important P₄ responsive genes observed in the endometrium of patients with endometriosis.**

Discussion

Many gynecological diseases are caused by abnormally reduced P₄ response; however, the underlying epigenetic aberration for the imbalanced steroid hormone responses has not been clearly elucidated. We demonstrate that CFP1-associated epigenetic regulation is required for maintaining appropriate P₄ responses for embryo implantation and decidualization in the uterus and inhibiting the ectopic growth of endometrial lesions outside the uterus. Cfp1^d/d mice exhibit a wide spectrum of infertility, including defective oviductal embryo transport, abnormal uterine cell proliferation, and complete failure of embryo implantation and decidualization (Fig. 1). These phenotypes suggest that epigenetic regulation through H3K4me3 is involved in the sequential events of female reproduction. A recent study supported those spatiotemporal dynamics of H3K4me3 in the uterine genome are needed to be well controlled for early pregnancy. In mice without MENIN (Men1^d/d mice), a member of the H3K4 methyltransferase complex, defective decidualization compromised fertility, whereas embryo implantation normally occurs⁴¹. Impaired decidualization in Men1^d/d mice was caused by reduced Bmp2 expression by abnormally increased FGF2 signaling⁴¹. In Cfp1^d/d uterus on Day 4, Fgf2 expression aberrantly increased because of a decrease in Ihh-dependent SSP that antagonizes FGF2 signaling, although the Bmp2 expression did not decrease (Fig. 3). While MLL1/2 complexes that contain MENIN catalyze H3K4 methylation in a gene- and cell-specific manner, SETD1–CFP1 complexes are the leading H3K4 methyltransferases among the six histone methyltransferases, SETD1A/B, MLL1/2, and MLL3/4^9,41. This is consistent with the fact that Cfp1^d/d mice show a wider spectrum of infertile phenotypes than Men1^d/d mice, suggesting that CFP1-dependent epigenetic regulation may work on the wider area of the genome. For example, we found an impairment of the oviductal embryo transport in Cfp1^d/d mice, which was not observed in Men1^d/d mice. Embryo transport in the oviduct could be interrupted by abnormalities in cilia movement, fluid secretion, and smooth muscle contraction^42,43. Since Cfp1 was mainly deleted in non-ciliated epithelial cells of the isthmus, but not in ciliated ones, which are positive for acetylated tubulin in the ampulla of Cfp1^d/d oviduct by Pgr-Cre (Supplementary Fig. 2e, f), embryo retainment in the oviduct could result from abnormalities in fluid secretion and smooth muscle contraction⁴⁴, but not from cilia movement. While the epigenetic changes for fluid secretion are largely unknown, histone marks to promote transcription, such as H3K4me3 and H3K27 acetylation (H3K27ac), are enriched at promoters of genes driving muscle contraction on the advance of labor onset in the myometrium⁴⁵.

CFP1-dependent epigenetic regulation has been investigated in various biological events. The conditional deletion of Cfp1 in mouse hematopoietic cells resulted in severe defects during hematopoiesis with complete loss of lineage-committed progenitors and mature cells⁴⁶. CFP1 is also required for thymocyte survival, the balanced differentiation between Th17 and Treg cells^7,8, and the phagocytic and bactericidal activity of macrophages⁴⁷. Since Pgr is expressed in various immune cells, including macrophages⁴⁸, dendritic cells⁴⁹, and T cells⁵⁰, Cfp1 was supposed to be deleted in immune cells as well as uterine cells in Cfp1^d/d mice. Immune cells play important roles during pregnancy⁵¹. However, immune-related gene sets were not significantly altered in Cfp1^d/d mouse uterus on Day 4 (Fig. 2g). Furthermore, normal fertility was observed in mice (Cfp1^f/f;LysM^cre/+) in which Cfp1 is deleted in the myeloid lineage cells, such as monocytes and macrophages, using LysM-Cre (Supplementary Table 2), suggesting that phenotypes observed in Cfp1^d/d mice are not directly associated with CFP1 functions in immune cells. Recently, the function of CFP1–SETD1 complexes for epigenetic reprogramming during germ cell development has been investigated. CFP1-mediated H3K4me3 is required for maintaining chromatin accessibility for transcriptional activities during oocyte development, and oocyte-specific deletion of Cfp1 caused reduced H3K4 methylation levels and globally downregulated transcription activities, in turn, leading to multiple defects in the meiotic division and maternal–to–zygotic transition¹⁰. Furthermore, CFP1 participates in regulating the expression of paracrine factors for communication between the oocyte and surrounding granulosa cells for follicle growth and ovulation⁵². The conditional deletion of Setd1b in the oocyte also caused the dysregulation of transcription factors for oogenesis, including Obox transcription factors, leading to oocyte maturation defects and infertility⁵³. CFP1 deletion before the onset of meiosis with Stra8-Cre in male mice caused complete infertility with the spermatogenic arrest at the MII stage¹¹, suggesting that CFP1-mediated H3K4me3 plays a role in meiosis and cell fate decision.

