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1 RYBP Expression Is Regulated by KLF4 and Sp1 and Is Related to Hepatocellular Carcinoma Prognosis Qiaojiajie Zhao1,4,Weihua Cai2,4,Xuan Zhang1,Shuo Tian1,Junwen Zhang1,Haibo Li3,Congcong Hou1,Xiaoli Ma1,Hong Chen1,Bingren Huang1*,and Deng Chen1*1State Key Laboratory of Medical Molecular Biology,Department of Biochemistry and Molecular Biology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences and School of Basic Medicine,Peking Union Medical College,Beijing 100005,China 2Department of Hepatobiliary Surgery,Nantong Third Hospital,Nantong University,Nantong,Jiangsu 226006,China 3Department of Clinical Laboratory Medicine,Nantong Maternal and Child Health Hospital,Nantong,Jiangsu 226018,China Running title:RYBP Expression Is Regulated by KLF4 and Sp1 in HCC 4These authors contributed equally to this work.*Corresponding authors:Deng Chen,Ph.D.,or Bingren Huang,Ph.D.,State Key Laboratory of Medical Molecular Biology,Department of Biochemistry and Molecular Biology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences and Peking Union Medical College,5 Dong Dan San Tiao,Beijing 100005,China.Tel:86-10-69156980,E-mail: or Keywords:RYBP,KLF4,Sp1,promoter,gene expression,gene regulation,hepatocellular carcinoma http:/www.jbc.org/cgi/doi/10.1074/jbc.M116.770727The latest version is at JBC Papers in Press.Published on December 27,2016 as Manuscript M116.770727 Copyright 2016 by The American Society for Biochemistry and Molecular Biology,Inc.by guest on December 30,2016http:/www.jbc.org/Downloaded from 2 ABSTRACT The expression of Ring1 and YY1 binding protein(RYBP)is reduced in several human cancers,but the molecular mechanism(s)have remained elusive.In this study,we used human HCC cell lines and tissue specimens to study the mechanism,and herein report several new findings.First,we cloned and characterized the basal promoter region of the human rybp gene.We found that the decreased RYBP expression in HCC tissues was not due to promoter sequence variation/polymorphisms or CpG dinucleotide methylation.We identified two transcription factors,KLF4 and Sp1,which directly bind the promoter region of rybp to respectively induce and suppress rybp transcription.We mapped the binding sites of KLF4 and Sp1 on the rybp promoter.Studies in vitro showed that KLF4 suppresses while Sp1 promotes HCC cell growth through modulating RYBP expression.Deregulated KLF4 and Sp1 contributed to decreased expression of rybp in HCC tumor tissues.Our studies of human HCC tissues indicated that a diminished RYBP level in the tumor(in association with altered KLF4 and Sp1 expression)was statistically associated with a larger tumor size,poorer differentiation and an increased susceptibility to distant metastasis.These findings help to clarify why RYBP is decreased in HCC and indicate that deregulated KLF4,Sp1,and RYBP may lead to a poorer prognosis.Our findings support the idea that RYBP may represent a target for cancer therapy,and suggest that it may be useful as a prognostic biomarker for HCC,either alone or in combination with KLF4 and Sp1.INTRODUCTION RYBP is becoming increasingly recognized as a central molecule involved in various processes.It interacts with Ring1A and Ring1B,making it a critical component of polycomb repressive complex 1(1-3).It promotes the monoubiquitination of Ring1B towards H2AK119,epigenetically regulating gene expression,and is involved in embryogenesis,stem cell self-renewal,cell differentiation,and X-chromosome inactivation(2).Mouse embryos with homozygously-deleted rybp die around embryonic day 5.5 to 6.0,implying that RYBP plays a crucial role during embryonic development(4).RYBP also interacts with a multitude of transcription factors,including YY1,E2F2/3/6,and E4TF1/hGABP,acting as a bridging factor to mediate the formation of transcription factor complexes,and therefore modulates gene expression independent of its polycomb group functions(1,5-7).RYBP has also been frequently reported to act as an adaptor protein to mediate interactions among death effector domain-containing proteins,such as caspase 8/10,FADD,and DEDD,as well as other apoptosis-associated proteins,including apoptin and Hippi,allowing it to induce apoptosis when localized in either the cytoplasm or nucleus.However,it did not show apparent cytotoxicity to non-tumorous cells(8-13).The genes and signaling pathways targeted by RYBP are still being elucidated.Our previous study indicated that RYBP formed a complex with MDM2 and p53,and that it inhibited MDM2-mediated p53 proteasome degradation,leading to p53 activation(14).In agreement with its apoptosis-inducing capacity,the expression of RYBP has been reported to be reduced in a variety of human cancers,including lung,cervical,prostate and liver cancers,and was recently shown to inhibit cancer growth,metastasis,and chemoresistance in vivo and in vitro(14-17),indicating that it is a potential candidate drug target for use against these