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. 2007 Mar 15;402(3):559-66.
doi: 10.1042/BJ20061194.

Histone acetyltransferase MOZ acts as a co-activator of Nrf2-MafK and induces tumour marker gene expression during hepatocarcinogenesis

Kumiko Ohta  1 , Megumi Ohigashi, Ayako Naganawa, Hiromi Ikeda, Masaharu Sakai, Jun-ichi Nishikawa, Masayoshi Imagawa, Shigehiro Osada, Tsutomu Nishihara
Affiliations

Histone acetyltransferase MOZ acts as a co-activator of Nrf2-MafK and induces tumour marker gene expression during hepatocarcinogenesis

Kumiko Ohta et al. Biochem J. .

Abstract

HATs (histone acetyltransferases) contribute to the regulation of gene expression, and loss or dysregulation of these activities may link to tumorigenesis. Here, we demonstrate that expression levels of HATs, p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] were decreased during chemical hepatocarcinogenesis, whereas expression of MOZ (monocytic leukaemia zinc-finger protein; MYST3)--a member of the MYST [MOZ, Ybf2/Sas3, Sas2 and TIP60 (Tat-interacting protein, 60 kDa)] acetyltransferase family--was induced. Although the MOZ gene frequently is rearranged in leukaemia, we were unable to detect MOZ rearrangement in livers with hyperplastic nodules. We examined the effect of MOZ on hepatocarcinogenic-specific gene expression. GSTP (glutathione S-transferase placental form) is a Phase II detoxification enzyme and a well-known tumour marker that is specifically elevated during hepatocarcinogenesis. GSTP gene activation is regulated mainly by the GPE1 (GSTP enhancer 1) enhancer element, which is recognized by the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2)-MafK heterodimer. We found that MOZ enhances GSTP promoter activity through GPE1 and acts as a co-activator of the Nrf2-MafK heterodimer. Further, exogenous MOZ induced GSTP expression in rat hepatoma H4IIE cells. These results suggest that during early hepatocarcinogenesis, aberrantly expressed MOZ may induce GSTP expression through the Nrf2-mediated pathway.

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Figures

Figure 1
Figure 1. Expression profiles of HAT during hepatocarcinogenesis
(A) The Solt–Farber protocol for chemically induced hepatocarcinogenesis in rats [37]. BD, basal diet; S, times at which rats were killed. (B) Expression profiles of HATs were investigated in control livers and those with hyperplastic nodules. Nuclear extracts were prepared from livers of various rats, and immunoblot analysis was performed with specific antibodies, as described in the Experimental section. GSTP in the cytosol fraction was also detected (bottom). The fractions shown in lanes 1 and 2 were from control rats; lanes 3–6, rats having livers with hyperplastic nodules; lane 7, rat treated with DEN only; lane 8, rat treated with AAF only; lane 9, rat underwent PH only.
Figure 2
Figure 2. Induction of the intact form of MOZ during hepatocarcinogenesis
(A) Nuclear extracts were prepared from control (lanes 1, 4 and 7) and livers with hyperplastic nodules (lanes 2, 3, 5, 6, 8 and 9), separated by SDS/PAGE (7.5% gel), and immunoblotted using polyclonal antibodies against the N- (lanes 1–3) or C- (lanes 7–9) terminal region of MOZ or the anti-MYST antibody (lanes 4–6). (B) The putative MOZ fusion partner, TIF-2, was detected with the anti-TIF-2 antibody. The fraction shown in lane 1 is from control; those in lanes 2 and 3 were from livers with hyperplastic.
Figure 3
Figure 3. MOZ activates the GSTP promoter activity through the GPE
(A) Diagram of the 5′-flanking region of the rat GSTP gene and the reporter constructs for observing the effect of MOZ on the promoter activity of the GSTP gene. (B) We co-transfected 100 ng of the reporter plasmid with (grey columns) or without (white columns) 1 μg of MOZ expression plasmid (pCI-MOZ) into H4IIE rat hepatoma cells. All transfection assays were repeated at least three times. The relative luciferase activity was calculated from mean values relative to the activity of −2.5GST-luciferase in the absence of MOZ. Each error bar indicates ±S.D. (C) Dose-dependent transactivation of −2.5GST-luciferase by MOZ. Relative luciferase activities are shown as in (B).
Figure 4
Figure 4. MOZ interacts with MafK in vitro and in vivo
(A) Structural domains of MOZ were indicated as follows: H15, histones H1- and H5-like module; MYST, MYST acetyltransferase domain; ED, glutamic acid/aspartic acid-rich acidic regions; S, serine-rich domain; P/Q, proline/glutamine-stretch; and M, methionine-rich domain. Also shown are the mutation positions in the PHD finger and MYST regions. Indicated wild-type and mutated in vitro-translated [35S]MOZ proteins were incubated with GST (lanes 2, 5 and 8) or GST–MafK (lanes 3, 6 and 9). MOZ protein retained on the GST-conjugated beads after extensive washing was analysed by SDS/PAGE and autoradiography. The amount of input (lanes 1, 4 and 7) is equivalent to 10% of the reaction volume in the assay. [35S]MOZ proteins are indicated by asterisks (*). (B) MOZ expression plasmid was co-transfected with HA-tagged MafK (lanes 1 and 3) or nontagged MafK (lanes 2 and 4) into HeLa cells, and nuclear extracts were prepared. Immunoprecipitation (IP) experiments were performed with anti-HA antibody. Immunoprecipitates (lanes 3 and 4) and 5% of input (lanes 1 and 2) were resolved by SDS/PAGE (7.5% gel) and detected by Western blotting using anti-N-terminal MOZ antibody. MOZ proteins are indicated by asterisks (*).
Figure 5
Figure 5. MOZ is a co-activator of Nrf2
Nrf2-mediated transactivation by MOZ was examined in mouse embryonic carcinoma F9 cells. We co-transfected 100 ng of the reporter plasmid (GPE1-luciferase, in the panel) and 5 ng of Renilla luciferase plasmid (phRL-tk) with 0, 0.3, 0.7 and 1 μg MOZ expression plasmid (pCI-MOZ) in the absence (–) or presence (+) of Nrf2 expression plasmid (pAβ2-Nrf2, 5 ng). The luciferase activity was normalized to Renilla luciferase activity. Relative luciferase activity was calculated from the mean values relative to the activity of GPE1-luciferase without Nrf2 and MOZ. Each error bar indicates ±S.D.
Figure 6
Figure 6. MOZ induces endogenous GSTP expression
H4IIE cells were transfected with 0, 0.4, 0.7 and 1.0 μg of MOZ expression plasmid (pCI-MOZ, lanes 1–4), and cell lysates were prepared. Endogenous GSTP and GAPDH (‘G3PDH’) were detected by immunoblotting.
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