P₄ antagonizes E₂ action on epithelial proliferation and promotes stromal cell proliferation to prepare uterine receptivity for embryo implantation and decidualization^13,54. P₄ increased the expression of Gata2, Sox17, and Ihh in epithelial cells to trigger SSP in the stroma on Day 4 in mice^13,33,34, all of which were downregulated in Cfp1^d/d mice (Figs. 2 and 3). As a result, uterine epithelial cells aberrantly proliferated and failed to prepare embryo implantation on Day 4 in Cfp1^d/d mice (Fig. 1). The expression of gene sets related to P₄ response, IHH-dependent SSP, and stromal cell stimulation was significantly reduced in Cfp1^d/d mice (Fig. 2g). Furthermore, SAG successfully rescued abnormal uterine cell proliferation in Cfp1^d/d mouse uterus (Fig. 3). They all indicate that disturbed P₄–PGR–SSP signaling pathway is the main cause of complete failure of embryo implantation and decidualization in Cfp1^d/d mice. In fact, mice without Gata2, Sox17, or Ihh phenocopied all these uterine defects observed in Cfp1^d/d mice^13,33,34. In addition, SAG restored decidualization and abnormal uterine cell proliferation in Ihh cKO mice³³. PGR is decreased in Gata2-deficient uteri, but it is necessary to mention that mice deficient in Sox17, Ihh, or Cfp1 had normal PGR expression. Although reduced PGR expression appears to be the main cause of P₄ resistance that contributes to the pathogenesis of endometriosis^29,38,55, some studies have reported comparable levels of PGR in eutopic^36,38 or ectopic⁵⁶ endometria of women with endometriosis. This suggests that the epigenetic aberration in PGR downstream pathways could be involved in the pathogenesis of endometriosis⁵⁷. Essentially, the activation of PGR downstream pathways with SAG rescued the abnormal epithelial proliferation in Cfp1^d/d uterus (Fig. 3) and suppressed the ectopic growth of Cfp1^d/d uterine lesions with P₄ resistance in the endometriosis model (Fig. 4). Aberrant epithelial proliferation was also found in the endometrium of patients with endometriosis^25,58. Considering impaired P₄-PGR signaling without PGR reduction (Supplementary Fig. 4) in Cfp1^d/d mice reflects P₄ resistance, disturbed CFP1-associated H3K4me3 could contribute to P₄ resistance that often leads to endometriosis in humans.

