Research Article Osteoponin Promoter Controlled by DNA ...2019-7-31 · Research Article Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine

Research ArticleOsteoponin Promoter Controlled by DNA MethylationAberrant Methylation in Cloned Porcine Genome

Chih-Jie Shen1 Yung-An Tsou123 Hsiao-Ling Chen4

Hung-Jin Huang5 Shinn-Chih Wu6 Winston T K Cheng7

Calvin Yu-Chian Chen28910 and Chuan-Mu Chen111

1 Department of Life Sciences National Chung Hsing University Taichung 402 Taiwan2 School of Medicine College of Medicine China Medical University Taichung 40402 Taiwan3Department of Otolaryngology Head and Neck Surgery China Medical University Taichung 40402 Taiwan4Department of Bioresources Da-Yeh University Changhwa 515 Taiwan5Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources College of Pharmacy China Medical UniversityTaichung 40402 Taiwan

6Department of Animal Science and Technology National Taiwan University Taipei 106 Taiwan7Department of Animal Science and Biotechnology Tung Hai University Taichung 407 Taiwan8Department of Biomedical Informatics Asia University Taichung 41354 Taiwan9Department of Medical Research Human Genetic Center China Medical University Hospital Taichung 40447 Taiwan10Research Center for Chinese Medicine amp Acupuncture China Medical University Taichung 40402 Taiwan11RongHsing ResearchCenter for TranslationalMedicine and the iEGGCenter National ChungHsingUniversity Taichung 402 Taiwan

Correspondence should be addressed to Calvin Yu-Chian Chen ycc929miteduand Chuan-Mu Chen cmchen7010978gmailcom

Received 24 February 2014 Accepted 5 March 2014 Published 2 July 2014

Academic Editor Chung Y Hsu

Copyright copy 2014 Chih-Jie Shen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming especially insome important organ development Osteoponin (OPN) is an extracellular-matrix protein involved in heart and bone developmentand diseases In this study we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay Aberrant methylation of porcine OPN was frequently found in differenttissues of somatic nuclear transferred cloning pigs and bisulfite sequence data suggested that the OPN promoter region minus2615to minus2239 nucleotides (nt) may be a crucial regulation DNA element In pig ear fibroblast cell culture study the demethylation ofOPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed theOPN mRNA reexpression In clonedpig study discrepant expression pattern was identified in several cloned pig tissues especially in brain heart and ear Promoterassay data revealed that four methylated CpG sites presenting in the minus2615 to minus2239 nt region cause significant downregulation ofOPN promoter activity These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPNexpression that we found in cloned pigs genome

1 Introduction

Nowadays many of pathogenesis of diseases have beendetermined [1ndash3] Methylation in the 51015840 cytosine in the CpGdinucleotides is crucial a mechanism that regulates geneexpression without changing DNA sequence and can beinherited to the offspring [4] The promoter region contains

various transcription factor binding motifs with numerousCpGdinucleotides Some transcription factors are blocked bymethylated CpG island resulting in inhibition of gene expres-sion [5] Somatic cell nuclear transfer (SCNT) technique isused to generate an identical genetic background offspring[6 7] However SCNT cloning animals usually showed lowsurvival rate and impropriate methylation reprogramming

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 327538 16 pageshttpdxdoiorg1011552014327538

2 BioMed Research International

process [8] This dilemma of SCNT animal may be caused bymethylation controlled genes such as imprinting genes [9]

OPN is an extracellular matrix protein and hydrophilicglycoprotein identified firstly in the bone as a sialoprotein Itcontains a thrombin and transglutaminase cutting site andthe molecular weight is about 25 kDa to 75 kDa in pig themolecular is about 67 kDa it contains numerous isoforms[10] OPN has a hydrophobic N terminal thus it can besecreted out of cell membrane the amino sequence ofOPN isfull of Asp Thr and Ser that can elevate the binding activitywith calcium glycosylation and phosphorylation respec-tively [11] Thus OPN plays numerous roles in many aspectssuch as bone remodeling cell migration iNOS regulationrepairment and leucocyte recruitment [12] And acquiredOPN expression has been found in a variety of cancer celltypes especially in the liver lung breast prostate colonbrain and spleen [13 14] OPN is cleaved by MMPs proteinto generate functional OPN that can bind to 120572v1205733 [15] Thisintegrin binding with OPN has influence on NF120581B signalingtransduction [16 17] Therefore overexpressed OPN is asso-ciated with tumorigenesis tumor invasion and metastasis[18 19] Previous study suggested that overexpressed OPNinduces the serious cardiac fibrosis [20 21] Thus our clonedpigs were also surrounded by various defects in heart fibrosisand retardation of growth of bones Therefore this studyfocuses on the methylation change of OPN promoter thatmay be disrupted by inappropriate reprogramming processConsequently aberrant methylation of promoter could leadto aberrant expression of OPN In the previous studies OPNexpression was induced with TSA (trichostatin A) in mouseundifferentiated mesenchymal cell line by AP1 site [22]The TSA is a histone deacetylase inhibitor It can lose thechromatin structure in order to let gene restore its expression5-aza-dc is also an analog with the same structure of cytosinewithout methyl group adding in the 51015840C end [23]

