Skip to main content
NCBI home page
Neoplasia (New York, N.Y.) logoLink to Neoplasia (New York, N.Y.)
. 2014 Sep 10;16(8):656–664. doi: 10.1016/j.neo.2014.07.008

Late Cornified Envelope Group I, a Novel Target of p53, Regulates PRMT5 Activity1

Zhenzhong Deng *,†, Koichi Matsuda †, Chizu Tanikawa †, Jiaying Lin *,†, Yoichi Furukawa ‡, Ryuji Hamamoto *, Yusuke Nakamura *,⁎
PMCID: PMC4234875  PMID: 25220593

Abstract

p53 is one of the most important tumor suppressor genes involved in human carcinogenesis. Although downstream targets of p53 and their biologic functions in cancer cells have been extensively investigated, it is still far from the full understanding. Here, we demonstrate that Late Cornified Envelope Group I (LCE1) genes, which are located in the LCE gene clusters encoding multiple well-conserved stratum-corneum proteins, are novel downstream targets of p53. Exogenous p53 overexpression using an adenoviral vector system significantly enhanced the expression of LCE1 cluster genes. We also observed induction of LCE1 expressions by DNA damage, which was caused by treatment with adriamycin or UV irradiation in a wild-type p53-dependent manner. Concordantly, the induction of LCE1 by DNA damage was significantly attenuated by the knockdown of p53. Among predicted p53-binding sites within the LCE1 gene cluster, we confirmed one site to be a p53-enhancer sequence by reporter assays. Furthermore, we identified LCE1 to interact with protein arginine methyltransferase 5 (PRMT5). Knockdown of LCE1 by specific small interfering RNAs significantly increased the symmetric dimethylation of histone H3 arginine 8, a substrate of PRMT5, and overexpression of LCE1F remarkably decreased its methylation level. Our data suggest that LCE1 is a novel p53 downstream target that can be directly transactivated by p53 and is likely to have tumor suppressor functions through modulation of the PRMT5 activity.

Introduction

p53 is the most frequently mutated tumor suppressor gene involved in human cancers [1], [2]. Its tetramer protein product can activate the transcription of a number of target downstream genes and mediate a variety of biologic functions through the transcriptional regulation of those targets [3]. To elucidate the critical roles of p53 in human carcinogenesis, we and others have attempted to identify p53 target genes through multiple approaches. We have mainly applied the expression profile analysis after the exogenous introduction of wild-type p53 into cancer cells using the adenovirus vector system and identified more than 50 p53 downstream candidate genes [4]. Among them, we have performed the functional analysis of more than a dozen of target genes including p53R2, p53AIP1, and p53RDL1 [5], [6], [7], [8]. Here, we report the characterization of the Late Cornified Envelope Group I (LCE1) as a novel downstream target of p53.

The LCE clusters contain multiple well-conserved genes encoding stratum-corneum proteins [9], [10] and are located on chromosome 1q21 in a region called as the epidermal differentiation complex [11], [12]. This region is enriched for genes, which are expressed during epidermal differentiation, including loricrin, involucrin, filagrin, the small proline-rich protein genes, and the LCE genes [9], [13]. In mice, members in the LCE1 group are expressed in the relatively late stage of epithelial development and incorporated into the cornified envelope through cross-linking by transglutaminases [10]. In addition, real-time quantitative polymerase chain reaction (qPCR) analysis demonstrated that human LCE1 and LCE2 genes were primarily expressed in skin, whereas LCE4 and LCE5 gene expressions were undetectable in any human tissues examined [9]. In general, physiological functions of LCE proteins, especially their involvement in human cancer are still largely unknown.

Protein arginine methyltransferases (PRMTs) constitute of a large family of enzymes having the arginine methyltransferases activity responsible for catalyzing the formation of monomethyl arginine, asymmetric dimethyl arginine, and symmetric dimethyl arginine (SDMA) [14]. PRMT5 is one of the most well-characterized family members with SDMA activity and catalyzes formation of SDMA in proteins with a glycine and arginine-rich motif [15]. PRMT5 was reported to regulate various cellular functions including apoptosis, Golgi structure, pluripotency, cell growth, and snRNP biosynthesis [16], [17], [18]. One important key marker of the PRMT5 activity is the symmetrical dimethylation of histone 3 arginine 8 (H3R8me2s) level. Through hypermethylation of histone H3R8 around the promoter regions, PRMT5 could cause the transcriptional silencing of cell cycle regulator genes [19], [20]. Since overexpression of PRMT5 has been reported in various types of human cancer, including melanoma, leukemia, lymphoma, glioma, as well as ovarian, breast, prostate, and lung cancers [16], [21], [22], [23], this enzyme is considered as a good molecular target for development of novel cancer therapy [16].

In the present study, we demonstrate that LCE is a novel direct target of p53, can interact with PRMT5, and might modulate histone H3 methylation by PRMT5. This mechanism may be important for the interplay of two important cancer-related genes, p53 and PRMT5, and our findings could indicate a possible role of LCE1 in human carcinogenesis.

Materials and Methods

Microarray Analysis

Replication-deficient recombinant adenovirus designed to express wild-type p53 (Ad-p53) or LacZ (Ad-LacZ) was generated and purified, as previously described [5], [24]. Microarray analysis was then carried out as previously described [1], [4], [24]. In brief, poly(A)+ RNAs were isolated from U373MG cells at different time points after infection with Ad-p53 or Ad-LacZ. Each RNA sample was labeled and hybridized to a microarray consisting of 36,864 genes or Expressed Sequence Tags (ESTs) (http://www.ncbi.nlm.nih.gov/geo/index.cgi, Accession No. GSE14953).

Cell Culture and Transfection

Human cell lines, U373MG (glioblastoma), H1299 (lung carcinoma), and Human Embryonic Kidney 293T (HEK293T), were purchased from American Type Culture Collection (ATCC, Manassas, VA). HCT116 p53−/− and HCT116 p53+/+ cell lines were obtained from Dr Bert Vogelstein (Johns Hopkins University, Baltimore, MD). U373MG glioblastoma cells and H1299 lung cancer cells were infected with Ad-p53 or Ad-LacZ at various multiplicity of infection (MOI) conditions and incubated at 37°C until the time of harvest. HEK293T cells were transfected with HA-Mock (empty vector of pCAGGSnHC) or HA-LCE1F (pCAGGSnHC-LCE1F) using FuGENE6. For protein-protein interaction experiments, HEK293T cells were transfected with HA-Mock or HA-LCE1F and FLAG-Mock or FLAG-PRMT5 using FuGENE6. To examine the co-localization, HCT116 p53+/+ cells were transfected by HA-Mock or HA-LCE1F using FuGENE6. For the gene reporter assay, U373MG (mutated p53) and H1299 (p53 null) cells were transfected with a reporter plasmid and either a Mock empty vector of pcDNA3.1 + or a wild-type p53 expression pcDNA3.1 + vector in combination with a pRL-CMV vector using FuGENE6 [25], [26]. Small interfering RNAs (siRNAs) that were commercially synthesized by Sigma-Aldrich (St Louis, MO) were transfected with Lipofectamine RNAiMAX reagent (Life Technologies, Carlsbad, CA). Two siRNA oligonucleotides of LCE1A-F were designed to target the region commonly conserved among LCE1A-F. Sequences of oligonucleotides are shown in Table W1. Western blot or real-time qPCR was applied to validate the efficiency of overexpression or knockdown experiments.