CFP1 mainly works to increase the expression of target genes through H3K4me3-dependent manners^7,52. Accordingly, 40.3% of DEGs in Cfp1^d/d uteri were downregulated with reduced H3K4me3 levels (Fig. 2d). However, a substantial portion of downregulated DEGs had even higher H3K4me3 levels (18.2%). Although CFP1 is an evolutionally conserved epigenetic regulator to work with SETDs for H3K4me3 from yeast to mammals, recent studies have suggested that CFP1 could collaborate with epigenetic modulators other than COMPASS (complex associated with SET1) complexes, such as DNA methyltransferases (DNMTs) and HDACs, in a context-dependent manner. When CFP1/SETD1 or MLL1/2 binds to DNA, they hamper DNA methylation by blocking the access of DNMT3A⁵⁹. CFP1 interacts with and recruits DNMT1 to suppress aberrant transcription re-initiation or silence specific genes^2,60. The expression of Dnmt1 and Dnmt3a increased gradually in the mouse uterus during early pregnancy⁶¹. They suggest that the H3K4me3-independent gene expression in Cfp1^d/d uteri could be associated with reduced DNA methylation. In addition to DNMTs, CFP1 could interact with HDAC1/2 complexes to regulate fertility and development in C. elegans. CFP1 recruits the HDAC complex to H3K4me3-rich promoter regions to deacetylate chromatin⁶². CFP1-dependent H3K4me3 is necessary to recruit histone acetylase(s) for H3K9ac dynamics in mouse embryonic stem cells⁶, suggesting that CFP1-associated H3K4me3 cross-talks with histone acetylation. Interestingly, higher P₄ levels in in vitro fertilization cycles on the day of hCG administration altered various epigenetic marks, including H3K9ac, H3K9me2, and H3K27me3, in the endometrium⁶³, suggesting that histone modifications and P₄-PGR signaling influence each other in the endometrium. The epithelial cells in endometriotic lesions expressed a higher level of EZH2, the enzyme responsible for a repressive mark H3K27me3, which P₄ upregulates⁶⁴. MLL1 is directly regulated by P₄–PGR signaling in the uterus and MLL1 and H3K4me3 both decreased in the eutopic endometrium of patients with endometriosis⁶⁵. Furthermore, the expression of HDAC3, one of HDACs that reduce gene expression, was significantly lower in the endometrium of patients with endometriosis. The loss of HDAC3 in mice leads to infertility that results from embryo implantation failures with defective decidualization possibly through the aberrant activation of Col1a1 and Col1a2 genes that promote fibrosis with decreased ESR and PGR in the uterus²⁵. In summary, our results suggest that aberrant epigenetic regulation in CFP1-deficient mice provides a uterine environment with P₄ resistance that leads to infertility caused by multiple failures and endometriosis (Fig. 6). This study is of great significance to provide an underlying epigenetic mechanism of P₄ resistance in the endometrium that could lead to endometriosis in humans.

**Fig. 6: A schematic illustration of CFP1 function in P₄-epigenome-transcriptome networks for uterine physiology.**

Methods

Animals

All mice used in this study were maintained in accordance with the policies of the CHA University Institutional Animal Care and Use Committee (No150083). Adult (8–10 weeks of age) C57BL/6 mice, provided by Orient Bio (Gapyeong, Gyeonggi, Korea), were housed under temperature- and light-controlled conditions with the light on for 12 h daily and fed ad libitum. Cfp1^f/f mice were kindly provided by Dr. David G. Shkolnik’s laboratory¹. First, Cfp1^f/f mice were mated to Pgr^cre/+ mice to generate Cfp1^f/+;Pgr^cre/+ mice⁶⁶. Then, these mice were crossed to generate Cfp1^d/d and Cfp1^f/f mice. Genotyping PCR was performed by genomic DNA extracts from tail biopsies (Supplementary Table 3).

Vaginal smear analysis and fertility test

Estrous cyclicity was evaluated in mature Cfp1^f/f and Cfp1^d/d female mice by daily analysis of vaginal smears over 2 weeks between 8:00 and 9:00 AM. The estrous cycle stage (proestrus, estrus, metestrus, and diestrus) was determined based on the presence of vaginal cornified epithelial cells and nucleated epithelial cells/total vaginal cells. To evaluate reproductive performance, mature Cfp1^f/f and Cfp1^d/d female mice (n = 4 per genotype) were individually bred to wildtype males with proven fertility. The numbers of litters and pups were recorded for 2 months.

Serum E₂ and P₄ level measurement

Blood samples were collected on Day 4 (9:00 AM) from the heart of Cfp1^f/f and Cfp1^d/d female mice (n = 5 to 9 per genotype). First, the mouse blood was sampled without anticoagulant and transferred to a sterile empty tube. Next, the mouse blood was centrifuged at 1500 × g for 15 min at 4 °C. Separated serum was transferred into a new sterile empty tube. Serum levels of mouse E₂ and P₄ were measured by radioimmunoassay⁶⁷.

