ORIGINAL PAPER
Comprehensive analysis of expression pattern and promoter regulation of human autophagy-related genes
Yusuke Kusama ÆKazuyuki Sato ÆNaoko Kimura ÆJun Mitamura ÆHiroaki Ohdaira ÆKenichi Yoshida
Published online:6August 2009
ÓSpringer Science+Business Media,LLC 2009
Abstract Autophagy is a highly conrved pathway for the degradation and recycling of long-lived proteins and cytoplasmic organelles.Similar to apoptosis,autophagy is a critical regulatory mechanism for determining cellular fate and various pathophysiological conditions in metazo-ans.So far,the systematic analysis of the expression pat-terns and transcriptional regulation of autophagy-related (ATG)genes has remained incompletely defined.In this study,we ud RT-PCR to analyze the expression patterns of 26human ATG genes simultaneously using cDNA derived from different adult and fetal tissues.As a result,we obrved a characteristic ubiquitous expression pattern for all the ge
nes except for ATG2A,ATG9B,and WIPI2.In particular,ATG2A was the only upregulated gene in the etoposide-induced apoptosis of HeLa cells.ATG2A mRNA was also upregulated by doxorubicin.Furthermore,we demonstrated that 13out of 23human ATG gene pro-moters were regulated by the transcription factor E2F1in HeLa cells,indicating that the constructs could be uful for examining how the autophagy pathway is involved in other cellular phenomena,such as apoptosis evoked by various stimuli.Taken together,the results suggest that autophagy might be regulated at both the transcriptional level and the post-translational level.
Keywords Autophagy ÁApoptosis ÁHuman ÁPromoter ÁExpression
Introduction
Autophagy,which literally means ‘‘lf-eating’’,is a unique and fundamental mechanism responsible for the degradation of cellular long-lived proteins and organelles that simultaneously recycles components to maintain cel-lular homeostasis;autophagy is often functionally related to apoptosis on a molecular level [1–5].Autophagy is induced in respon to nutrient starvation and has tissue-specific functions,though a basal level of autophagy is maintained as a houkeeping process in most cells [6,7].More than thirty autophagy-related (ATG)genes have been identified by yeast geneti
cs,and recent advances in our understanding of the molecular mechanism underlying autophagy have enabled the spatiotemporal regulation of the formation of specific protein complexes during autophagy to be revealed.The protein complexes are mainly involved in three well-characterized process:microautophagy,macroautophagy (generally referred to as autophagy),and chaperone-mediated autophagy [8–11].In mammals,approximately two dozen homologs of the yeast ATG genes have been identified,strongly implying that the genes might also be involved in the pathogenesis of various dias including cancer,neurodegenerative dis-ea,and cardiac dia [12–14].To date,whether autophagy promotes or hinders the progression of such dias remains uncertain [15,16].
To reveal the functional aspects of the human autophagy pathway,we focud on uncovering the mechanism responsible for the transcriptional regulation of the fol-lowing human ATG genes:ULK1and ULK2(UNC51-like
Electronic supplementary material The online version of this article (doi:10.1007/s10495-009-0390-2)contains supplementary material,which is available to authorized urs.
Y.Kusama ÁK.Sato ÁN.Kimura ÁJ.Mitamura ÁH.Ohdaira ÁK.Yoshida (&)
Department of Life Sciences,Faculty of Agriculture,Meiji University,1-1-1Higashimita,Tama-ku,Kawasaki,Kanagawa 214-8571,Japan e-mail:iji.ac.jp
Apoptosis (2009)14:1165–1175DOI 10.1007/s10495-009-0390-2
kinas1and2,respectively),ATG2A,ATG2B,ATG3, ATG4A(AUTOPHAGIN2),ATG4B(AUTOPHAGIN1), ATG4C,ATG4D,ATG5,BECN1(BECLIN1),ATG7, GABARAP(gamma-aminobutyric acid type-A receptor-associated protein),GABARAPL1(APG8L),GABA-RAPL2(GATE16),MAP1LC3A and MAP1LC3B (microtubule-associated protein1,light chain3,alpha and beta,respectively),ATG9A,ATG9B,ATG10,ATG12, ATG16L1,WIPI1(WD40repeat protein interacting with phosphoinositides1),WIPI2,WIPI3(WDR45L),WIPI4 (WDR45),and DRAM(damage-regulated autophagy modulator).Most of the numbered genes were named in accordance with their homologous counterparts found in model organisms,but exceptional gene names also exist. ULK1and ULK2have been identified as mammalian Atg1 homologs[17,18].BECN1has been shown to be struc-turally similar to the yeast Vps30(Atg6)gene[19]. GABARAP,GABARAPL1,GABARAPL2,and MAP1LC3have been recognized as yeast Atg8homologs [20].And WIPIs are considered to be homologous to yeast Atg18[21].
