Methylation of C/EBPa by PRMT1 Inhibits Its Tumor ... · Molecular Cell Biology Methylation of C/EBPa by PRMT1 Inhibits Its Tumor-Suppressive Function in Breast Cancer Li-Ming Liu1,2,Wen-Zheng

Molecular Cell Biology

Methylation of C/EBPa by PRMT1 Inhibits ItsTumor-Suppressive Function in Breast CancerLi-Ming Liu1,2,Wen-Zheng Sun1,2, Xue-Zhe Fan1,2, Ya-Li Xu3, Mo-Bin Cheng1,2,and Ye Zhang1,2

Abstract

C/EBPa is an essential transcription factor involved inregulating the expression or function of certain cell-cycleregulators, including in breast cancer cells. Although proteinarginine methyltransferases have been shown to play onco-genic roles in a variety of cancers, little is known about therole of arginine methylation in regulating the antiprolifera-tion activity of C/EBPa. Here, we report that the proteinarginine methyltransferase 1 (PRMT1) is overexpressed inhuman breast cancer and that elevated PRMT1 correlateswith cancer malignancy. RNA-sequencing analysis revealedthat knockdown of PRMT1 in breast cancer cells is accom-panied by a decrease in the expression of pro-proliferativegenes, including cyclin D1. Furthermore, tandem affinitypurification followed by mass spectrometry identifiedPRMT1 as a component of the C/EBPa complex. C/EBPa

associated with and was methylated by PRMT1 at threearginine residues (R35, R156, and R165). PRMT1-depen-dent methylation of C/EBPa promoted the expression ofcyclin D1 by blocking the interaction between C/EBPa andits corepressor HDAC3, which resulted in rapid growth oftumor cells during the pathogenesis of breast cancer. Inhi-bition of PRMT1 significantly impeded the growth of cancercells from patients with triple-negative breast cancer. Thisevidence that PRMT1 mediates C/EBPa methylation shedslight on a novel pathway and potential therapeutic target inbreast cancer.

Significance: This study provides novel mechanistic insightof the role of the arginine methyltransferase PRMT1 in breastcancer pathogenesis.

IntroductionProtein arginine methylation catalyzed by the protein arginine

methyltransferase (PRMT) family is a major posttranslationalmodification that regulates multiple cellular processes (1–3).PRMT1 is thought to perform 85% of PRMT activity in mamma-lian cells (4). Many nonhistone substrates of PRMT1 are involvedin biological processes such as transcriptional regulation and cellsignaling (1, 5), and methylation of histone H4 at arginine 3(H4R3me2a) by PRMT1 activates gene regulation (6, 7). PRMT1-mediated arginine methylation of FOXO1 enhances its transacti-vation functionby stabilizing the FOXO1protein (8). PRMT1 also

functions as a coactivator of RUNX1 to activate its target genes bydisrupting the interaction between RUNX1 and SIN3A (9). Giventhe extensive substrates of PRMT1 in cells, aberrant expression ofPRMT1 has been implicated in the pathogenesis of several dis-eases, including cancer (3). PRMT1 upregulation (10–12) oraberrant splicing (13) has been observed in many types ofmalignancies. However, the function of PRMT1 in breast cancerand the mechanism of its effect on gene transcription are stillincompletely understood.

CCAAT/enhancer binding proteins (C/EBP) are a family ofbasic leucine zipper (bZIP) DNA-binding proteins that regulatethe transcription of several tissue-specific genes and some growth-related genes (14). C/EBPa is the foundingmember of the C/EBPsfamily and contains a C-terminal bZIP and N-terminal transacti-vation domain (TAD1–3; refs. 15, 16). C/EBPa has two distinctisoforms, p42 and p30, with varying N-terminus length due totranslation from different initiation codons in a single mRNA. C/EBPa-p30 lacks themajor transactivation domain (1–120 aa) andcan neutralize the transcriptional activity of C/EBPa-p42 (17, 18).C/EBPa is a strong inhibitor of cell proliferation and has beenimplicated as a tumor suppressor in various malignant tumors.The antimitotic function of C/EBPa depends on protein–proteininteractions and regulation of its binding proteins, such asp21 (19), CDK4/6 (20), and E2F (21, 22). C/EBPa also functionsas a transcriptional factor to activate or repress some growth-related genes, such as N-Myc (23), Sox4 (24), and BMI1 (25), tomodulate cell proliferation.

A key component of C/EBPa regulation is posttranslationalmodifications (PTM). C/EBPa is decorated with various PTMs,including phosphorylation (26–29), SUMOylation (30–32), andacetylation (33). Most of these modifications repress C/EBPafunction by interferingwith itsDNA-binding function or blocking

1State Key Laboratory of Medical Molecular Biology, Chinese Academy ofMedical Sciences & Peking Union Medical College, Beijing, China. 2Departmentof Biochemistry and Molecular Biology, Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing,China. 3Department of Breast Surgery, Peking Union Medical College Hospital,Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing,China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

L.-M. Liu and W.-Z. Sun contributed equally to this article.

Corresponding Authors: Ye Zhang, Peking Union Medical College & ChineseAcademy of Medical Sciences, 5 Dongdan Santiao, Beijing 100005, China.Phone: 8610-6915-5939; Fax: 8610-6526-9665; E-mail:[email protected]; and Mo-Bin Cheng,[email protected]

Cancer Res 2019;79:2865–77

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interactions with partners. However, little is known about the roleof argininemethylation in regulating the antiproliferation activityof C/EBPa.

Here, we report that C/EBPa directly interacts with and isarginine methylated by PRMT1; methylated C/EBPa impairs thebinding of HDAC3 to derepress the expression of cyclin D1followed by cell growth in breast cancer cells. PRMT1 is signifi-cantly upregulated in breast carcinoma samples, and PRMT1knockdown impairs the proliferation capability of breast cancercells. Importantly, a specific inhibitor of PRMT1 profoundlydecreases the growth of cancer cells from patients.

Materials and MethodsCell cultures

MDA-MB-231 (catalog no. 3111C0001CCC000014), MDA-MB-435 (catalog no. 3111C0001CCC000351), MDA-MB-468 (catalogno. 3111C0001CCC000249), MCF7 (catalog no. 3111C0001-CCC000013), SK-BR-3 (catalog no. 3111C0001CCC000085),HEK293T (catalog no. 3111C0001CCC000091), HeLa (catalog no.3111C0001CCC000011), BT549 (catalog no. 3111C0001-CCC000336), and MCF10A (catalog no. 3111C0001CCC000406)cell lines were purchased from Chinese National Infrastructure ofCell Line Resource. Before the experiments, all the cell lines wereauthenticated on cell micrograph compared with the cell lines onATCC.HEK293T cells showed 90% transfect efficiencywithGFP-tagplasmid. Mycoplasma contamination was detected by the EZ-PCRMycoplasma Test Kit (catalog no. 20-700-20) as described previ-ously (34). MDA-MB-231, MDA-MB-435, MDA-MB-468, MCF7,SK-BR-3, HEK293T, HeLa, and BT549 cells were grown in DMEMsupplemented with 10% FBS and 100 U/mL penicillin–streptomy-cin. MCF10A cells were maintained in DMEM/F12 (1:1) mediumsupplemented with 5% horse serum, 0.5 mg/mL hydrocortisone, 10mg/mL insulin, 20 ng/mL EGF, 0.1 mg/mL cholera toxin, 100 U/mLpenicillin–streptomycin.