Ovulation, fertilization, and preimplantation embryo development

Cfp1^f/f and Cfp1^d/d female mice were bred to wildtype males with known fertility. The morning of the vaginal plug observation was designated as Day 1. The mice were sacrificed on Day 2, and their oviducts were flushed to evaluate the number and fertilization potential of ovulated oocytes (n = 7 per genotype). In addition, fertilized 2-cell embryos were cultured up to the blastocyst stage in 20 ml droplets of KSOM (Millipore, Danvers, MA, USA) covered with oil (SAGE In-Vitro Fertilization, Inc., Trumbulla, CT, USA) in a petri dish.

Hormone treatments

To investigate time-dependent actions of E₂ or P₄ on the expression of Cfp1 in C57BL/6 mice uterus, adult (8–10 weeks of age) female mice were OVX, rested for 14 days, and then subcutaneously injected with either vehicle (sesame oil, 0.1 mL/mouse; Acros, NJ, USA), E₂ (100 ng/mouse, Sigma–Aldrich, St. Louis, MO, USA) or E₂ + P₄ (2 mg/mouse, Sigma–Aldrich). After hormone injection, the mice were sacrificed at various time points (0–24 h) and the uterus was collected for real-time RT-PCR (n = 4 to 6 per each group).

Tissue preparation

Female reproductive organs under various conditions, such as early pregnancy, ovarian steroid hormone treatment, and artificial decidualization, were dissected and then fixed in 4% paraformaldehyde. Fixed tissues were washed, dehydrated, and embedded in paraplast (Merck KGaA, Darmstadt, Germany). Paraffin-embedded tissues were sectioned to 5 μm thickness using a microtome, stained with hematoxylin and eosin (Sigma–Aldrich), and observed by light microscopy.

Early pregnancy and embryo implantation

Pregnancy was evaluated by the presence of a vaginal plug on the next morning after breeding with a fertile male. The pregnant mice were sacrificed on various days of pregnancy from Day 1 to 5, and their uterine horns were collected and processed for the following experiments (n = 4 to 6 per each group). IS were detected by intravenous (i.v.) injection of 1% Chicago Sky Blue (in saline) on the morning of Day 5, and the number of IS demarcated by blue spots was recorded⁶⁸.

Embryo transfer

Embryo transfer was performed as previously described with some modifications⁶⁹. Four-week-old C57BL/6 mice were given intraperitoneal (i.p.) injections of 5 IU PMSG (Sigma–Aldrich) followed by i.p. injections with 5 IU hCG (Sigma–Aldrich) and then mated with fertile male mice to obtain the embryos for embryo transfers. The blastocysts were transferred to the uteri of either pseudopregnant day 4 Cfp1^f/f and Cfp1^d/d recipient mice, which were mated with vasectomized C57BL/6 male mice. Embryo implantation was evaluated by an i.v. injection (0.1 mL/mouse) of Chicago Sky Blue (1% in saline, Sigma–Aldrich) 24 h after embryo transfer.

Artificial decidualization

Artificial decidualization was experimentally induced, as previously described, with some modifications⁶⁹. Adult (8–10 weeks of age) Cfp1^f/f and Cfp1^d/d female mice were OVX and rested for 14 days and then received daily injections of 100 ng E₂ for 3 days (n = 3 to 4 per genotype). After 2 days of resting, the mice were then treated with daily injections of 1 mg P₄ and 10 ng E₂ for 3 days. At 6 h after the last injection, one uterine horn was traumatized by the injection of 50 μL of sesame oil. Mice were given daily injections of P₄ (1 mg) + E₂ (10 ng) following trauma. The weight of stimulated and non-stimulated uterine horns was recorded 4 days after the oil infusion. The fold increase in uterine weights and alkaline phosphatase staining served as an index of decidualization.