DNA-Damaging Treatments

Cells were seeded 24 hours before treatment. When cells reached 60% to 70% confluency, cells were incubated with 1 μg/ml adriamycin for 2 hours followed by further incubation of the drug-free medium. Then, the cells were harvested at different time points as indicated in the figure legends. For UV irradiation (UVR) experiments, the cells were washed twice with phosphate-buffered saline (PBS) and exposed to UV rays at different doses using XL-1500 Spectrolinker (Spectronics, Westbury, NY; peak emission, 254 nm). Cells were harvested 36 hours after UVR treatment.

Real-Time qPCR

Total RNA was isolated from cultured cells using RNeasy mini-spin column kits (Qiagen, Hilden, Germany) according to the manufacturer’s procedure. cDNAs were synthesized with the SuperScript Pre-amplification System (Life Technologies). Real-time qPCR was conducted using the SYBR Green I Master on the LightCycler 480 (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. The primer sequences used in this manuscript are shown in Tables W1 and W2. Primers for LCE1A, LCE1C, LCE1E, and LCE1F were described previously [9]. Primers for LCE1B, LCE1D LCE3A, LCE3B, LCE3C, LCE4A, XP33, and C1orf45 genes were designed by us. Except LCE1D, the specificity of all primers was confirmed by DNA sequencing of amplicons. For graphic representation of transcript data, all expression of target genes was shown relative to the housekeeping gene β2-microglobulin expression in the same sample.

Prediction of Putative p53-Binding Sites

DNA sequences of an entire genomic region of LCE1 including 10 kb of the 5′ upstream sequence were downloaded from the University of California Santa Cruz (UCSC) website (http://genome.ucsc.edu/), and the putative p53-binding sites (p53BSs) were screened according to the following criteria; at least 80% matched with the 20 nucleotides of consensus sequence RRRCWWGYYY__RRRCWWGYYY (R, purine; W, A, or T; Y, pyrimidine); we started the screening of 11 consensus sequences without any spacers between the two halves of p53BSs (Figure W1) and confirmed one site that was likely to be a direct p53-binding sequence.

Gene Reporter Assay

DNA fragments including each of the potential p53BSs of the LCE1 gene cluster were amplified by KOD-Plus-DNA polymerase (Toyobo, Osaka, Japan) and subcloned into the pGL3-promoter (pGL3-pro) vector. The primers for amplification are indicated in Table W1. To make a series of mutant vectors, a point mutation “T” was inserted into the site of the fourth and the fourteenth nucleotide “C” and into the seventh and the seventeenth nucleotide “G” of the consensus p53-BS using the KOD-Plus-Mutagenesis Kit (Toyobo). Since a functional p53BS is known in the Fas promoter region, a wild-type Fas promoter construct, pGL3-Fas, was used as a positive control. U373MG (mutated p53) and H1299 (p53 null) cells were plated in 12-well culture plates (5 × 104 cells per well) 24 hours before co-transfection of 125 ng of a reporter plasmid and either 125 ng of a Mock vector or a wild-type p53 expression vector in combination with 25 ng of a pRL-CMV vector. Cells were rinsed with PBS 36 hours after transfection and lysed in 250 μl of lysis buffer. Twenty microliters and 5 μl of lysates from U373MG and H1299, respectively, were sequentially measured using the PGD-S Dual Luciferase assay system following the manufacturer’s procedure (Toyo Ink, Tokyo, Japan). The firefly luciferase value was normalized by the Renilla luciferase activity.

Expression Plasmid Construction

An entire coding sequence of PRMT5 was amplified using cDNA generated from mRNA of HEK293T cells and cloned into pCAGGSn3FC (PRMT5) vector. The entire coding sequences of LCE1A-F were amplified by the use of cDNA generated from mRNA that was extracted from HCT116 p53+/+ cells after 70 J/m2 of UVR treatment. Due to unknown reasons, we have been unsuccessful in cloning the LCE1D cDNA. An HA-epitope tag was placed at the C-terminus of the pCAGGSnHC vector and three-tandem FLAG epitope tags were placed at the C-terminus of the pCAGGSn3FC vector. The DNA sequences of expression constructs for LCE1 (pCAGGSnHC-LCE1) and PRMT5 (pCAGGSn3FC-PRMT5) were confirmed by DNA sequencing using ABI PRISM 3730XL Genetic Analyzer (Life Technologies).

Antibodies

The following primary antibodies were deployed: rabbit anti-HA (Y-11; Santa Cruz Biotechnology, Santa Cruz, CA; dilution used in WB: 1:1000), rabbit anti-PRMT5 (07-415; Millipore, Billerica, MA; dilution used in WB: 1:1000, ICC: 1:400), rabbit anti-p53 (sc6243, Santa Cruz; dilution used in WB: 1:1000), mouse anti-p53 [Ab-1, Calbiochem, San Diego, CA, dilution used in chromatin immunoprecipitation (ChIP): 1:100], rabbit anti-H3R8me2s (ab130740, Abcam, Cambridge, United Kingdom; dilution used in Western blot (WB): 1:1000), mouse anti–α-tubulin (clone DM1A, Millipore; dilution used in WB: 1:1000), rat anti-HA (3F10, Roche; dilution used in Immunocytochemistry (ICC): 1:800), and anti-FLAG (F7425, Sigma-Aldrich; dilution used in ICC: 1:1000).

Immunocytochemistry

Forty-eight hours after transfection with HA-Mock (empty vector of pCAGGSnHC) or pCAGGSnHC-HA-LCE1F into HEK293T or HCT116 p53+/+ cells in four-well chambers, the cells were fixed by 1.7% formaldehyde or 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 in PBS. After covering with blocking solution (3% BSA in 0.2% Triton X-100) for 1 hour at room temperature, cells were incubated with a rat anti-HA antibody and a rabbit PRMT5 antibody overnight under humidified atmosphere at 4°C. Further, the cells were stained with fluorescence-conjugated secondary antibodies and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in VECTASHIELD Mounting Media (HT1200; Vector Laboratories, Burlingame, CA).