Among the above-mentioned ATG genes,the tran-scriptional regulations of ULK1,ATG5,MAP1LC3B,and DRAM are known to be governed by the transcription factor E2F1,and DRAM is also regulated by p53[22,23]. Thefindings suggest that transcriptional regulation might be involved in the link between autophagy and apoptosis, since E2F1and p53together play a pivotal role in the induction of apoptosis by orchestrating the expressions of their target genes[24].In addition,the possible role of autophagy in cancer has been partly explained by the fact that DRAM is inactivated in certain cancers[25].More-over,human cells carrying mono-allelic deletions of the BECN1gene and Becn1heterozygous mice clearly showed a tendency toward tumorigenesis[19,26,27],indicating that subtle transcriptional deregulation might be a rate-limiting step in autophagy-involved human pathogenesis.
天下乌鸦
In the prent study,we conducted a transcriptional analysis of human ATG genes by examining the distribu-tions of their mRNA expressions during tissue develop-ment and etoposide-induced apoptosis.Our results clearly suggest that the majority of human ATG genes are ubiq-uitously expresd in adult and fetal tissues;however,a few genes are expresd in a tissue-specific manner. Interestingly,ATG2A was uniquely upregulated during the etoposide-and doxorubicin-induced apoptosis of HeLa cells,suggesting that ATG2A could be a novel biomarker of topoisomera II inhibi
tor-mediated apoptosis.We also succeeded in establishing promoter-lucifera constructs of 23human ATGs,and this t of constructs might be uful for examining how autophagy is involved in other cellular process in human somatic cells,at least at the tran-scriptional level.Materials and methods
Cell culture and chemicals
Human HeLa cervical adenocarcinoma cells were cultured in Earle’s modified Eagle’s medium(MEM)(Invitrogen, Carlsbad,CA)supplemented with10%fetal bovine rum (FBS),1%non-esntial amino acids(Invitrogen),and antibiotic-antimycotics(Invitrogen)in a5%CO2humidi-fied atmosphere at37°C.Etoposide and doxorubicin hydrochloride were purchad from Wako Pure Chemical Industries(Osaka,Japan).Chemicals were dissolved in dimethyl sulfoxide(DMSO)as100mM stock solutions and kept at-20°C until dilution before u.Thefinal concentrations of etoposide and doxorubicin were 1910-5and1910-6M,respectively.
In silico analysis
马家荡The expression profile was determined(May29,2009) bad on the EST(expresd quence tag)counts in human tissues and organs in the UniGene databa(EST Profile Viewer,NCBI).The dat
a ts ud for the human ATG genes are listed in Supplementary Table2.The number of transcripts per million(TPM)was calculated bad on the gene EST/total EST in the pool,and this value was exported to an Excelfile.Transcription factor binding motif was arched by TRANSFAC algorithm(threshold[85)sup-ported by jp/). Rever transcription-polymera chain reaction
(RT-PCR)
Total RNA was purified using an RNeasy mini kit(Qiagen, Valencia,CA)according to the manufacturer’s instruc-tions.Two micrograms of total RNA were rever tran-scribed using High-Capacity cDNA Rever Transcription kits(Applied Biosystems,Foster City,CA).The PCR was carried out in25l l of a mix consisting of19buffer, 200l M dNTPs,400nM primers,1mM MgSO4,5% DMSO and1unit of KOD plus DNA polymera(Toyobo, Osaka,Japan).Hot-start PCR was then performed as fol-lows:denaturation for3min at94°C followed by ad libi-tum cycles at94°C for15s,a gene-specific annealing temperature(Table1)for30s,and68°C for30s,followed by an extension step of2min at68°C.The PCR results were verified by varying the number of PCR cycles for each cDNA and t of primers.The target gene primer pairs were listed in Table1.The amplified products were parated on 1.0*1.5%agaro gels and visualized under ultraviolet transillumination.The gel images were pro
cesd using Quantity One(Bio-Rad Laboratories, Hercules,CA).Briefly,each specific band was quantified
by volume analysis using the background subtraction method and the resulting density was reprented as the ratio of etoposide/DMSO.The target gene bands were also compared to the corresponding GAPDH(glyceraldehyde-3-phosphate dehydrogena;R&D Systems,Minneapolis, MN)band.For the cDNA panel analysis,2.5l l of cDNA purchad from Clontech(Mountain View,CA)was ud (Human MTC panel I and II,Fetal MTC panel,and MCF7 apoptosis cDNA panel).The G3PDH primer in the kit was ud as a control to amplify983-bp.