Primary breast cancer cells (TNBC-1 and TNBC-2) were grownin DMEM/F12 (1:1) medium supplemented 5% FBS, 0.4 mg/mLhydrocortisone, 5 mg/mL insulin, 10 ng/mL EGF, 10 ng/mLcholera toxin, 25 mg/mL adenine, 5 mmol/L Rock inhibitor, and100 U/mL penicillin–streptomycin. These cells were treated withfuramidine dihydrochloride (SML1559, Sigma), a specific inhib-itor of PRMT1 (35) at concentration of 0, 20, 40, and 60 mmol/Lfor 6 days.

AntibodiesAntibodies for PRMT1 (07-404) and asymmetric dimethyl-

arginine (ASYM25, anti-Rme2, 09-814) were from Millipore;antibody for C/EBPa (2295)was fromCell Signaling Technology;antibody for cyclin D1 (ab134175) was from Abcam; antibodiesfor actin (sc-47778), GAPDH(sc-166574), andGST (sc-138)werefrom Santa Cruz Biotechnology; antibody for FLAG (F3165) wasfrom Sigma; antibodies for FLAG (PM020), Myc (M047-3, and562), and GFP (598) were from MBL; antibody for HDAC3(A2139) was from Abclonal.

PlasmidsExpression plasmids of C/EBPa-pcDNA6, C/EBPb-pcDNA6, C/

EBPd-pcDNA6, C/EBPg-pcDNA6, HDAC1-pcDNA6, HDAC2-pcDNA6, and HDAC3-pcDNA6 were described previously (36).HDAC1 andHDAC3 coding region were further cloned intoMyc-tagged pCMV-3tag7 vector. C/EBPa was cloned into pCMV-

3tag7 and pGEX4T-1 vectors. Deletion mutants of C/EBPa(DTAD1, 1-120, DbZIP, p30, bZIP) were amplified by PCRusing C/EBPa-pcDNA6 as template and inserted into pcDNA6.Expression vectors of C/EBPa deletions DTAD2 and DTAD3were generated using Seamless Cloning kit (Biomed). PRMT1cDNA was amplified by PCR from HEK293T cells cDNAs andcloned into pcDNA6 and pCMV-tag3B vector. PRMT1 mutant(E153Q) and C/EBPa mutants (R35K, R156K, R165K, R35/156/165K, R156F, R35/156/165F, R289A) were generated byPCR-based site-directed mutagenesis. Cyclin D1 promoter,extending from �808 to þ133 relative to the transcriptionstart site, was cloned into the pGL3-basic luciferase reportervector, and was used as templates to subclone and generate aseries of promoter deletions of cyclin D1 (�304/þ133, �113/þ133, �8/þ133, �113/þ15). Promoter of cyclin D1 withdisrupted potential C/EBPa-binding site was constructed usingPCR-based site-directed mutagenesis. The primers were listed inSupplementary Table S1.

Reporter gene assayReporter gene assays were performed as described

previously (37).

PRMT1 knockdown (shPRMT1) cell linesPRMT1 shRNAs were cloned into pLKO.1 vector, and control

shRNA was a hairpin designed against GFP. HEK293T cells weretransfected with indicated pLKO.1 plasmid and lentiviral con-structs (psPAX2 and pMD2.G). Lentiviruses were collected 48hours posttransfection. MDA-MB-231 cells were infected usinglentivirus expressing shPRMT1 or shGFPwith 8 mg/mL polybrene.Infected cells were selected with 1.5 mg/mL puromycin 36 hoursafter infection. Multiple monoclonal cultures were screened forshPRMT1 by Western blotting and RT-PCR analysis. The primerswere listed in Supplementary Table S2.

Colony formation assayCellswere seeded in35-mmdishes at adensity of 103 cells/dish.

After continued culturing for about 10 days, cells were washed byPBS twice, then stained with 0.05% crystal violet. Cells were thenair dried and photographed.

MTS assayCells were seeded at a density of 3 � 103 cell/well in quintu-

plicate in 96-well plates. Proliferationwas assessed using CellTiter96 AQueousOne Solution Cell Proliferation Assay (MTS). Briefly,solution reagent was added to eachwell and incubated at 37�C for1 hour at various time points. Subsequently, cell proliferationwasassessed by measuring the absorbance at 490 nm. Wells withoutcells (only medium) were used as blanks.

Anchorage-independent growth assaySoft-agar assays were constructed in 35-mm dishes. The base

layer of cell growth matrix consisted of 2 mL of 1 �DMEM, 10%FBS, and 0.6% low melting point agarose. Plates were chilled at4�C until solid. Upon solidification, the top layer was poured,consisting of 3 � 103 shPRMT1 or shGFP MDA-MB-231 cellssuspended in 2 mL of 1 � media and 0.35% low melting pointagarose. A further 1 mL 1 �media without of agarose was addedwhen needed. Cells were cultured for 4 weeks, colonies werestained with 0.005% crystal violet and counted (>200 mm indiameter) in five random 1-cm2 areas.

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Wound-healing assayCells were plated in growth medium in 6-well plates. After

confirming the formation of a completemonolayer, the cells werewounded by scratching lines with a standard 200 mL plastic tip.Cell migration throughout the wound area was observed andmeasured at different time points.

Xenograft assayFour-week-old female BALB/c nude mice were purchased and

maintained at animal center for 1 week prior to injection to allowthe mice adjusting the new environment. The assay was per-formed according to ethical approval. A total of 5� 105 shPRMT1or shGFP MDA-MB-231 cells suspended in 100 mL PBS wereinjected subcutaneously into nude mice (8 mice for each group).The size of the resultant tumors was measured every 5 days for 45days. On day 45 after implantation, the tumors were harvestedand tumor weights were measured. The tumor volume wascalculated with the formula V ¼ L �W2/2 (V, volume; L, length;W, width of tumor). All studies involving mice were approved bythe Animal Care and Use Committee of Chinese Academy ofMedical Sciences.

Flow cytometry assayFor cell-cycle analysis, 2 � 106 cells were trypsinized, washed

twice in ice-cold PBS, and fixed in cold 75% alcohol at 4�Covernight. After that, the cells were suspended in PBS with 100mg/mL RNAase A for 30 minutes at 37�C, stained with 50 mg/mLpropidium iodide (PI) for 15 minutes in the dark at roomtemperature, measured for DNA content with Accuri C6 (BDBiosciences). A total of 20,000 cells were counted and all analyseswere performed in triplicate.

Western blotting, coimmunoprecipitation, andGST-pulldown

Western blotting, coimmunoprecipitation (co-IP), and GST-pulldown assays were performed as described previously (37).

