Epigenetic mechanisms regulate Mallory Denk body formation in the livers of drug-primed mice

Abstract

The mechanism of Mallory Denk body formation is still not fully understood, but growing evidence implicates epigenetic mechanisms in MDB formation. In a previous study the epigenetic memory of MDB formation remained intact for at least 4 months after withdrawal from the DDC diet. In the present study, mice were fed a diet containing DDC or a diet containing DDC and S-adenosylmethionine (SAMe) to investigate the epigenetic memory of MDB formation. DDC feeding caused an increase in histone 3 acetylation, a decrease in histone 3 trimethylation, and an increase in histone ubiquitinylation. The addition of SAMe to the DDC diet prevented the DDC induced decrease of H3K4 and H3K9 trimethylation and the increase in histone ubiquitinylation. Changes in histone modifying enzymes (HATs and HDACs), were also found in the liver nuclear extracts of the DDC/SAMe fed mice. Data mining of microarray analysis confirmed that gene expression changed with DDC refeeding, particularly the SAMe metabolizing enzymes, Mat2a, AMD, AHCY and Mthfr. SAMe supplementation prevented the decrease of AHCY and GNMT, and prevented the increase in Mthfr, which provides a mechanism to explain how DDC inhibits methylation of histones. The results indicate that SAMe prevented the epigenetic cellular memory involved in the MDB formation.

Figure 1

Mouse liver sections showing MDB formation due to DDC feeding and a significant decrease of MDB formation when SAMe was added to the diet. (A): H&E liver section staining, ×400; (B, C, and D) Double stained for ubiquitin and cytokeratin 8 which colocalized in the MDBs (yellow) (arrows). The DDC Refed + SAMe showed only a few small MDBs (arrow) ×800

Figure 2

Histone 3 Acetylation analysis. Histones were isolated using acid extraction and then analyzed by SDS-Page electrophoresis and Coomassie Blue staining (A). The same amount of protein was used for Western blot to assess histone modifications (loading control). (B) Immunostaining for acetylated histone 3 at lysine 9, and (C) acetylated histone 3 at lysine 18.

Figure 3

(A) Histone acetyltransferase GCN5 levels in the liver of mice refed DDC (DDC R) and the liver of mice refed DDC with SAMe (DDC R SAMe). (B) Histone Deacetylase Sirtuin 1 (Sirt1) levels in the liver of mice DDC R and the liver of mice DDC R with SAMe. (Mean ± SE, n = 3)

Figure 4

H3K9me3 and K9me3 trimethylation. The histones were isolated from the nuclear extracts of mouse livers. Western blots were performed using antibodies against H3K9me3 (A) and H3K4me3 (B). (Mean ± SEM, n = 3).

Figure 5

(A) Histone methyltransferase (HAT) SUV39H1; (B) SET 7/9; (C) demethylase LSD1. Western blot; and (D) LSD1 Gene expression in the liver of DDC refed mice and the liver of DDC refed +SAMe mice. (Mean ±SE, n = 3).

Figure 6

Histone ubiquitinylation. Histones were isolated from the nuclear extracts of livers. Western blots were performed using antibodies against ubiquitin. DDC increased H2A (arrow) and this was prevented by SAMe. (Mean ± SEM, n = 3).

Figure 7

(A) Microarray analysis of SAMe metabolizing enzymes comparing DDC refed mice to the controls; (B) Mat2a increase in the liver of mice fed DDC; and (C) AHYC, S-adenosylhomocysteine hydrolase, decrease were confirmed by PCR and Western blot analysis. (D) The methyltransferase GNMT was significantly decreased in the liver of mice fed DDC and this decrease was restored by SAMe supplementation.

Figure 8

DDC Refeeding Increases the AMD Pathway (Red) and decreases the GNMT Pathway (green). DDC Refeeding Increases AMD and MTA Pathway, which Inhibits Methylation of H3K4me3 and decreases AHCY, which decreases methionine and SAMe methylation of histones. SAMe treatment reverses these two pathways to favor a histone trimethylation. (Modified from (Berger 2003)).