Fig 1: Intermittent fasting (IF) modulates aberrant changes in the global DNA methylation landscape under chronic cerebral hypoperfusion (CCH). (A) Heatmap of differentially methylated genes in the ad libitum (AL) CCH mice (BCAS) compared to the respective AL control mice (Sham) at the corresponding timepoints of 7, 15 and 30 days. Global changes in the gene methylation patterns were observed to deviate in the AL CCH mice compared to its respective Sham controls. (B) Heatmap of differentially methylated genes in the IF CCH mice compared to the respective AL CCH mice at the corresponding timepoints of 7, 15 and 30 days. The global trends in the gene methylation patterns was consistently distinct between the two groups as represented by the heatmap. Hypomethylated genes are indicated in blue while hypermethylated genes in red. The colour scale represents the mean methylation levels. (C) The circos representation graphs of the AL CCH mice across the 3 different timepoints showed a temporal increase in hypomethylated status as represented by the blue dots in the internal rim of the graphs. The methylation status of the differentially methylated genes was compared to the methylation levels of the respective time-specific AL Sham group. (D) The circos representation graphs of the IF CCH mice showed a temporal decrease in hypomethylated status with decreasing number of blue dots represented in the internal rim of the graphs. (E) A simplified diagram to capture the DNA methylation process and expression of DNA methyltransferases and TET1. Representative immunoblots and quantification illustrating significantly higher levels of DNA methyltransferases (DNMT1, DNMT3A, DNMT3B) and TET1 protein abundance in the IF CCH mice compared to the AL CCH mice and their respective Sham mice at the 30-day timepoint. The relative protein expression was all normalised to AL Sham samples. Vinculin was used as a loading control. Data are represented as mean ± SD. n=5-7 mice in each experimental group. Unpaired t-test, Bonferroni correction, *p<0.05 when compared with ALB30D.