Quantitative real-time RT-PCR
Real-time PCR was performed using a MiniOpticon machine(Bio-Rad Laboratories)with Power SYBR Green PCR Master Mix(Applied Biosystems).All the primers were tested to produce proportional changes in the threshold cycle with various initial quantities of cDNA. The measured threshold cycles(Ct)were converted to relative copy numbers using primer-specific standard curves.The Ct values were ud to plot a standard curve in which Ct decread in linear proportion to the log of the template copy number.The correlation values of the standard curves were always[90
%.The gene expression levels induced by etoposide were normalized by the aver-age levels of the mean-centered houkeeping gene GAPDH. All the samples were assayed in duplicate.All the val-ues in the experiments were expresd as the mean±SD (standard deviation)and were compared using two-tailed Student t-tests.
Promoter plasmids
Promoter fragments were generated using the PCR method from human genomic DNA(Promega,Madison,WI)and were ligated into the respective enzyme sites(Table2)of the pGL3-Basic vector(Promega).The PCR primers are listed in Table2.All the plasmid quences were verified by quencing at the Takara facility(Mie,Japan).Differ-ences found by quencing were compared with the
Table1Sequence of primers ud for RT-PCR
学习弹钢琴
Gene name Sen primer quence(50–30)Antin primer quence(50–30)Annealing
temperature(°C)PCR product
length(bp)
GenBank诗经汉广
accession number
ULK1GTCCAGCTTTGACAGTCAGT TCCCAGGACTCAGGATTCCA52480NM_003565 ULK2TGTTTCTAGGGTGTAGCTGG CACCATCTACCCCAAGACCT52450NM_014683 ATG2A ACCTACACACGAGTGAGCGG TGCCATGGTAATCCAGCCAG63420NM_015104 ATG2B GTGAGAGTCCTCTCTGGCGA CGAGAATCCAAATCGTACCC53460NM_018036 ATG3CCAACATGGCAATGGGCTAC ACCGCCAGCATCAGTTTTGG58420NM_022488 ATG4A GGAGGAGTTCATTGCTTCCC GCATGGCCTGCAAAGCCCAA54500NM_052936 ATG4B TGCCTGTCCTGGGAAAGTAT GGTCAAGGTGTCCCACACAT52390NM_013325 ATG4C TGCACATTGAGAACTGGCCA ATGCCTTGCTTCTTCAACTG55420NM_032852 ATG4D CAGCCCACTGTGGATGTCAG GCTCAAGATCCCACACCCGA60420NM_032885 ATG5AGTATCAGACACGATCATGG TGCAAAGGCCTGACACTGGT59590NM_004849 BECN1ACTGTGTTGCTGCTCCATGC CCCAAGCAAGACCCCACTTA60400NM_003766 ATG7CACTGTGAGTCGTCCAGGAC CGCTCATGTCCCAGATCTCA60380NM_006395 GABARAP ACATTGCCTACAGTGACGAA TTTCAGTCCCTTCCAACTAC58420NM_007278 GABARAPL1GTTGAGCATCCCTGTCTAAC CTGACAGAACTGACACACAC53500NM_031412 GAB
