Epigenetic control via histone methylation is important in transcriptional regulation and occurs in the nucleus. However, this process can be regulated spatially by a protein localized in the Golgi apparatus. Subcellular compartmentalization can therefore affect epigenetic modification.

INTRODUCTION

Histone methylation at lysine residues plays important roles in many biological processes such as heterochromatin formation, X-chromosome inactivation, transcriptional regulation and cancer formation [1,2]. Methylation of histone 3 at lysine 4 (H3K4) can occur in monomethylated, dimethylated or trimethylated forms [35]. H3K4 trimethylation (H3K4me3) is a prevalent mark that is exclusively associated with actively transcribed genes such as HOX genes that are essential for vertebrate development [6].

The first identified H3K4 methyltransferase is the large macromolecular complex of proteins associated with Set1 (COMPASS) from Saccharomyces cerevisiae and the SET domain-containing protein Set1 is the catalytic subunit in the complex [79]. Mixed-lineage leukaemia (MLL) is predicted to be a functional homologue of Set1 in mammalian cells and was found in COMPASS-like complexes capable of methylating H3K4 in metazoan cells [10,11]. Translocations of MLL gene are associated with a variety of aggressive acute leukaemia in humans [2,10]. In fact, humans encode at least six different Set1 family members: MLL1–MLL4, SET1A and SET1B [12]. In addition to the catalytic subunit, COMPASS complexes require a number of regulatory subunits to achieve full histone methyltransferase activity. Four regulatory subunits are shared between the metazoan COMPASS-like complexes and the yeast COMPASS complexes, including ASH2 (absent, small or homoeotic discs 2; related to Cps60 in yeast), WDR5 (WD40 repeat-containing protein 5; related to Cps30 in yeast), RBBP5 (retinoblastoma-binding protein 5; related to Cps50 in yeast) and DPY30 (related to Cps25 in yeast) [11]. These four critical regulatory subunits form a complex coined the WRAD sub-complex (WDR5–ASH2–RBBP5–DPY30) [11]. The WRAD sub-complex is critical for the full enzyme activity of H3K4 methyltransferase. It was reported that the minimal H3K4 methyltransferase complex required for di- and tri-methylation of H3K4 in mammalian cells includes MLL1, ASH2L, WDR5, and RBBP5 [13]. In the absence of any of the core regulatory members such as ASH2L, WDR5 or RBBP5, the methyltransferase activity of MLL is severely lessened [13]. WDR5 plays a critical role in the WARD sub-complex as it is required for binding of the COMPASS-like complexes to the K4-dimethylated H3 tail and essential for global H3K4me3 and HOX gene activation in human cells [14]. In addition, WDR5 is required to maintain MLL complex integrity, including the stability of ASH2L within the complex [15]. Recently, DPY30 was found to directly regulate H3K4 methylation in both in vitro and in vivo studies [16].

H3K4 methylation is a prevalent marker for transcriptionally activated genes and is implicated in developmental processes and human diseases such as cancers [1]. A genome-wide study has revealed that active promoters are marked by trimethylation of H3K4 and enhancers are marked by monomethylation, but not trimethylation of H3K4. Such study has led to a prediction that there are over 200 promoters and 400 enhancers within the 30-Mbp region in human genome [17]. Using a single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells, it was reported that trimethylation of H3K4 and H3K27 effectively discriminates genes that are either expressed or repressed, reflecting cell state and lineage potential [18]. During carcinogenesis, cancer cells usually experience severe hypoxia resulting from reduced oxygen supply from blood vessels and hypoxia activates the major transcription factor hypoxia-inducible factor 1 (HIF-1), as well as other important transcription factors that are actively involved in tumour progression and metastasis. Interestingly, it was recently found that hypoxia-mediated regulation of H3K4 methylation is implicated in hypoxia-induced epithelial–mesenchymal transition (EMT) that is important for metastasis and cancer stemness [19]. Hypoxia could induce the expression of WDR5 and histone deacetylase 3 (HDAC3) and both of them synergistically facilitate H3K4 methylation, leading to activation of mesenchymal gene expression and EMT features in tumour cells. In addition, hypoxia may alter H3K4 methylation via changes in demethylase activities. Hypoxia could induce H3K4me3 via inhibition of JARID1A demethylase in cancer cells [20]. The histone demethylase LSD1 (lysine-specific demethylase 1) was also found to be implicated in modulation of H3K4 methylation and silencing of a DNA mismatch repair gene MLH1 in tumour cells [21].

PAQR3 (progestin and adipoQ receptors member 3) is a member of the PAQR family which are predicted to have seven transmembrane domains similar to G-protein-coupled receptors (GPCRs) [22]. PAQR1 and PAQR2 function as cell-surface receptors for adiponectin, an adipocyte-secreted cytokine that regulates glucose and lipid metabolism [23]. Although PAQR3 shares high sequence homology with PAQR1 and PAQR2, it is exclusively localized in the membrane of the Golgi apparatus rather than the plasma membrane [24]. PAQR3 was named RKTG (Raf kinase trapping to Golgi) due to its spatial regulation of Raf kinase [24]. Our previous studies have revealed that PAQR3 functions as a tumour suppressor due to its inhibitory activity on Raf–MEK (mitogen-activated protein kinase/ERK kinase)–ERK (extracellular-signal-regulated kinase) signalling [2529]. Subsequent characterization of PAQR3 indicates that it also regulates GPCR signalling pathways by sequestering the Gβ-subunit to the Golgi apparatus [30]. In addition, PAQR3 is markedly down-regulated in human gastric cancers. The expression level of PAQR3 is closely associated with the progression and metastasis of gastric cancers, indicating that PAQR3 is a new genetic signature that can predict the prognosis of the patients with gastric cancer [31]. In the present study, we provide evidence for a unique role of PAQR3 in the regulation of H3K4 methylation via sequestration of the core regulatory subunits of the COMPASS-like complexes in mammalian cells.

