Differential expression analysis of genes on WD. (A) Venn diagram showing that the expression of 395 genes changes at WD8, whereas the mRNA levels of 761 transcripts were altered at WD12 (overlap of 147 genes). (B) Ingenuity pathway analysis (IPA) of differential gene expression identified multiple upstream regulators. Genes activated by the presence of cholesterol were activated, whereas cholesterol synthesis and other enzymes regulated by SREBF2 were repressed in a negative feedback loop in both WD8 and WD12. Despite increased transcription of cholesterol-regulated genes, activity of NR1H3, activated by binding of oxysterol, a derivative of cholesterol, is only partially activated in WD8. Changes at the nuclear lamina associated with derepression of additional NR1H3 targets lead to full activation of LXR-dependent gene expression in WD12. Although cholesterol controls gene expression in WD8 and WD12 in the same direction, the activity of SREBF1 is repressed in WD8 but activated in WD12. (C) IPA also identified a network relating increased activity of lamin B1 (LMNB1) with reduced expression of cholesterol-synthesizing enzymes in WD8. (D) mRNA levels of Fasn and Acaca genes, SREBF1 targets crucial for lipogenesis, by quantitative RT–PCR. Expression of Fasn and Acaca does not change in livers of mice on WD8 but significantly increases in livers of mice on WD12, correlating to phenotypic changes (severe steatosis at WD12). (*) P-value < 0.05). (E) Pathways identified by IPA analysis of differential gene expression include “cholesterol biosynthesis” and “NRF2 stress response,” which are enriched in WD8, and “liver steatosis” and “FXR/RXR activation,” which are equally significant in both conditions. “Hepatic fibrosis,” “TGF β signaling,” and “liver damage” are highly enriched at WD12 before the fibrosis phenotype. (F) Lamin B1 ChIP-seq signal (RPKM) calculated at loci with gene expression changes in WD8 (left) and WD12 (right) is reduced in both conditions; hence, changes at the nuclear lamina are directly linked to altered gene expression in WD.
