VPA inhibitor

Gestational valproic acid exposure induces epigenetic modifications in murine decidua

Sidra Shafique 1, Louise M Winn 2

Abstract

Introduction: Valproic acid (VPA), a widely prescribed antiepileptic drug and an effective treatment for bipolar disorder and neuropathic pain, results in multiple developmental defects following in utero exposure. Uterine decidua provides nutritional and physical support during implantation and early embryonic development. Perturbations in the molecular mechanisms within decidual tissue during early pregnancy might affect early embryonic growth, result in early pregnancy loss or cause complications in the later gestational stage. VPA is a known histone deacetylase inhibitor and epigenetic changes such as histone hyperacetylation and methylation have been proposed as a mechanism of VPA-induced teratogenesis.
Methods: This study investigated the effects of in utero VPA exposure on histone modifications in murine decidual tissue. Pregnant CD-1 mice were exposed to 400 mg/kg VPA or saline on GD9 via subcutaneous injection. Decidual tissue from each gestational sac was harvested at 1, 3 and 6 h following exposure. Levels of acetylated histones H3, H4 and H3K56, as well as methylated histones H3K9 and H3K27 were acid extracted and assessed by western blotting followed by acid histone extraction.
Results: VPA exposure induced a significant increase (p < 0.05) in the levels of acetylated H3 at 1, 3 h; acetylated H4 at 1, 3 and 6 h and trimethylated H3K9 at 6 h. In contrast, no significant perturbations were noted in the levels of monomethylated H3K9, trimethylated H3K27 and acetylated H3K56. Discussion: The results from this study suggest that VPA-induced decidual histone modifications might play an important role as a mechanism of VPA-induced teratogenesis during early embryonic growth. Keywords: Valproic acid Decidua Histones Acetylation Methylation 1. Introduction Epigenetics is a combination of molecular modifications and the resulting processes that regulate genome activity independent of any alteration in DNA sequence [1,2]. Epigenetic-induced alterations in gene expression have a critical role in regulating cellular genomic activity and the differentiation and development of an organism [1,2]. Euchromatin or loosely packaged DNA is actively transcribed while the genes in the heterochromatin or condensed DNA are repressed [3]. Structurally, DNA is wrapped around histone octamers to form a nucleosome [4]. Each nucleosome contains approximately 146 bp of DNA combined with a histone octamer having two of H2A, H2B, H3 and H4 histones [1] [Fig. 1A]. The histone amino (N)-terminal tails protrude out from their own nucleosome and modification of these tails affect inter-nucleosomal interactions thus modifying the overall chromatin configuration [5,6]. The most studied histone modifications include methylation, acetylation and phosphorylation [6], with euchromatin generally associated with high levels of histone acetylation [3]. The interaction between the wrapped DNA and the nucleosomes is reduced by the acetylation of lysine residues and the overall positive charge on basic histone proteins thus increasing the accessibility of the transcription factors [7]. On the other hand, the heterochromatin state is attained through either deacetylation or methylation [8]. Regulation of gene expression through methylation is determined by the location of methylated amino acids in a specific histone such as the trimethylation at lysine 4 of histone H3 (H3) activates transcription while the trimethylation at lysine 9 of H3 represses gene expression [8]. Thus, the altered epigenetic landscape would predict the trends in the gene expression induced any environmental toxicant such as valproic acid (VPA). VPA is clinically used for the treatment of epilepsy and mood disorders in women of reproductive age [9,10]. At the same time, VPA is a known teratogen and its gestational exposure can cause birth defects such as neural tube, cardiac and musculoskeletal defects in the offspring [11]. VPA can induce epigenetic modifications through the inhibition of histone deacetylases (HDACs) [12]. VPA-induced HDAC inhibition as a mechanism of epigenetic modification is an example of an epigenetic pathway. In this epigenetic pathway, VPA could be considered as an epigenator, HDAC as an epigenetic initiator while