ARAPL2CGTGGAGTCCGCGAAGATTC AGGCATGAGGACAATGCACA63530NM_007285 MAP1LC3B AAAGCTGTGGATGATCCACG AGCAGGTGACAGGAACTCCT52480NM_022818 ATG9A ATGGGCAGTCGGCATCAAGG CCCTGCTTGGCAGCTTCTAT65440NM_001077198 ATG9B CCACCTTGGGCAGTTCTTCT CCTGCATGGTGAGAATGGTG60480NM_173681 ATG10CCAAGAGTTTACCTGGCCAG CCTGGGTTAAAGCCAACCTC59380NM_031482 ATG12GAGACCAGCCTGGTTAGCAA CTGAACCCTCAGTGGCAAAC56420NM_004707 ATG16L1AGAAGAAGCACATGGGCTCC CAGGGAGGGGTCTGTAGTTC60460NM_030803 WIPI1CAGTAACACCGAGACGGTAC GCACTTGGTCTGGCTACGGT54420NM_017983 WIPI2CGCACTGTAAGATGAGGCAG ACTGGCGCAGCAGTAAGAGT58410NM_015610 WIPI3TGGCTGTGACCAGCTCGACT GTGACGTCACGGGCACAGAT60420NM_019613 WIPI4TGACATAGCCTGTGTGTCTC TCAAGGTACACGTCGAAAGC59480NM_007075 DRAM TGAGCCTGGGACTTCGAGAC CTCAAGGAGCTCCCAATCTA54520NM_018370
The annealing temperatures,PCR product lengths,and GenBank Accession numbers are also indicated
deposited reference quence in GenBank(Accession numbers are shown in Table2).Differences in the fol-lowing plasmid quences were identified:ATG3(G was changed to C at-221position),ATG7(T
was changed to A at-199position),GABARAPL1(T was changed to C at-144position),MAP1LC3B(A was changed to G at -474position),ATG10(T was deleted from T stretch at -479position),and DRAM(G was changed to A at-417 position),where the minus position is relative to a tran-scriptional start site at?1.
Lucifera assay
For the promoter assay,29104cells were transfected with FuGENE6(Roche,Bal,Switzerland)according to the manufacturer’s instructions.Briefly,200ng of the expres-sion plasmid(pcDNA3and pcDNA3-E2F1),200ng of the firefly lucifera reporter plasmids pGL3-Basic(Promega) or pTA-Luc,pE2F-TA-Luc(Clontech),and0.6ng of the Renilla lucifera reporter plasmid pRL-TK(Promega)per 24-well dish were ud for each transfection.Cells were lyd24h after transfection by applying100l l of Passive Lysis Buffer of the Dual Lucifera Reporter Assay Kit (Promega)into each well of the24-well plate.Five micro-liters of cell lysate were ud for the lucifera reporter assay with the same kit,according to the manufacturer’s protocol.Light intensity was quantified in a GloMax20/20n Luminometer(Promega).The experiments were performed at least in triplicate.As a control for the transfection effi-ciency,thefirefly lucifera activity values were normalized to the Renilla lucifera activity values.Data are prented as the mean valu
e±SD.Statistical differences were ana-lyzed using two-tailed Student t-tests.A value of P\0.05 was considered statistically significant.
