Elsevier

Placenta

Volume 60, December 2017, Pages 64-73
Placenta

DNA methylation of amino acid transporter genes in the human placenta

https://doi.org/10.1016/j.placenta.2017.10.010Get rights and content

Highlights

  • This study implicates DNA methylation in the regulation of amino acid transporter gene expression.

  • In trophoblast-like cells, inhibition of DNA methylation altered the expression of specific amino acid transporters.

  • Promoter DNA methylation of human placental amino acid transporter genes remains low across gestation.

  • Specific amino acid transporter genes showed changes in both enhancer region methylation and expression across gestation.

Abstract

Introduction

Placental transfer of amino acids via amino acid transporters is essential for fetal growth. Little is known about the epigenetic regulation of amino acid transporters in placenta. This study investigates the DNA methylation status of amino acid transporters and their expression across gestation in human placenta.

Methods

BeWo cells were treated with 5-aza-2′-deoxycytidine to inhibit methylation and assess the effects on amino acid transporter gene expression. The DNA methylation levels of amino acid transporter genes in human placenta were determined across gestation using DNA methylation array data. Placental amino acid transporter gene expression across gestation was also analysed using data from publically available Gene Expression Omnibus data sets. The expression levels of these transporters at term were established using RNA sequencing data.

Results

Inhibition of DNA methylation in BeWo cells demonstrated that expression of specific amino acid transporters can be inversely associated with DNA methylation. Amino acid transporters expressed in term placenta generally showed low levels of promoter DNA methylation. Transporters with little or no expression in term placenta tended to be more highly methylated at gene promoter regions. The transporter genes SLC1A2, SLC1A3, SLC1A4, SLC7A5, SLC7A11 and SLC7A10 had significant changes in enhancer DNA methylation across gestation, as well as gene expression changes across gestation.

Conclusion

This study implicates DNA methylation in the regulation of amino acid transporter gene expression. However, in human placenta, DNA methylation of these genes remains low across gestation and does not always play an obvious role in regulating gene expression, despite clear evidence for differential expression as gestation proceeds.

Introduction

The placenta selectively mediates transfer of nutrients to the fetus, which is vital for optimal fetal development. Impaired placental amino acid transport results in reduced fetal growth [1], [2], which is associated with increased risk of adulthood disease [3]. Regulation of amino acid transporters occurs in response to short-term maternal stimuli [4], but could also occur across longer timescales. Epigenetic modification provides a potential mechanism for regulation of placental amino acid transporters, mediating early environmental influences across gestation [5]. The placenta has a unique DNA methylation profile with large regions of hypomethylation, containing developmental and tissue specific genes, interspersed with regions of high methylation [6]. The extent of epigenetic regulation by DNA methylation of functionally related genes such as amino acid transporters is unclear.

Accumulative, uniporter and facilitated amino acid transporters act in a coordinated way to transfer amino acids across the microvillous and basal plasma membrane (BM) of the placental syncytiotrophoblast [7], [8]. Placental amino acid metabolism also contributes to the rate of amino acid transfer [9], [10]. There are multiple classes of amino acid transporters, all of which are members of the SLC (solute carrier) gene series as outlined in Table 1 [11]. Accumulative transporters from the SLC1 (EAAT) [12], SLC7 (CAT) [15] and SLC38 (SNAT) [13], [14] families transport maternal amino acids into the syncytiotrophoblast. Amino acids are exchanged between the intracellular and extracellular pools by antiporters from the SLC1 (ASC) and SLC7 (LAT, y+LAT) families [16]. SLC16A10 (TAT1), SLC43A1 (LAT3) and SLC43A2 (LAT4) encode the BM facilitated transporters that mediate net amino acid transfer to the fetus, with SLC16A10 expression correlating with fetal and neonatal growth [7], [17].

Expression and activity of specific placental amino acid transporters increase across gestation [18], [19], to help meet increasing fetal demand as gestation progresses [4], [20]. Maternal factors such as diet, smoking and vitamin D levels can alter placental amino acid transporter gene expression [17], [21], potentially via DNA methylation. Indeed, maternal nutrition [22], obesity [23], stress [24], toxin exposure [25] or smoking [26] during pregnancy, influence placental DNA methylation.

DNA methylation could mediate the placental adaptations to early exposures and increased fetal demand for nutrients across gestation [27]. Placentas from both small and large for gestational age babies have altered methylation profiles for specific genes [28], [29]. In addition, global methylation in human placenta is reduced with gestational diabetes or preeclampsia and increased with maternal obesity [30], [31]. The early environment influences placental glucose transport, potentially by epigenetic regulation of human placental glucose transporters. Indeed, associations between DNA methylation and glucose transporter expression have previously been demonstrated across gestation [32], [33], [34].

Taken together these studies suggest that DNA methylation may have a role in regulating placental nutrient transport. We therefore investigated whether this epigenetic mechanism is involved in regulating amino acid transporters in the human placenta.

Section snippets

Amino acid transporter DNA methylation in BeWo and HEK293 cells

Cell culture: BeWo human choriocarcinoma cells from the HPA Culture Collections (Salisbury, UK) or Human Embryonic Kidney 293 cells (HEK293) were cultured in DMEM/Ham's F12 (1∶1) media (with HEPES and l-glutamine, with phenol red) supplemented with 500 iu/ml penicillin, 500 iu/ml streptomycin, 1 mM glutamine and 10% fetal bovine serum (FBS; All Lonza). Cells were cultured in 32 mm wells at a density of 2.5 × 105 at 37 °C in a humidified incubator (5% CO2 in air). At 24 h cells were incubated

Results

This study aimed to investigate the DNA methylation and expression of 25 amino acid transporter genes (Table 1) in human placenta as follows. Accumulative transporter genes: SLC1A1, SLC1A2, SLC1A3, SLC1A6, and SLC1A7 (EAATs); SLC7A1 (CAT1); and SLC38A1, SLC38A2, SLC38A3, SLC38A4 and SLC38A6 (SNATs). Antiporter genes: SLC1A4 and SLC1A5 (ASCs); SLC7A5 and SLC7A8 (LATs); SLC7A6 and SLC7A7 (y+LATs); SLC7A9, SLC7A10 and SLC7A11; and the auxiliary proteins SLC3A1, and SLC3A2. Facilitated transporter

Discussion

This study investigated DNA methylation of amino acid transporter genes in the placenta. In a trophoblast derived cell line, we observed changes to the expression of specific amino acid transporters following inhibition of DNA methylation. However, it is unclear whether these observations are due to direct effects on DNA methylation within the transporter genes themselves or methylation of indirect regulatory factors. Cohort data revealed that promoter DNA methylation of the placental amino

Funding

CS was supported by the Gerald Kerkut Charitable Trust.

Authors' roles

BN, KL, NC, RS, RL, JC: participation in study design. CS, BN, RS, JC: participation in study execution and analysis. CS, BN, KL, CB, NH, CC, RS, RL, JC: participation in manuscript drafting and critical discussion.

Conflict of interest

None to declare.

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