title: Activation of endocrine-related gene expression in placental choriocarcinoma cell lines following DNA methylation knock-down.
abstract: Increasingly, placental DNA methylation is assessed as a factor in pregnancy-related complications, yet the transcriptional impact of such findings is not always clear. Using a proliferative in vitro placental model, the effect of DNA methylation loss on gene activation was evaluated at a number of genes selected for being differentially methylated in pre-eclampsia-associated placentae in vivo. We aimed to determine whether reduced DNA methylation at specific loci was associated with transcriptional changes at the corresponding gene, thus providing mechanistic underpinnings for previous clinical findings and to assess the degree of transcriptional response amongst our candidate genes. BeWo and JEG3 choriocarcinoma cells were exposed to 1 μM 5-Aza-2'-deoxycytidine (5-Aza-CdR) or vehicle control for 48 h, and re-plated and cultured for a further 72 h in normal media before cells were harvested for RNA and DNA. Bisulphite pyrosequencing confirmed that DNA methylation was reduced by ∼30-50% points at the selected loci studied in both cell lines. Gene activation, measured by qRT-PCR, was highly variable and transcript specific, indicating differential sensitivity to DNA methylation. Most notably, loss of DNA methylation at the leptin (LEP) promoter corresponded to a 200-fold and 40-fold increase in LEP expression in BeWo and JEG3 cells, respectively (P < 0.01). Transcripts of steroidogenic pathway enzymes CYP11A1 and HSD3B1 were up-regulated ∼40-fold in response to 5-Aza-CdR exposure in BeWo cells (P < 0.01). Other transcripts, including aromatase (CYP19), HSD11B2, inhibin (INHBA) and glucocorticoid receptor (NR3C1) were more moderately, although significantly, affected by loss of associated DNA methylation. These data present a mixed effect of DNA methylation changes at selected loci supporting cautionary interpretation of DNA methylation results in the absence of functional data.
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada Child & Family Research Institute, Vancouver, BC, Canada khogg@cfri.ca.
authors.forename: K
authors.initials: K
authors.lastname: Hogg
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada Child & Family Research Institute, Vancouver, BC, Canada.
authors.forename: W P
authors.initials: WP
authors.lastname: Robinson
authors.affiliation: Child & Family Research Institute, Vancouver, BC, Canada Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, BC, Canada.
authors.forename: A G
authors.initials: AG
authors.lastname: Beristain
mesh_terms: D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2 / Q000235:genetics; D001141:Aromatase / Q000235:genetics; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D002822:Choriocarcinoma / Q000235:genetics* / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D000077209:Decitabine; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D020738:Leptin / Q000235:genetics; D010920:Placenta / Q000187:drug effects / Q000378:metabolism*; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000235:genetics; D014594:Uterine Neoplasms / Q000235:genetics* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D020738:Leptin; C497713:NR3C1 protein, human; D011965:Receptors, Glucocorticoid; D000091262:TEA Domain Transcription Factors; C106169:TEAD3 protein, human; D014157:Transcription Factors; D000077209:Decitabine; D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; C468387:HSD11B2 protein, human; D001141:Aromatase; D001374:Azacitidine
keywords: DNA methylation; inhibin A; leptin; placenta; pre-eclampsia