Thus 5-aza-dc addition leads to low methylation per-centage in the CpG sites rich region The hypomethylationstatus in the promoter may contribute its gene transcriptionactivity Porcine fetal fibroblasts in 5th passage cultures weretreated with 05 10 20 and 30 120583M 5-aza-dc for 96 h 5-aza-dc inhibited the growth of cell at all concentrations 5-aza-dc induced a reduction of transcripts level in DNMT1 andincreasing expression in imprinted gene IGF2 [24] Further-more human OPN promoter sequence is similar to porcinein the front 400 nt of the porcine promoter Therefore weinvestigatedOPN RNA and promotermethylation changes inthe porcine ear fibroblast cell Data showed that the elevatedOPN expression and in 5-aza-dc treated fibroblast cell is dueto the decreased methylation of OPN promoter Cloned pigssamples had found extremelymethylation changes especiallyin the brain (9975 upregulation) heart (1150 down-regulation) and ear (1803 down-regulation) Deletionanalysis of the promoter region revealed 5-aza-dc inducedluciferase response that was regulated by minus2615 to minus2239 ofthe OPN promoter These data suggested that methylation inthe OPN promoter plays a crucial role in the regulation ofOPN expression Methylation of OPN promoter may be anepigenetic marker of diagnosis of cancer

2 Materials and Methods

21 CpG Island Prediction The sequence of a putative CpGisland in OPN promoter was analysed by using MethPrimersoftware (httpwwwurogeneorgmethprimerindex1html)

22 Cell Culture The porcine fibroblast cell line was grownin Dulbeccorsquos modified Eaglersquos medium (DMEM Gibco-BRL Gaithersburg MD USA) supplemented with 10 fetalbovine serum (FBS Gibco BRL) and containing 100UmLpenicillin and streptomycinThe cells were incubated at 37∘Cin humidified incubator with 5 CO

2

23 5-aza-dc Demethylation Drug Treatment For 5-aza-dctreatment porcine fibroblast cell in 5th passage cultures wastreated with 5-aza-dc (sigma) at various concentrations thatis 0 (control) 05 15 and 20120583M for 72 h Medium waschanged every 24 h and then cells were collected for RNA andDNA extraction and stored at minus80∘C [24]

24 Quantitative Real Time-PCR 2 120583g RNA of ear fibroblastcell was used to be transformed to cDNA 05 120583L of cDNAwas performed for quantitative real time-PCR with Rotor-Gene 6000 (Corbett) 120573-actin was the internal control fornormalize target gene OPN The calculated gene expressionfold from CT value was according to the previous study119875 value less than 05 exhibited the obviously significantdifference

25 Methylation Analysis by Combined Bisulfite Restric-tion Analysis (COBRA) For amplification of porcine OPNpromoter methylation analysis site PCR was performedusing 2120583L of bisulfite-converted genomic DNA as tem-plate The primer sets of COBRA were OPN-C sense 51015840-TTTTTTGAGGGAGATTAGTTTTTG-31015840 and antisense 51015840-ATTCTACTAAAATCCAACCACCC-31015840 The COBRA-PCRproducts were purified by phenolchloroform followed byethanol precipitationThe DNAwas resuspended in 85 120583L ofdistilled deionized water Purified PCR products were thendigested with 10U BstUI restriction enzyme (New EnglandBiolabs MA USA) at 65∘C Products were electrophoresedon 6 native acrylamide gel stained with 200 gmL ethidiumbromide and visualized using a Kodak 1D software

26 Methylation Specific-PCR Genomic DNA (05 120583g) wastreated with sodium bisulfite according to the manufacturersquosrecommendations (EZDNAMethylationKit Zymo researchCA USA) and amplified with specific primers for methylatedor unmethylated DNA The primer sets of MS-PCRwereOPN-M sense 51015840-AAGCGGGGAAGGAGTTATTACGT-31015840 antisense 51015840-TCCGACAAAACGAAACGATCATACA-31015840OPN-U sense 51015840-GAAGTGGGGAAGGAGTTTATTATGT-31015840 and antisense 51015840-CAATAACTCCAACAAAACAAAACAATC-31015840 All PCR reactions were performed on PTC 200thermocyclers (MJ Research MA USA) and in 25 120583Lvolume using the PlatiumTaq DNA polymerase system(Invitrogen CA USA) PCR products were separated on15 agarose gels The M-set primers contained at least three

BioMed Research International 3

CpG sites to distinguish themethylation status of investigatedregion And U-set primers overlapping the M-set primerswere used to amplify the unmethylated region

