Regulation and function of histone acetyltransferase MOF

Abstract

The mammalian MOF (male absent on the first), a member of the MYST (MOZ, YBF2, SAS2, and Tip60) family of histone acetyltransferases (HATs), is the major enzyme that catalyzes the acetylation of histone H4 on lysine 16. Acetylation of K16 is a prevalent mark associated with chromatin decondensation. MOF has recently been shown to play an essential role in maintaining normal cell functions. In this study, we discuss the important roles of MOF in DNA damage repair, apoptosis, and tumorigenesis. We also analyze the role of MOF as a key regulator of the core transcriptional network of embryonic stem cells.

Keywords

MOF / histone acetyltransferase / DNA damage repair / tumorigenesis / embryonic stem cells

Introduction

Males absent on the first (MOF), also known as MYST1 or KAT8, is a highly conserved histone acetyltransferase (HAT) of the MOZ, YBF2, SAS2, and TIP60 (MYST) family. MOF has strict substrate specificity for lysine 16 of nucleosomal histone H4 (H4K16) when in a complex with several evolutionarily conserved proteins [1–3]. MOF was originally described as an essential component of the X chromosome dosage compensation complex in Drosophila [4–6] and initially purified from an MSL-containing complex [1]. Loss of MOF in Drosophila and mammals results in a global reduction in H4K16 acetylation and causes genetic instability, chromosomal abnormalities, defects in the cell cycle, and decreased levels of gene transcription [7–9]. The defects caused by the loss of MOF suggest that MOF is the major HAT for this site [2–5] (Fig. 1). In addition, loss of MOF leads to the severe arrest of the cell cycle at the G2/M phase, massive chromosome aberrations, and defects in the repair of DNA damage induced by ionizing radiation. These findings indicate that MOF has a crucial role in embryogenesis, tumorigenesis, cell proliferation, and DNA damage repair (DDR) [9,10]. MOF may be essential for vertebrate development, because loss of MOF in mice caused peri-implantation lethality [9].

In vitro studies have shown that MOF participates in two distinct and evolutionarily conserved complexes in mammals: MOF-MSL and MOF-MSL1v1 [11–14]. MOF-MSL comprises three components: MSL1, MSL2, and MSL3. Studies have revealed that the specific and efficient acetylation of H4K16 by MOF depends on its interaction with MSL1 and MSL3 [15]. By contrast, the MOF-MSL1V1 complex, which includes WDR5 (the crucial component of the H3K4 methyltransferase MLL), MOF, MSL1V1, PHF20, and MCRS1/2, acetylates not only the histone substrate H4 but also the non-histone substrates p53 and YY1. The MOF-MSL1V1 complex physically interacts with the MLL complex through the common component, WDR5, and associates with MLL to activate transcription [11,14].

MOF has been reported to participate in DDR, apoptosis, and tumorigenesis [13]. In previous studies, MOF-MSL1v1 complex, not the MOF-MSL complex, was found to be specifically required for the acetylation of K120 on p53, a tumor suppressor protein, and regulates apoptosis independent of transcription [14].

Recent studies have shown that MOF mutant embryonic stem cells (ESCs) are incapable of self-renewal and form defective embryonic bodies. These defects are accompanied by a global reduction in H4K16 acetylation and a global change in the ESC transcriptome [16]. MOF plays an essential role in the maintenance of ESC self-renewal and pluripotency. MOF, as a key regulator, directly regulates the expression of the core ESC transcription factors Nanog, Oct4, and Sox2, and the phenotypes of MOF null mice can be partially rescued by ectopic expression of Nanog [16].