tumors.However,little is currently known about by guest on December 30,2016http:/www.jbc.org/Downloaded from 3 the molecular mechanism(s)responsible for the downregulation of RYBP in these tumors,and this has limited the understanding of its regulation,and consequently,the development of an optimal approach for targeting RYBP expression as a therapeutic strategy for human cancers.In this study,we investigated the molecular mechanism(s)underlying the downregulation of RYBP using a normal liver cell line,tumor cell lines,and hepatocellular carcinoma(HCC)tissue samples as models.We herein report several important results,including the cloning and characterization of the previously uncharacterized promoter region of the human rybp gene,the discovery of the direct binding of two transcription factors(KLF4 and Sp1)to this region of rybp,as well as the specific binding sites of these transcription factors,the involvement of RYBP in KLF4-and Sp1-modulated liver cancer cell growth,and also demonstrate that the deregulation of KLF4,Sp1 and RYBP is related to a more malignant phenotype of HCC.MATERIALS AND METHODS Patients,tissue microarray(TMA)and immunohistochemistry(IHC)A total of 77 liver cancer patients who underwent curative surgery between January 2012 and May 2013 at Nantong Third Hospital were recruited for this study.This study was approved by the ethics board of the Institute of Basic Medical Sciences,Chinese Academy of Medical Science,and Nantong Third Hospital,and informed consent was provided by the patients.All of the patients were pathologically diagnosed to have HCC,and their detailed clinicopathological characteristics are described later in the text.TMA was constructed from tumor and adjacent normal tissues from each patient as described previously(18).Then,4-m sections were obtained and incubated with antibodies from Sigma against RYBP,or KLF4,or Sp1 at a 1:200 dilution,and then washed and incubated with a goat anti-rabbit or anti-mouse secondary antibody labeled with biotin.After washing step,the sections were incubated with SABC and diaminobenzidine,and finally counterstained with hematoxylin,dehydrated,and mounted.The expression levels of the three target proteins in each tissue specimen were evaluated as described previously with minor modifications(19).Cell lines,cell culture and reagentsThe immortalized human hepatocyte(IHH)cell line was a kind gift from Dr.Jerome Torrisani(Cancer Research Center of Toulouse,France).HEK293T，Hep3B and HepG2 cell lines were from Cell Resource Center,PUMC(Beijing,China),and Huh7,PLC/PRF/5 and SK-Hep-1 cell lines were from the Cell Bank of Chinese Academy of Sciences(Shanghai,China).All of the cell lines were maintained in DMEM supplemented with 10%(v/v)fetal bovine serum.PrimeStar HS DNA polymerase and SYBR Premix Ex TaqTM II were purchased from TaKaRa(Dalian,China).Lipofectamine 2000 transfection reagent and the Trizol reagent were from Invitrogen(Carlsbad,USA).The CellTiter 96 AQueous One Solution Cell Proliferation Assay kit,GoScript Reverse Transcription System and dual-luciferase reporter assay kit were from Promega(Wisconsin,USA).The control siRNA and siRNAs against KLF4 or Sp1 were from Ribobio(Guangzhou,China).Genomic DNA extraction and gel extraction kits were from Sangon(Shanghai,China).EZ DNA Methylation-Gold Kit and ZymoTaq DNA Polymerase were from Zymo Research(Irvine,USA).Protein A-Sepharose beads were purchased from GE Healthcare(Piscataway,USA).Propidium Iodide,control rabbit IgG,anti-Flag M2(F1804),anti-actin(A5441),anti-RYBP(PRS2227),anti-KLF4(SAB5300069),and anti-Sp1(SAB1404397)antibodies were from Sigma(St.Louis,USA).by guest on December 30,2016http:/www.jbc.org/Downloaded from 4 Anti-KLF4(H-180)and anti-Sp1(D4C3)antibodies for Chromatin immunoprecipitation(ChIP)were obtained from Santa Cruz(Santa Cruz,USA)and CST(Beverly,USA),respectively.Preparation of transcription factor expression vectorsThe full-length open reading frames(ORF)for Ets-1(441),MZF-1(734),NKX2-5(112),NKX2-5(324)and YY-1 were amplified by proof-reading PCR from cDNAs of HEK293T cells,and were cloned into the pFlag-CMV-2 vector(note:the numbers in parentheses represent the amino acid numbers of different protein isoforms).The human GV227-KLF4 cloning vector was purchased from Genechem(Shanghai,China),and was subcloned into the pFlag-CMV-2 vector.The human pcDNA3.1-His-Sp1 template was a generous gift from Dr.Xiaozhong Peng(the Institute of Basic Medical Sciences,Chinese Academy of Medical Science,Beijing,China),and its full-length ORF was re-subcloned into the pFlag-CMV-2 vector.pcDNA3.1-HNF1A was a kind gift from Dr.Xiaoming Yang(Beijing Institute of Radiation Medicine,Beijing,China).The plasmids for GFP-RYBP,shCtrl and shRYBP were described previously(20).All of the constructed clones were confirmed by sequencing,and detailed cloning primer information is provided in Table 1.Generation of wild-type,truncated and site-directed mutated reporter vectors for the human RYBP promoterPCR was used