E3 ligase UHRF2 stabilizes the acetyltransferase TIP60 and regulates H3K9ac and H3K14ac via RING finger domain
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UHRF2 is a ubiquitin-protein ligase E3 that regulates cell cycle, genomic stability and epigenetics. We conducted a co-immunoprecipitation assay and found that TIP60 and HDAC1 interact with UHRF2. We previously demonstrated that UHRF2 regulated H3K9ac and H3K14ac differentially in normal and cancer cells. However, the accurate signal transduction mechanisms were not clear. In this study, we found that TIP60 acted downstream of UHRF2 to regulate H3K9ac and H3K14ac expression. TIP60 is stabilized in normal cells by UHRF2 ubiquitination. However, TIP60 is destabilized in cancer cells. Depletion or inhibition of TIP60 disrupts the regulatory relationship between UHRF2, H3K9ac and H3K14ac. In summary, the findings suggest that UHRF2 mediated the post-translational modification of histones and the initiation and progression of cancer.
KEYWORDSUHRF2 TIP60 ubiquitination acetylation hepatocellular carcinoma
Post-translational modifications (PTMs) of histones regulate gene expression. They are induced by several families of proteins including histone deacetylases (HDACs), histone acetyltransferases (HATs), physphorylases of the aurora kinases family, and histone ubiquitinases of the E3 ligase family (Basu et al., 2009; Pokholok et al., 2005; Tan et al., 2011; Wang et al., 2008). These modifications involving the interaction between histone and DNA regulate gene transcription either synergistically or antagonistically (Arnaudo and Garcia 2013; Su et al., 2016). UHRF2 is a multi-domain E3 ubiquitin ligase, which is related to mouse Np95 and human ICBP90. It comprises UBL, PHD, TTD, SET and RING-associated (SRA/YDG) and RING finger domains (Bronner et al., 2007; Qian et al., 2012; Wang et al., 2012). Several studies have demonstrated that UHRF2 represents a nodal point in the cell cycle network and regulates the epigenetics (Mori et al., 2011). UHRF2 interacts with the DNA methyltransferases (DNMT1, DNMT3a and DNMT3b), HDAC1, G9a and H3K9me2/me3 (Li et al., 2004; Mori et al., 2004; Pichler et al., 2011). Recently we found that UHRF2 influenced the acetylation of histone H3 on lysine 9 (H3K9ac) and lysine 14 (H3K14ac). However, the molecular mechanism was unclear. We investigated the mechanism underlying UHRF2-mediated regulation of H3K9ac and H3K14ac expression and the factors associated with differential regulation in normal and cancer cells.
Histone acetyltransferase TIP60 attracted our interest. It belongs to the MYST family and controls the acetylation of histone H2A on lysine 5 (H2AK5), histone H3 on lysine 14 (H3K14) and histone H4 (lysines 5, 8, 12, and 16) (Dai et al., 2013; Ikura et al., 2015; Jacquet et al., 2016; Renaud et al., 2016). TIP60 also participates in DNA repair, cellular growth, apoptosis and response to DNA double-strand breakage (Grezy et al., 2016; Jang et al., 2015; Sun et al., 2015). Recently, it was demonstrated that UHRF1 recruits histone acetyltransferase TIP60 and controls its expression and activity (Achour et al., 2009). In this study, the interactions between TIP60 and UHRF2 were detected using co-immunoprecipitation. It is suggested that TIP60 regulates H3K9ac and H3K14ac as downstream signal molecules.
We identified a previously undefined mechanism associated with TIP60-mediated UHRF2-regulation of H3K9ac and H3K14ac expression. In normal cells, UHRF2 overexpression enhances the expression of H3K9ac and H3K14ac, which is reversed in cancer cells. The regulatory mechanism is disrupted following deletion of the YDG or RING finger domains of UHRF2. In HEK293 cells, the interaction between UHRF2, TIP60 and HDAC1 was detected with co-immunoprecipitation, which was mediated via PHD and RING finger domains of UHRF2. Further, we found that TIP60 was a key intermediate in this molecular mechanism. Inhibition of TIP60 expression or activity disrupted the regulatory relationship. We also determined the levels of UHRF2, H3K9ac, H3K14ac, TIP60 and H2AK5ac in human hepatocellular carcinoma (HCC) tissues immunohistochemically. The results showed that all the aforementioned proteins were decreased under the high expression of UHRF2 in HCC tissues.
In summary, we identified a new mechanism underlying UHRF2-TIP60-H3K9ac and H3K14ac signaling axis. UHRF2 may contribute to the initiation and development of primary hepatocellular cancer by TIP60, which warrants further investigation.