In vitro methylation assayFLAG-C/EBPa, PRMT1-WT, andPRMT1-E153Qwere expressed

and purified from HEK293T cells with anti-FLAG M2 agarosebeads. Purified C/EBPa was incubated with PRMT1-WT orPRMT1-E153Q in buffer [50 mmol/L Tris-HCl (pH 8.0), 1mmol/L phenylmethylsulfonylfluoride, and 0.5 mmol/L DTT]supplemented with S-adenosyl-L-[methyl-3H]methionine at37�C for 1 hour. Reactions were stopped by adding 5 � SDS-PAGE loading buffer and heating. Samples were analyzed bySDS-PAGE and fluorography.

Electrophoretic mobility shift assayElectrophoretic mobility shift assay (EMSA) were per-

formed as described previously (36). Briefly, oligonucleotidesfrom cyclin D1 promoter (þ62/þ93, GTTTTGTTGAAGTTG-CAAAGTCCTGCAGCCTC) were labeled with biotin at 50-end.C/EBPa proteins were purified from HEK293T cells trans-fected with FLAG-C/EBPa-pcDNA6. For specific competition,unlabeled oligonucleotide was added to the binding re-action mixtures at a 200-fold molar excess. For supershiftassays, 2 mg of specific anti-C/EBPa antibody was used to thereactions.

qRT-PCR and chromatin immunoprecipitationqRT-PCR and chromatin immunoprecipitation (ChIP) assays

were performed as described previously (37). The primers werelisted in Supplementary Tables S3 and S4.

Mass spectrometry for C/EBPa-associated proteinsAbout 4 � 108 HeLa cells overexpressed with C/EBPa were

harvested for assays. FLAG-tagged C/EBPa proteins were immu-noprecipitated using anti-FLAGM2beads. Thebeadswerewashed8 times using RIPA buffer and eluted with 3 � FLAG peptide.Elutes were resolved on SDS-PAGE, stained with Coomassiebrilliant blue, and bands were excised and digested with chymo-trypsin and subjected to LC/MS-MS sequencing and data analysis.

Mass spectrometry for C/EBPa methylationExpression plasmids of C/EBPa and PRMT1 were transfected

into HEK293T cells. C/EBPa was purified using anti-FLAG M2beads and eluted with 3� FLAG peptide. Elutes were resolved onSDS-PAGE and stained with Coomassie brilliant blue. The bandof C/EBPa was excised and subjected to LC/MS-MS analysis.

IHC analysisTissue microarray containing 30 normal breast tissues (N), 10

fibroadenoma (FA), 9 fibrocystic mastopathy (FM), 7 mild dys-plasia (MD), 4 severe dysplasia (SD), 10 ductal carcinaoma in situ(DCIS), 2 invasive lobular carcinoma (ILC-I), 3 invasive ductalcarcinoma (IDC-I), 23 IDC-II, and 5 IDC-III were purchased fromSuperbiotek. IHC was performed using anti-PRMT1 (1:500) anti-body. Each sample was assigned with a score according to theintensity of the staining (0 ¼ no staining, 1 ¼ weak staining, 2 ¼moderate staining, and 3¼ strong staining) and the proportion ofthe stained cells (0¼ 0%, 1¼ 1%–25%, 2¼ 25%–50%, 3¼50%–

75%, 4 ¼ 75%–100%). The stained tissues were scored by threeindividuals blinded to the clinical parameters.

Patient samplesThis study was approved by The Clinical Research Ethics

Committee of the Institute of Basic Medical Sciences, ChineseAcademy of Medical Sciences. The written informed consent wasobtained from the participants. Primary breast cancer tissues andtheir corresponding adjacent noncancerous tissues were obtainedfrom patients treated at Peking Union Medical College Hospitalafter receiving their written informed consent. Specimens weresnap-frozen in liquid nitrogen at time of surgery and stored at�80�C.

RNA-seqTotal RNAs isolated from shPRMT1 (sh1 and sh2) or shGFP

MDA-MB-231 cells using TRIzol reagent (Invitrogen). RNA integ-rity and concentration were determined using Nanodrop andagarose gel electrophoresis. The purified RNA samples, with RIN(RNA Integrity Number) over 8.0, determined by Agilent 2100Bioanalyzer (Agilent) were sequenced at ThorGene corporation,who constructed their digital gene expression libraries andsequenced by means of Illumina HiSeq 2500 platform to obtainthe expression libraries of 150-nt read length. Independent trip-licate cultureswere sampled. The clean readsweremapped againsthuman genome hg19with less than two-basemismatching, usingTophat (version 2.1.1b). The normalized expression values foreach gene were calculated by FPKM (expected number of Frag-ments per Kilo base of transcript sequence per Million base pairs

PRMT1 Methylates C/EBPa to Regulate Cyclin D1

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sequenced). Differential expression gene was determined byCuffdiff package (v2.2.1). The transcript levels of genes havinga P value of less than 0.05 were significantly differential betweentwo samples. RNA-seq data files are available from theNCBIGeneExpression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) data-base (accession number GSE121168).

Statistical analysisThe two-tailed, Student t test was used to compare two groups.

All data are shown as the mean with SDs from at least threeindependent experiments. Probabilities of P > 0.05 were consid-ered as not significant (#), P < 0.05 as significant (�), and P < 0.01as highly significant (��). Survival analysis was conducted usingthe Kaplan–Meier method. P < 0.05 indicated significantdifference.

ResultsElevated PRMT1 correlates with malignancy of breast cancer

To define the clinical relevance of PRMT1 in breast cancer, weanalyzed the correlation of PRMT1 expression and breast cancerpatients' clinical behaviors using Kaplan–Meier survival analysis

with an online tool. Patients whose tumors expressed higherlevels of PRMT1 had significantly worse overall survival dura-tion (Fig. 1A). Moreover, we analyzed a published clinicaldataset (GSE29044), and statistical analysis revealed signifi-cantly positive correlations of the expression of PRMT1 and thepro-proliferative gene cyclin D1 (Fig. 1B). We then determinedthe expression of PRMT1 in breast tumor samples (T) andadjacent nontumor (N) tissues from 8 patients (including fourgroups, Erb-B2, Basal, Luminal-B, and Luminal-A). Westernblotting showed that the expression levels of PRMT1 were highrelative to the adjacent nontumor tissue in 7 of 8 cancersamples (lanes 1, 5, 7, 9, 11, 13, and 15 of Fig. 1C). Further-more, a breast cancer tissue microarray containing 103 samplesfrom normal tissues, benign tumors, and malignant tumorsexcised prior to treatment was employed to detect PRMT1expression by IHC staining (Fig. 1D; Supplementary Fig. S1;Supplementary Table S5). Significantly greater PRMT1 stainingwas observed for malignant tumors, while weaker IHC stainingwas observed for normal and benign tumor tissues (Fig. 1E).The data revealed that the expression of PRMT1 is elevated inbreast cancer tissues and that the level of its expression ishighest in grade III of invasive ductal carcinoma.

Figure 1.