Immunoprecipitation and Mass-Spectrometric Analysis

Forty-eight hours after transfection with HA-Mock or HA-LCE1F, HEK293T cells were lysed in lysis buffer [50 mM Tris-HCl (pH 8.0), 0.4% NP-40, and 150 mM NaCl] containing Protease Inhibitor Cocktail Set III (Calbiochem). Whole-cell lysates were pre-cleared by incubation with normal mouse IgG (sc2025, Santa Cruz Biotechnology) and recombinant protein G-sepharose 4B (Life Technologies) at 4°C for 1 hour, followed by incubation with anti-HA agarose (A2095, Sigma-Aldrich) overnight. The proteins were separated in Mini-PROTEAN 5% to 20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis precast gels (Bio-Rad Laboratories, Hercules, CA) and stained with the silver-staining kit (Life Technologies). Protein bands that were specifically observed in the cell extracts transfected with HA-LCE1F compared with Mock were excised and analyzed by liquid chromatography–tandem mass spectrometry as previously described [27]. For the co-immunoprecipitation experiments, HEK293T cells were co-transfected with HA-LCE1F and FLAG-PRMT5 or FLAG-Mock as a control. An HA pull-down experiment is exactly the same as mentioned above. As for FLAG pull-down experiment, whole-cell lysates were pre-cleared by incubation with normal mouse IgG (sc2025, Santa Cruz Biotechnology) and recombinant protein G-sepharose 4B (Life Technologies) at 4°C for 1 hour, followed by incubation with mouse anti-FLAG M2 agarose (F3165, Sigma-Aldrich) overnight. Rat anti-HA (3 F10, Roche) and rabbit anti-Flag (F7425, Sigma-Aldrich) antibodies were used for Western blot. For the histone methylation analysis, histones were extracted by the histone purification mini kit (40026, Active Motif, Carlsbad, CA) following the manufacturer’s procedure strictly.

ChIP Assay

ChIP assays were performed using ChIP Assay kit (17-295; Millipore) according to the manufacturer’s protocol [28]. Briefly, HCT116 p53+/+ cells were treated with 1 μg/ml adriamycin for 2 hours, and cells were cultured in the drug free-medium for 24 hours. Then, cells were harvested and the fragment of p53 and chromatin complexes was immnoprecipitated with an anti-p53 antibody (Ab-1, Calbiochem). After DNA fragments bound to p53 were eluted out, an aliquot was subjected to real-time qPCRs. Protein A agarose/Salmon Sperm DNA (16-157; Millipore) was used as a negative control. Primers were designed to amplify the region containing the p53BS2-binding site, and their sequence information is shown in Table W1.

Results

Identification of a Novel p53 Downstream Target

We had previously performed expression profile analysis to compare U373MG glioblastoma cells infected with wild-type p53 (Ad-p53) with those infected with LacZ (Ad-LacZ) to screen possible downstream genes that are regulated by p53 and identified more than 50 genes that were likely to be induced by wild-type p53. Among them, we confirmed that the transcriptional levels of LCE1B and LCE1C were elevated more than seven-fold higher in the cells infected with wild-type p53 than those with LacZ (data not shown). LCE1B and LCE1C belong to the LCE gene cluster containing multiple well-conserved genes that encode stratum-corneum proteins. Within the LCE cluster, multiple genes form “groups” at chromosome 1q21 and are known to respond “group-wise” to various environmental stimuli like calcium and UV light [9], suggesting that other LCE members that were not included in our microarray might also be regulated by p53. We first compared the sequence similarity of the LCE1B and LCE1C transcripts with other members in the LCE cluster (Table W3) and selected genes showing high similarity (> 80%) for further validation by real-time qPCR because the microarray results might reflect the cross-hybridization of other LCE members that were possibly induced by p53. Interestingly, expression levels of LCE1 group genes were significantly increased in both U373MG (Figure 1A) and H1299 (Figure 1B) cells after 8 MOIs of Ad-p53 infection (Table W4), although the induction levels are very different. However, other genes in the LCE cluster were not induced by wild-type p53 introduction. In addition, endogenous p53, which was activated by adriamycin and UVR, could also induce expressions of the LCE1 group genes in HCT116 p53+/+ cancer cells but not in HCT116 p53−/− cancer cells (Figure 1, C and D), indicating that the LCE1 group genes can respond group-wise to the genotoxic stress condition in a p53-dependent manner [9].

Figure 1.

Figure 1

Open in a new tab

LCE1 is upregulated by p53. Real-time qPCR analysis of LCE1 mRNA expression in U373MG (p53 mutant) (A) and H1299 (p53 null) (B) cells at indicated time points after infection with Ad-p53 or Ad-LacZ at 8 MOIs. (C) Endogenous p53-dependent induction of LCE1 mRNA transcription in p53−/− and p53+/+ HCT116 cells after treatment with adriamycin (1 μg/ml) at various time points. (D) Cells were treated with UVR at different doses and harvested 36 hours after UVR. β2-Microglobulin was used for the normalization of expression levels.

LCE1 Is a Direct Target of p53

We then attempted to clarify whether LCE1 is directly or indirectly regulated by p53. First, 11 putative p53BSs of 20 nucleotides with at least 80% match to the consensus p53-binding sequence (see Materials and Methods section) were selected from the LCE1 cluster region (Figure W1A). We then constructed serial reporter vectors containing each of the 11 predicted p53BSs. Luciferase activity of p53BS2, p53BS6, and p53BS7 was likely to be enhanced by co-transfection of each luciferase vector and a p53 wild-type expression vector into U373MG (Figure W1B) and H1299 cells (Figure W1C) in comparison with those with the mock vector. Hence, these three p53BSs were further investigated by replacement of critical nucleotides in the core consensus sequence with other nucleotides (Figure 2A). As a result, we observed that substitutions in either of the p53BS2, p53BS6, and p53BS7 sequences significantly diminished the enhancement of the luciferase activity in both U373MG and H1299 cells (Figure 2, B and C).These results imply that p53 directly binds to these p53BSs in the LCE1 cluster and regulates their transcriptions.

Figure 2.