Intrauterine delivery of SAG

SAG (Abcam, Cambridge, UK), a smoothened agonist, was applied to rescue aberrant uterine cell proliferation and gene expression in Cfp1^d/d female mice³³. Adult (8–10 weeks of age) Cfp1^f/f and Cfp1^d/d female mice were OVX and rested for 14 days. Each OVX mouse was treated with 2 mg P₄ for 2 days and then 2 mg P₄ and E₂ 60 ng/mouse on the third day. At 3 h after E₂ and P₄ injection, 10 μL of vehicle (PBS) and SAG (1000 nM) in PBS were injected intraluminally in each horn of the uterus. Mice were sacrificed at 3 or 21 h after SAG injection depending on experimental conditions, and the uterus was collected for real-time RT-PCR and immunofluorescence staining (n = 8 per each group).

RNA extraction, RT-PCR, and real-time RT-PCR

Total RNA was extracted from mouse uteri under various conditions (n = 3 to 8 per each group) using Trizol Reagent (Invitrogen Life Technologies, San Diego, CA, USA) according to the manufacturer’s protocols. The first-strand cDNA was synthesized from 1 µg of total RNA using M–MLV reverse transcriptase (Promega, Madison, WI, USA) and RNasin Ribonuclease inhibitor (Promega). Synthesized cDNA (10 ng) was utilized for PCR with specific primers at optimized cycles (Supplementary Table 4). To quantify expression levels, real-time RT-PCR was performed using the SYBR green dye (iQ SYBR Green Supermix, Bio-Rad, Hercules, CA, USA), as previously described⁶⁸. To compare transcript levels between samples, a standard curve of cycle thresholds for several serial dilutions of a cDNA sample was established and then used to calculate the relative abundance of each gene. Then, values were normalized to the relative amounts of Rpl7 cDNA. All reactions were performed in duplicates.

Western blotting

Uterus tissues (n = 4 to 6 per each group) were lysed in lysis buffer including PRO-PREP (iNtRON, Seongnam, Korea) solution and 1X phosphatase inhibitor (Roche Applied Sciences, Indianapolis, IN, USA). The protein samples (20 μg/lane) were then separated by 8% SDS-PAGE, transferred onto nitrocellulose membrane (Bio-Rad), and blocked with 5% non-fat milk (Bio-Rad) in TBS (Bio-Rad) containing 0.1% Tween 20 (Sigma–Aldrich). Antibodies used for Western blotting, immunostaining, and/or ChIP were summarized in Supplementary Tables 5 and 6. The signals were developed using an ECL Western blotting substrate kit (Bio-Rad) and detected using a Chemidoc XRS+ (Bio-Rad) with Image Lab software.

Immunohistochemistry and immunofluorescence analysis

Paraffin-embedded tissues were sectioned to 5 µm thickness using a microtome. Uterine sections were deparaffinized and rehydrated. Endogenous peroxidase was inactivated with 3% H₂O₂. Sections were subjected to antigen retrieval in 0.01 M sodium citrate buffer (pH 6.0). Nonspecific staining was blocked using protein block serum (Dako, Carpinteria, CA, USA) for 1 h. Sections were incubated with primary antibodies at 4 °C overnight. On the following day, sections were incubated with appropriate secondary antibodies for 1 h at room temperature. Sections were counterstained using Topro-3-iodide (TOPRO; Life Technologies, Carlsbad, CA, USA) and mounted. For immunohistochemistry, DAB reagent (Vector Laboratories, Inc., Burlingame, CA, USA) was applied to visualize signals. Images were observed under a microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using ZEN software (Carl Zeiss).

mRNA-seq and data analysis

Libraries were prepared from 2 μg of total RNA in Cfp1^f/f and Cfp1^d/d mouse uterus on Day 4 using the SMARTer stranded mRNA-Seq kit (Clontech Laboratories, Inc., USA). mRNAs were used for the cDNA synthesis and shearing, following the manufacturer’s instructions (n = 3 pools: 3 mice per each pool). The Illumina indexes were used, and the enrichment step was conducted with PCR. The RT (read count) data were processed based on the quantile normalization method using EdgeR within R⁷⁰ utilizing bioconductor. The alignment files were also used for assembling transcripts, estimating their abundances, and detecting differential expression of genes or isoforms using cufflinks. These are performed using Bowtie2 software. We also used the fragments per kilobase of exon per million (FPKM) fragments to determine the expression levels of the gene regions. Gene classification was based on searches made by GSEA, and heatmaps of the unsupervised hierarchical clustering and DEGs were produced using MeV software.