PRMT1 correlates tumor grade in breast cancer. A, Kaplan–Meier survival analysis of the correlation between PRMT1 expression levels and survivaltime in patients with breast cancer using the online tool (http://kmplot.com/analysis/; P < 0.05). B, Correlation analysis of public dataset (GEO:GSE29044) for the mRNA expression of PRMT1 and cyclin D1 by R programming. C, Representative Western blot analysis showing 8 pairs of breastcancer (T) and adjacent noncancer tissues (N). Anti-PRMT1 antibody and anti-actin were used as control (top). The panels were scanned with ImageJ,a software for scanning grayscale, and the relative density of PRMT1 relative to actin is presented (bottom). D and E, IHC staining of breast tissuearray for PRMT1 expression. D, Representative images in each group are shown. E, PRMT1 score shown is the mean � SD, and statistical significancewas determined by two-tailed t test. #, P > 0.05; �, P < 0.05; �� , P < 0.01. F, Comparing endogenous expression of PRMT1 in human breast cancer celllines by Western blot analysis. Breast cell line (MCF10A) and GAPDH served as a control and reference gene, respectively (top). The panels werescanned as in C and the relative expression of PRMT1 relative to MCF10A without EGF is presented (bottom).

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Next, we detected the expression of PRMT1 in multiple breastcancer cell lines. TheMCF10A cells, derived fromnontumorigenicbreast epithelial cells, were cultured in growth medium with EGFor without EGF, and Western blotting showed that EGF wasresponsible for PRMT1 expression (lane 1 vs. lane 2, Fig. 1F).Consistent with literature (38), PRMT1 levels inMCF7,MDA-MB-435, and SK-BR-3were increased relative toMCF10A (�EGF), butsimilar to the MCF10A cells (þEGF), the levels of PRMT1 expres-sion were higher in MDA-MB-231, -468, and BT-549 cells thanthat in MCF7 and SK-BR-3 cells (Fig. 1F). Given that MDA-MB-231, -468, and BT-549 cells are more aggressive than MCF7 andSK-BR-3 cell lines, the result suggested that the expression ofPRMT1 correlated with the malignance of breast cancer.

PRMT1 regulates the expression of cell-cycle genes inMDA-MB-231 cells

To gain insight into the mechanism by which PRMT1 regulatesbreast cancer cell growth, we employed MDA-MB-231, an aggres-sive breast cancer cell line, to establish two stably transfectedclones in which PRMT1 was knocked down by cotransduction ofthe cells with lentivirus encoding shRNAs specific for PRMT1,designated sh1 and sh2. RNA sequencing was performed tosystematically determine the differential gene expression profilein PRMT1-knockdown MDA-MB-231 cells. A total of 1,493 dif-ferentially expressed genes (DEG) were identified in both sh1 andsh2 cells compared with shGFP cells (P < 0.05), including 932downregulated genes (Fig. 2A and B) and 561 upregulated genes

Figure 2.

PRMT1 knockdown affects the expression of cell-cycle genes. A, The Venn diagram of overlapping downregulated genes in two independent shPRMT1 MDA-MB-231 cell lines (sh1 and sh2) compared with shGFP control. B, Heatmap of the downregulated genes (852) overlapping between sh1 and sh2. Each columnrepresents one indicated sample, and each row indicates a transcript. Expression values are depicted as a ratio relative to cells expressing shGFP and arerepresented as a red–blue color scale. P < 0.05. C,GO analysis of shPRMT1 downregulated genes showing the top 20 significant pathways in PRMT1-knockdowncells. P < 0.05. D, The bioinformatics analysis with Enrichr was carried out. Enriched terms, columns; input genes, rows; and the cells in the matrix indicate if agene is associated with a term. E, IGV browser views of RNA-seq reads covering indicated genes in shPRMT1 (sh1 and sh2) and shGFP MDA-MB-231 cells. The x-axis indicates the genomic location, and y-axis represents the normalized scale of RNA-seq reads. PRMT5, CARM1, and GAPDHwere used as negative controls,GDF15 as a positive control. F and G, The expression of indicated genes was measured byWestern blot (F) and RT-qPCR (G) analysis in shPRMT1 (sh1 and sh2) orshGFP MDA-MB-231 cells. H, The promoter activity of cyclin D1 was tested using luciferase reporter assay in HEK293T cells. Cells were transfected with cyclin D1promoter-luciferase reporter, Renilla, and PRMT1-WT, -E153Q, or vector control. I, Reporter gene assay showing a decreased luciferase activity in PRMT1knockdown cells (sh1 and sh2) or shGFP cells transfected with reporter construct carrying cyclin D1 promoter and Renilla. Each reporter plasmid was transfectedat least three times, and each sample was assayed in triplicate. J, ChIP-qPCR analysis of specific binding of PRMT1-WT or -E153Q to the promoter (�10/þ100) ofcyclin D1 in MDA-MB-231 cells.�3400/�3300 region of the gene served as a negative control. IgG was used as an IP control. Data of 2G-J are the meanvalue�SD from at least three independent experiments (#, P > 0.05; ��, P < 0.01).






(Supplementary Table S6). Top 20 pathways for the genes down-regulated in PRMT1-knockdown cells were shown in GO enrich-ment analyses, in which the cell-cycle pathway was most signif-icant (Fig. 2C). A bioinformatics analysis with Enrichr (39) wascarried out and revealed that cell-cycle genes were enriched in thedownregulated genes provided in Supplementary Table S6,including CCNA2, CCNB1, CCND1, and CCNE2, CDK6, TP53,

RBL1, CDC20, and CDC23 genes (Fig. 2D). Representative datafrom Integrative Genomics Viewer 2.0 (IGV2.0) showed that theexpression of cyclin D1 was decreased in sh1 and sh2 cellscompared with shGFP cells, with confirmation of the downregu-lation of PRMT1 and no changes in PRMT5 and CARM1/PRMT4(Fig. 2E). We next focused on cyclin D1 due to its crucial role incell proliferation. Western blotting and real-time RT-PCR assays

Figure 3.

C/EBPa interacts with and is methylated by PRMT1. A, Immunopurification andmass spectrometry analysis of C/EBPa binding proteins. Cell extracts from HeLacells ectopic expressing FLAG (Ctrl) or FLAG-C/EBPawere purified with anti-FLAGM2 beads and eluted with FLAG peptide. The elution was resolved bySDS-PAGE and silver-stained, the protein bands were excised and analyzed by mass spectrometry. B and C, The interaction between PRMT1 and C/EBPawasdetected by Co-IP and GST-pulldown assays. B, FLAG immunoprecipitates from HEK293T cells expressing FLAG-PRMT1 and Myc-C/EBPawere analyzed byWestern blotting with anti-FLAG and anti-Myc antibodies. C, GST-pulldown assay was performed using FLAG-PRMT1 containing HEK293T whole-cell lysatesincubated with GST or GST-fused C/EBPa, followed byWestern blotting with anti-FLAG and anti-GST antibodies. D, Autoradiography of in vitromethylationassay using purified C/EBPa and PRMT1-WT or PRMT1-E153Q from HEK293T cells ectopically expressed with indicated plasmids. Total amounts of C/EBPa andPRMT1 are shown by Coomassie Brilliant Blue (C.B.B) staining. E, Detection of C/EBPamethylation byWestern blotting using anti-Rme2. C/EBPawasimmunoprecipitated with anti-FLAG M2 beads from PRMT1-knockdown MDA-MB-231 cells expressed FLAG-C/EBPa. F, Schematic drawing of the wild-type andtruncated FLAG-C/EBPa fragments. G,Domain mapping of C/EBPa regions methylated by PRMT1. FLAG-tagged C/EBPa delete truncations were expressed inHEK293T cells and purified by anti-FLAG M2 agarose. Methylation of these truncations was analyzed byWestern blotting using anti-Rme2. H,Mass spectrumanalysis of the chymotryptic C/EBPa peptide mixture to identify methylated sites in vivo. FLAG-tagged C/EBPawas purified from HEK293T cells cotransfectedwith C/EBPa and PRMT1. Red, methylated arginine of C/EBPa. I,Western blotting assay by anti-Rme2 with a series of arginine mutants of FLAG-C/EBPa purifiedfrom HEK293T cells transfected with indicated vectors. Less exposed means exposing shorter time.