Regulation of the acetyltransferase activities of MOF

Several studies have shown that MOF alone cannot function as an acetyltransferase. As previously stated, MOF is a component of two distinct and evolutionarily conserved complexes: MOF-MSL and MOF-MSL1v1 [13]. The acetyltransferase activity of MOF is strictly regulated by these two complexes, which have distinct compositions and very different substrate specificities. For instance, MOF-MSL1v1 interacts with H3K4 methyltransferase MLL to promote the reciprocal recruitment of each protein and initiate transcription of the p53 gene [14]. This coordination is likely mediated by WDR5 [14]. Recent studies have shown that MOF can be autoacetylated at K274, and this modification considerably affects the catalytic activity of MOF [17]. The autoacetylation activity of MOF is decreased by mutations at C316 or E350, which suggests that these two residues also participate in the autoacetylation of MOF [18,19]. MOF is a substrate of SIRT1, which is a histone H4K16 deacetylase and can also deacetylate non-histone proteins [20]. Lu et al. further found that modulation of MOF autoacetylation by SIRT1 can affect the recruitment of MOF and its activity on chromatin [20]. Moreover, SIRT1 also promotes the ubiquitination of MOF at K432 and K444 and the subsequent proteasome-dependent degradation of MOF protein [21]. FOXP3 is a key transcriptional regulator of the development and function of regulatory T cells. Hiroto et al. recently determined that targeted mutation of FOXP3 disrupts the nuclear localization of MOF [22]. FOXP3 overexpression increases both histone H4K16 acetylation and histone H3K4 trimethylation on the chromatin of multiple FOXP3 target genes by recruiting MOF and displacing PLU-1 [22].

MOF and DDR

Double-strand breaks are the most serious form of DNA damage. The pathways for DDR include homologous recombination (HR) and non-homologous end joining (NHEJ). Several studies have confirmed that MOF is involved in the repair of IR-induced DNA damage through both NHEJ and HR [23,24]. Evidence suggests that the underlying mechanism of MOF in DDR could involve the interaction with DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which participates in the NHEJ pathway. However, the activity of MOF does not affect the expression of the DDR-related protein [24]. Although the results of some studies using RNA interference-mediated knockdown of MOF suggest that MOF affects signaling by ATM and phosphorylation of H2A.X [23], other studies have obtained contrasting results [25]. Li et al. recently observed severe arrest of the cell cycle at the G2/M phase in MOF-knockout cells and genome instability in cell lines derived from embryonic fibroblasts of MOF-conditional knockout mice. Although MOF deletion does not influence ATM signaling or H2A.X foci formation, it remarkably affects the recruitment of Mdc1 and other downstream proteins that mediate repair, such as 53BP1 and Brca1, to the DNA damage foci [10].

MOF regulates apoptosis

The tumor suppressor p53 plays an important role in DDR during apoptosis. Inactivation of the p53 pathway is prevalent in many cancers. In addition to acetylation of histone H4, MOF is also inferred to regulate the activity of the p53 pathway through acetylation of p53 on K120 [26]. Blocking the acetylation of p53, either by knockout of MOF or K120 mutation, inhibits the ability of p53 to activate the downstream gene expression of BAX and PUMA, which can promote cancer [27]. Li et al. demonstrated that the MOF-MSL1v1 complex, not the MOF-MSL complex, is specifically required for the acetylation of p53K120 in vitro and in vivo [3]. In another study, the acetylation of p53K120 was also found to regulate apoptosis independent of transcription [28].

Loss of H4K16 acetylation is a common feature of tumor cells

Recent studies have demonstrated that MOF is involved in the development of a variety of human tumors. Loss of MOF and its substrate, acetylated H4K16, is now recognized as a common marker of human tumors [29]. For most primary breast carcinomas, MOF expression and H4K16 acetylation are reduced or lost completely [30]. Moreover, MOF expression is negatively correlated with tumor stage and tumor cell proliferation, as measured by Ki67 expression, and positively correlated with the expression of the estrogen receptor (ER) and progesterone receptor (PR) [31]. These data suggest that MOF plays an important role in the occurrence and progression of breast cancer, and MOF expression is associated with the prognosis of breast cancer. Stefan et al. conducted a similar study on glioma and concluded that MOF expression can serve as an independent prognostic marker to evaluate the clinical outcomes for patients with glioma [30]. In cervical cancer, the MOF expression is downregulated as the cervical lesion progresses [32]. Hence, these findings indicate that MOF may function as a tumor suppressor. However, the role of MOF in tumorigenesis may be more complex, given that a recent study has determined that MOF can significantly inhibit the growth of Calu-6 cells [33].

Given the critical roles of MOF in regulating biological processes such as DNA transcription, replication, DNA damage response, DDR, cell cycle, and apoptosis, the involvement of MOF in malignant transformation may occur through one or several mechanisms. First, H4K16 acetylation by MOF alters the conformation of chromatin and activates the expression of specific genes. The loss of H4K16 acetylation and MOF protein can affect other chromatin modifications, such as methylation, and lead to abnormal expression of key genes involved in tumorigenesis. Second, the absence of MOF affects DDR through various pathways and promotes the occurrence of a tumor. Third, MSL2, one component of the MOF-MSL complex, functions as an E3 ligase and has a zinc finger structure. MSL2 mediates p53 ubiquitination and the subcellular localization of p53; these processes may be related to tumor progression [34].