CFP1 immunoprecipitation

Immunoprecipitation of CFP1 protein using CFP1 antibodies was performed with the manufacturer’s instructions using immunoprecipitation kit (Abcam). In addition, CFP1 Western blotting following immunoprecipitation was performed to evaluate the specificity of CFP1 antibodies used for CFP1 ChIP-seq for Cfp1^f/f uterine samples (Supplementary Fig. 10). On Day 4, uterine horns of Cfp1^f/f and Cfp1^d/d mice were cut vertically, and epithelial and stromal cells were separated from the smooth muscles (n = 4 to 5 per each group). Then, tissues were incubated in a lysis buffer with protease inhibitors and mixed on a rocker at 4 °C for 1 h. The tissue extracts were transferred to a fresh tube, and a predetermined amount of antibodies was added on a rocker at 4 °C for 4 h. After antibody binding, protein A/G Sepharose beads were added on a rocker at 4 °C for 1 h. Next, protein A/G Sepharose beads were collected, washed, and eluted. The protein of interest was purified by low-speed centrifugation at 4 °C and used for Western blotting.

ChIP and real-time ChIP PCR

ChIP analysis was performed with a slight modification of the manufacturer’s instructions using the ChIP-IT Express Enzymatic kit (Active Motif, Carlsbad, CA, USA). On Day 4, Cfp1^f/f and Cfp1^d/d mouse uterus horns were cut, and epithelial and stromal cells were separated from the uterine smooth muscles (n = 3 pools: 3 mice per each pool). Then, epithelial and stromal cells were fixed in DMEM high-glucose media containing 1% formaldehyde and then made into single cell slurry through an electric homogenizer. DNA fragments between 150 and 500 bp were obtained by enzymatic shearing cocktail after lysis cell and nuclei isolation through lysis buffer and Dounce homogenizer. Antibodies were added in sheared chromatin, and immunoprecipitation was performed overnight at 4 °C. Immunoprecipitated DNA was utilized for real-time ChIP PCR with specific primers (Supplementary Table 1). To quantify the enrichment level in ChIP-seq data, real-time ChIP PCR was performed with iQTM SYBR Green Supermix (Bio-Rad) on BIO-RAD iCycler using immunoprecipitated DNA. All PCR reactions (10% input, CFP1, SETD1, H3K4me3, and normal IgG) were duplicated (n = 3 to 4 per each group).

ChIP-seq and data analysis

The library was constructed using NEBNext® UltraTM DNA Library Prep Kit for Illumina (New England Biolabs, UK) according to the manufacturer’s instructions. Briefly, the chopped DNA was ligated with adaptors. After purification, the PCR reaction was conducted with adaptor-ligated DNA and index primer for multiplexing sequencing. The library was purified using magnetic beads to remove all reaction components. The library size was assessed by Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, Netherlands). High-throughput sequencing was performed as paired-end 100 sequencing using HigSeq 2500 (Illumina, Inc. USA). ChIP-seq metaplots and heatmaps were analyzed using DeepTools software.

Experimentally induced mouse model of endometriosis

Recipient (wild type) and donor (Cfp1^f/f and Cfp1^d/d) female mice (8–10 weeks of age) were primed with 5 IU PMSG (Sigma–Aldrich) for 48 h to stimulate uterine growth. After inhalation anesthesia, the uterine horn was collected from donor mice and vertically opened with scissors in a petri dish containing warmed Dulbecco’s phosphate-buffered saline (37 °C). The uterine horn was cut into small pieces of approximately 1 mm² and injected into the peritoneum of the OVX recipient mice. After transplantation of uterine tissues in recipient mice, E₂ (100 ng/mouse), P₄ (2 mg/mouse), PBS, and/or SAG were injected every 3 days. At 15 days after transplantation, the volume and number of ectopic lesions were measured, and ectopic lesions were collected for real-time RT-PCR.