UHRF2 regulates the expression of H3K9ac and H3K14ac
UHRF2 interacts with TIP60 and HDAC1
UHRF2 enhances the expression and activity of TIP60
UHRF2 ubiquitinates and stabilizes TIP60
UHRF2 regulates H3K9ac and H3K14ac expression via interaction with TIP60
Inverse correlation between TIP60 and UHRF2 in HCC tissues
TIP60 is a transcriptional co-factor involved in several essential physiological processes in cells. Its aberrant expression has been reported in several cancers (Feng et al., 2016; Mo et al., 2016). TIP60 plays a major role in histone acetylation to potentially trigger cancer-related gene expression (Sun et al., 2015). A previous study showed that TIP60 co-localizes with the UHRF1/DNMT1 complex, which is involved in ubiquitination as a substrate of E3 ligase (Dai et al., 2013). Our findings strongly suggested that TIP60 was a new substrate for UHRF2. Protein modification via ubiquitination is of great importance in many regulatory processes inside the cells (Hershko et al., 2000). Ubiquitination alters protein turnover via proteasome-mediated degradation of proteins (Harrison et al., 2016; Leithe 2016; Yamano et al., 2015; Lechtenberg et al., 2016). Altered ubiquitination may prolong the half-life of specific proteins, while other proteins are rapidly degraded (Vinther-Jensen et al., 2015). In ubiquitination pathway, the E3 ligase plays an important role in substrate recognition. In this study, we found that E3 ligase UHRF2 ubiquitinated TIP60 and further stabilized its expression and elevated its activity via RING finger domain in normal cells. Conversely, the expression and activity of TIP60 were decreased in tumor cells. These results are of utmost significance, with two diametrically opposite responses in different cells. We conjectured that a series of unknown signal transduction factors mediate this pathway, which remain to be investigated.
PTMs associated with gene expression usually occur at the amino acid level, including methylation, phosphorylation, ubiquitination, SUMOylation, plamitoylation, and acetylation (Holt et al., 2015; Kouzarides 2007; Li 2002). Histone acetylation is one of the reversible PTMs catalyzed by HATs and HDACs, which play a key role in the regulation of specific gene expression, chromosome segregation and leukemia (Basu et al., 2009; Wang et al., 2008). Histone acetylation alters the chromosome structure and gene activity, and triggers the initiation and progression of tumors via regulation of cellular proliferation and apoptosis (Pokholok et al., 2005). The proliferation and differentiation levels are balanced under normal conditions through s series of molecular regulatory mechanisms (Tan et al., 2011). Normal cells transform into tumor cells when the balance of histone acetylation and deacetylation is disrupted by physical, chemical and biological factors to trigger abnormal differentiation and chaotic proliferation (Su et al., 2016). Histone H3 is a core component of eukaryotic nucleosome octamer (Karmodiya et al., 2012; Yamada et al., 2013). The two lysine sites 9 and 14 are the focus of intensive study in histone acetylation (Karmodiya et al., 2012). H3K9ac is associated with meiotic recombination hotspots and DNA damage response, and plays a key role in recombination with other factors (Grezy et al., 2016). H3K14ac interacts with transcription factors in tumor initiation and progression (Pichler et al., 2011). In this study, we found that UHRF2 regulated H3K9ac and H3K14ac expression via YDG and RING finger domain, and contrasting results were found in normal and cancer cells. In addition, we found that TIP60 elevated the expression of H3K9ac and H3K14ac. Based on these results, we investigated the UHRF2-mediated regulation of H3K9ac and H3K14ac via interaction with TIP60, and ubiquitination of TIP60 as a new substrate of E3 ligase UHRF2. Interestingly, the differential regulatory mechanism of UHRF2 against TIP60 in normal and cancer cells attracted our attention and investigation.
In summary, we suggest that UHRF2 regulated H3K9ac and H3K14ac expression via the TIP60 downstream signal. It is widely believed that cellular protein-protein interactions are universal. However, in this study, contrasting results were repeatedly found in normal and cancer cells. We used immunohistochemical analysis of HCC tissues and similar results were obtained. The differences in normal and cancer cells were the focus of this study, although we found that an intermediate signaling molecule “TIP60” participated in this mechanism. However, several unknown factors remain to be further investigated. We hypothesized that this mechanism includes specific signal transduction pathways, which trigger carcinogenesis in normal cells, and promote or co-promote malignant transformation of cells.