hyperacetylation and other histone modifications as epigenetic maintainers [13]. VPA-induced hyperacetylation is proposed as one potential teratogenic mechanism of action of VPA, as evidenced by somite hyperacetylation and associated axial abnormalities following VPA exposure in a mouse model [12,14]. In addition to H3 and H4 hyperacetylation, VPA exposure results in alterations in methylated histone levels such as H3K9me1 and H3K4me2,3 in gestational day (GD)9 mouse embryos [15] [Fig. 1B]. VPA-induced H3 and H4 hyperacetylation is also associated with dysregulated gene expression in embryonic stem cells [16]. Similarly, a study has shown an increase in the levels of acetylated histones H3 and H4 and altered mRNA expression of major carriers for folic acid, glucose, choline, thyroid hormones, and serotonin in human placenta [17]. Histologically, VPA induces cell damage in rat decidua and placenta following in utero exposure [18], thus indicating its effect on these tissues. However, the effects of VPA on epigenetic modifications in the decidua remains unknown. Decidua plays a key role in the growth and nutrition of the growing embryo during the period between implantation and placental development [18]. The process of “decidualization” consists of uterine endometrial stromal cells remodeling to decidual cells [19]. In the mouse model, the decidualization extends from the antimesometrial pole to the mesometrial end in the uterine wall [19]. Formation of decidua is stimulated on GD4.5 and remains as functional tissue until the development of the placenta [19]. A well-established decidua is a prerequisite for the successful implantation of the growing embryo and the formation of a functional placenta. By GD10.5, a placenta with a complete structure has been formed in mice [20]. The present study aimed at examining VPA-induced epigenetic modifications following gestational exposure in mouse decidua. The selected experimental dose of VPA (400 mg/kg) has already been established as a teratogenic dose that would induce histone modifications following gestational exposure on GD9 in the CD-1 mouse model [15]. Moreover, the decidual tissue is fully functional and established in the pregnant uterus by GD9. In this study, acetylation and methylation histone modifications were studied at the 1, 3 and 6 h post-exposure time points. 2. Methods and materials 2.1. Experimental animals CD-1 female virgin mice aged six to eight weeks were purchased from Charles River Laboratories (St. Constant, QC, Canada) and housed in the Queen’s University Animal Care Facilities. Mice were maintained in a temperature-controlled room with a 12 h light/dark cycle and acclimatized for one week before breeding. Animals were provided with standard rodent chow (Purina Rodent Chow, Ralston Purina International, Strathroy, ON, Canada), and tap water ad libitum. Breeding was done by housing together a maximum of two female mice with one male CD-1 mice (Charles River Laboratories Inc., St. Constant, QC, Canada) overnight. The presence of a vaginal plug the next morning was designated as GD1 and females with a positive vaginal plug were housed together. The housing and breeding practices were conducted in accordance with the guidelines of the Canadian Council on Animal Care. All the experimental procedures were approved by the Queen’s University Animal Care Committee and in compliance with the guidelines of the Canadian Council on Animal Care. 2.2. Animal treatment Pregnant CD-1 mice were injected subcutaneously with 400 mg/kg of VPA (Sigma-Aldrich Canada Ltd., Oakville, Canada) or the vehicle control (0.9% saline) on the morning of GD9. The selected VPA dose had already been established as a teratogenic dose and shown to induce histone modifications in GD9 VPA-exposed embryos [15,21]. This study was aimed to further investigate VPA-induced epigenetic changes in the decidua on the same gestational day of GD9 and 1, 3 and 6 h post-exposure time points as the previous study. Following exposure pregnant mice were sacrificed on GD9 by cervical dislocation followed by puncturing the pleural cavity at the designated post-exposure time point. The decidual swellings were dissected out as previously described [22]. Briefly, the uterine wall was incised along the antimesometrial border and the decidual swellings carefully separated from the uterine wall. Embryos were removed with extraembryonic tissues [23]. and the empty decidual swelling s, three to five per litter, were then pooled together for the histone extraction. The decidual tissue was harvested from both the treated and control groups with each n value denoting one pregnant dam or one litter. 