MATERIALS AND METHODS

Plasmid construction

The Myc-tagged PAQR3 and GFPc1-tagged PAQR3 were described previously [32]. The full-length human ASH2, hDPY30, RBBP5 and WDR5 cDNA were cloned by reverse transcription (RT)-PCR from total RNA isolated from human cell lines and confirmed by sequencing. These cDNAs were fused with a FLAG–epitope tag at the N-termini in p3XFlag–CMV10 plasmid (Sigma–Aldrich).

Cell culture, transfection and hypoxic treatment

Human embryonic kidney (HEK) 293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. AGS cells were cultured in F-12K medium containing 10% FBS. Transient transfection was performed with the polyethyleneimine (PEI) method for HEK293T and Polyjet (Invitrogen) for HeLa cells according to the manufacturer's instructions. Hypoxia was achieved by culturing the AGS cells for 24 h in an airtight chamber containing 1% oxygen, 5% carbon dioxide and 94% nitrogen using a protocol previously described [27].

Antibodies, immunoprecipitation and immunoblotting

The antibodies were purchased as follows: antibody against Myctag was from Santa Cruz Biotechnology; antibody against FLAG-tag was from Sigma–Aldrich; antibodies against mono-, di- and tri-methylated histone H3K4 were from Cell Signaling Technology, antibody against histone H3 was from Beyotime Institute of Biotechnology; antibodies against Golgin-97, Alexa Fluor 488-conjugated donkey anti-mouse IgG, Alexa Fluor 546-conjugated goat anti-mouse, rabbit IgG and Hoechst 33342 were from Molecular Probes; Cy5-labelled goat anti-mouse IgG was from GE Healthcare. The cells were lysed in a buffer containing 20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EGTA and 1% Nonidet P-40 with a mixture of protease inhibitors (Sigma–Aldrich) before immunoprecipitation and immunoblotting assays using protocols as previously described [24].

ChIP and PCR analysis

ChIP was performed using the Chromatin Immunoprecipitation Assay Kit (Upstate Biotechnology) and the anti-H3K4me3 antibody (9751) from Cell Signaling Technology. Primers used for real-time PCR detection are listed as follows: 5′-GTGCT-GGGTGCTAAGGT-3′ and 5′-CAGACGCCAGGATCCCA-3′ for HOXC8 (promoter region −600 to −400); 5′-CCAGG-CGGGTAGCTGTG-3′ and 5′-GTTTGGTTGGCGAGCTC-3′ for HOXA9 (promoter region −800 to −600).

Analysis of HOX gene expression

Total RNA was isolated using TRIzol reagent from Invitrogen. The RNA was treated with RNase-free DNase I and reverse-transcribed with oligo(dT) primer using the SuperScript First-Strand Synthesis System for RT–PCR (Invitrogen). Quantitative real-time PCR was done with the SYBR Green PCR system (Applied Biosystems), using actin as an internal control for normalization. Primers used for each gene are listed as follows: 5′-GATGGCATTGGATTATGCAG-3′ and 5′-AAGCACGGTGA-TCAGGTACA-3′ for PAQR3, 5′-GATCATTGCTCCTCCTG-AGC-3′ and 5′-ACTCCTGCTTGCTGATCCAC-3′ for actin, 5′-AGGAACCTGATGGAAACCTG-3′ and 5′-AAACAGCGA-AGGAGAGGAAG-3′ for HOXC8, 5′-CATCGATCCCAAT-AACCCA-3′ and 5′-CCCTGGTGAGGTACATGTTG-3′ for HOXA9.

Confocal microscopy

The confocal analyses with overexpression experiments were performed as previously reported [24]. The Golgi localization was determined by immunostaining with antibodies against either Golgin-97 or GM130, two well-characterized Golgi markers.

Preparation of nuclear extracts and H3K4 methyltransferase activity assay

Nuclear extracts were prepared from HEK293T cells 48 h after transfection by using EpiQuik™ Nuclear Extraction Kit I (Epigentek) and stored at −80°C until being analysed. The nuclear extracts were used to analyse H3K4 methyltransferase activity using EpiQuik™ Histone Methyltransferase Activity/Inhibition Assay Kit (H3-K4; Epigentek) according to the manufacturer's instructions and detected by a microplate reader at 450 nm.

Statistical analysis

The unpaired and two-tailed Student's t test is used for all the statistical analyses.

RESULTS

PAQR3 is able to regulate trimethylation of H3K4

To address whether PAQR3 had a global effect on the steady-state level of H3K4 methylation, HEK293T cells were transfected with various amounts of Myc-tagged PAQR3. Whole-cell extracts were prepared 3 days after the transfection and analysed by immunoblotting with antibodies against different forms of H3K4 methylation. The global levels of H3K4 methylation were significantly reduced in PAQR3-overexpressing cells, shown as an apparent decrease in mono- and tri-methylation of H3K4 (Figure 1A). However, the global level of H3K4 dimethylation (H3K4me2) was unaffected (Figure 1A).