to amplify the rybp promoter region from the genomic DNAs prepared from the six liver cell lines(see Table 2 for primer information).The products were cloned into pGL3-Basic vector.pGL3-P(I-R)and pGL3-P(P-R)represent the origin of the genomic DNA from IHH and PLC/PRF/5 cells,respectively.To generate serial truncated constructs of the rybp promoter region,the pGL3-P(I-R)vector was used as a template to amplify a series of rybp promoter truncated fragments(-507/+1040,-294/+1040,-91/+1040,+122/+1040,+312/+1040,+513/+1040,+820/+1040,+312/+512,+413/+512,+513/+819,+614/+819 and+715/+819)using the fragment-specific primers listed in Table 2,and cloned into pGL3-Basic vector.For site-directed mutagenesis,truncated constructs(pGL3-P-M1,pGL3-P-M2,pGL3-P-M3 and pGL3-P-M4)and corresponding full-length constructs(pGL3-M1,pGL3-M2,pGL3-M3 and pGL3-M4)were generated from the pGL3-P(I-R)plasmid using specific mutant primer sets(Table 3).All of the constructs were confirmed by DNA sequencing.Transient transfection and luciferase reporter assaysAssayed cells were grown to about 90%confluence in 24-well plates,then were co-transfected with 1 g of promoter reporter vectors and 50100 ng of Renilla luciferase expression vector(pRL-TK)together with either the empty vector or a KLF4 or Sp1 expression vector using the Lipofectamine 2000 reagent.After 24 h,the cells were lysed and the promoter activities were assessed as described by the manufacturer.RNA interference analysisAssayed cells were grown to about 4050%confluence in six-well plates,then were transfected with either control siRNA or siRNAs against KLF4 or Sp1 using Lipofectamine 2000.The cells were harvested 24 or 48 h after transfection and were used for quantitative real-time reverse transcription-PCR(qRT-PCR)or Western blotting.qRT-PCRTotal RNA was extracted from cultured cells using the TRIzol reagent.cDNA was synthesized using a GoScript Reverse Transcription System with oligo d(T)12-18 primers.qRT-PCR was performed using SYBR Premix Ex TaqTM II on a CFX-96 system(Bio-Rad,Hercules,USA).The SYBR signal was normalized to that of by guest on December 30,2016http:/www.jbc.org/Downloaded from 5 endogenous glyceraldehyde-3-phosphate dehydrogenase.All primers used for qRT-PCR are shown in Table 4.DNA methylation analysisGenomic DNA was isolated using genomic DNA extraction kit according to the manufacturers protocol.Subsequently,bisulfate conversion of the extracted DNA was performed using the EZ DNA Methylation-GoldTM Kit according to the manufacturers instructions.The modified DNA was used as a template and amplified by ZymoTaq DNA Polymerase following the manufacturers manual.The amplified PCR products were gel-extracted and sequenced directly using PCR primers and internal oligonucleotide sequencing primers.The primer sequences are listed in Table 5.Western blot analysisCells were harvested and lysed in ice-cold lysis buffer 50 mM Tris-HCI(pH 7.6),150 mM NaCl,1%NP40,10%(v/v)glycerol,1 mM EDTA supplemented with a protease inhibitor mixture,and were rotated at 4 C for 20 min followed by centrifugation at 12,000g at 4 C for 15 min.The protein concentration was measured using a BCA kit.40 g of proteins were separated by SDS-PAGE and the targeted proteins were probed with corresponding antibodies.Cell survival assayHuh7 cells were seeded into 96-well plates at a density of 3 103/well and transfected next day with indicated plasmids for 72 h.The viable cells were assayed using the MTS reagent according to the manufacturers protocol.Cell cycle distribution assayHepG2 cells were seeded into 60-mm dishes at a density of 4 105/dish.On the next day,cells were transfected with different combinations of indicated plasmids for 48 h.The cells were collected and fixed in 75%alcohol overnight,and the cell pellets were digested with RNase A at 37 C for 20 min and stained with Propidium Iodide.Then,the cell cycle distribution was analyzed by Coulter Epics XL Flow Cytometer(Coulter Corporation,USA).Colony formation assayCells were seeded into 6-well plates at 300 cells per well,and were transfected with pFlag-CMV-2 or pFlag-Sp1 together with either shCtrl or shRYBP expression vector for 24 h.The medium was replaced every three days.After two weeks culture,the medium was removed and cell colonies were stained with crystal violet(0.1%in 20%methanol).Pictures were taken using a digital camera.ChIP assayCells were seeded into 10-cm dishes and cultured to about 90%confluence.The cells were fixed with 1%formaldehyde and the cross-linking reaction was quenched by the addition of glycine.Cellular lysates were collected and genomic DNAs were sonicated to lengths around 500 bp.The DNA solution was clarified by centrifugation,and 15 l of the supernant was de-crosslinked by heating at 65 C for 5 h and used as an input.Equal amounts of the rest of the supernatants were immunoprecipitated with control rabbit IgG,anti-KLF4 or anti-Sp1 antibodies and de-crosslinked by heat.The DNA samples were purified and resuspend