2.3. Histone extraction and western blotting The harvested and pooled decidual tissue was homogenized in triton extraction buffer consisting of the PBS with 0.5% Triton X (v/v), 0.02% sodium azide (w/v) and ROCHE cOmpleteTM Protease Inhibitor Cocktail tablet (Thomas Scientific, New Jersey, United States). The homogenate was cold centrifuged, resuspended in 0.2 N HCl overnight and centrifuged again the following morning to perform histone acid extraction. The supernatant was removed and the protein concentration was quantified by the Bradford assay. Samples were then stored at − 80 ◦C till further use. Histones (2.5ug) were separated on a 15% polyacrylamide gel at 100 V for 1 h and 40 min. The proteins were transferred to a 0.45 μm Low Fluorescence Polyvinylidene fluoride (LF- PVDF) membrane by a wet transfer at 100 V for 1 h at 4 ◦C. Membranes were then blocked using 5% bovine serum albumin (BSA) and probed overnight with the primary antibodies. The primary antibodies used for probing included anti-histone 3 mouse (1:10 000; Abcam, Cambridge, Massachusetts), anti-histone H4 mouse (1:10 000; Abcam, Cambridge, Massachusetts), anti-acetyl- histone H3 rabbit (1:10 000; Millipore Sigma, Temecula, California), anti-hyperacetylated histone H4 rabbit (1:10 000; 06-946, Millipore Sigma, Temecula, California), anti-monomethyl histone H3K9 rabbit (1:1000; Abcam, Cambridge, Massachusetts), anti-trimethyl histone H3K9 rabbit (1:10 000; Abcam, Cambridge, Massachusetts), anti- trimethyl histone H3K27 rabbit (1:40000; Millipore Sigma, Temecula, California) and anti-acetyl histone H3K56 rabbit (1:1000; Millipore Sigma, Temecula, California). The membranes were washed four times for 5 min each in tris-buffered saline containing Tween (TBST) (25 mM tris–HCl, 140 mM NaCl, 2 mM KCl, 0.1% Tween) followed by incubation with the appropriate secondary antibody for 1 h. The secondary antibodies used were Goat anti-rabbit IgG H&L (Cy3®) (1:10 000; ab6939, Abcam, Cambridge, Massachusetts) and goat anti-mouse IgG H&L (Cy5®) (1:10 000; ab6563, Abcam Cambridge, Massachusetts) in the blocking solution. The membranes were then washed four times for 5 min each in TBST and imaged using the Azure Biosystems c600 machine. The protein of interest was matched to the loading control depending on the histone tail being detected. The proteins were quantified by the densitometry using the ImageJ and the protein of interest normalized to the respective loading control. 2.4. Statistical analysis GraphPad Prism (Version 8) was used to conduct the statistical analysis. Unpaired t-tests were used to determine the statistical difference between control and treated groups while p < 0.05 was deemed as a statistically significant difference. The data has been presented as the protein of interest levels relative to control values. Each datapoint in the scatter plots indicates an independent n value while the error bars represent the standard deviation (SD). 3. Results 3.1. Acetylated histone H3 levels are upregulated following VPA exposure As VPA is an HDACi and induces histone hyperacetylation following in vitro and in vivo exposure [14,24], hyperacetylation is considered a hallmark of VPA effects on tissue. Following gestational exposure to VPA on GD9, levels of AcH3 were assessed by Western blotting on decidual tissue from CD-1 mice. A significant increase in the acetylation of histone H3 by VPA was noted at 1 and 3 h post-exposure in the treated group (Fig. 2A, B). The elevated levels of AcH3 observed at 1 h post-exposure (Fig. 2A) (p = 0.0003) remained upregulated at 3 h (Fig. 2B) (p = 0.0055), but returned to control levels by 6 h post-VPA exposure (Fig. 2C). 3.2. Acetylated histone H4 levels are upregulated following VPA exposure Similar to AcH3 levels, acetylated histone H4 (AcH4) levels were also upregulated in decidual tissue following VPA exposure (Fig. 3). However, it was noted that the increase in AcH4 levels remain persistent at all the examined timepoints. The noted VPA-induced hyperacetylation in decidual tissue is consistent with previous results reported in GD9 embryonic tissue (Tung 2010). The examined elevated acetylation of histone H4 was highly significant at 1 h (Fig. 3A) (p = 0.0001) and 3 h (Fig. 3B) (p = 0.0004) post-exposure. The increased levels of AcH4 remained persistent at 6 h (Fig. 3C) (p = 0.0022) in contrast to those of AcH3 (Fig. 2C). 