Figure 2

Open in a new tab

Identification of p53BSs in the LCE1 cluster genes. (A) In the LCE1 cluster genes, we identified 11 potential p53BSs that have at least 80% match to the consensus p53-binding sequence without any spacer nucleotides between the two halves of p53BSs. Among them, three possible p53 enhancer sequences are shown in the figure. Bold horizontal arrows indicate the locations and relative sizes of each LCE1 gene. Vertical arrows show the potential p53BS locations. Identical nucleotides to the p53-binding sequence are written in capital letters. The underlined cytosine and guanine were substituted with thymine to introduce a mutant-type reporter vector at each p53BS. (B, C) Reporter assays of wild-type p53 and mutant-type p53 in U373MG (B) and H1299 (C) cells. Cells were co-transfected with a Mock (empty pcDNA3.1 +) or a wild-type p53 vector and each reporter plasmid, and luciferase assays were conducted 36 hours after transfection. Results are shown as the firefly luciferase activity normalized by the Renilla luciferase activity with 1 SD. (D) Real-time qPCR of LCE1 family genes. HCT116 p53+/+ cells were transfected with siRNAs targeting EGFP, p53, or LCE1 for 24 hours and then treated with 1 μg/ml adriamycin. Total RNA was extracted from the cells 36 hours after adriamycin treatment.

We subsequently transfected siRNA targeting p53 or that commonly targeting LCE1A-F into HCT116 p53+/+ cells and then the cells were treated with adriamycin. As shown in Figure 2D, induction of LCE1 genes by adriamycin treatment was significantly attenuated in the cells transfected with si-p53, also supporting that LCE1 family genes are p53-direct targets. Additionally, ChIP analysis showed the direct binding of p53 to the LCE1 cluster region in HCT116 p53+/+ cells after adriamycin treatment (Figure W2). We then examined the expression of LCE1F in 82 cancer cell lines by real-time qPCR (Table W5) and confirmed that LCE1F was significantly downregulated in p53 mutant cells compared with p53 wild-type cells in colon and lung cancer cell lines (Figure W3). This is consistent with the induction of LCE1F by p53 in HCT116 colon and H1299 lung cancer cells as shown in Figure 1.

LCE1F Interacts with PRMT5

To further analyze possible biologic functions of LCE1F in cancer cells, we constructed an expression vector designed to express full-length LCE1F protein (pCAGGS-nHC-LCE1F) and transfected it into HEK293T cells. After confirmation of the LCE1F protein expression by Western blot analysis (Figure W4A), we performed immunocytochemical analysis to examine the subcellular localization of LCE1F. As shown in Figure W4B, LCE1F was strongly stained in both nucleus and cytoplasm.

In addition, we attempted to identify the interacting protein(s) of LCE1F. An HA-tagged LCE1F expression vector or an empty mock vector was transfected into HEK293T cells, and cell lysates were immunoprecipitated with anti–HA-conjugated agarose beads. Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the gel was silver stained. We found a 70-kDa protein, which was strongly stained only in HA-LCE1F–overexpressing cells, conducted liquid chromatography–tandem mass spectrometry analysis of this protein, and identified it to be an arginine methyltransferase, PRMT5. This result was confirmed using an anti–PRMT5-specific antibody (Figures 3A and W5). Reversibly, cell lysates of HA-LCE1F–overexpressed cells were immunoprecipitated with an anti-PRMT5 antibody, and the immunoprecipitates were blotted with an anti-HA antibody, which also validated the interaction between LCE1F and PRMT5 (Figure 3B). Moreover, we co-transfected either FLAG-PRMT5 or FLAG-Mock with HA-LCE1F or HA-Mock, and cell lysates were immunoprecipitated with an anti-HA antibody (Figure 3C) or an anti-FLAG antibody (Figure 3D). Subsequent Western blot analysis clearly indicated the interaction between PRMT5 and LCE1F. These results imply that LCE1F interacts with PRMT5 in the cells. Interestingly, we confirmed that all LCE1 family proteins examined could interact with PRMT5 (Figure W6), suggesting that the interaction with PRMT5 is likely to be a common characteristic among LCE1 family members.

Figure 3.

Figure 3

Open in a new tab

Interaction between LCE1F and PRMT5. (A) HEK293T cells were transfected with either a Mock (empty) or an HA-LCE1F expression vector and immunoprecipitated with anti-HA agarose. Samples were immunoblotted with an anti-PRMT5 antibody (Millipore). (B) HA-LCF1F–transfected HEK293T cells were immunoprecipitated with an anti-PRMT5 antibody or normal rabbit IgG (negative control). Samples were immunoblotted with an anti-PRMT5 or anti-HA antibody. (C, D) HEK293T cells were co-transfected with either FLAG-Mock or FLAG-PRMT5 and either HA-Mock or HA-LCE1F. Cells were immunoprecipitated with anti-HA agarose (C) or anti-FLAG antibody (D), and samples were immunoblotted with anti-FLAG or anti-HA antibodies.

LCE1F Suppresses PRMT5 Activity

Since PRMT5 was reported to regulate the transcription of various genes through the methylation of arginine 8 on histone H3 (H3R8), which is considered to play an important role in human carcinogenesis [16], [20], [21], we further investigated the biologic significance of the interaction of PRMT5 and LCE1 members. We first performed immunocytochemical analysis and detected co-localization of LCE1F and PRMT5 in both the nucleus and the cytoplasm of the HCT116 p53+/+ cells as shown in Figure 4. We then examined the methylation status of H3R8 after standardizing the quantity of PRMT5 using the anti-PRMT5 antibody. Using an antibody for symmetric dimethylation of H3R8 (H3R8me2s), we conducted Western blot analysis after transfection with siRNAs targeting Enhanced green fluorescent protein (EGFP), p53, or LCE1 under treatment with adriamycin (Figure 5A) and found that knockdown of p53 and LCE1 significantly enhanced the methylation levels of H3R8 in HCT116 p53+/+ cells, compared with the control cells, suggesting that LCE1 may modulate the PRMT5-dependent H3R8 methylation. To further validate this result, we co-transfected either FLAG-PRMT5 or FLAG-Mock and HA-LCE1F or HA-Mock into HEK293T cells and purified histone proteins from cell lysates. As shown in Figure 5B, in the presence of LCE1F, H3R8 methylation by PRMT5 protein was significantly suppressed. Taken together, our data indicate that LCE1F may negatively regulate PRMT5-dependent H3R8 methylation through the direct association to PRMT5.

Figure 4.

Figure 4

Open in a new tab

Co-localization of LCE1F and PRMT5. HCT116 p53+/+ cells were transfected with HA-LCE1F. Cells were fixed by 1.7% formaldehyde and stained with an anti-HA antibody (red) and an anti-PRMT5 antibody (green).

Figure 5.