MATERIALS AND METHODS
Cell lines and cell culture
Human normal liver cells (LO2) were cultured in RPMI-1640 medium (Gibco, USA) with 15% fetal bovine serum (Gibco). Human embryonic kidney 293 cells (HEK293) and human hepatocellular liver carcinoma cells (HepG2) were routinely cultured in DMEM/HIGH glucose medium (Hyclone, USA) with 10% fetal bovine serum (Gibco). In this study, HEK293 and LO2 cells were regarded as normal cells. HepG2 cells were regarded as cancer cells.
Plasmids and siRNAs
Full-length and mutant UHRF2 genes (Flag-UHRF2, ΔUBL, ΔYDG, ΔPHD, ΔRING) were obtained by cloning the PCR fragment corresponding to UHRF2 into the pCMV-3×Flag vector. The pcDNA3.0-HA-Ub was a kind gift obtained from Lu Bai (Fudan University). The pcDNA3.1-His-TIP60, short hairpin RNA (shRNA) against UHRF2 and small interfering RNA (siRNA) against TIP60 were purchased from Invitrogen (USA) and GenePhama (China), respectively. Transfection was performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.
Total protein was isolated with RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with phenylmethanesulfonyl fluoride (PMSF) (Roche, Germany) and a protease inhibitor cocktail (Roche) after 48 h transfection. Protein concentrations were measured using a BCA protein assay Kit (Beyotime Biotechnology) and boiled in loading buffer containing 1% SDS before infection. Proteins were separated by SDS-PAGE followed by Western blot with the indicated antibodies. The primary and secondary antibodies are listed in Table S1.
Tissue samples and immunohistochemistry
Forty-five cases of HCC tissue samples were collected from patients who underwent surgical resection at the department of the First Affiliated Hospital of Chongqing Medical University. The study was approved by the Ethics Committee of Chongqing Medical University (reference number: 2015018). All subjects signed informed consent. Patients’ information will not be disclosed completely.
Immunohistochemistry was performed as described elsewhere (Das et al., 2016).
Cells were lysed with NP40 lysis buffer (Beyotime Biotechnology) supplemented with PMSF and a protease inhibitor cocktail at 48 h after transfection. The whole cell extracts were immunoprecipitated overnight at 4°C with the indicated antibody. After extensive washing with lysis buffer, the immune complexes were boiled in a loading buffer containing 1% SDS to deplete the interference of poly-ubiquitin chain on the other molecules in the same complex, and analyzed by Western blot.
To detect ubiquitinated TIP60 proteins, cells in a 10 mL cell bottle were transiently transfected with 4 μg HA-ubiquitin-expressing plasmids together with the indicated plasmid. Twelve hours before collection, cells were treated with 20 μmol/L of MG132 (Sigma-Aldrich, USA). Cells were lysed using NP40 lysis buffer supplemented with PMSF and protease inhibitor cocktail. The following procedures were identical to the immunoprecipitation assay except for the use of monoclonal HA antibody.
Cells were treated with 100 μg/mL of cycloheximide (CHX) (ABmole Bioscience, USA) for the indicated times, to inhibit protein synthesis. The cellular lysates were analyzed by Western blot.
Cells seeded on the coverslips were fixed in 4% paraformaldehyde, and incubated with primary antibodies and secondary antibodies before staining with DAPI, mounted and observed, as described elsewhere (Liang et al., 2016; Takase et al., 2016).
All statistical data are described in the figure legends. Two-sided unpaired Student’s t-test was used for experimental comparisons, after quantification with Image J software. Significance was represented as *P < 0.05, and non-significance was indicated as n.s P > 0.05.
This work was supported byNational Natural Science Foundation of China (Grant No. 30872248). We acknowledge Lu Bai for providing the HA-Ub plasmid. We thank Yan Sun, Ming Li and Yanyan Xuan for their excellent technical assistance.
CHX, cycloheximide; H3K9ac, the acetylation of histone H3 on lysine 9; H3K14ac, the acetylation of histone H3 on lysine 14; H2AK5ac, the acetylation of histone H2A on lysine 5; HATs, histone acetyltransferases; HCC, hepatocellular carcinoma; HDACs, histone deacetylases; PMSF, phenylmethanesulfonyl fluoride; PTMs, post-translational modifications; PVDF, polyvinylidene difluoride; RT, room temperature; h, hour; TBST, Tris-buffered saline Tween-20.
COMPLIANCE WITH ETHICS GUIDELINES
Shengyuan Zeng, Yangyang Wang, Ting Zhang, Lu Bai, Yalan Wang and Changzhu Duan declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study
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