3.3. Monomethylated histone H3K9 levels remain stable following VPA exposure In addition to acetylation, the effect of VPA exposure on the levels of monomethylated histone H3K9 (H3K9me1) was also assessed in the decidual tissue (Fig. 4). Results demonstrated that there were no significant VPA-induced changes in the expression of H3K9me1at any time point including 1 h (Fig. 4A) 3 h (Fig. 4B) and 6 h (4C). 3.4. Trimethylated histone H3K9 levels are upregulated following VPA exposure The levels of the trimethylated histone H3K9 (H3K9me3) were also examined in GD9 mouse decidual tissue following gestational VPA exposure (Fig. 5). There was no significant difference in H3K9me3 expression at 1 and 3 h (Fig. 5A, B). However, an increase in the expression of H3K9me3 was noted at 6 h post-exposure (Fig. 5C) (p = 0.0260). 3.5. Trimethylated histone H3K27 levels remain stable following VPA exposure Western blot assessment of trimethylated histone H3K27 (H3K27me3) indicated that VPA exposure had no effect on the status of H3K27me3 in the treated group as compared to controls (Fig. 6). The levels of H3K27me3 in the decidual tissue remained stable at 1 h (Fig. 6A), 3 h (Fig. 6B) and 6 h (Fig. 6C). 3.6. Acetylated histone H3K56 levels remain stable following VPA exposure To understand the effects of VPA on histone H3 acetylation at lysine 56, the levels of acetylated histone H3K56 (AcH3K56) was examined (Fig. 7). The data indicated that gestational VPA exposure does not alter the AcH3K56 levels at any of the examined post-exposure time point including 1 h (Fig. 7A), 3 h (Fig. 7B) and 6 h (Fig. 7C). 4. Discussion The present study informs on the early and significant epigenetic modifications that occur in murine decidua following gestational VPA exposure. The decidual tissue is present between the embryonic compartment and uterine myometrium and plays a key role in the implantation, growth and survival of the embryo through the provision of nutrients, protection against maternal immune cells and structural support [25]. As the decidual tissue has an interim role in embryonic growth following implantation and before the placental development, we were interested to investigate whether VPA induced epigenetic modifications in this tissue that might affect the ultimate pregnancy outcome. We are the first to report that gestational VPA exposure induces a significant upregulation in acetylated histone H3 (AcH3), acetylated histone H4 (AcH4) and trimethylated histone H3K9 (HK9me3) [Figs. 2, 3, 5] in CD-1 mouse decidual tissue. Decidua, a tissue that lies between the chorionic vesicle and the uterine wall, contains abundant sinusoids and arterial channels [25]. and it has been reported that gestational VPA exposure causes distinct histopathological changes in the decidua [26]. Following VPA exposure in a rat model, the cells lining the maternal sinusoids in the decidua basalis are damaged, desquamated, and washed away by the arterial circulation through the afferent channels [26]. In contrast, the necrosed cells with their walls still intact occlude the lumen of these arterial channels at the point of their entry into the giant cell-trophospongial zone [26]. Ultimately, these VPA-induced histopathological changes in the decidual tissue are thought to lead to ischemia and disruption of the homeostasis of blood circulation followed by a defective placentation [26]. VPA-induced morphological changes in the decidual development during early pregnancy might result in a pregnancy loss or associated complications at a later gestational stage [25,27]. Epigenetic modifications are proposed as one potential mechanism of VPA’s underlying teratogenic mechanism [15], therefore we speculated that VPA-induced morphological changes in the decidual tissue might be concurrently associated with VPA-induced histone modifications. Therefore, in the present study, we used the previously established CD-1 mouse model which develops neural tube defects in the embryos and has dysregulated gene expression and histone modifications following in utero VPA exposure on GD9 [15,28]. Acetylation of histone tails regulates gene expression by neutralizing the positive charge of the lysine residue by increasing the local and specific DNA sequence accessibility for transcription factors [29]. The acetylation of the four lysines (K5, K8, K12, and K16) on the N-terminal tail results in increased transcription as a cumulative outcome effect [29]. Histone H4 acetylation has been associated with specific chromatin activities such as gene regulation, DNA repair and chromatin remodeling [30]. Evidence suggests a key role for histone modifications in the dynamic environment of the decidual tissue [31]. Within days after implantation of the embryo, the stromal cells of the endometrium undergo a striking transformation called the decidual reaction [25]. A study using decidual cell reaction as a model of cellular differentiation showed a strong association between stromal cell differentiation and histone modifications including acetylation and phosphorylation of histones [31]. Specifically, the study showed the preferential acetylation of H2B and H4 along with the phosphorylation of H1, H2A and H3 in differentiating stromal cells within the decidua, indicative of the profound role of epigenetics in decidual function [31]. As previously indicated, VPA induces epigenetic modifications of histones including acetylation and methylation during mouse embryonic development [15]. Specifically, VPA induced hyperacetylation dysregulates gene expression of functional genes related to cell growth and proliferation [32]. In the current study, we investigated the effects of VPA on the overall acetylation of histone H3 and H4 in the mouse decidua [Fig. 3]. Our results demonstrating histone H3 and H4 hyperacetylation [Figs. 2 and 3] in decidua tissue following VPA exposure are in agreement with previously reported hyperacetylation in the mouse embryo [14,15]. It is worth noting that over the investigated time points of 1, 3 and 6 h following VPA exposure, H3 hyperacetylation returns to baseline by 6 h post-exposure [Fig. 2c] while that of H4 remains persistent till the latest point of examination i.e. 6 h [Fig. 3c]. As a future direction, it would be interesting to study H4 hyperacetylation at farther time points beyond 6 h to know the temporal persistence of VPA’s effects on decidual tissue. In addition to examining the overall acetylated status of H3 [Fig. 2], we also investigated the specific acetylation status of H3 on K56 [Fig. 7]. H3K56 acetylation (AcH3K56) is conserved from yeast to mammals including mouse embryonic stem cells, rat (Rat 1 a) and human (H1299) cells [33]. Importantly, AcH3K56 is a transient chromatin signal demonstrating rapid turnover and is closely linked to active DNA replication and transcription [34]. In spite of the noted significant increase in acetylated H3 levels [Fig. 2], the AcH3K56 levels remained unchanged at all time points [Fig. 7]. Given that H4 hyperacetylation is directly involved in programmed cell death [30]. the observed unchanged H3K56ac while hyperacetylated H4 status by VPA could be related to halted cell proliferation and cell death in the decidua, which would be consistent with the previously observed data demonstrating VPA-induced histopathological cell necrosis in the rat decidua [26]. Along with the known VPA-induced histone acetylation changes, previous studies have also found that histone methylation levels are perturbed following VPA exposure in mouse embryos as demonstrated by increased methylated H3K4 and decreased monomethylated H3K9 levels [15]. Therefore, here we investigated the levels of trimethylated H3K9 (H3K9me3) [Fig. 5], monomethylated H3K9 (H3K9me1) [Fig. 4] and trimethylated H3K27 (H3K27me3) [Fig. 6] following VPA exposure in decidua tissue in. Our data indicate a significant increase in H3K9me3 levels [Fig. 5] while H3K9me1 [Fig. 4] and H3K27me3 [Fig. 6] levels remain unchanged. H3K9me3 is the epigenetic modification that has been best identified with heterochromatin where the heterochromatin state of genome is associated with restricted transcription and gene silencing [35] [Fig. 1]. It is known that H3K9me3-dependent heterochromatin undergoes dramatic reprogramming during early embryonic development [36] and plays an important role in the process of stem cells transitioning to differentiating cells [37]. As development progresses, the pluripotency-associated