Figure 5

Open in a new tab

Possible mechanism of LCE1F on regulation of PRMT5 activity. (A) HCT116 p53+/+ cells were transfected with siRNAs targeting EGFP, p53, or LCE1 for 24 hours and then treated with 1 μg/ml adriamycin. Proteins were extracted from the cells 36 hours after adriamycin treatment and immunoblotted with anti-p53, anti-H3R8 SDMA (H3R8me2s), or anti–α-tubulin (control) antibodies. (B) HEK293T cells were co-transfected with either FLAG-Mock or FLAG-PRMT5 and either HA-Mock or HA-LCE1F. Cells were harvested 48 hours after transfection and immunoblotted with anti-H3R8me2s or histone H3 (control) antibodies after histone purification. (C) A possible model of the network among p53, LCE1F, and PRMT5. p53 induced by DNA damage activates transcription of LCE1F, which suppresses the PRMT5 methyltransferase activity through their interaction.

Discussion

In this study, we showed that LCE1 is a novel p53 target gene and that the LCE1F protein interacts with the arginine methyltransferase PRMT5. Through the interaction with PRMT5, LCE1F may suppress the PRMT5 methylation activity on arginine 8 of histone H3 (Figure 5C). Since PRMT5 is overexpressed in a wide range of human cancer and plays a critical role in tumorigenesis through the regulation of histone methylation, we unveiled a novel mechanism of tumor suppression mediated by p53.

The LCE gene cluster contains multiple conserved genes encoding stratum-corneum proteins [9], [10], [12], [29]. Our study demonstrated that most of the members in the LCE1 group are transcriptionally regulated by the tumor suppressor p53 although the induction levels varied. Concordantly, LCE1 genes were reported to be significantly upregulated in response to UVB irradiation of the skin cells [9]. UVR causes DNA damage, photoperoxidation of lipids, protein cross-linking, and isomerization of urocanic acid that lead to immunosuppression, photo-induced aging and cancer. p53 protein acts as a molecular sensor for the damages generated by UVR through mediating cell cycle arrest and apoptosis in damaged keratinocytes [30], [31], [32], [33], [34]. Taken together, cells may possess the function to express LCE1 family genes through p53 activation to eliminate dangerous cells with DNA damages. Importantly, LCE1 family proteins show a high level of sequence similarity and LCE1F has more than 90% homology with other members (LCE1A-E; Table W6). In fact, as mentioned above, we confirmed the interaction between all of LCE1 proteins examined and PRMT5 (Figure W6). Since the expression of LCE1 cluster genes is regulated by p53 as a whole (may not be all of the members), this protein family members might play important roles to complementally or redundantly function as a tumor suppressor.

We identified PRMT5, a histone methyltransferase, as a key binding partner of the LCE1 proteins. Current progress of molecular medicine revealed that the enzymes relevant to histone methylation play critical roles in human carcinogenesis [28], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45]. PRMT5 is one of the type II arginine methyltransferases, which catalyze the formation of symmetric dimethylation of arginine residues (SDMA) and regulates various cellular pathways [16], [17], [21]. A number of reports described the importance of this arginine methyltransferase in tumorigenesis [16], [18], [21], [46], [47], [48], [49]. Intriguingly, PRMT5 was reported to interact with BRG1- and hBRM-based hSWI/SNF chromatin remodelers and methylate arginine 8 on histone H3 [20]. The H3R8 methylation mediated by PRMT5-containing BRG1 and hBRM complexes directly repressed the expression of suppressor of tumorigenicity 7 and nonmetastatic 23, tumor suppressor genes [20]. This line of pathway seems to be a key mechanism in the PRMT5-dependent tumorigenesis. Our data presented here have implied that LCE1, which is regulated by the tumor suppressor p53, negatively regulates H3R8 methylation mediated by PRMT5 (Figure 5, A and B), suggesting a new role of p53 in the regulation of histone modification. Since PRMT5 was reported to methylate p53 through the direct interaction and this methylation prevents p53-dependent apoptosis in cancer cells [50], we suggest an interesting feedback mechanism among p53, LCE1, and PRMT5.

Overall, our study elucidates a novel function of p53 as a tumor suppressor through the transcriptional regulation of the LCE1 cluster genes. Further functional analysis may explore the importance of the LCE1 group proteins as tumor suppressors and the physiological relevance among p53 downstream genes.

Acknowledgment

We thank Koji Ueda and Yuki Funauchi for helpful discussion and their technical supports. This work was supported by Project for Development of Innovation Research on Cancer Therapeutics. Disclosure of potential conflicts of interest: Y.N. is a stockholder and a scientific advisor of Oncotherapy Science and also has research grants from Oncotherapy Science. No potential conflicts of interest were disclosed by the other authors.

Footnotes

1

This article refers to supplementary materials, which are designated by Tables W1 to W6 and Figures W1 to W6 and are available online at www.neoplasia.com.

Appendix A

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.neo.2014.07.008.

Appendix A. Supplementary Materials

Figure W1. Screening of p53BSs in the gene regulatory region of the LCE1 cluster. (A) Eleven potential p53BSs of 20 nucleotides in the gene regulatory region of the LCE1 gene cluster were predicted to match the consensus p53-binding sequence with over 80% similarity. Bold horizontal arrows indicate the locations and relative sizes of each LCE1 gene. Vertical arrows show the potential p53BS locations. Identical nucleotides to the p53-binding sequence are written in capital letters. (B, C) Reporter assays of wild-type p53 in U373MG (B) and H1299 (C) cells. Cells were co-transfected with a mock (empty) or a wild-type p53 and each reporter plasmid, and luciferase assays were conducted 36 hours after transfection. Results were shown as relative values compared to the activity of the pGL3-promoter empty vector.

Figure W2. ChIP analysis using primer pairs of the p53BS2 region as described in the Materials and Methods section. Cross-linked and sheared chromatin was immunoprecipitated with an anti-p53 antibody (Ab-1; Calbiochem). Protein A agarose/Salmon Sperm DNA (Millipore) was used as a negative control. Results are the mean ± SD of three independent experiments and shown as a percentage of the input chromatin. The P value was calculated using Student’s t test.

Figure W3. Expression levels of LCE1F in cancer cell lines. Real-time qPCR confirmed the mRNA expression of LCE1F in 82 cancer cell lines (Table W5). β2-Microglobulin (B2M) was used for the normalization of expression levels. Asterisks indicate P value calculated by Student’s t test: *P < .05.

Figure W4. Subcellular localization of LCE1F. HEK293T cells were transfected with a vector designed to express HA-tagged LCE1F (pCAGGSnHC-LCE1F). (A) Cell lysates were immunoblotted with an anti-HA antibody. CBB staining was shown as the control of loading protein amount. (B) Subcellular localization of LCE1F in HEK293T cells analyzed by immunocytochemistry. Cells were fixed with 4% paraformaldehyde and stained with an anti-HA antibody (green) and DAPI for staining the nucleus (blue).