Human endometrial sampling

Control (n = 7) and endometriotic endometrial tissues (n = 5) were collected from patients who underwent hysteroscopy–laparoscopy surgery to evaluate endometrial abnormalities, including endometriosis in the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Xiamen University. The sample collection and studies reported here were approved by the ethics committee of Hospital of Xiamen University (XMYY-2021KYSB044), and all participants signed the informed consent. Endometriotic lesions were obtained from women (aged 25–35 years) suffering from ovarian endometriosis, confirmed by laparoscopy and histopathology. These patients had regular menstrual cycles and were recruited without hormone treatment for at least 3 months before surgery. All samples were collected during the luteal phase of the menstrual cycle.

Statistical analysis

GraphPad Prism version 8 software (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. All values are represented as mean ± standard deviation. Statistical analyses were performed using the unpaired Student’s t-tests, and p < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Raw data files are deposited in the NCBI Gene Expression Omnibus database under Super Series accession number GSE219104. The Supplementary Fig. 7 data re-analyzed in this study are available in the GSE118264 database¹³, GSE40661 database³⁴, and GSE178541 database³⁵. All other data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF- 2019R1A6A1A03032888 to H.S.), by the NRF grant funded by the Korea government (the Ministry of Science and ICT) (NRF-2015R1A2A2A01006714 to H.S., NRF- 2020R1C1C1008317 to H.R.K., NRF-2020R1A2C2005012 to H.S., and NRF-2020R1A6A3A01100338 to M.P.), by Bio & Medical Technology Development Program of the NRF and funded by the Korean government (MSIT) (NRF-2022M3A9E4016936 to S.-H.H.).

Author information

These authors contributed equally: Seung Chel Yang, Mira Park.

Authors and Affiliations

Department of Biomedical Science, CHA University, Seongnam, Gyeonggi, 13488, Korea
Seung Chel Yang, Mira Park, So Hee Park, Yeon Sun Kim, Hye-Ryun Kim & Haengseok Song
Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, 05029, Korea
Kwon-Ho Hong, Hyeonwoo La, Chanhyeok Park & Youngsok Choi
Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xiamen, School of Medicine, Xiamen University, Xiamen, Fujian, 361102, China
Peike Wang, Gaizhen Li & Qionghua Chen
Department of Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 12233, USA
Francesco J. DeMayo
Department of Molecular and Cellular Biology and Center for Reproductive Medicine, Baylor College of Medicine, Houston, TX, 77030, USA
John P. Lydon
Department of Biology, School of Science, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202, USA
David G. Skalnik
Department of Veterinary Science, Konkuk University, Seoul, 05029, Korea
Hyunjung J. Lim
Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do, 24431, Korea
Seok-Ho Hong
KW-Bio Co., Ltd, Wonju, 26493, Korea
Seok-Ho Hong

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Seung Chel Yang
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Mira Park
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Kwon-Ho Hong
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Hyeonwoo La
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Chanhyeok Park
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Peike Wang
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Gaizhen Li
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Qionghua Chen
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Francesco J. DeMayo
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John P. Lydon
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Hyunjung J. Lim
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Seok-Ho Hong
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So Hee Park
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Contributions

S.C.Y. and H.S. conceived and designed the experiments. S.C.Y., M.P., H.L., P.W., C.P., G.L., Q.C., S.H.P., and Y.S.K. performed experiments. S.C.Y., M.P., K.-H.H., Y.C., F.J.D., J.P.L., D.G.S., H.J.L., S.-H.H., H.-R.K. and H.S. analyzed the results. S.C.Y., M.P., and H.S. discussed the results and wrote the manuscript.

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Correspondence to Haengseok Song.

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Yang, S.C., Park, M., Hong, KH. et al. CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis. Nat Commun 14, 3220 (2023). https://doi.org/10.1038/s41467-023-39008-0

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Received: 14 November 2022
Accepted: 24 May 2023
Published: 03 June 2023
DOI: https://doi.org/10.1038/s41467-023-39008-0

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