genes are silenced, while the genes involved in the alternative cell fates become activated [38]. Given that the cells within the decidua tend to differentiate into developing placenta related cell lineages, the endometrial stromal cells in the decidual tissue experience a remarkable transformation to support embryonic development, placental formation, and the maintenance of normal pregnancy in both mice and human [19]. In fact, cells with more heterochromatin tend to differentiate and at the same time have reduced levels of cell proliferation [37]. Thus, the observed increased level of H3K9me3 in VPA exposed decidual tissue found in our study could influence the requirements for adequate cell proliferation for angiogenesis and stromal growth in the dynamic intrauterine environment, potentially leading to early or premature cellular differentiation. Together, decidual changes leading to defective angiogenesis followed by restricted placental development could be an underlying mechanism of the reported VPA-associated intrauterine growth retardation [39]. We also speculate that upregulated H3K9me3 following VPA exposure might also affect the precursor cells of the placenta, their proliferation and invasion thus resulting in the deterioration of placental tissue impacting the nutritional support of the embryo. Results from future studies would help strengthen these ideas. The methylation status of H3K9 alternates from trimethylated to di- or mono-methylated and is regulated by methyltransferases during embryonic development [35,40]. A study on the human genome indicated that H3K9me3/me2 signals were prominent in silenced genes while H3K9me1 levels were higher in active gene promoters, indicating that H3K9me1 might be associated with transcriptional activation [41]. In the current study, in contrast to a significant upregulation of H3K9me3 levels at 6 h [Fig. 5], H3K9me1 levels remained stable at all time points following VPA exposure [Fig. 4]. Thus, in context with the upregulated H3K9me3 and expected repressed transcription, the stable H3K9me1 levels suggest an overall suppression of gene transcription via increased methylation of H3K9 by VPA in the decidual tissue. Previous studies support the notion that H3K27me3 dynamics in mouse decidual stromal cells (DSCs) during early gestation serve to provide protection to the conceptus against maternal tissue immune reactions [42]. In the mouse decidua, the active methylation and demethylation states of H3K27me3 have been shown to regulate target gene upregulation and decidual activation particularly with respect to markers or positive regulators of fibroblast activation, myofibroblast formation, or fibrosis [42]. In spite of the known distinct role of H3K27me3 in murine decidua, we observed no changes in the levels between control and VPA-exposed decidual tissue at any time point [Fig. 6], which is in agreement with the already reported unchanged levels in VPA-exposed mouse brains [43]. Taken together, the upregulated status of H3K9me3 with unchanged H3K9me1 and H3K27me3 levels, may might indicate an overall trend of gene repression following VPA exposure in murine decidua. Interestingly our reported epigenetic changes induced by VPA exposure indicate that there is an early hyperacetylation at 1 and 3 h post-exposure [Figs. 2 and 3] and a relatively late methylation induction at 6 h [Fig. 5]. As mentioned before, the hyperacetylation of histones H3 and H4 is associated with activate transcription while the upregulated H3K9me3 with repressed transcription. Therefore, it can be inferred that in the murine decidua, early VPA induced transcription is suppressed later on possibly leading to limited gene expression, cell growth and cell proliferation. Given that VPA is an HDAC inhibitor and causes hyperacetylation [24], the phenomenon of temporal VPA induced gene repression might be adaptive as well. Moreover, VPA-induced acetylation of H4 persists till 6 h which is the latest examined time point in this study [Fig. 3C], however, it is possible that this could persist for a longer period, therefore investigating AcH4 levels at later time points would be informative. Similarly, we have reported that H3K9me3 levels are only affected at the 6-h time-point [Fig. 5C], thus warranting studies investigating later time points. 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