PAQR3 negatively regulates trimethylation of H3K4

Figure 1
PAQR3 negatively regulates trimethylation of H3K4

(A) Effect of PAQR3 overexpression on H3K4 methylation. HEK293T cells were transfected with Myc-tagged control plasmid (Myc-control) or Myc-tagged PAQR3 (Myc–PAQR3). The whole-cell extracts 3 days after transfection were used in immunoblotting with the antibodies indicated. The same experiments were performed five times with similar results. (BD) PAQR3 affects H3K4me3 by confocal analysis. HeLa cells were transfected with GFP-fused PAQR3 or an shRNA specific for PAQR3 (PAQR3-shRNA) and used in immunofluorescence staining using the anti-H3K4me3 antibody. The arrows indicate cells with overexpression of PAQR3. The arrowheads denote cells with down-regulation of PAQR3. The same experiments were performed three times. The relative intensity of H3K4me3 fluorescence signals assayed by ImageJ software (NIH) is shown in (D). The data are shown as means±S.D. *P<0.05 and **P<0.01 between the two groups. (E and F) Effect of PAQR3 on hypoxia-induced H3K4me3. AGS cells were transfected with a scrambled shRNA control (control-shRNA), PAQR3-specific shRNA (PAQR3-shRNA), a Myc-containing empty vector (Myc-control) or Myc-tagged PAQR3 (Myc-PAQR3), as previously reported [31]. At 48 h after transfection, the cells were cultured under normoxia (N) or hypoxia (H) for 24 h before immunoblotting using the antibodies indicated. The same experiments were performed three times with similar results.

Figure 1
PAQR3 negatively regulates trimethylation of H3K4

(A) Effect of PAQR3 overexpression on H3K4 methylation. HEK293T cells were transfected with Myc-tagged control plasmid (Myc-control) or Myc-tagged PAQR3 (Myc–PAQR3). The whole-cell extracts 3 days after transfection were used in immunoblotting with the antibodies indicated. The same experiments were performed five times with similar results. (BD) PAQR3 affects H3K4me3 by confocal analysis. HeLa cells were transfected with GFP-fused PAQR3 or an shRNA specific for PAQR3 (PAQR3-shRNA) and used in immunofluorescence staining using the anti-H3K4me3 antibody. The arrows indicate cells with overexpression of PAQR3. The arrowheads denote cells with down-regulation of PAQR3. The same experiments were performed three times. The relative intensity of H3K4me3 fluorescence signals assayed by ImageJ software (NIH) is shown in (D). The data are shown as means±S.D. *P<0.05 and **P<0.01 between the two groups. (E and F) Effect of PAQR3 on hypoxia-induced H3K4me3. AGS cells were transfected with a scrambled shRNA control (control-shRNA), PAQR3-specific shRNA (PAQR3-shRNA), a Myc-containing empty vector (Myc-control) or Myc-tagged PAQR3 (Myc-PAQR3), as previously reported [31]. At 48 h after transfection, the cells were cultured under normoxia (N) or hypoxia (H) for 24 h before immunoblotting using the antibodies indicated. The same experiments were performed three times with similar results.

We next employed confocal analysis to investigate H3K4me3 at the single-cell level. HeLa cells were transfected with GFP-fused PAQR3 and subjected to immunofluorescence analysis to detect H3K4me3 levels. The cells with PAQR3 overexpression had a reduced level of H3K4me3 compared with adjacent cells without PAQR3 overexpression (Figures 1B and 1D). We also investigated the H3K4me3 level under the condition of PAQR3 down-regulation. Knockdown of PAQR3 was performed by a specific shRNA that had a high efficiency to silence the expression of endogenous PAQR3 [27]. The cells with down-regulation of PAQR3 had a profound increase in H3K4me3 signals (Figures 1C and 1D). Collectively, these results indicate that PAQR3 is able to negatively regulate H3K4me3.

PAQR3 modulates hypoxia-induced H3K4 trimethylation

To further investigate the potential function of PAQR3 on H3K4me3, we analysed whether PAQR3 could alter H3K4me3 under hypoxia as a previous study has revealed that hypoxia was able to elevate the cellular H3K4me3 level [19]. In AGS gastric cancer cells, the H3K4me3 level was markedly increased by hypoxia (Figures 1E and 1F). Knockdown of PAQR3 could increase the H3K4me3 level under normoxia and further facilitate hypoxia-induced H3K4me3 (Figure 1E). In contrast, overexpression of PAQR3 reduced the H3K4me3 level under normoxia and abolished the increment of H3K4me3 under hypoxia (Figure 1F). These data therefore indicate that PAQR3 could regulate hypoxia-induced H3K4me3 in cancer cells.

PAQR3 regulates HOX gene expression

The transcription of the HOX gene family has been well characterized to be tightly regulated by H3K4 methylation during embryonic development [6]. We examined whether HOX gene expression could also be regulated by PAQR3. Quantitative RT–PCR was performed with HEK293T cells under the condition of PAQR3 overexpression or PAQR3 down-regulation. We found that the mRNA levels of HOXC8 and HOXA9 were significantly reduced by PAQR3 overexpression (Figure 2A). In contrast, the mRNA levels of these two genes were significantly elevated by PAQR3 knockdown (Figure 2B).

PAQR3 regulates HOXC8 and HOXA9 gene expression and alters the H3K4me3 level in the promoter region

Figure 2
PAQR3 regulates HOXC8 and HOXA9 gene expression and alters the H3K4me3 level in the promoter region

(A and B) Expression levels of HOXC8 and HOXA9 mRNA in HEK293T cells 24 h after transfection with a scrambled shRNA control (control-shRNA), PAQR3-specific shRNA (PAQR3-shRNA), a Myc-containing empty vector (Myc-control) or Myc-tagged PAQR3 (Myc-PAQR3) as indicated. Total RNA was isolated from these cells and used in reverse transcription and quantitative PCR. The data are shown as mean±S.D. **P<0.01 as compared with the controls. The same experiments were performed three times. (B) Results of ChIP assay using HEK293T cells transfected with Myc-tagged control plasmid or Myc-tagged PAQR3. The antibody against H3K4me3 was used in a ChIP assay and the genomic DNA was used in real-time PCR to amply the promoter regions of HOXC8 and HOXA9 genes. The same experiments were performed two times. The data are shown as means±S.D. *P<0.05 as compared with the controls.