27 Plasmid Constructs A full length pig OPN promoter(minus2615-luc) was amplified from wild-type pig heart tissuecDNAThis fragment was cloned into a luciferase fusion plas-mid pGL3-Enhancer vector (Promega) to generate pOPN-full-lucHindIII and NcoI cutting sites were used for cloningThree truncated forms of pOPN promoter were prepared byPCR using the pOPN-full-luc as a template and using syn-thesized oligonucleotides as follows pOPN-full-luc sense 51015840-AAGCTTGAATTCACTCGTCTTTCCTTTGAGA-31015840 andantisense 51015840-CCATGGGCTGACAGCCTGGACCTCCCC-31015840 minus2239-luc sense 51015840-AAGCTTCCTATAACTGTCTAC-GTTCATATTAGAC-31015840 and antisense 51015840-CCATGGGCT-GACAGCCTGGACCTCCCC-31015840 minus1505-luc sense 51015840-AAG-CTTAATTTTCATTTAAGTAACCAACTTTATATATC-31015840 and antisense 51015840-CCATGGGCTGACAGCCTGGAC-CTCCCC-31015840 minus495-luc sense 51015840-AAGCTTGCCTGAACA-ATATAGCCTTGTCGC-31015840 and antisense 51015840-CCATGG-GCTGACAGCCTGGACCTCCCC-31015840 The sequence of con-structs was confirmed by DNA sequencing There were two-pointmutation different fromNCBI one is 287A to T and theother is 957T to A

28 Transient Transfection and Luciferase Assay Pig earfibroblast cells were transfected using the Lipofectamine 2000(Invitrogen) Fibroblast cells were incubated at a density of8 times 105 cells into 35mm diameter dishes After 24 h when cellwas adherent to the dishes 3120583g of reporter plasmidDNAwastransfected for 6 h in Lipofectamine mixture (Invitrogen)24 h after the transfection cell lysates were collected fora luciferase assay The luciferase activity of the cell lysateswas detected by Dual-light system (Applied biosystems) Theactivity data wasmeasured with PARADIGMDetection Plat-forms (BeckmanCoulter) Luciferase activity was normalizedwith 1 120583g 120573-gal plasmid All luciferase assays were carried outin triplicate

29 In Vitro Methylation of the OPN Promoter Region TheOPN reporter construct minus495-luc was methylated by incu-bation with SssI methyltransferase (New England BioLabs)The minus2615-luc construct was methylated by HhaI and HpaIImethyltransferase (New England BioLabs) for 16 h at 37∘CThe methylation status was also verified by digested withHhaI and HpaII enzyme

210 Electrophoresis Mobile Shift Assays Nuclear extractswere prepared from HEK293T cells Two probes weredesigned for methylation binding activity test Two probescontaining either 6ndash8CpG sites or 13th CpG site in theminus2615 to minus2239 of the OPN promoter were generatedby annealing two complementary oligonucleotides(OPN 6ndash8 51015840-TGCATGATCGTTCCGTCC -TGCCGGAGTCACTGACGGAACCAGACCGAGGT-3101584051015840-ACCTCGGTCTGGTTCCGTCAGTGACTCCGGCAG-GACGGAACGATCATGCA-31015840 the predicted core

WT pig Cloned pig

L In Ov Mu KiLuMaHeStBr Bl Sp Li Ea Br Mu Ea He Li Li WC1 C1 C2 C2 C3 C4

OPN

120573-Actin

(a)

Liver

Heart

Ear

Muscle

Brain

Cloned pigWT pig

0

10

20

30

40

90

100

OPN

mRN

A ex

pres

sion

leve

l

C1 WT C1 WT C2 WT C2 WT C4 WTTissues

(b)

Figure 1 (a) (b) The semiquantitative RNA expression of OPNC1ndashC4 indicated four different cloned pigs The black arrowsrepresent the contrary expression in the cloned pig tissues relativeto WT tissues Br brain Ea ear He heart Ki kidney Li liver LulungMumuscle Sk skin In intestine Sp spleen Pl placenta Umumbilical cord B blood S blood treated with SssI and W ddH

2O

TSS

ExonICpG islandCOBRA primer set

minus2615

minus2500

minus2000

minus1500

minus1000

minus500

+1

(a)

M-set

U-set

Br MuPl Ea Mu PlHeLi Ea Li He Ea Mu LiPlLi Ea B WS

Copy1 Copy2 Copy3 Copy4 Control

214 bp

222 bp

(b)

Figure 2 MS-PCR analysis of OPN promoter (a) The distributionof CpG sites in the pigOPN promoterThe two arrows indicated theMS-PCR primer sets (b) The MS-PCR results of OPN promoter incloned pigsThe arrows represent both existence of methylation andunmethylation DNA element Br brain Ea ear He heart Li liverLu lung Mu muscle Pl placenta B blood S blood treated withSssI and W ddH

2O


1 SB8765432

Mock Control

287

Methylation ()9084512413350759775296

35

30

25

20

15

10

5

0

0 05 15 20

Met

hyla

tion

()

243bp

144bp

387bp

05 120583M 15 120583M 20 120583M

lowast

5-aza-dc (120583M)

(a)

35

30

25

20

15

10

5

0

0 05 15 20

Met

hyla

tion

()

