Methylation of SOD2 in FHR PASMC is reversible by 5-AZA. (a) Schematic of the CG dinucleotide percentage and CpG islands within the SOD2 promoter and first 2 kb after the transcriptional start site. Seven amplicons were surveyed within the SOD2 gene. Their approximate locations are represented by solid horizontal lines. The positions of individual CpG dinucleotides are shown as vertical tick marks below the amplicon map. No methylated CpG pairs were identified in amplicons 3 to 6. *Differentially methylated CpG dinucleotides in FHRs within amplicon 7 vs BN1 tissue. (b) Corresponding methylation percentage of the differentially methylated CpG in intron 2. Results are expressed as a frequency of cytosine methylation in PAs from consomic control BN1 rats (n=2), FHRs (n=3), and FHRs treated with 5-AZA (FHR-Tx; n=3). (c) Representative sequencing traces of genomic DNA from cultured PASMCs. Only methylated cytidines are protected against bisulfite-mediated deamination of cytidine into uridine (which is recognized as thymidine when the polymerase chain reaction product is amplified). As indicated by the arrow, the cytidine in FHR PASMCs was methylated (and therefore remains a cytidine; top left); this is reversed by 5-AZA. The site is not methylated in FHR aortic SMCs or in SDR PASMCs. The bar graph shows the mean data indicating the reversibility and tissue specificity of this SOD2 methylation in intron 2 in cultured PASMCs. (d) FHR PASMCs have lower SOD2 mRNA levels vs consomic PASMCs. 5-AZA causes a dose-dependent increase in SOD2 expression. Reproduced with permission from Archer et al ;(1)Archer SL,Marsboom G,Kim GH,Zhang HJ,Toth PT,Svensson EC,Dyck JRB,Gomberg-Maitland M,Thbaud B,Husain AN, et al.Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target.Circulation. 2010 ;121(24):2661-71.
Additional keywords: SOD2,methylation,PASMC,fawn hooded rat,sequencing