Figure W5. Validation of an anti-PRMT5 antibody. HEK293T and HCT116 p53+/+ cells were transfected by one control siRNA (si-EGFP) and two si-PRMT5s (#1 and #2). Western blot was performed using a rabbit PRMT5 antibody 60 hours after transfection (left panel). The membrane was stained with Simple Blue staining solution to visualize protein loading amount (right panel).

Figure W6. Interaction between LCE1A-F and PRMT5. HEK293T cells were transfected with either a mock (empty) or HA-LCE1A-F (except LCE1D) expression vector and immunoprecipitated with anti-HA agarose (Sigma). Samples were immunoblotted with an anti-PRMT5 antibody (Millipore) or an anti-HA antibody (Roche 3F10).

Table W1. Primers and Oligonucleotides Designed

Table W2. Primer Sequences of Real-Time qPCR for 11 LCE Members

Table W3. mRNA Similarity of LCE Family

Table W4. p53 Induction Status of 11 LCE Members

Table W5. List of 82 Cancer Cell Lines

Table W6. Homology of LCE1 Protein Family

mmc1.docx (2.6MB, docx)

References

  • 1.Soussi T. TP53 mutations in human cancer: database reassessment and prospects for the next decade. Adv Cancer Res. 2011;110:107–139. doi: 10.1016/B978-0-12-386469-7.00005-0. [DOI] [PubMed] [Google Scholar]
  • 2.Leroy B, Fournier JL, Ishioka C, Monti P, Inga A, Fronza G, Soussi T. The TP53 website: an integrative resource centre for the TP53 mutation database and TP53 mutant analysis. Nucleic Acids Res. 2013;41:D962–D969. doi: 10.1093/nar/gks1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Beckerman R, Prives C. Transcriptional regulation by p53. Cold Spring Harb Perspect Biol. 2010;2:a000935. doi: 10.1101/cshperspect.a000935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Tanikawa C, Furukawa Y, Yoshida N, Arakawa H, Nakamura Y, Matsuda K. XEDAR as a putative colorectal tumor suppressor that mediates p53-regulated anoikis pathway. Oncogene. 2009;28:3081–3092. doi: 10.1038/onc.2009.154. [DOI] [PubMed] [Google Scholar]
  • 5.Oda K, Arakawa H, Tanaka T, Matsuda K, Tanikawa C, Mori T, Nishimori H, Tamai K, Tokino T, Nakamura Y. p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell. 2000;102:849–862. doi: 10.1016/s0092-8674(00)00073-8. [DOI] [PubMed] [Google Scholar]
  • 6.Tanaka H, Arakawa H, Yamaguchi T, Shiraishi K, Fukuda S, Matsui K, Takei Y, Nakamura Y. A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature. 2000;404:42–49. doi: 10.1038/35003506. [DOI] [PubMed] [Google Scholar]
  • 7.Tanikawa C, Matsuda K, Fukuda S, Nakamura Y, Arakawa H. p53RDL1 regulates p53-dependent apoptosis. Nat Cell Biol. 2003;5:216–223. doi: 10.1038/ncb943. [DOI] [PubMed] [Google Scholar]
  • 8.Nakamura Y. Isolation of p53-target genes and their functional analysis. Cancer Sci. 2004;95:7–11. doi: 10.1111/j.1349-7006.2004.tb03163.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Jackson B, Tilli CM, Hardman MJ, Avilion AA, MacLeod MC, Ashcroft GS, Byrne C. Late cornified envelope family in differentiating epithelia—response to calcium and ultraviolet irradiation. J Invest Dermatol. 2005;124:1062–1070. doi: 10.1111/j.0022-202X.2005.23699.x. [DOI] [PubMed] [Google Scholar]
  • 10.Marshall D, Hardman MJ, Nield KM, Byrne C. Differentially expressed late constituents of the epidermal cornified envelope. Proc Natl Acad Sci U S A. 2001;98:13031–13036. doi: 10.1073/pnas.231489198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Mischke D, Korge BP, Marenholz I, Volz A, Ziegler A. Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex (“epidermal differentiation complex”) on human chromosome 1q21. J Invest Dermatol. 1996;106:989–992. doi: 10.1111/1523-1747.ep12338501. [DOI] [PubMed] [Google Scholar]
  • 12.Bergboer JG, Tjabringa GS, Kamsteeg M, van Vlijmen-Willems IM, Rodijk-Olthuis D, Jansen PA, Thuret JY, Narita M, Ishida-Yamamoto A, Zeeuwen PL. Psoriasis risk genes of the late cornified envelope-3 group are distinctly expressed compared with genes of other LCE groups. Am J Pathol. 2011;178:1470–1477. doi: 10.1016/j.ajpath.2010.12.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhao XP, Elder JT. Positional cloning of novel skin-specific genes from the human epidermal differentiation complex. Genomics. 1997;45:250–258. doi: 10.1006/geno.1997.4952. [DOI] [PubMed] [Google Scholar]
  • 14.Bedford MT, Clarke SG. Protein arginine methylation in mammals: who, what, and why. Mol Cell. 2009;33:1–13. doi: 10.1016/j.molcel.2008.12.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Gkountela S, Li Z, Chin CJ, Lee SA, Clark AT. PRMT5 is required for human embryonic stem cell proliferation but not pluripotency. Stem Cell Rev. 2014;10:230–239. doi: 10.1007/s12015-013-9490-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Nicholas C, Yang J, Peters SB, Bill MA, Baiocchi RA, Yan F, Sif S, Tae S, Gaudio E, Wu X. PRMT5 is upregulated in malignant and metastatic melanoma and regulates expression of MITF and p27(Kip1.) PLoS One. 2013;8:e74710. doi: 10.1371/journal.pone.0074710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Powers MA, Fay MM, Factor RE, Welm AL, Ullman KS. Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4. Cancer Res. 2011;71:5579–5587. doi: 10.1158/0008-5472.CAN-11-0458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bao X, Zhao S, Liu T, Liu Y, Liu Y, Yang X. Overexpression of PRMT5 promotes tumor cell growth and is associated with poor disease prognosis in epithelial ovarian cancer. J Histochem Cytochem. 2013;61:206–217. doi: 10.1369/0022155413475452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Majumder S, Alinari L, Roy S, Miller T, Datta J, Sif S, Baiocchi R, Jacob ST. Methylation of histone H3 and H4 by PRMT5 regulates ribosomal RNA gene transcription. J Cell Biochem. 2010;109:553–563. doi: 10.1002/jcb.22432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Pal S, Vishwanath SN, Erdjument-Bromage H, Tempst P, Sif S. Human SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and negatively regulates expression of ST7 and NM23 tumor suppressor genes. Mol Cell Biol. 2004;24:9630–9645. doi: 10.1128/MCB.24.21.9630-9645.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Pal S, Baiocchi RA, Byrd JC, Grever MR, Jacob ST, Sif S. Low levels of miR-92b/96 induce PRMT5 translation and H3R8/H4R3 methylation in mantle cell lymphoma. EMBO J. 2007;26:3558–3569. doi: 10.1038/sj.emboj.7601794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Gu Z, Gao S, Zhang F, Wang Z, Ma W, Davis RE, Wang Z. Protein arginine methyltransferase 5 is essential for growth of lung cancer cells. Biochem J. 2012;446:235–241. doi: 10.1042/BJ20120768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Gu Z, Li Y, Lee P, Liu T, Wan C, Wang Z. Protein arginine methyltransferase 5 functions in opposite ways in the cytoplasm and nucleus of prostate cancer cells. PLoS One. 2012;7:e44033. doi: 10.1371/journal.pone.0044033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Tanikawa C, Nakagawa H, Furukawa Y, Nakamura Y, Matsuda K. CLCA2 as a p53-inducible senescence mediator. Neoplasia. 2012;14:141–149. doi: 10.1593/neo.111700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Hamamoto R, Furukawa Y, Morita M, Iimura Y, Silva FP, Li M, Yagyu R, Nakamura Y. SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat Cell Biol. 2004;6:731–740. doi: 10.1038/ncb1151. [DOI] [PubMed] [Google Scholar]
  • 26.Hamamoto R, Silva FP, Tsuge M, Nishidate T, Katagiri T, Nakamura Y, Furukawa Y. Enhanced SMYD3 expression is essential for the growth of breast cancer cells. Cancer Sci. 2006;97:113–118. doi: 10.1111/j.1349-7006.2006.00146.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Piao L, Nakagawa H, Ueda K, Chung S, Kashiwaya K, Eguchi H, Ohigashi H, Ishikawa O, Daigo Y, Matsuda K. C12orf48, termed PARP-1 binding protein, enhances poly(ADP-ribose) polymerase-1 (PARP-1) activity and protects pancreatic cancer cells from DNA damage. Genes Chromosomes Cancer. 2011;50:13–24. doi: 10.1002/gcc.20828. [DOI] [PubMed] [Google Scholar]