15 35302520

Cycles

The fl

uro

amou

nt o

f syb

er g

reen

05 120583M15 120583M20 120583M10 120583M

lowast

5-aza-dc (120583M)

10minus05

10minus07

10minus09

10minus11

10minus13

10minus15

(b)

Figure 3 OPN RNA expression and DNA methylation of 5-aza-dc treated pig fibroblast cell (a) The schematic showed the methylationpercentage and mRNA expression of OPN promoter in different concentrations 5-aza-dc (b) The COBRA analysis of OPN promoter Belowthe square is the methylation percentage of OPN promoter methylation

sequence of the AP1 binding site is underlinedOPN13th 51015840-CCTCCGTGTTCCCTGTTAATGTGT-AGCGCGTCGTTGTTGGGAAATAGTTC-31015840 51015840-GAACTATTTCCCAACAACGACGCGCTACAC-ATTAACAGGGAACACGGAGG-31015840 the predicted coresequence of the ADR1 binding site is underlined) Thetranscription factor prediction software is TFSEARCH30 version The probes were labeled with 120574-32P-ATP byusing T4 kinase (Promega) Annealing probes also weremethylated with SssI methyltransferase (NEB) Nuclearextracts containing 56 120583g of the protein were preincubatedin 20120583L of binding buffer (50mM Tris-HCl (pH 80)750mM KCl 25mM EDTA 05 Triton-X 100 625glycerol (vv) and 1mM DTT) with or without unlabeledcompetitor (10-fold molar excess) For supershift assayantibody of AP1 was added to the preincubation buffer After10min of preincubation on ice the DNA probe labeled with[120574-32P]-ATP was added and the mixtures were incubatedat room temperature for 30min The reaction mixtureswere resolved on 6 polyacrylamide gels The gels weredried and subjected to PhosphorImager analysis using aTyphoon system and ImageQuant TL software (AmershamBiosciences Sunnyvale CA USA)

211 Molecular Modelling The molecular docking was thenfurther analyzed for proving the further mechanism of our

findings we further surveyed the interaction of AP1 (c-Jun)and TFIIB by computational biology The AP1 (c-Jun) ispossible higher spot for hypermethylation in OPN promoterarea and provides the binding domain for RNA-polymeraseII initial binding transcription factor (TFIIB) in this studyTherefore we first utilized the Z-DOCK program to simulatethe structures of c-Jun and TFIIB After that we further usedmolecular dynamics (MD) to validate the stability of c-Junand TFIIB complex under the GROMACS 455 program[25] with charmm27 force field The model is set in theTIP3Pwatermodeling in 12 nm distance of box for water boxsetting Na and Cl ions in the concentration of 0145M NaClmodel are used for system neutralization All bonds are fixedby linear constraint solver (LINCS) algorithm to constrainall bonds lengths in the simulation system Newtonrsquos Law isutilized for calculating the motion of molecular dynamics asfollows

119889

2119903

119889119905

= 119872

minus1119865

(1)

The Particle mesh Ewald (PME) is also used for calculatethe coulomb type of electrostaticsTheVan derWaals (VDW)interactions are set as 14 nm cut-off distance for nonboundinteraction The first step is set on the 5000 cycle stepsperformed in the manner of Steepest Descent algorithm forenergyminimization And then equilibrationwas performed


WT brain WT heart WT ear WT liver

Copy1 brain Copy2 heart Copy2 ear Copy4 liver

Total 57400 = 143CpG1-10 49200 = 245

Total 57400 = 29CpG1-10 49200 = 47

Total 57400 = 117CpG1-10 49200 = 233

Total 57400 = 85CpG1-10 49200 = 115

Copy5 brain Copy3 heart

Total 57400 = 133CpG1-10 49200 = 222

Total 57400 = 133CpG1-10 49200 = 261

Total 57400 = 181CpG1-10 49200 = 35

Total 57400 = 50CpG1-10 49200 = 94

Total 57400 = 181CpG1-10 49200 = 328

Total 57400 = 53CpG1-10 49200 = 94

Figure 4 The bisulfite sequencing of OPN minus2610sim minus2400 nt upstream the promoter in cloned pigsrsquo tissues The closed circles represent themethylation CpG sites The hollow circles represent the unmethylated CpG sites The bottom number indicated the methylation percentageof each sample The range of square showed that the region may be the methylation controlled region of OPN promoter

Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc

++

minus2615-luc

minus2239-luc

minus1505-luc

minus495-luc

Luciferase fluro normalized with 120573-gal

(a)

Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc

lowastlowastminus2615-luc

minus2239-luc

minus1505-luc

minus495-luc


(b)

