Alterations in the hepatocyte epigenetic landscape in steatosis

Fatty liver disease or the buildup of fat in the liver, has been reported to affect the global population. This comes with an increased risk for the development of fibrosis, cirrhosis, and hepatocellular carcinoma. Yet, little is known about the effects of a diet containing high fat and alcohol towards epigenetic aging, with respect to changes in transcriptional and epigenomic profiles. In this study, we took up a multi-omics approach and integrated gene expression, methylation signals, and chromatin signals to study the epigenomic effects of a high-fat and alcohol-containing diet on mouse hepatocytes. We identified four relevant protein-protein interaction network clusters that were associated with relevant pathways that promote steatosis. Using a machine learning approach, we predict specific transcription factors that might be responsible to modulate the functionally relevant clusters. Finally, we discover four additional CpG loci and validate aging-related differential CpG methylation. Differential CpG methylation linked to aging showed minimal overlap with altered methylation in steatosis. Reduced representation bisulfite sequencing (RRBS): Library preparation was conducted as described previously in a one-tube reaction [19]. Briefly, 500 ng genomic DNA was digested with MspI, repaired and A-tailed using Klenow fragment enzyme (NEB), subsequently Illumina TruSeq universal adaptors were ligated (T4 ligase, NEB). Bisulfite treatment was performed using the EZ DNA Methylation-Gold Kit (Zymo Res.) according to the manufacturer's protocol. Library preparation was accomplished by amplification with indexed TruSeq adaptor sequences (12 cycles) to add sample-specific 6bp identifiers. Sequencing was conducted on the HiSeq2500 (Illumina) on a 100 bp single-read flow cell aiming at 25-30 Mio reads per sample.