  • 28.Kogure M, Takawa M, Saloura V, Sone K, Piao L, Ueda K, Ibrahim R, Tsunoda T, Sugiyama M, Atomi Y. The oncogenic polycomb histone methyltransferase EZH2 methylates lysine 120 on histone H2B and competes ubiquitination. Neoplasia. 2013;15:1251–1261. doi: 10.1593/neo.131436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Tong L, Corrales RM, Chen Z, Villarreal AL, De Paiva CS, Beuerman R, Li DQ, Pflugfelder SC. Expression and regulation of cornified envelope proteins in human corneal epithelium. Invest Ophthalmol Vis Sci. 2006;47:1938–1946. doi: 10.1167/iovs.05-1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Benjamin CL, Ullrich SE, Kripke ML, Ananthaswamy HN. p53 tumor suppressor gene: a critical molecular target for UV induction and prevention of skin cancer. Photochem Photobiol. 2008;84:55–62. doi: 10.1111/j.1751-1097.2007.00213.x. [DOI] [PubMed] [Google Scholar]
  • 31.Ziegler A, Jonason AS, Leffell DJ, Simon JA, Sharma HW, Kimmelman J, Remington L, Jacks T, Brash DE. Sunburn and p53 in the onset of skin cancer. Nature. 1994;372:773–776. doi: 10.1038/372773a0. [DOI] [PubMed] [Google Scholar]
  • 32.Yamaizumi M, Sugano T. U.v.-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes independent of the cell cycle. Oncogene. 1994;9:2775–2784. [PubMed] [Google Scholar]
  • 33.Gujuluva CN, Baek JH, Shin KH, Cherrick HM, Park NH. Effect of UV-irradiation on cell cycle, viability and the expression of p53, gadd153 and gadd45 genes in normal and HPV-immortalized human oral keratinocytes. Oncogene. 1994;9:1819–1827. [PubMed] [Google Scholar]
  • 34.Maltzman W, Czyzyk L. UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol Cell Biol. 1984;4:1689–1694. doi: 10.1128/mcb.4.9.1689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Cho HS, Shimazu T, Toyokawa G, Daigo Y, Maehara Y, Hayami S, Ito A, Masuda K, Ikawa N, Field HI. Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B. Nat Commun. 2012;3:1072. doi: 10.1038/ncomms2074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Hayami S, Kelly JD, Cho HS, Yoshimatsu M, Unoki M, Tsunoda T, Field HI, Neal DE, Yamaue H, Ponder BA. Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers. Int J Cancer. 2011;128:574–586. doi: 10.1002/ijc.25349. [DOI] [PubMed] [Google Scholar]
  • 37.Hayami S, Yoshimatsu M, Veerakumarasivam A, Unoki M, Iwai Y, Tsunoda T, Field HI, Kelly JD, Neal DE, Yamaue H. Overexpression of the JmjC histone demethylase KDM5B in human carcinogenesis: involvement in the proliferation of cancer cells through the E2F/RB pathway. Mol Cancer. 2010;9:59. doi: 10.1186/1476-4598-9-59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Takawa M, Masuda K, Kunizaki M, Daigo Y, Takagi K, Iwai Y, Cho HS, Toyokawa G, Yamane Y, Maejima K. Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker. Cancer Sci. 2011;102:1298–1305. doi: 10.1111/j.1349-7006.2011.01958.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Toyokawa G, Cho HS, Masuda K, Yamane Y, Yoshimatsu M, Hayami S, Takawa M, Iwai Y, Daigo Y, Tsuchiya E. Histone lysine methyltransferase Wolf-Hirschhorn syndrome candidate 1 is involved in human carcinogenesis through regulation of the Wnt pathway. Neoplasia. 2011;13:887–898. doi: 10.1593/neo.11048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Yoshimatsu M, Toyokawa G, Hayami S, Unoki M, Tsunoda T, Field HI, Kelly JD, Neal DE, Maehara Y, Ponder BA. Dysregulation of PRMT1 and PRMT6, Type I arginine methyltransferases, is involved in various types of human cancers. Int J Cancer. 2011;128:562–573. doi: 10.1002/ijc.25366. [DOI] [PubMed] [Google Scholar]
  • 41.Takawa M, Cho HS, Hayami S, Toyokawa G, Kogure M, Yamane Y, Iwai Y, Maejima K, Ueda K, Masuda A. Histone lysine methyltransferase SETD8 promotes carcinogenesis by deregulating PCNA expression. Cancer Res. 2012;72:3217–3227. doi: 10.1158/0008-5472.CAN-11-3701. [DOI] [PubMed] [Google Scholar]
  • 42.Cho HS, Kelly JD, Hayami S, Toyokawa G, Takawa M, Yoshimatsu M, Tsunoda T, Field HI, Neal DE, Ponder BA. Enhanced expression of EHMT2 is involved in the proliferation of cancer cells through negative regulation of SIAH1. Neoplasia. 2011;13:676–684. doi: 10.1593/neo.11512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Cho HS, Toyokawa G, Daigo Y, Hayami S, Masuda K, Ikawa N, Yamane Y, Maejima K, Tsunoda T, Field HI. The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G1/S transition in cancer cells via transcriptional regulation of the HOXA1 gene. Int J Cancer. 2012;131:E179–E189. doi: 10.1002/ijc.26501. [DOI] [PubMed] [Google Scholar]
  • 44.Toyokawa G, Cho HS, Iwai Y, Yoshimatsu M, Takawa M, Hayami S, Maejima K, Shimizu N, Tanaka H, Tsunoda T. The histone demethylase JMJD2B plays an essential role in human carcinogenesis through positive regulation of cyclin-dependent kinase 6. Cancer Prev Res (Phila) 2011;4:2051–2061. doi: 10.1158/1940-6207.CAPR-11-0290. [DOI] [PubMed] [Google Scholar]
  • 45.Kogure M, Takawa M, Cho HS, Toyokawa G, Hayashi K, Tsunoda T, Kobayashi T, Daigo Y, Sugiyama M, Atomi Y. Deregulation of the histone demethylase JMJD2A is involved in human carcinogenesis through regulation of the G1/S transition. Cancer Lett. 2013;336:76–84. doi: 10.1016/j.canlet.2013.04.009. [DOI] [PubMed] [Google Scholar]
  • 46.Wang L, Pal S, Sif S. Protein arginine methyltransferase 5 suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells. Mol Cell Biol. 2008;28:6262–6277. doi: 10.1128/MCB.00923-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Cho EC, Zheng S, Munro S, Liu G, Carr SM, Moehlenbrink J, Lu YC, Stimson L, Khan O, Konietzny R. Arginine methylation controls growth regulation by E2F-1. EMBO J. 2012;31:1785–1797. doi: 10.1038/emboj.2012.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Yan F, Alinari L, Lustberg ME, Katherine Martin L, Cordero-Nieves HM, Banasavadi-Siddegowda Y, Virk S, Barnholtz-Sloan J, Bell EH, Wojton J. Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma. Cancer Res. 2014;74:1752–1765. doi: 10.1158/0008-5472.CAN-13-0884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Shilo K, Wu X, Sharma S, Welliver M, Duan W, Villalona-Calero M, Fukuoka J, Sif S, Baiocchi R, Hitchcock CL. Cellular localization of protein arginine methyltransferase-5 correlates with grade of lung tumors. Diagn Pathol. 2013;8:201. doi: 10.1186/1746-1596-8-201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Jansson M, Durant ST, Cho EC, Sheahan S, Edelmann M, Kessler B, La Thangue NB. Arginine methylation regulates the p53 response. Nat Cell Biol. 2008;10:1431–1439. doi: 10.1038/ncb1802. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure W1. Screening of p53BSs in the gene regulatory region of the LCE1 cluster. (A) Eleven potential p53BSs of 20 nucleotides in the gene regulatory region of the LCE1 gene cluster were predicted to match the consensus p53-binding sequence with over 80% similarity. Bold horizontal arrows indicate the locations and relative sizes of each LCE1 gene. Vertical arrows show the potential p53BS locations. Identical nucleotides to the p53-binding sequence are written in capital letters. (B, C) Reporter assays of wild-type p53 in U373MG (B) and H1299 (C) cells. Cells were co-transfected with a mock (empty) or a wild-type p53 and each reporter plasmid, and luciferase assays were conducted 36 hours after transfection. Results were shown as relative values compared to the activity of the pGL3-promoter empty vector.

