Promoter hypermethylation of the AE2/SLC4A2 gene in PBC

Abstract

Graphical abstract Highlights• Patients with PBC have higher AE2 CpG methylation in upstream AE2a and/or AE2b2/AE2b1 promoter regions in liver and PBMCs.Background & Aims: Patients with primary biliary cholangitis (PBC) exhibit reduced AE2/SLC4A2 gene expression in the liver and peripheral blood mononuclear cells (PBMCs). AE2 encodes a Cl -/HCO 3 exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Reduced AE2 expression in PBC may be pathogenic, as Ae2-knockout mice reproduce characteristic PBC features. Herein, we aimed to identify CpG-methylation abnormalities in AE2 promoter regions that might contribute to the reduced gene transcription in PBC livers and PBMCs.Methods: CpG-cytosine methylation rates were interrogated at 1-base pair resolution in upstream and alternate AE2 promoter regions through pyrosequencing of bisulphite-modified genomic DNA from liver specimens and PBMCs. AE2a and alternative AE2b1 and AE2b2 mRNA levels were measured by real-time PCR. Human lymphoblastoid-T2 cells were treated with 5-aza-2´deoxycytidine for demethylation assays.Results: AE2 promoters were found to be hypermethylated in PBC livers compared to normal and diseased liver specimens.Receiver operating characteristic (ROC) curve analysis showed that minimal CpG-hypermethylation clusters of 3 AE2a-CpG sites and 4 alternate-AE2b2-CpG sites specifically differentiated PBC from normal and diseased controls, with mean methylation rates inversely correlating with respective transcript levels. Additionally, in PBMCs a minimal cluster of 3 hypermethylated AE2a-CpG sites distinguished PBC from controls, and mean methylation rates correlated negatively with AE2a mRNA levels in these immune cells. Alternate AE2b2/AE2b1 promoters in PBMCs were constitutively hypermethylated, in line with absent alternative mRNA expression in diseased and healthy PBMCs. Demethylation assays treating lymphoblastoid-T2 cells with 5aza-2´-deoxycytidine triggered AE2b2/AE2b1 expression and upregulated AE2a-promoter expression.Conclusions: Disease-specific hypermethylation of AE2 promoter regions and subsequent downregulation of AE2-gene expression in the liver and PBMCs of patients with PBC might be critically involved in the pathogenesis of this complex disease.

The Pathogenesis of Primary Biliary Cholangitis: A Comprehensive Review

“…Recently, Arenas et al discovered there is significant increase in CpG methylation in the promoter regions of AE2α, AE2b1, and AE2b2 in livers of patients with PBC when compared with controls. 118 Consequently, AE2 messenger RNA levels in the liver were also lower in PBC than controls. Since AE2 is involved in bicarbonate secretion, 33 reduction in AE2 expression can negatively affect the bicarbonate umbrella in the biliary tree, leading to bile duct injury.…”

Biliary Epithelial Senescence in Liver Disease: There Will Be SASP

“…(Hisamoto et al, 2016). Since loss of the bicarbonate umbrella, specifically through reduced AE2 expression, is a hallmark of PBC, this data is quite compelling (Arenas et al, 2019;Banales et al, 2012). In human samples, decreased expression of AE2 is correlated with increased bile duct senescence during PBC (Sasaki et al, 2018a), thus identifying that the bicarbonate umbrella may be an important component of PBC progression via increased senescence.…”

Primary biliary cholangitis: pathogenic mechanisms

“…Recently, we reported that there is a significant increase in CpG-cytosine methylation affecting selective promoter regions of the AE2 gene both in liver and PBMCs from PBC individuals [ 81 ▪ ]. Moreover, mean methylation rates inversely correlate with mRNA levels [ 81 ▪ ]. Of note, selective CpG-hypermethylation clusters in PBC affected relevant nuclear factor sites/motifs [ 81 ▪ , 82 – 84 ].…”

“…Moreover, mean methylation rates inversely correlate with mRNA levels [ 81 ▪ ]. Of note, selective CpG-hypermethylation clusters in PBC affected relevant nuclear factor sites/motifs [ 81 ▪ , 82 – 84 ].…”