Figure 5 Methylation and deletion analysis of OPN promoter in 293T cells The match-like bar with black circle represents the methylationCpG site white circle of match bar indicated the unmethylation CpG sites PGL3 vector as standard in 293T cell line PGL3-enhance vectoras negative control cell lysate as the background pCMV-b-gal as internal control The relative value is adjusted by cell lysate minus495M-lucindicated the methylation in PGL3 backbone with HhaI and HpaII methyltransferase (lowastlowast119875 lt 001) minus2615-luc and minus2615M-luc 119899 = 3minus495-luc and minus495M-luc 119899 = 4 The experiments were repeated three times and the results were analyzed and presented as the mean plusmn SE


ttcctttgagggagaccagctcttgAGCGAGTGTGGGAAGCGGGGAAGGAGCCCATCAC

GTCCACCTGCGGTTGCTAAAGACAACAGAGCAGAAAAGAACGCTCTGCT

TCTCTTGGCCTCCGTGTTCCCTGTTAATGTGTAGCGCGTCGTTGTTGGGAA

ATAGTTCCTCACCTGACTTTCCAAGAAATGGAGGGCCTCACAGTTGTTTGA

TGGCTCGGTCATTAAATGCATGATCGTTCCGTCCTGCCGGAGTCACTGAC

GGAACCAGACCGAGGTCTCAGGTCCTTCTCCGAAATGCTGCCATCGTGTG

GCACCTCGGAGCCATGACCGGAAGAGCCCTATGGGTCATATGGTTCAGCG

CAgggtggctggactccagcagaatct

1 2 3

4 5

6 7 8 9

10 11 12 13 14

15 16 17

18 19 20

HhaI

HhaI

HpaII

HpaII

AP1-like

AP1-like

Figure 6 The sequence and CpG sties distribution of pig OPN promoter 20 CpG sites exist in the front region of OPN promoter Theunderline indicates the predicted AP1-like binding site The gray marker indicates two methyltransferase sites of HhaI (GCGC) and HpaII(CCGG)

Methylated UnmethylatedN PCompetitor

1st CpG

100()

75353466526693912

++++++minus minus

(a)

13th CpG

100()

157239589676844561

(b)

20th CpG

100()

352493400820837800

(c)

Figure 7 Electrophoresis mobile shift assay in porcine OPN promoter CpG sites (a) Using CpG1 contained DNA element binding withSH-SY5Y nuclear extract Methylated and unmethylated competitors were used as 2X 5X and 10X concentration than isotope labeled probe(b) CpG 13th probe ofOPN promoter binding with HEK-293 nuclear extract (c) CpG 20th ofOPN promoter element binding with HEK-293nuclear extract There was no difference between methylated or unmethylated competitors in competition experiment

c-Junc-fos

Premix with antibodyCpG 13 CpG 20

Binding ()

758

816

51448100

963

994

555

627

100

++ +

+ ++ + +

++

+ +

minusminusminusminus

minusminus

minusminus

minus

minus

minusminusminus

minusminus

minusminus

minus

Figure 8 The CpG 13 and CpG 20 showed competition bindingactivity characteristics with c-Jun and c-Fos The competitionreduced the binding activity between transcription factors andlabeled OPN EMSA probe The amount of EMSA c-Jun and c-Fosantibody is 3 120583g The EMSA probe is 30 120583g Adding the Ab with apremix way can reduce the binding effects with the transcriptionfactors It is suggested that the premix with antibody blocks theaccess to its binding site (antibody-transcription factor-DNA)

with a time period of 1 ns for position restraints set underthe constant temperature dynamics (NVT type) conditionsThe third step is calculating the production run for 5000 psunder constant pressure and temperature dynamics (NPTtype) All theMD systems are set by 310 K temperature duringall simulation times MD frames data were saved every 20 psfor all production runs

212 Molecular Dynamics Analysis First we survey the sta-bility of all atoms performed by using the GROMACS 455software though the commands of g rms and g gyrate tocalculate root mean square deviation (RMSD) and radius ofgyration (Rg) respectively Secondly we calculate the totalenergy for all the systems by the command of g energyThirdly we further calculate root mean squared fluctua-tion (RMSF) for each protein residue by commands ofg rmsf Fourthly the distance between c-Jun and TFIIB andmovement analysis are calculated by the g dist programFifthly the migration of dock protein (c-Jun and TFIIB) is


Chain A of c-Jun

TFIIB

Chain B of c-Jun

181

180

179

178

177

176

175

174

1730 1 2 3 4 5 6 7 8 910 11

Docking pose

Zdoc

k sc

ore

Figure 9The best docking pose of c-Jun and TFIIB with 1804 Zdock ScoreThe structures of c-Jun and TFIIB are colored in red and orangerespectively

0 50 100 150 200 250 30000

02

04

06

08

10

12TFIIB

Diso

rder

disp

ositi

on

Residue index

c-Jun

Figure 10 Disorder prediction of c-Jun and TFIIB complex thevalue of disorder disposition below 05 indicates order foldingregionThe sequence of c-Jun is in the region from residue index 0 to54 and the sequence of TFIIB is in the region from residue index 55to 258The folded structure of c-Jun reveals disorder theN-terminaland C-terminal of TFIIB structure display folded disorder

presented by mean square displacement (MSD) under thecommand of g msd module in GROMACS during all thesimulation times Sixthly the g cluster program is selected

for further calculation of the representative structure fromall MD frames and the representative structure is taken forfurther snapshot analysis We also employed DSSP analysisandmatrices of the smallest distances between each residue toinvestigate the stability of the protein structureThe principlecomponent analysis (PCA) is then applied to observe theprotein motion changes during all the MD frames Finally inorder to observe the compactness between c-Jun and TFIIBCaver 30 software [26] was used to predicted space in thecomplex