Caspa-3/7assay
The induction of apoptosis was assd using a Caspa-Glo3/7assay according to the manufacturer’s instructions (Promega).Briefly,the cells were inoculated into a96-well plate at a concentration of19104cells/well24h before treatment.The cells were then treated with chemicals for
Table2Sequence of primers ud for promoter-lucifera constructs
Gene name Sen primer quence(50–30)Antin primer quence(50–30)Flanking
enzyme site GenBank accession
number
Amplified
region(bp)
ULK1TGTTCGTCACGCCCGGCTCT GGATCCGACTCCGACTCCGA KpnI/BglII AC131009-184*6 ULK2GCCTGTCACCGTCCCTCTAG CGACGTCCGCGCGCCTTGAA KpnI/BglII AC015726-478*-32 ATG2A TCCTCTGGGGAAGGCTTCTA CCATCGTGACATCTCGGAGA KpnI/BglII AC000159-402*68 ATG2B TCTTCTTCCTTCCTCCGGAG CCCTATTTGGTGCCGGGAGT KpnI/BglII AL359240-210*250 ATG3CCAGCGTACTGTAGGAATAG GCCACCGACTCGCATCAGCA KpnI/BglII AC092692-866*24 ATG4A TTCGCCGACGTCGCCGACTG TGTGAGGGCAGCAAAGGCGC KpnI/BglII AL031177-341*19 ATG4B CACGTCCGTACCGCAAGATG CCAATAGGTGTGCCGGCGCA KpnI/BglII AC133781-261*19 ATG4C CAGTTCCTACGCCAGAGGCA CACACGCAGACGTTCCGACC KpnI/BglII AC103923-345*45 ATG4D TGCCCCGGAGGTCACACTAG GCCATCGCCATCTTAGCGGG KpnI/BglII AC011461-460*-41 ATG5AATGCCTGCGGTGGTTCCAA TCCGTGTTCTGCCTAACCCA KpnI/BglII AL138917-378*-29 BECN1AATAGCGGAGCCTCCCCATT CTCTGTCTTCAGCGACTTCC KpnI/BglII AC055866-207*13 ATG7AGGTCCTGGGGGTAAGGAAA GTAGCTGCCGCCATTATTTC KpnI/HindIII AC020750-325*25 GABARAP CCTACTTCGCGCACCCGCCA GATCCACGAATTTGCGCCAC KpnI/HindIII AC120057-653*-1 GABARAPL1GAGGCTGGATCCCAACCAGC GGGAGCACAAAAACAGCTGG KpnI/BglII AC115676-339*1 GABARAPL2ATGAACCGCTCTAAGAGCCC CGGCTGTCGGAGCCTAGCAA KpnI/BglII AC025287-321*9 MAP1LC3A TCGCCCTAAGCCCCGTGAAG CCCGGTGACGTCAGGTC
ACA KpnI/BglII AL118520-384*6 MAP1LC3B ACTGCGCCCAGAATGAAGGC ATGGGCGAAACGCGCGCTCT KpnI/BglII AC010531-568*-49 ATG9A AACTGGCCCGCGTAAACTCG ACTCACTGTCACCCGCAGCC KpnI/BglII AC068946-303*7 ATG9B ACACCTGGCTCTAACACGAT GATGGGAGCTGTTGTTGCTT KpnI/BglII AC010973-295*45 ATG10CCTTTCACTTCTTCGCCTGC GCCTCCTTCAGTCAGGTCCG KpnI/BglII AC114969-673*17 ATG12GCGAGAAGAACGAGTTCCAC GGTCGAATGCGCACACTCCA KpnI/BglII AC022111-745*5 ATG16L1CCCTGTACCAAGCACAGTGC CTACGCAGGGCGCTCGCTAG KpnI/BglII AC013726-389*21 DRAM GTGATGGCTATTCACGGCGC AAGCGGACGCGACTACGGAG KpnI/BglII AC005409-444*166
The amplified genomic region is indicted by numbering relative to the transcription initiation site(at?1)described in the NCBI UniGene Databa(Genome View).The GenBank Accession numbers of the genomic clones were ud to designate the PCR primers
48h,followed by1h of incubation with Caspa-Glo3/7 substrate at room temperature,and the resulting activity was measured using a GloMax20/20n Luminometer (Promega).The activity was prented as relative light units(RLU,9106)in the treated cells relative to the control value(treated with0.1%DMSO).Blanks were measured in wells containing0.1%DMSO or chemicals without cells. T
he data are prented as the mean values±SD.Statis-tical differences were analyzed using a two-tailed Student t-test.A value of P\0.05(n=3)was considered statis-tically significant.婴儿奶量
Results
Expression patterns of human ATG genes in adult
and fetal tissues
Reports on the mRNA expression patterns of human ATG genes have been increasing in number;however,the reports have utilized different detection methods,such as Northern blot or RT-PCR,and different mRNA resources (summarized in Supplementary Table1).Alternatively, EST counts for quenced cDNA pools can be utilized as predictable information on the mRNA distribution patterns in various tissues and developmental stages for most of the human ATG genes(summarized in Supplementary Table2);however,the data should be verified using a single detection method and a uniform cDNA resource.To achieve this goal,we performed RT-PCR for23human ATG genes in16adult tissues(heart,brain,placenta,lung, liver,skeletal muscle,kidney,pancreas,spleen,thymus, prostate,testis,ovary,small intestine,colon,and leuko-cyte)and8fetal tissues(brain,lung,liver,kidney,heart, spleen,thymus,and skeletal muscle)(Fig.1).Most