Figure 2
PAQR3 regulates HOXC8 and HOXA9 gene expression and alters the H3K4me3 level in the promoter region

(A and B) Expression levels of HOXC8 and HOXA9 mRNA in HEK293T cells 24 h after transfection with a scrambled shRNA control (control-shRNA), PAQR3-specific shRNA (PAQR3-shRNA), a Myc-containing empty vector (Myc-control) or Myc-tagged PAQR3 (Myc-PAQR3) as indicated. Total RNA was isolated from these cells and used in reverse transcription and quantitative PCR. The data are shown as mean±S.D. **P<0.01 as compared with the controls. The same experiments were performed three times. (B) Results of ChIP assay using HEK293T cells transfected with Myc-tagged control plasmid or Myc-tagged PAQR3. The antibody against H3K4me3 was used in a ChIP assay and the genomic DNA was used in real-time PCR to amply the promoter regions of HOXC8 and HOXA9 genes. The same experiments were performed two times. The data are shown as means±S.D. *P<0.05 as compared with the controls.

Next, we used a ChIP assay to directly analyse the status of H3K4me3 at the promoters of HOXC8 and HOXA9 genes. Immunoprecipitated genomic DNA was subjected to PCR using probes specific for HOXC8 and HOXA9 genes. As shown in Figure 2(C), PAQR3 overexpression resulted in an obvious decrease in H3K4me3 at the HOXC8 promoter, whereas there was a slight reduction in H3K4me3 at the HOXA9 promoter (Figure 2C). Collectively, our results demonstrate that PAQR3 has a regulatory effect on the expression of HOX genes that are controlled by H3K4 methylation.

Interaction of PAQR3 with the core regulatory subunits of COMPASS-like complexes

The metazoan COMPASS-like complexes are composed of a SET-domain containing catalytic subunit (including MLL1–MLL4, SET1A, SET1B) and four core regulatory subunits including WDR5, ASH2, RBBP5 and DPY30 (the WRAD sub-complex). Our earlier studies indicated that PAQR3 was able to interact with the Gβ-subunit of GPCRs [30]. As Gβ is a WD40-containing protein, we postulated that PAQR3 might also interact with other WD40-containing proteins. Within the WRAD sub-complex, there are two WD40-containing proteins, WDR5 and RBBP5. We used co-immunoprecipitation assays to determine the potential interaction of PAQR3 with WDR5 and RBBP5. When both PAQR3 and WDR5 were overexpressed, immunoprecipitation of PAQR3 could pull down WDR5 (Figure 3A). Meanwhile, when both PAQR3 and RBBP5 were overexpressed, immunoprecipitation of RBBP5 could pull down PAQR3 (Figure 3B). As expected, the WD40 domains of both WDR5 and RBBP5 could also interact with PAQR3 by co-immunoprecipitation assays (results not shown).

Interaction of PAQR3 with WDR5, RBBP5, ASH2 and DPY30

Figure 3
Interaction of PAQR3 with WDR5, RBBP5, ASH2 and DPY30

(AE) HEK293T cells were transiently transfected with the plasmids indicated. The cell lysate was used in immunoblotting (IB) and immunoprecipitation (IP) using the antibodies indicated. The same experiments were performed two times with similar results.

Figure 3
Interaction of PAQR3 with WDR5, RBBP5, ASH2 and DPY30

(AE) HEK293T cells were transiently transfected with the plasmids indicated. The cell lysate was used in immunoblotting (IB) and immunoprecipitation (IP) using the antibodies indicated. The same experiments were performed two times with similar results.

We also analysed the potential interaction of PAQR3 with two other subunits of the WRAD sub-complex. To our surprise, PAQR3 could also interact with ASH2 by the co-immunoprecipitation assay (Figure 3C), but did not bind DPY30 (Figure 3D). However, in the presence of overexpressed WDR5, immunoprecipitation of PAQR3 could pull down DPY30 (Figure 3E), indicating that PAQR3 might indirectly interact with DPY30 through WDR5. Collectively, these data indicate that PAQR3 could interact with the four core regulatory subunits of the COMPASS-like complexes directly or indirectly in mammalian cells.

PAQR3 tethers the WRAD sub-complex to the Golgi apparatus

As PAQR3 is a membrane protein specifically localized in the Golgi apparatus [33], we next investigated whether PAQR3 could change the subcellular localization of the subunits of the COMPASS-like complexes. When WDR5, RBBP5, ASH2 and DPY30 were overexpressed in HeLa cells, all of them were localized in the nucleus (Figures 4A–4D). Intriguingly, when co-expressed with PAQR3, the four regulatory subunits were almost completely mobilized to the Golgi apparatus (Figures 4A–4D). In contrast, overexpression of PAQR3 had no effect on the distribution of endogenous MLL1, one of the catalytic subunits of COMPASS-like complexes (Figure 4E). These data therefore indicate that PAQR3 could tether the WRAD sub-complex, but not MLL1, to the Golgi apparatus, leading to reduced distribution these subunits in the nucleus.

PAQR3 tethers members of WRAD sub-complex to the Golgi apparatus

Figure 4
PAQR3 tethers members of WRAD sub-complex to the Golgi apparatus

(AD) PAQR3 sequestrates WDR5, RBBP5, ASH2 and DPY30 to the Golgi apparatus. HeLa cells were transiently transfected with the plasmids indicated, followed by immunofluorescence staining and confocal analysis. The arrow indicates co-localization of the core regulatory subunits of COMPASS-like complexes with PAQR3 at the Golgi apparatus. The Golgi apparatus was stained with an antibody against GM130. The nuclei were stained with Hoechst 33342. The same experiments were performed three times. (E) PAQR3 cannot co-localize with endogenous MLL1. HeLa cells were transiently transfected with GFP-fused PAQR3, followed by immunostaining with an antibody against endogenous MLL1. The Golgi apparatus was stained with an antibody against Golgi-97. The same experiments were performed two times.