3 Results

31 Distribution of Porcine OPN CpG Island There are denseCpG sites existing in the front of the OPN promoter regionOne putative CpG island was found (CpG island size gt 100GC Percent gt 500 ObsExp gt 06) by MethPrimer program

32 Methylation and Expression Analysis of OPN in ClonedPig Various Tissues Firstly the OPN mRNA expressionwas investigated in the WT pig tissues and cloned pigtissues Data showed the various expression levels in differenttissues Particularly Copy1 brain overexpressed the OPNand Copy2 ear with no expression of OPN relative to itswild-type tissue respectively (Figure 1) The unique aberrant


0 1000 2000 3000 4000 500000

02

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06

08Pr

otei

n RM

SD (n

m)

Time (ps)

(a)

0 1000 2000 3000 4000 500019

20

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Radi

us o

f gyr

atio

n (n

m)

Time (ps)

(b)

0 1000 2000 3000 4000 5000

Tota

l ene

rgy

(kJm

ol)

Time (ps)

minus910

minus912

minus914

minus916

minus918

minus920

times105

(c)

Figure 11 The trajectory analysis of c-Jun and TFIIB during 5000 ps simulation times (a) RMSD values of all atoms of c-Jun and TFIIBcomplex (b) radius of gyration of c-Jun and TFIIB complex for identifying the compactness of protein structure (c) total energy of allsimulated systems of c-Jun and TFIIB complex the total energy is sum of potential energy and kinetic energy

expression patterns exhibited the different control way ofthe OPN expression We proposed that OPN expressionmay be a tissue-specific manner MS-PCR primers weredesigned to estimate themethylation status ofOPN promoterHypomethylation generally appeared in the various tissuesof cloned pigs However there were still some tissues thatshowed the methylated region in OPN promoter (Figure 2)

33 5-aza-dc IncreasesOPNmRNAandDecreasesMethylationof OPN Promoter in Pig Ear Fibroblast Cell In order torealize whether the methylated OPN promoter affects the

activity of OPN promoter The 5-aza-dc treated porcine earfibroblast cells showed that when the concentration of 5-aza-dc level increased it will decrease the methylation of OPNpromoter and restore the OPN RNA expression at 05 to20 120583M (Figure 3) The results suggested that the activity ofOPN promoter can be affected by DNA methylation directlyor indirectly COBRA assay was also used to investigate themethylation status of OPN promoter in WT tissues Brainear liver and lung tissues exhibited little part methylation ofthe OPN promoter (data not show) This is thought that themethylation of OPN promoter in the aforementioned tissuesmay be involved withOPN transcript regulation mechanism


250 255 260 265 270 275 28000

01

02

03

04

05

c-Jun chain A

RMSF

(nm

)

Residue

(a)

250 255 260 265 270 275 28000

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RMSF

(nm

)

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(b)

100 120 140 160 180 200 220 240 260 280 300 32000

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TFIIB

RMSF

(nm

)

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(c)

Figure 12 RMSF analysis of protein resides on (a) chain A of c-Jun (b) chain B of c-Jun and (c) TFIIB during simulation time of 5000 psThe residue index of chain A and chain B of c-Jun is from 254 to 280 and the residue index of TFIIB is from 113 to 316 The high values ofRMSF indicated the high fluctuation of residue during all simulation times

34 Bisulfite Sequencing Analysis of the Whole CpG SitesMethylation Profile in Cloned Pigs To investigate whichregion of the OPN promoter is affected by methylation inthe CpG site bisulfite sequencing was performed to dissectthe methylation status of CpG sites in the OPN promoterIn the brain tissue bisulfite sequencing of Copy5 and Copy1brain exhibited the saturated status in theirmethylated regionwhile WT brain exhibited fragmentary methylated CpGsites The 1806 methylation percentage of Copy5 brainwas more than the WT brain 1425 (Figure 4) In the

heart tissues bisulfite sequencing showed that the Copy2heart had extremely hypermethylated percentage with 1333more than WT heart 294 (Figure 4) Particularly the datashowed the inhibition of Copy2 heart mRNA (Figure 1) Inthe liver the different methylation pattern also appeared inthe Copy4 liver relative to WT liver Copy2 ear with 1805methylation in the OPN analyzed region was higher than theWT ear 1167 the result was proved by the experiment ofOPNmRNAexpression (Figure 1)These bisulfite sequencingresults matched our previous hypothesis that methylation in


0 1000 2000 3000 4000 5000

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ue

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(a)

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Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)