of the human ATG genes as well as G3PDH were ubiquitously expresd in the adult and fetal tissues that we examined, with a few exceptions(Fig.1).Of note,the expression of ATG2A was not detected in adult tissues including the heart,brain,placenta,lung,liver,and skeletal muscle (Fig.1).To our knowledge,the expression pattern of ATG2A has not been previously reported.ATG7was expresd faintly in adult heart(Fig.1),though the EST count both in literature and the UniGene databa predicted that ATG7would be expresd ubiquitously(Supplemen-tary Tables1and2)[28].ATG9B showed a characteristic expression pattern in veral RT-PCR analys.Namely, ATG9B was abundantly expresd in some adult(placenta and testis)and fetal(brain,lung,and thymus)tissues, whereas none or a minimal expression level was obrved in other adult(heart,lung,liver,skeletal muscle,kidney, pancreas,ovary,and colon)and fetal(liver,heart,and skeletal muscle)tissues(Fig.1),though previously reported Northern blot and RT-PCR analys predicted an ubiquitous pattern with the highest expression level thought to occur in the placenta(Supplementary Table1) [29,30].The ATG9B expression pattern predicted by the EST count was tissue specific,with abundant expression in the placenta and ovary and medium or minimal expression in the lung,testis,liver,muscle,brain,and pancreas (Supplementary Table2).The results support the notion that ATG9B mRNA exists in specific tissues and is most abundantly obrved in the placenta.WIPI2has been reported to exhibit a ubiquitous expression pattern,with the highest leve
ls obrved in the heart,skeletal muscle,and pancreas(Supplementary Table1)[21];however,our repeated RT-PCR experiments showed that WIPI2was undetected in adult tissues including the heart,brain,pla-centa,lung,skeletal muscle,and kidney(Fig.1).At pres-ent,we have no explanation for this discrepancy.DRAM expression was ubiquitous,with the highest level obrved in the pancreas(Fig.1),whereas the EST counts failed to show a high count in the pancreas and no counts were obrved for the spleen(Supplementary Table2).Toge-ther,thefindings suggest that our comprehensive mRNA analysis of human ATG genes might be superior to EST counts and also to previous reports of individual experi-ments that were incomplete for most human ATG genes. Changes in the expressions of human ATG genes员工信息登记表
怎样煮元宵in apoptotic cells
绮丝碧
DRAM is a reprentative example of a gene that links autophagy and apoptosis,since DRAM is an esntial factor for p53-mediated apoptosis and is also involved in autophagy[22].Therefore,we checked the mRNA levels of human ATG genes regulated by apoptosis-inducing etoposide.HeLa cells were treated with1910-5M of etoposide for48h,which was sufficient to induce casp-as-3/7activity(Fig.2a).In addition,the accumulation of p53protein accompanying an elevated phosphorylation level of rine15was detected48h after etoposide treat-ment,compared with the leve
l in a DMSO vehicle treat-ment group(data not shown).Amongst the genes that were examined,the ATG2A and ATG4C mRNA levels were uniquely upregulated and downregulated,respectively, with a statistically significant difference(Fig.2b,c).The expression level of DRAM was unchanged(Fig.2b,c).We focud on ATG2A as an upregulated gene and further examined changes in the ATG2A transcript levels in HeLa cells using real-time PCR.Etoposide treatment incread the ATG2A mRNA level by2.1-fold(SD=0.0007,two-tailed Student t-test,P=0.02,n=3).
We reasoned that the upregulation of ATG2A mRNA might be a common phenomenon in apoptotic HeLa cells. Conquently,we treated the cells with doxorubicin;