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revealed that the mRNA and protein expression of cyclin D1 weredecreased by PRMT1 knockdown in sh1 and sh2 cells (Fig. 2F andG). To verify the role of PRMT1 in the transcription level of cyclinD1, a luciferase reporter assay was employed to examine the effectof PRMT1 on cyclin D1 promoter activity driven by a DNAfragment of -806/þ133 of the gene. The reporter assay showedthat cyclin D1 transcriptional activity in MDA-MB-231 cells wasinduced by ectopically expressed PRMT1 but not by overexpres-sion of the catalytically inactive mutant PRMT1-E153Q (Fig. 2H).Accordingly, we also conducted the reporter assay in PRMT1knockdown cell lines. Consistently, knocking down PRMT1repressed the activity of the promoters (Fig. 2I). ChIP assays wereperformed by using anti-FLAG antibody from FLAG-PRMT1-WT

or -E153Q–transfectedMDA-MB-231 cells, and the result showedthat both ectopicWT-PRMT1 and E153Q-PRMT1occupied on thepromoter of cyclin D1 (Fig. 2J). Our findings suggested thatPRMT1 regulates the expression of cell-cycle genes dependent onits enzyme activity, but not on its occupancy of the promoter.

C/EBPa associates with and is methylated by PRMT1C/EBPa has a well-established role as a repressor of the pro-

proliferative E2F target genes and has been shown to play a tumorsuppressive role in breast cancer (40, 41). Interestingly, ourPRMT1 knockdown RNA-seq data revealed an enrichment ofE2F-binding sites in the promoters of downregulated genes (Sup-plementary Fig. S2A; Supplementary Table S6). We hypothesized

Figure 4.

C/EBPa downregulates cyclin D1 transcription by recruiting HDAC3. A and B, Effect of C/EBPa overexpression on cell proliferation was measured by colonyformation assay (A) and MTS assay (B) in MAD-MB-231 cells overexpressed with vector (Ctrl) or C/EBPa plasmids. C, Cell-cycle profile analysis by flowcytometry in MAD-MB-231 cells transfected with vector (Ctrl) or C/EBPa expression construct. The percentage of cells in G1, S, and G2–M phases is shown. D, Theexpression of the indicated proteins in MAD-MB-231 cells transfected with C/EBPa expression plasmid was analyzed byWestern blotting assay. E, Reporter geneassays were performed in HEK293T cells transfected with luciferase reporter plasmid containing cyclin D1 promoter and different amounts of plasmidsexpressing C/EBPa. F, The effects of truncated C/EBPa on reporter gene assays. The expression of truncations of C/EBPa is shown at bottom. G, Reporter geneassays were performed to map the cis-acting elements of C/EBPa in cyclin D1 promoter. HEK293T cells were cotransfected with C/EBPa or control plasmid,together with the indicated cyclin D1-luciferase reporter plasmids. Left, schematic diagrams of the cyclin D1-luciferase reporter constructs. H, Luciferase activitieswere measured in HEK293T cells transfected with reporter vectors carrying wild-type or site-mutated cyclin D1 promoter with an empty vector (Ctrl) orC/EBPa-expressing plasmid. I, EMSA assay using biotin-labeled probe containing a potential C/EBPa-binding site from cyclin D1 promoter was performed withpurified C/EBPa from HEK293T cells ectopic expressing FLAG-C/EBPa. S, shift band of C/EBPa-DNA complex; SS, supershifted band with C/EBPa antibody;free probe, migration of free probe. ChIP-qPCR analysis of C/EBPa (J) or HDAC3 (K) recruitment on indicated region of cyclin D1 promoters in MDA-MB-231cells transfected with FLAG-C/EBPa or HDAC3 plasmid. IgG was used as an IP control. Data of C, E–H, J, and K are the mean� SD from at least threeindependent experiments. #, P > 0.05; � , P < 0.05; �� , P < 0.01.






Figure 5.

PRMT1-mediated C/EBPamethylation promotes the disassociation of HDAC3 from C/EBPa.A,Overexpression of PRMT1 impaired the inhibitory activity ofC/EBPa in HEK293T cells by luciferase reporter gene assay. B, Colony formation assay in MDA-MB-231 cells double knocking down of PRMT1 and C/EBPa. C,Luciferase activities were measured using reporter assay in HEK293T cells cotransfected with expression vectors of wild-type C/EBPa (WT) or mutated C/EBPa(R156K and R156F) together with cyclin D1 reporter. D, Reporter gene assay in HEK293T cells transfected with expression plasmids of wild-type (WT) or mutatedC/EBPa (3RF, 3RK), or together with or without PRMT1. E, Colony formation assay in MDA-MB-231 cells transfected with wild type C/EBPa (WT) or mutatedC/EBPa of R156F and R156K (left). F, RepresentativeWestern blot analysis showing 8 pairs of breast cancer (T) and adjacent noncancer tissues (N). Anti-C/EBPaantibody and GAPDHwere used as control (top). G, C/EBPa bound to a specific sequence within cyclin D1 promoter. EMSA assay using biotin-labeled probe fromcyclin D1 promoter was performed with purified C/EBPa from HEK293T cells overexpressing wild-type (�WT) or mutated C/EBPa (�3RK,�3RF). S, shift bandof C/EBPa-DNA complex; SS, supershifted band with C/EBPa antibody; free probe, migration of free probe. (Continued on the following page.)