Figure 13 The secondary structure analysis for (a) c-Jun and (b) TFIIB over all simulation times Each secondary type of the structure suchas Bend Turn alpha helix (A-Helix) and 3

10-helix (3-helix) is colored in green yellow blue and gray respectively The ldquoChain Seoaratorrdquo in

(a) is used to differentiate between chain A and chain B of c-Jun

the OPN promoter region regulates the activity of OPN pro-moter Moreover the hypermethylated OPN promoter maydirectly affect the activity of OPN transcription especially inheart and ear tissues

35 Analysis of Methylation Implication of OPN Transcriptionby Promoter Assay The analyzed OPN promoter regionmay involved with methylated control of gene transcrip-tion Promoter assay was designed to explain the directinhibition of OPN promoter activity by methylation on theOPN CpG sites Four different truncated forms of OPN fulllength (26 kb) were used to prove the hypothesis (Figure 5)Particularly the truncated form 22 kb deleted the 377 bppromoter region (minus2615simminus2239 nt) This region is the ana-lyzed region for bisulfite sequencing profile And this regionis thought to be the most possible element that regulatesthe OPN transcription Moreover four CpG sites were invitro added to the methyl group by methyltransferase thatcan provide important evidence how methylation affect theOPN transcription Figure 5(b) shows that theOPN promoteractivity was significantly decreased in methylated minus2615-lucplasmid However minus2239-luc that deleted the 377 bp con-taining methylated characteristic DNA element leads to lessinhibition of promoter activity than the methylated vectorminus2615M-luc (Figures 5(a) and 5(b)) The results indicatedthat methylation in the front OPN promoter is not onlydecreasing the promoter activity to the basal level but alsorecruiting the inhibition factors to enhance the inhibitionability In order to avoid the effects of CpG sites in PGL3-enhancer backbone minus495M-luc that have no methylatedCpG sites in the OPN promoter but it can be methylatedin the vector backbone CpG sites compared to minus495-lucData showed that there is no difference in the promoteractivity between methylation or unmethylation in the PGL3-enhancer backbone CpG sites (Figure 5(b)) It is suggestedthat methylation in the critical region such asOPN promoter

front end may lead to the rearrangement of chromatinstructure Otherwise deletion of the OPN promoter to the495 bp with significant promotion of the promoter activityindicated that in the middle part of promoter DNA elementmay able to inhibit the activity of OPN promoter

36 Methylation in CpG 13th and CpG 1st of OPN Pro-moter Blocks the Binding Access of Transcription Factors Weinvestigate that the methylated CpG sites in the minus2615simminus2239 nt of theOPN promoter region affect the transcriptionfactor binding activity Electrophoresis mobile shift assay wasperformed with nuclear extracts from human HEK293T andSH-SY5Y cell line Four EMSA probes that contain the CpG 1CpG 3-4 CpG 6ndash8 CpG 11ndash15 and CpG 19-20 were designedaccording to the CpG sites in our analyzed region (Figure 6)EMSA data suggested that CpG 13th and 1st sites showedmethylation noncompetition phenomenon which had influ-ence on binding with transcription factor (Figure 7) 19-20thCpG sites containing EMSA probe showed no competitionability in the methylated or unmethylated status The premixwith antibody and nuclear extracts by EMSA assay indicatedthat the c-Jun and c-Fos were involved in the binding to CpGsites 13 and 20 (Figure 8) However the adding of antibodyin the mixture of probe and nuclear extracts showed nosignificant shift bands It is indicated that other transcriptionfactors may also participate in the transcription activity OPNpromoter Thus the c-Jun and c-Fos could be involved inthe partialOPN transcriptional activity in a competition wayIn addition the c-Jun had higher binding affinity than c-Fos in the EMSA probe analysis We therefore did followedcomputational survey to see why c-Juc affects the consequentOPN transcription

37 The Computational Biology Results After surveying thepossible zone forOPN promoter hypermethylation we foundthat the AP1 (c-Jun) sequence frequently appeared in our


5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)

Figure 14 Matrices of smallest distance between each residue on(a) chain A of c-Jun and (b) chain B of c-Jun The value of distancebetween residues is represented by rainbow bar and the value ofdistance with longer than 15 nm is colored in red The indexes ofresidues from0 to 27 indicate residues from 254 to 280 on each chainof c-Jun

molecular laboratory study Then we further validated thatthe c-Jun methylation will cease the further mRNA pro-duction by inhibiting the binding of RNA-polymerase IIinitiation factor TFIIB We do the further computationalmodelling for mechanism survey between the c-Jun andTFIIB By the Z-DOCK analysis we found the c-Jun andTFIIB could combine tightly (Figure 9)We chose the highestdocking pose (dock score = 1804) for further MD analysis

Then the disorder predication was employed to observethe protein folding analysis and the result is shown inFigure 10 We found that the c-Jun has relative high disorderin folding than TFIIB we suppose that the flexibility of c-Jun

structure could easier bound to TFIIB This finding could bean explanation why the c-Jun bound to TFIIB by Z-DOCKprogram To confirm the stability of the c-Jun and TFIIBcomplex the series of molecular dynamic studies furthervisualize their interactions