Figure 4
PAQR3 tethers members of WRAD sub-complex to the Golgi apparatus

(AD) PAQR3 sequestrates WDR5, RBBP5, ASH2 and DPY30 to the Golgi apparatus. HeLa cells were transiently transfected with the plasmids indicated, followed by immunofluorescence staining and confocal analysis. The arrow indicates co-localization of the core regulatory subunits of COMPASS-like complexes with PAQR3 at the Golgi apparatus. The Golgi apparatus was stained with an antibody against GM130. The nuclei were stained with Hoechst 33342. The same experiments were performed three times. (E) PAQR3 cannot co-localize with endogenous MLL1. HeLa cells were transiently transfected with GFP-fused PAQR3, followed by immunostaining with an antibody against endogenous MLL1. The Golgi apparatus was stained with an antibody against Golgi-97. The same experiments were performed two times.

PAQR3 inhibits H3K4 methyltransferase activity and reduces the interaction of WDR5 with MLL1

Based on our studies, we hypothesized that PAQR3 might negate H3K4 methyltransferase activity by reducing complex formation between the four subunits with the SET domain-containing catalytic subunit. To test this hypothesis, we analysed the effect of PAQR3 on the interaction of WDR5 with the SET domain of MLL1, as WDR5 is the major component that mediates the interaction of the WRAD sub-complex with the catalytic subunit [34]. The full-length MLL1 contains 3969 amino acid residues with a conserved SET domain at the C-terminus together with other conserved domains including a Win (WDR5-interaction) motif [35,36]. Previous studies have shown that the C-terminus of MLL1 (C-ter) is the minimal domain required for the assembly of a full MLL1 core complex in vitro [36]. As measured by a co-immunoprecipitation assay, the C-terminal domain of MLL1 was able to interact with WDR5 (Figure 5A). Interestingly, overexpression of PAQR3 could dose-dependently compete off the binding of WDR5 with the C-ter (Figure 5A).

PAQR3 decreases the interaction between WDR5 with MLL1 and reduces H3K4 methyltransferase activity

Figure 5
PAQR3 decreases the interaction between WDR5 with MLL1 and reduces H3K4 methyltransferase activity

(A) PAQR3 interferes with the interaction of WDR5 with MLL1. Full-length MLL1 contains 3969 amino acid residues. The C-ter used in the experiment consists of residues 3745–3969 and includes the evolutionarily conserved SET domain and the Win motif. HEK293T cells were transiently transfected with the plasmids indicated. The cell lysate was used in immunoblotting (IB) and immunoprecipitation (IP) using the antibodies indicated. The same experiments were performed two times with similar results. (B) Effect of PAQR3 on H3K4 methyltransferase activity. Nuclear protein was extracted in HEK293T cells 48 h after transfection with a Myc-tagged control plasmid or Myc-tagged PAQR3. The extracted nuclear protein was used to determine H3K4 methyltransferase activity. The data are shown as means±S.D. **P<0.01 as compared with the controls. The same experiments were performed two times. (C) A model to depict the modulation of COMPASS-like complexes by PAQR3. The COMPASS-like complexes responsible for H3K4 methylation is composed of a SET domain-containing catalytic subunit (shown as MLL) and various regulatory subunits in which WDR5, RBBP5, ASH2 and DPY30 (the WRAD sub-complex) are the core ones. PAQR3 interacts with members of the WRAD sub-complex and tether them to the Golgi apparatus, resulting in reduced availability of these subunits in the nucleus and decreased H3K4 methylation.

Figure 5
PAQR3 decreases the interaction between WDR5 with MLL1 and reduces H3K4 methyltransferase activity

(A) PAQR3 interferes with the interaction of WDR5 with MLL1. Full-length MLL1 contains 3969 amino acid residues. The C-ter used in the experiment consists of residues 3745–3969 and includes the evolutionarily conserved SET domain and the Win motif. HEK293T cells were transiently transfected with the plasmids indicated. The cell lysate was used in immunoblotting (IB) and immunoprecipitation (IP) using the antibodies indicated. The same experiments were performed two times with similar results. (B) Effect of PAQR3 on H3K4 methyltransferase activity. Nuclear protein was extracted in HEK293T cells 48 h after transfection with a Myc-tagged control plasmid or Myc-tagged PAQR3. The extracted nuclear protein was used to determine H3K4 methyltransferase activity. The data are shown as means±S.D. **P<0.01 as compared with the controls. The same experiments were performed two times. (C) A model to depict the modulation of COMPASS-like complexes by PAQR3. The COMPASS-like complexes responsible for H3K4 methylation is composed of a SET domain-containing catalytic subunit (shown as MLL) and various regulatory subunits in which WDR5, RBBP5, ASH2 and DPY30 (the WRAD sub-complex) are the core ones. PAQR3 interacts with members of the WRAD sub-complex and tether them to the Golgi apparatus, resulting in reduced availability of these subunits in the nucleus and decreased H3K4 methylation.

As PAQR3 could interfere with the interaction of WDR5 with MLL1, we postulated that such action of PAQR3 should directly inhibit the enzyme activity of H3K4 methyltransferase. We examined the histone methyltransferase activity in HEK293T cells with PAQR3 overexpression. Compared with the control group, PAQR3 overexpression could significantly reduce the enzyme activity of H3K4 methyltransferase (Figure 5B).