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that PRMT1 might directly regulate C/EBPa activity and we firstperformed a tandem affinity purification combined with massspectrometry from FLAG-tagged C/EBPa-overexpressing HeLacells. We indeed identified PRMT1 as a potential partner of C/EBPa in this setting (Fig. 3A; Supplementary Table S7). To confirmthe association between C/EBPa and PRMT1, we then performeda co-IP assaywithHEK293T cells cotransfectedwith FLAG-PRMT1and Myc-C/EBPa. C/EBPa blotted with anti-Myc was found tocoexist with PRMT1 IPed by anti-FLAG (Fig. 3B). In addition,pulldown assays showed that FLAG-PRMT1 was pulled down bypurified GST-C/EBPa but not by GST alone (Fig. 3C), whichindicated that PRMT1 interacts with C/EBPa. Most importantly,in vitromethylation assays showed that recombinant C/EBPawasmethylated by PRMT1 based on 3H labeling but not by PRMT1-E153Q, a catalytically inactive mutant of PRMT1 (Fig. 3D). Fur-thermore, a commercial antibody against asymmetric dimethyl-arginine (ASYM25, Rme2) was employed to detect the methyla-tion of C/EBPa. There was a strong signal for FLAG-C/EBPaimmunoprecipitated from cells but not for C/EBPa purified fromE. coli (Supplementary Fig. S2B), and the methylation signal wasdiminished in the presence of a PRMT1-specific inhibitor (Fur,furamidine dihydrochloride; Supplementary Fig. S2C), whichconfirmed its specificity for arginine methylation. Knockdownof PRMT1 significantly inhibited argininemethylation of C/EBPain cells (Fig. 3E).

To determinewhich regionofC/EBPa ismethylated byPRMT1,we purified a series of C/EBPa deletion mutants from transfectedHEK293T cells (Fig. 3F).Western blotting with Rme2 showed thatfull-length C/EBPa (Fig. 3G, lane 1), and DbZIP (1–281 aa), weremethylated in IPed C/EBPa (Fig. 3G, lane 2), but no signals weredetected for the fragments of 1–120 aa and bZIP (282–358aa; Fig. 3G, lanes 4 and 5). A weak signal was detected for thep30 fragment (120–358 aa), suggesting that argininemethylationby PRMT1 mainly occurs in the 120–281 aa region. Next, wecarried out immunoprecipitation of C/EBPawith anti-FLAG fromtransfectedHEK293T cells (Supplementary Fig. S2D), followedbymass spectrometric analysis to identify the arginine methylationsites on C/EBPa. Three arginine residues (R35, R156, and R165)of C/EBPa were found to be monomethylated (SupplementaryFig. S2E–S2G), while only R156 was dimethylated (Fig. 3H).Western blotting with Rme2 showed that the methylation signaldecreased markedly when these two or three arginine residueswere individually substituted with lysine (R2K, R156/165K; R3K,R35/156/165K), and each of the single mutants R35K, R156K, orR165K also displayed a decreased signal at least to some extent(Fig. 3I). These results demonstrated that C/EBPa could bemethylated by PRMT1.

C/EBPa represses the transcription of cyclin D1 via HDAC3Because of the inhibitory effect of C/EBPa on cell growth, an

MDA-MB-231 cell line stably expressing C/EBPa could not be

constructed. Transiently expressing C/EBPa inMDA-MB-231 cellssignificantly inhibited cell growth according to colony formationassays (Fig. 4A) and MTS assays (Fig. 4B). FACS assays indicatedthat ectopic C/EBPa induced G1–S arrest in MDA-MB-231 cellscompared with the vector control (Fig. 4C). Western blottingshowed that ectopically expressed C/EBPa repressed cyclinD1 expression (Fig. 4D). Knockdown of C/EBPa was accompa-nied by activation of cyclin D1 at themRNA level (SupplementaryFig. S3A). Reporter assays showed that ectopic C/EBPamarkedlyreduced cyclin D1 promoter activity inHEK293T cells (Fig. 4E). Tofurther analyze the domains of C/EBPa that were implicated inrepressive activity, we constructed a series of deletion mutantsof C/EBPa in which the TAD1, TAD2, TAD3, or bZIP domainwas removed individually or in combination. HEK293T cellswere then transfected by the cyclin D1 reporter along with theindicated C/EBPa truncations or an empty vector as a control.The result showed that removal of TAD1 or TAD3 did notrepress cyclin D1 to the same extent as full-length C/EBPa,while TAD2 did (Fig. 4F), implying that TAD1 and TAD3 aremore essential than TAD2 for repression of cyclin D1 activity.Moreover, the bZIP deletion of C/EBPa abolished its repressiveactivity (Fig. 4F), which indicated that the DNA binding activityof C/EBPa is essential for repression of cyclin D1. To identifythe response element for C/EBPa on the cyclin D1 promoter, aseries of 50 truncated promoter-luciferase constructs werecotransfected with C/EBPa (Fig. 4G, right), and then the lucif-erase activities were measured. We found that the �808/þ133,�304/þ133, �113/þ133, or �8/þ133 fragment but not the�113/þ15 fragment of the promoter was repressed by ectopi-cally expressed C/EBPa (Fig. 4G, left), which indicated thatthere is a critical response element for C/EBPa-mediatedrepression within the þ15/þ133 region of the cyclin D1 gene.Analyzing the DNA sequence in the þ15/þ133 region revealeda consensus C/EBPa binding site, AGTTGCAAA, at þ72/þ80 inthe cyclin D1 gene regulatory region, which is identical with therespective sequences of the cyclin D1 genes in mouse and in ratas well (Supplementary Fig. S3B). Mutation of this site toAGTGTCCGA in a reporter driven by the �8/þ133 promotereliminated C/EBPa-mediated transcriptional repression(Fig. 4H). Furthermore, EMSA was performed by using synthe-sized 32-mer oligonucleotides (þ62/þ93 of the cyclin D1gene) labeled with biotin at the 50end as probe. As a specificcompetitor, the unlabeled probe eliminated the specificband (S), and adding anti-C/EBPa antibody to the incubationmedium clearly elicited a novel supershift band (SS) at thetop of lane 5 (Fig. 4I). ChIP data confirmed that C/EBPacould efficiently target the region (�10/þ100) downstreamfrom the transcriptional start site of the cyclin D1 gene(Fig. 4J). The results demonstrated that C/EBPa can directlybind to the promoter of the cyclin D1 gene, which is critical forC/EBPa-mediated negative regulation.

(Continued.) H,Domain mapping of C/EBPa region interaction with HDAC3. Myc-HDAC3 was coexpressed with FLAG-C/EBPa or its deletions in HEK293T cellsand immunoprecipitated by anti-Myc antibody conjugated to agarose. Coprecipitated C/EBPa deletions were detected byWestern blotting using anti-FLAGantibody. Bottom, input. I, Co-IP assay was performed using HEK293T cells transfected with FLAG-C/EBPa-WT or C/EBPa-3RF and Myc-HDAC3 plasmids. IPedwithout (Input; bottom) or with anti-FLAG (top) and then immunoblotting with anti-HDAC3 or anti-FLAG individually. J, Co-IP assay of C/EBPa and HDAC3proteins from HEK293T cells cotransfected with FLAG-HDAC3, Myc-C/EBPa, with PRMT1-WT or PRMT1-E153Q expressing vectors. IPed without (Input; bottom)or with anti-Myc (top) and then immunoblotting with anti-HDAC3, anti-Myc, or anti-FLAG for PRMT1. K, ChIP-reChIP assay showing that C/EBPa-mediatedHDAC3 recruitment to the promoter of cyclin D1 is arginine methylation dependent. MDA-MB-231 cells were cotransfected with FLAG- C/EBPa-WT, -R156F/K, or-3RF and Myc-HDAC3. Anti-FLAG antibody was used as the initial ChIP (1st) to gain the C/EBPa-associated chromatin fragments. Then, these fragments weresubjected to reChIP (2nd) using anti-Myc antibody. IgG was used as a ChIP control. Data of A–D and J are the mean� SD from at least three independentexperiments. #, P > 0.05; �� , P < 0.01.