Figure W2. ChIP analysis using primer pairs of the p53BS2 region as described in the Materials and Methods section. Cross-linked and sheared chromatin was immunoprecipitated with an anti-p53 antibody (Ab-1; Calbiochem). Protein A agarose/Salmon Sperm DNA (Millipore) was used as a negative control. Results are the mean ± SD of three independent experiments and shown as a percentage of the input chromatin. The P value was calculated using Student’s t test.

Figure W3. Expression levels of LCE1F in cancer cell lines. Real-time qPCR confirmed the mRNA expression of LCE1F in 82 cancer cell lines (Table W5). β2-Microglobulin (B2M) was used for the normalization of expression levels. Asterisks indicate P value calculated by Student’s t test: *P < .05.

Figure W4. Subcellular localization of LCE1F. HEK293T cells were transfected with a vector designed to express HA-tagged LCE1F (pCAGGSnHC-LCE1F). (A) Cell lysates were immunoblotted with an anti-HA antibody. CBB staining was shown as the control of loading protein amount. (B) Subcellular localization of LCE1F in HEK293T cells analyzed by immunocytochemistry. Cells were fixed with 4% paraformaldehyde and stained with an anti-HA antibody (green) and DAPI for staining the nucleus (blue).

Figure W5. Validation of an anti-PRMT5 antibody. HEK293T and HCT116 p53+/+ cells were transfected by one control siRNA (si-EGFP) and two si-PRMT5s (#1 and #2). Western blot was performed using a rabbit PRMT5 antibody 60 hours after transfection (left panel). The membrane was stained with Simple Blue staining solution to visualize protein loading amount (right panel).

Figure W6. Interaction between LCE1A-F and PRMT5. HEK293T cells were transfected with either a mock (empty) or HA-LCE1A-F (except LCE1D) expression vector and immunoprecipitated with anti-HA agarose (Sigma). Samples were immunoblotted with an anti-PRMT5 antibody (Millipore) or an anti-HA antibody (Roche 3F10).

Table W1. Primers and Oligonucleotides Designed

Table W2. Primer Sequences of Real-Time qPCR for 11 LCE Members

Table W3. mRNA Similarity of LCE Family

Table W4. p53 Induction Status of 11 LCE Members

Table W5. List of 82 Cancer Cell Lines

Table W6. Homology of LCE1 Protein Family

mmc1.docx (2.6MB, docx)

Articles from Neoplasia (New York, N.Y.) are provided here courtesy of Neoplasia Press

RESOURCES