DISCUSSION

Our studies reveal for the first time that H3K4 methylation is regulated by PAQR3 in a spatial manner by alterations of the subcellular compartmentalization of the WRAD sub-complex of the COMPASS methyltransferase (Figure 5C). PAQR3 is a transmembrane protein specifically localized in the Golgi apparatus. It can directly or indirectly interact with the four core regulatory subunits of the COMPASS-like complexes and sequestrate them to the Golgi apparatus (Figures 2 and 3). In the absence of PAQR3 overexpression, the WRAD sub-complex members including ASH2, WDR5, RBBP5 and DPY30 were all localized in the nucleus. However, overexpressed PAQR3 could tether most portions of these four subunits to the Golgi apparatus (Figure 3). As COMPASS-like complexes-mediated H3K4 methylation can only occur in the nucleus, the reduced availability of the WRAD sub-family would render a decrease in the H3K4 methyltransferase activity in the nucleus, leading to reduced level of H3K4me3. Consistently, we found that the H3K4me3 level, HOX gene expression and H3K4 methyltransferase activity are all reduced by PAQR3 overexpression (Figures 1, 2 and 5A). Importantly, we also found that down-regulation of endogenous PAQR3 had an effect on H3K4me3 (Figure 1C). Knockdown of PAQR3 also facilitated hypoxia-induced H3K4me3 (Figures 1D and 1F). These results indicate that the basal level of H3K4me3 is dependent on the expression level of PAQR3 in a particular cell. In other words, the endogenous PAQR3 level may set the tone for the degree of H3K4me3. Collectively, our studies have pinpointed a new mode of regulation on the activity of mammalian COMPASS-like complexes, leading to alterations in H3K4me3 levels in the cells. It is noteworthy that H3K4 monomethylation (H3K3me1) is also affected by PAQR3 (Figure 1A), consistent with a previous report showing that down-regulation of WDR5 and RBBP5 severely reduced the tri- and mono-methylation of H3K4 but with little or no significant effect on H3K4me2 [37].

In addition to its critical roles on activation of development-related genes such as the HOX gene family, the mammalian COMPASS-like complexes play an important role in cancer formation. It is known that EMT is critical for cancer metastasis. It was recently discovered that hypoxia, a condition that commonly occurs during cancer progression, is able to induce EMT and metastatic phenotypes through modulation of H3K4 methylation [19]. Under hypoxia, HDAC3 is able to interact with hypoxia-induced WDR5 and then recruit the histone methyltransferase complex to increase H3K4 methylation, leading to activation of mesenchymal gene expression. Meanwhile, knockdown of WDR5 could abolish mesenchymal gene activation under hypoxia [19]. In the present study, we found that hypoxia could induce the H3K4me3 level in AGS cancer cells, and PAQR3 knockdown further increased hypoxia-induced H3K4me3 (Figures 1D and 1F). Such findings are consistent with our previous observation that PAQR3 has a negative effect on EMT and tumour metastasis [29,31]. Deletion of the PAQR3 gene in the mice is associated with an increase in EMT features in collaboration with loss of p53 [29]. In human gastric cancers, reduced expression of PAQR3 is highly correlated with increased EMT markers and accelerated tumour progression and metastasis [31]. It is likely that the reduced expression of PAQR3 in human tumours could relieve the inhibitory effect of PAQR3 on H3K4 methylation, leading to facilitation of hypoxia-induced H3K4 methylation and activation of mesenchymal gene expression. Therefore, it is worth investigating the functional roles of PAQR3 regulation on H3K4 methylation and the associated EMT and cancer development in the future.

Abbreviations

     
  • ASH2

    absent, small or homoeotic discs 2

  •  
  • COMPASS

    complex of proteins associated with Set1

  •  
  • C-ter

    C-terminus of MLL1

  •  
  • EMT

    epithelial–mesenchymal transition

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • H3K4

    histone H3 lysine 4

  •  
  • H3K4me2

    H3K4 dimethylation

  •  
  • H3K4me3

    H3K4 trimethylation

  •  
  • HDAC

    histone deacetylase

  •  
  • HEK

    human embryonic kidney

  •  
  • MLL

    mixed-lineage leukaemia

  •  
  • PAQR

    progestin and adipoQ receptors member

  •  
  • RBBP5

    retinoblastoma-binding protein 5

  •  
  • RT

    reverse transcription

  •  
  • WDR5

    WD40 repeat-containing protein 5

  •  
  • Win

    WDR5-interaction

  •  
  • WRAD

    WDR5–ASH2–RBBP5–DPY30

AUTHOR CONTRIBUTION

Chunchun Liu, Yuxue Zhang, Yongfan Hou, Liqiang Shen, Yinlong Li, Weiwei Guo, Daqian Xu, Gaigai Liu, Zilong Zhao and Kaiyang Man performed the experiments. Yi Pan and Zhenzhen Wang provided critical advice. Yan Chen planned the experiments. Chunchun Liu, Yuxue Zhang and Yan Chen analysed the results and wrote the paper.

FUNDING

This work was supported by the Ministry of Science and Technology of China [grant numbers 2012CB524900 (to Y.C.) and 2013BAI04B03 (to Z.W.)]; and the National Natural Science Foundation of China [grant numbers 81130077, 81390350 and 81321062] to Y.C.