To further elucidate the cofactors recruited by C/EBPa tomediate transcriptional inhibition of cyclin D1, we focused onthe HDAC family of corepressors based on our previous reportthat the recruitment of HDAC3 byC/EBPa at its response elementin the SIRT7 promoter is sufficient and prerequisite for repressionof the SIRT7 gene (36). We found that transfection of HDAC1,HDAC2 or HDAC3 reduced cyclin D1 promoter activity inHEK293T cells (Supplementary Fig. S3C) and that cotransfectionwith C/EBPa led to a more effective repression of the cyclin D1promoter compared with transfection of C/EBPa alone (Supple-mentary Fig. S3D). Importantly, ChIP assays further proved thatHDAC3 can be recruited to the promoter of cyclin D1 (Fig. 4K).Taken together, these data suggest that C/EBPa directly repressescyclinD1gene expression, at least, in part, by recruitingHDAC3 toits promoter.

PRMT1-mediated methylation negatively regulates thetranscriptional activity of C/EBPa

On the basis of above results, we hypothesized that enhancedPRMT1 could disturb the transcriptional repressive activity of C/EBPa in breast cancer cells.

To confirm this hypothesis, we first transfected expressionvectors encoding C/EBPa and/or PRMT1 in HEK293T cells andfound thatC/EBPa-mediated repression of the cyclinD1promoterwas impaired by coexpressing PRMT1 (Fig. 5A). Colony forma-tion assay showed that knock down of C/EBPa promoted thegrowth of MDA-MD-231 breast cancer cells, importantly, knockdown of C/EBPa partially rescued the effect of PRMT1 knockingdown in sh1 cells (Fig. 5B). When arginine 156 was mutated tophenylalanine (R156F), a mimic of methylated arginine (42), therepressive activity of C/EBPa on the cyclin D1 promoter wasreduced compared with wild-type C/EBPa, while the R156Kmutant retained repressive activity (Fig. 5C). The combinedmutant C/EBPa-3RF (R35F, R156F, and R165F) further disturbedC/EBPa-mediated repression of the cyclin D1 reporter (Fig. 5D,lane 4), especially comparedwith 3RK (R35K, R156K, andR165K;Fig. 5D, lane 6). Cotransfection with PRMT1 had no effect oneither 3RF or 3RK mutant (Fig. 5D). We next examined thegrowth-inhibitory effects of the C/EBPa mutants in MDA-MB-231 cells. The cells transfected with the R156F mutant grew fasterthan those transfected with wild-type C/EBPa, while cells trans-fectedwith theR156Kmutant didnot (Fig. 5E). Thedata indicatedthat PRMT1 mediates methylation of C/EBPa on R156 to disturbthe transcriptional repressive activity of C/EBPa on the cyclin D1gene in breast cancer cells.

To investigate the molecular mechanisms by which methyla-tion of C/EBPa by PRMT1 regulates C/EBPa repressive activity oncyclin D1, we first detected the expression of C/EBPa in thesamples of breast cancer as described in Fig. 1C and found thatthe expression of C/EBPa was not reduced in tumor samples (T)compared with adjacent normal tissues (N; Fig. 5F). EMSA wasthen performed by using purified WT- and 3RF-C/EBPa fromtransfected HEK293T cells. The mimic of arginine methylatedC/EBPa (3RF) did not affect its DNA-binding activity on thecyclin D1 promoter comparing with the WT-CEBPa, implyingthat the recruitment of cofactors might be affected (Fig. 5G). Wefound that HDAC3 interacted with C/EBPa in the 118–281 aaregion (Fig. 5H), in which the methylation sites R156/165 mod-ified by PRMT1 are located. Furthermore, compared with WT-C/EBPa, ectopic 3RF-C/EBPa disassociated with HDAC3 (Fig. 5I).Compared with WT-PRMT1, the presence of E153Q-PRMT1

resulted in an increased association of HDAC3 and C/EBPa(Fig. 5J). Most intriguingly, ChIP/reChIP assays demonstratedthat C/EBPa cooccupied the promoter of cyclin D1 with HDAC3;the R156F or 3RFmutant of C/EBPa diminished the cooccupancywithHDAC3, while R156K did not affect the occupancy (Fig. 5K).These data indicated that HDAC3 preferentially interacts withunmethylated C/EBPa and that this interaction is weakened onceC/EBPa is methylated by PRMT1, subsequently leading to atten-uation of the transcriptional repressive activity of C/EBPa on thecyclin D1 promoter.

Loss of function of PRMT1 inhibits cell growth andtumorigenesis

We found that both mRNA and protein expression of PRMT1were much lower in sh1 and sh2 cells than in cells mock-trans-fected with shRNA against GFP (shGFP; Fig. 6A). Knockdown ofendogenous PRMT1 inMDA-MB-231 cells significantly decreasedcell growth in the colony formation assay and MTS assay (Fig. 6Band C). The FACS assay indicated that PRMT1 knockdownresulted in G1–S arrest compared with the shGFP control(Fig. 6D). Knockdown of PRMT1 in MDA-MB-231 cells attenu-ated cell migration ability in the wound-healing assay (Fig. 6E)and decreased anchorage-independent growth of cells in the soft-agar colony formation assay (Fig. 6F). To assess the role of PRMT1in breast tumorigenesis in vivo, we implanted shPRMT1 or shGFPMDA-MB-231 cells in female BALB/c nude mice. The resultsindicated that PRMT1 knockdown led to dramatic reductions oftumor volume and weight (Fig. 6G). Overall, these results suggestthat PRMT1 plays an oncogenic role in breast cancer cells. Toexplore the possibility of using a PRMT1 inhibitor to treat breastcancer, we selected two triple-negative breast cancer cell lines(TNBC1 and TNBC2) for treatment of Fur, a specific inhibitor ofPRMT1 (35), and found that cancer cell growth was significantlyinhibited by the inhibitor in a concentration-dependent manner(Fig. 6H). This result indicates that PRMT1 inhibitor is a potentialdrug for treatment of human breast cancer.

DiscussionIn contrast to C/EBPb, there have been few reports of PTMs of

C/EBPa. Regarding C/EBPa, the early discovered PTMs mainlyincluded phosphorylation (26–29), SUMOylation (30–32), andacetylation (33). Although C/EBPa is expressed in a number oftissues, its function has largely been characterized in adipocytesand the hematopoietic system (16). Here, we demonstrated thatthe transcriptional factor C/EBPa interacts with and ismethylatedby PRMT1. Methylated C/EBPa loses its repressive function oncellular growth and cyclin D1 transcription via interfering with therecruitment of HDAC3 to the cyclin D1 promoter in breast cancer.The schematic model is summarized in Fig. 6I. This report is thefirst to demonstrate that C/EBPa arginine methylation can reg-ulate its function.