The function of MOF in ESCs

The modification of histone H3K4me3 and H3K27me3 was found to play an important role in the regulation of ESC self-renewal and pluripotency. The interaction of MOF with MLL [11] is related to the H3K4me3 and H4K16ac levels at the sites of active transcription [35,36]. To determine whether MOF plays an important role in maintaining stemness in mammalian ESCs, Li et al. compared the expression of MOF and the H4K16 acetylation level between ESCs and mouse embryonic fibroblasts (MEFs) [16] and discovered that the levels of MOF and acetylated H4K16 were significantly higher in ESCs than in MEFs. However, levels of methylated H3K4 and H3K27 were not significantly different. In addition, the mRNA and protein levels of MOF decreased in the ESCs that were induced to differentiate by retinoic acid treatment and in natural embryoid bodies. At the same time, the expression of key pluripotency genes such as Pou5f1, Nanog, and Sox2 decreased [16].

To illustrate the function of MOF in ESCs, Li et al. derived several lines of ESCs with conditional mutation in MOF: Cre-ER⁺, MOF^flox/flox; Cre-ER⁺, MOF^{flox/ +}, and Cre-ER⁺, MOF^+/+ [10,16]. The MOF-knockout cell lines displayed marked morphological changes, abnormal chromatin condensation, considerably increased amounts of nuclear heterochromatin, and loss of alkaline phosphatase activity and the ability to form embryoid bodies; these defects eventually led to cell death (Fig. 2). MOF-knockout ESCs lost the ability to self-renew and the potential to differentiate into various cell types. The changes in phenotype and their extensive influence on the patterns of transcription in ESCs illustrate that MOF plays an essential role in maintaining ESC function.

To further understand the role of MOF in ESCs, Li et al. conducted a microarray-based analysis and demonstrated that the loss of pluripotency and ability to self-renew in the MOF-knockout ESCs was due to a common defect in cell proliferation and an increase in apoptosis [16]. The absence of MOF has a remarkable impact on the ESC transcriptome. Consistent with the fact that MOF plays an important role in ESCs, the expression of Oct4, Nanog, and their conserved common targets are also changed in MOF mutant ESCs [37]. With the exception of Klf4 and Myc, the expression of most genes is significantly decreased.

To verify whether changes in gene expression are directly related to MOF, Li et al. performed a ChIP-based analysis of MOF and H4K16 acetylation. The pluripotency genes that MOF directly interacts with were found to include Nanog, Pou5f1, Sox2, Fgf4, Lefty1, and Tcl1. These results indicate that MOF directly regulates key pluripotency genes, and MOF is a unique HAT. MOF is different from other HATs because other HATs, such as Tip60, Gcn5, and p300/CBP, have little impact on the expression of Oct4, Nanog, and Sox2 in ESCs. On the contrary, in ESCs, these HATs control the downstream processes of differentiation [38–40].

ChIP-seq analysis shows that MOF plays an extensive role in the modulation of the ESC transcriptome. In MOF-knockout ESCs, the expression of approximately 65% of downstream genes decreased, while the expression of 62.5% of downstream genes increased. Both upregulated and downregulated genes share the same MOF interaction site [16].

Conclusions and perspectives

In mammals, MOF resides in two complexes: MOF-MSL and MOF-MSL1v1. MOF, a HAT, catalyzes the acetylation of histone H4K16 and also partially acetylates H4K5 and H4K8. The MOF-MSL1v1 complex can also acetylate the non-histone substrates p53 and YY1. Acetylation can affect the activation of gene transcription. Thus, MOF plays an irreplaceable role in the proliferation and differentiation of normal cells and in embryogenesis, DDR, and tumorigenesis. The current findings suggest that MOF is involved in the development of a normal organism, and this protein plays a crucial role in the development of human disease. However, the direct effects on tumors and the concrete mechanisms of regulation are still unclear and should be elucidated. Furthermore, MOF, a key regulator in the core transcriptional network of ESCs, is important for both ESC stemness and differentiation. Whether these two processes use the same MOF-containing complexes should be examined in future studies.

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