References

References
1
Martin
C.
Zhang
Y.
The diverse functions of histone lysine methylation
Nat. Rev. Mol. Cell Biol.
2005
, vol. 
6
 (pg. 
838
-
849
)
[PubMed]
2
Krivtsov
A.V.
Armstrong
S.A.
MLL translocations, histone modifications and leukaemia stem-cell development
Nat. Rev. Cancer
2007
, vol. 
7
 (pg. 
823
-
833
)
[PubMed]
3
Santos-Rosa
H.
Schneider
R.
Bannister
A.J.
Sherriff
J.
Bernstein
B.E.
Emre
N.C.
Schreiber
S.L.
Mellor
J.
Kouzarides
T.
Active genes are tri-methylated at K4 of histone H3
Nature
2002
, vol. 
419
 (pg. 
407
-
411
)
[PubMed]
4
Schneider
R.
Bannister
A.J.
Myers
F.A.
Thorne
A.W.
Crane-Robinson
C.
Kouzarides
T.
Histone H3 lysine 4 methylation patterns in higher eukaryotic genes
Nat. Cell Biol.
2004
, vol. 
6
 (pg. 
73
-
77
)
[PubMed]
5
Schneider
J.
Wood
A.
Lee
J.S.
Schuster
R.
Dueker
J.
Maguire
C.
Swanson
S.K.
Florens
L.
Washburn
M.P.
Shilatifard
A.
Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression
Mol. Cell
2005
, vol. 
19
 (pg. 
849
-
856
)
[PubMed]
6
Milne
T.A.
Briggs
S.D.
Brock
H.W.
Martin
M.E.
Gibbs
D.
Allis
C.D.
Hess
J.L.
MLL targets SET domain methyltransferase activity to Hox gene promoters
Mol. Cell
2002
, vol. 
10
 (pg. 
1107
-
1117
)
[PubMed]
7
Miller
T.
Krogan
N.J.
Dover
J.
Erdjument-Bromage
H.
Tempst
P.
Johnston
M.
Greenblatt
J.F.
Shilatifard
A.
COMPASS: a complex of proteins associated with a trithorax-related SET domain protein
Proc. Natl. Acad. Sci. U.S.A.
2001
, vol. 
98
 (pg. 
12902
-
12907
)
[PubMed]
8
Briggs
S.D.
Bryk
M.
Strahl
B.D.
Cheung
W.L.
Davie
J.K.
Dent
S.Y.
Winston
F.
Allis
C.D.
Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae
Genes Dev.
2001
, vol. 
15
 (pg. 
3286
-
3295
)
[PubMed]
9
Bernstein
B.E.
Humphrey
E.L.
Erlich
R.L.
Schneider
R.
Bouman
P.
Liu
J.S.
Kouzarides
T.
Schreiber
S.L.
Methylation of histone H3 Lys 4 in coding regions of active genes
Proc. Natl. Acad. Sci. U.S.A.
2002
, vol. 
99
 (pg. 
8695
-
8700
)
[PubMed]
10
Shilatifard
A.
Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression
Annu. Rev. Biochem.
2006
, vol. 
75
 (pg. 
243
-
269
)
[PubMed]
11
Shilatifard
A.
The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis
Annu. Rev. Biochem.
2012
, vol. 
81
 (pg. 
65
-
95
)
[PubMed]
12
Shilatifard
A.
Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation
Curr. Opin. Cell Biol.
2008
, vol. 
20
 (pg. 
341
-
348
)
[PubMed]
13
Dou
Y.
Milne
T.A.
Ruthenburg
A.J.
Lee
S.
Lee
J.W.
Verdine
G.L.
Allis
C.D.
Roeder
R.G.
Regulation of MLL1 H3K4 methyltransferase activity by its core components
Nat. Struct. Mol. Biol.
2006
, vol. 
13
 (pg. 
713
-
719
)
[PubMed]
14
Wysocka
J.
Swigut
T.
Milne
T.A.
Dou
Y.
Zhang
X.
Burlingame
A.L.
Roeder
R.G.
Brivanlou
A.H.
Allis
C.D.
WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development
Cell
2005
, vol. 
121
 (pg. 
859
-
872
)
[PubMed]
15
Steward
M.M.
Lee
J.S.
O'Donovan
A.
Wyatt
M.
Bernstein
B.E.
Shilatifard
A.
Molecular regulation of H3K4 trimethylation by ASH2L, a shared subunit of MLL complexes
Nat. Struct. Mol. Biol.
2006
, vol. 
13
 (pg. 
852
-
854
)
[PubMed]
16
Jiang
H.
Shukla
A.
Wang
X.
Chen
W.Y.
Bernstein
B.E.
Roeder
R.G.
Role for Dpy-30 in ES cell-fate specification by regulation of H3K4 methylation within bivalent domains
Cell
2011
, vol. 
144
 (pg. 
513
-
525
)
[PubMed]
17
Heintzman
N.D.
Stuart
R.K.
Hon
G.
Fu
Y.
Ching
C.W.
Hawkins
R.D.
Barrera
L.O.
Van Calcar
S.
Qu
C.
Ching
K.A.
, et al. 
Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome
Nat. Genet.
2007
, vol. 
39
 (pg. 
311
-
318
)
[PubMed]
18
Mikkelsen
T.S.
Ku
M.
Jaffe
D.B.
Issac
B.
Lieberman
E.
Giannoukos
G.
Alvarez
P.
Brockman
W.
Kim
T.K.
Koche
R.P.
, et al. 
Genome-wide maps of chromatin state in pluripotent and lineage-committed cells
Nature
2007
, vol. 
448
 (pg. 
553
-
560
)
[PubMed]
19
Wu
M.Z.
Tsai
Y.P.
Yang
M.H.
Huang
C.H.
Chang
S.Y.
Chang
C.C.
Teng
S.C.
Wu
K.J.
Interplay between HDAC3 and WDR5 is essential for hypoxia-induced epithelial-mesenchymal transition
Mol. Cell
2011
, vol. 
43
 (pg. 
811
-
822
)
[PubMed]
20
Zhou