C/EBPa contains three independent transactivation elementsnamed TAD1, TAD2, and TAD3, which can cooperate with oneanother to achieve protein function (15, 43). TAD1 and TAD2interact with the basal transcriptional apparatus (44, 45). TAD3contains a transcriptional regulatory domain that can inhibit theactivity of an adjacent transcriptional activation domain (15). Inaddition, TAD3 plays an important role in modulating C/EBPafunction, and it recruits cofactors or allows PTMs to enhance,repress, or even convert the activity of C/EBPa. We demonstrated

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that C/EBPa inhibits the transcription of cyclin D1 and that theTAD1 and TAD3 domains but not TAD2 are required for thisactivity. HDAC3 binding to the TAD3 domain participates in therepressive process mediated by C/EBPa.

Deregulation of C/EBPa can occur at the posttranscriptionallevel through PTMs. ERK1/2-mediated phosphorylation ofC/EBPa at Ser 21 inhibits its activity in FLT3-activated humanAML (46). C/EBPa phosphorylation on Ser 193 is required forthe growth-inhibitory function in hepatic carcinoma (29).

We found that elevated PRMT1 in breast cancer is a novelregulator of C/EBPa transcriptional activity and that methylatedC/EBPa results in loss of control of the cell cycle in breastcancer cells.

C/EBPamodulates cell growth by regulating the expression ofseveral proliferation-related genes. Our data showed that over-expression of C/EBPa leads to a decrease in the expression levelsof cyclin D1 in breast cancer cells. A previous study showed thatC/EBPa reexpression in gastric carcinoma cells leads to a

Figure 6.

Knockdown or inhibitor of PRMT1 restrains cell growth and tumorigenesis of breast cancer cells. A, RT-PCR and Western blotting analysis wereperformed to validate the knockdown efficiency of PRMT1 in MDA-MB-231 cells (sh1 and sh2). RT-PCR, relative PRMT1 expression levels werenormalized to GAPDH and are represented as percentage of shGFP (top). Western blotting, GAPDH served as internal reference (bottom). B, Colonyformation assay shown in shPRMT1 (sh1 and sh2) or shGFP MDA-MB-231 cells. C, MTS assays shown (sh1 and sh2) in shPRMT1 or shGFP MDA-MB-231cells. D, Cell-cycle analysis of MDA-MB-231 cells with PRMT1 knockdown (sh1 and sh2). The percentage of cells at different phases (G1, M, and G2–M)is shown. E, Wound-healing assays were performed to value the migration ability of MDA-MB-231 cells with PRMT1 knockdown (sh1 and sh2; n ¼ 6).Images present the wound closure rate at different time points (0, 12, 24 hours). F, Soft-agar assays show anchorage-independent growth ability ofPRMT1 knockdown cells (sh1 and sh2) compared with shGFP cells. G, Xenograft model in athymic mice injected with shPRMT1 (sh1 and sh2) or shGFPMDA-MB-231 cells (n ¼ 8). Tumors were excised from the mice and weighed. H, Proliferation ability was assessed in primary breast cancer cellstreated with increasing concentrations of PRMT1 inhibitor (0, 20, 40, or 60 mmol/L) for 6 days. I, Schematic of C/EBPa methylated by PRMT1 toregulate cyclin D1 expression via preventing HDAC3 recruitment in human breast cancer. Data of D and F are the mean � SD from at least threeindependent experiments. �� , P < 0.01. Data of E and G are the mean � SD, and statistical significance is shown.






reduction of cyclin D1 (47), but the mechanism has not beendescribed in detail. We identified one consensus C/EBPa bindingsite in the cyclin D1 promoter. Cyclin D1 is a direct target gene oftranscriptional repression byC/EBPa and that inhibition of cyclinD1 is at least partially required for C/EBPa-mediated growtharrest. Previous reports from our lab and others have shown thatC/EBPa causes repression of target genes through the recruitmentof an HDAC-dependent repression complex (36, 48). This studyrevealed that HDAC3 is involved in C/EBPa-mediated repressionof cyclin D1.

PRMT1 has been implicated in various types of human tumors,including breast cancer (1, 3), however, the underlying mechan-isms have not been completely elucidated. In this report, we findthat PRMT1 is dramatically upregulated in primary breast cancersamples, and patients with higher PRMT1 expression show ahigher malignancy grade. Silencing PRMT1 in MDA-MB-231 cellsleads to significant cell-cycle arrest and a reduced ability oftransformation and migration, suggesting that PRMT1 plays akey role in breast tumorigenesis. On the basis of RNA-seq data, wedetermined that PRMT1 knockdown in breast cancer cells canactivate or repress several proliferation-related genes, includingcyclin D1. We showed that PRMT1 methylates the tumor suppres-sor C/EBPa, and methylation of C/EBPa at least partially alle-viates its inhibitory effects on its target gene, cyclin D1.

In breast cancer, CARM1, another protein arginine methyl-transferase, methylates chromatin remodeler BAF155 to enhancetumor progression andmetastasis (49). Here, we have shown thatC/EBPa can be specifically methylated by PRMT1 and that thismethylation functions to impair the transcriptional repressiveactivity of C/EBPa by decreasing its interaction with anHDAC3-containing corepressor complex. These findings further

confirm the oncogenic role of PRMT1 and the tumor suppressorfunction of C/EBPa during breast tumorigenesis, and thus poten-tial drugs targeting PRMT1-C/EBPa pathway may have utility asbreast cancer therapeutics.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Zhang, L.-M. Liu, M.-B. ChengDevelopment of methodology: L.-M. Liu, W.-Z. Sun, X.-Z. Fan, M.-B. ChengAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.-M. Liu, W.-Z. Sun, Y.-L. XuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Zhang, L.-M. Liu, X.-Z. Fan, M.-B. ChengWriting, review, and/or revision of the manuscript: Y. Zhang, L.-M. Liu, W.-Z. Sun, M.-B. ChengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. ZhangStudy supervision: Y. Zhang

AcknowledgmentsY. Zhang received grants from National Natural Science Foundation of

China (91519301, 31871310) and CAMS Initiative for Innovative Medicine(2016-I2M-3-002). This work was supported in part by the CAMS Initiativefor Innovative Medicine (2017-I2M-3-009 to M. Cheng).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedOctober 11, 2018; revised January 24, 2019; acceptedApril 17, 2019;published first April 23, 2019.

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2019;79:2865-2877. Published OnlineFirst April 23, 2019.Cancer Res Li-Ming Liu, Wen-Zheng Sun, Xue-Zhe Fan, et al. Function in Breast Cancer

by PRMT1 Inhibits Its Tumor-SuppressiveαMethylation of C/EBP

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Methylation of C/EBPa by PRMT1 Inhibits Its Tumor ... · Molecular Cell Biology Methylation of C/EBPa by PRMT1 Inhibits Its Tumor-Suppressive Function in Breast Cancer Li-Ming Liu1,2,Wen-Zheng