X.
Sun
H.
Chen
H.
Zavadil
J.
Kluz
T.
Arita
A.
Costa
M.
Hypoxia induces trimethylated H3 lysine 4 by inhibition of JARID1A demethylase
Cancer Res.
2010
, vol. 
70
 (pg. 
4214
-
4221
)
[PubMed]
21
Lu
Y.
Wajapeyee
N.
Turker
M.S.
Glazer
P.M.
Silencing of the DNA mismatch repair gene MLH1 induced by hypoxic stress in a pathway dependent on the histone demethylase LSD1
Cell Rep.
2014
, vol. 
8
 (pg. 
501
-
513
)
[PubMed]
22
Tang
Y.T.
Hu
T.
Arterburn
M.
Boyle
B.
Bright
J.M.
Emtage
P.C.
Funk
W.D.
PAQR proteins: a novel membrane receptor family defined by an ancient 7-transmembrane pass motif
J. Mol. Evol.
2005
, vol. 
61
 (pg. 
372
-
380
)
[PubMed]
23
Yamauchi
T.
Kamon
J.
Ito
Y.
Tsuchida
A.
Yokomizo
T.
Kita
S.
Sugiyama
T.
Miyagishi
M.
Hara
K.
Tsunoda
M.
, et al. 
Cloning of adiponectin receptors that mediate antidiabetic metabolic effects
Nature
2003
, vol. 
423
 (pg. 
762
-
769
)
[PubMed]
24
Feng
L.
Xie
X.
Ding
Q.
Luo
X.
He
J.
Fan
F.
Liu
W.
Wang
Z.
Chen
Y.
Spatial regulation of Raf kinase signaling by RKTG
Proc. Natl. Acad. Sci. U.S.A.
2007
, vol. 
104
 (pg. 
14348
-
14353
)
[PubMed]
25
Xie
X.
Zhang
Y.
Jiang
Y.
Liu
W.
Ma
H.
Wang
Z.
Chen
Y.
Suppressive function of RKTG on chemical carcinogen-induced skin carcinogenesis in mouse
Carcinogenesis
2008
, vol. 
29
 (pg. 
1632
-
1638
)
[PubMed]
26
Fan
F.
Feng
L.
He
J.
Wang
X.
Jiang
X.
Zhang
Y.
Wang
Z.
Chen
Y.
RKTG sequesters B-Raf to the Golgi apparatus and inhibits the proliferation and tumorigenicity of human malignant melanoma cells
Carcinogenesis
2008
, vol. 
29
 (pg. 
1157
-
1163
)
[PubMed]
27
Zhang
Y.
Jiang
X.
Qin
X.
Ye
D.
Yi
Z.
Liu
M.
Bai
O.
Liu
W.
Xie
X.
Wang
Z.
, et al. 
RKTG inhibits angiogenesis by suppressing MAPK-mediated autocrine VEGF signaling and is downregulated in clear-cell renal cell carcinoma
Oncogene
2010
, vol. 
29
 (pg. 
5404
-
5415
)
[PubMed]
28
Wang
X.
Li
X.
Fan
F.
Jiao
S.
Wang
L.
Zhu
L.
Pan
Y.
Wu
G.
Ling
Z.Q.
Fang
J.
Chen
Y.
PAQR3 plays a suppressive role in the tumorigenesis of colorectal cancers
Carcinogenesis
2012
, vol. 
33
 (pg. 
2228
-
2235
)
[PubMed]
29
Jiang
Y.
Xie
X.
Li
Z.
Wang
Z.
Zhang
Y.
Ling
Z.Q.
Pan
Y.
Wang
Z.
Chen
Y.
Functional cooperation of RKTG with p53 in tumorigenesis and epithelial-mesenchymal transition
Cancer Res.
2011
, vol. 
71
 (pg. 
2959
-
2968
)
[PubMed]
30
Jiang
Y.
Xie
X.
Zhang
Y.
Luo
X.
Wang
X.
Fan
F.
Zheng
D.
Wang
Z.
Chen
Y.
Regulation of G-protein signaling by RKTG via sequestration of the G betagamma subunit to the Golgi apparatus
Mol. Cell. Biol.
2010
, vol. 
30
 (pg. 
78
-
90
)
[PubMed]
31
Ling
Z.Q.
Guo
W.
Lu
X.X.
Zhu
X.
Hong
L.L.
Wang
Z.
Wang
Z.
Chen
Y.
A Golgi-specific protein PAQR3 is closely associated with the progression, metastasis and prognosis of human gastric cancers
Ann. Oncol.
2014
, vol. 
25
 (pg. 
1363
-
1372
)
[PubMed]
32
Wang
X.
Wang
L.
Zhu
L.
Pan
Y.
Xiao
F.
Liu
W.
Wang
Z.
Guo
F.
Liu
Y.
Thomas
W.G.
Chen
Y.
PAQR3 modulates insulin signaling by shunting phosphoinositide 3-kinase p110alpha to the Golgi apparatus
Diabetes
2013
, vol. 
62
 (pg. 
444
-
456
)
[PubMed]
33
Luo
X.
Feng
L.
Jiang
X.
Xiao
F.
Wang
Z.
Feng
G.S.
Chen
Y.
Characterization of the topology and functional domains of RKTG
Biochem. J.
2008
, vol. 
414
 (pg. 
399
-
406
)
[PubMed]
34
Ernst
P.
Vakoc
C.R.
WRAD: enabler of the SET1-family of H3K4 methyltransferases
Brief. Funct. Genom.
2012
, vol. 
11
 (pg. 
217
-
226
)
[PubMed]
35
Patel
A.
Dharmarajan
V.
Cosgrove
M.S.
Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
32158
-
32161
)
[PubMed]
36
Patel
A.
Vought
V.E.
Dharmarajan
V.
Cosgrove
M.S.
A conserved arginine-containing motif crucial for the assembly and enzymatic activity of the mixed lineage leukemia protein-1 core complex
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
32162
-
32175
)
[PubMed]
37
Higa
L.A.
Wu
M.
Ye
T.
Kobayashi
R.
Sun
H.
Zhang
H.
CUL4-DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation
Nat. Cell Biol.
2006
, vol. 
8
 (pg. 
1277
-
1283
)
[PubMed]

Author notes

1

These authors contributed equally to this work.