Protein complex RMSD analysis proved that the c-Junand TFIIB were stable from 3000 ps to 5000 ps In additionwe found the TFIIB are easier to be stable during the molec-ular dynamics (Figure 11(a)) We also found the radius ofgyration tend to be stable for all simulation timeswith averageof 205 nm (Figure 11(b)) suggesting that the two proteinstructures are compact after binding together Figure 11(c)also shows the binding complex in a stable fluctuation andthe energy of the binding complex is stable around minus915 times105 (kJmol)

38 Stability Analysis of Residues on the Major Binding Regionduring MD Simulation To analyze the flexibility of residueson protein structure the RMSF calculation was used toobserve the flexibility of each residue Figure 12(a) showsthat the chain A of c-Jun had high frequency of fluctuation(binding site 200ndash210 binding resides) However the chainB of the c-Jun has relative fewer frequency of fluctuation andthemajor binding region (from 228 to 240 residues) showed aless fluctuation as shown in Figure 12(b) Figure 12(c) revealsthe binding regions (200ndash210 binding residues) for chain Aof c-Jun that is more fluctuated and unstable compared to thebinding regions (228ndash240) for chain B of c-Jun binding site inthe TFIIB binding region Figure 13 is the result for secondarystructure variation calculated by DSSP analysis Most of themain scaffold belong to alpha Helix there are no significantchanges during the whole MD simulation All helices of thesecondary structure for c-Jun and TFIIB binding remainedstable during a 5000 ps simulation time (Figure 13) Wethereafter surveyed the distance between each residue of c-Jun for 5000 ps The variation of distances between residuesin c-Jun chain is wider than the distances of residues in c-Jun chain BTherefore the chain B of c-Jun is more stable forTFIIB binding (Figure 14)

The hydrophobic area was then calculated by SASAin Figure 15(a) the value of hydrophobic area decreasedduring the last 1000 ps This indicated the compactness ofthe c-Jun and TFIIB binding increased by the MD timeperiod in our study It is worthy to know that the distancebetween centrals ofmasses of c-Jun andTFIIBwas decreasingmore and more after time goes by in the 5000 ps survey(Figure 15(b)) In the migration analysis of c-Jun and TFIIBthe MSD was employed to count the migration of c-Jun andTFIIB The c-Jun is more unstable than TFIIB during thebinding interaction throughout the whole MD simulationin 5000 ps period (Figure 15(c)) Besides we further utilizedthe principal component analysis (PCA) to measure all MDframes over all simulation times The first two eigenvectors(PC1 and PC2) were shown in Figure 18 most of framesare ranged in the short range of eigenvalues minus10 and 10 inPC1 (Figure 16(a)) and arranged in eigenvalues from minus5 to5 in PC2 (Figure 16(b)) The phase space comparing for PC1and PC2 was shown in Figure 17 we found that each frame


0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)

Figure 15 The area of solvent and protein migration analyses during simulation time of 5000 ps (a) The total solvent accessible surface areaof c-Jun and TFIIB complex (b) the distance between the centrals of masses of of c-Jun and TFIIB (c) trajectory analysis of MSD of c-Junand TFIIB The high values of MSD indicated the longer distance of migration from the initial binding position

could be grouped into two clusters This suggests that themotion of each frame was not changed significantly over allsimulation times In order to select the most representativestructure for snapshot investigation we did cluster analysis(Figure 18) We found that the last group (cluster 14) isthe predominant cluster and is also displayed in the timerange from 4000 to 5000 ps the cluster 14 also appears mostpredominant in frame numbers (Figure 18) and the middlestructure (4260 ps) of cluster 14 is regarded as representativeframe For snapshot analysis the comparison of initial andrepresentative frames is shown in Figure 19 we found that

the chain A of c-Jun is more encompassed by TFIIB at4260 ps through the inward rotation of TFIIB This madethe bindings between chain A of c-Jun and TFIIB morecompacted through 0 ps to 4260 ps along with time Theelevated activation between TFIIB and chain A of c-Jun isalso confirmed in RMSF analysisTherefore we supposed theinitiated transcription factor on RNA polymerase II (TFIIB)is closed interaction to the chain A of c-Jun (AP1) from 0 psto 4260 ps This hypothesis was also confirmed by Figure 20There were more spaces between chain B and TFIIB thanchainAHence we could see that the TFIIB actsmore close to


0 5 10 150

5

10

15

20

25

Num

ber

Eigenvaluesminus20 minus15 minus10 minus5

(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)

Figure 16 The number of MD frames of the first two eigenvectors(PC1 and PC2) by PCA analysis during simulation time of 5000 psThe higher range of eigenvalue denotes the wider motion of proteinstructure over all simulation times

the chain A of c-Jun (Figure 20) Overall we presume that theinitiation ofOPN transcription started fromTFIIB binding tochain A of c-Jun

4 Discussion

Previous study has shown that DNA element (GGGT-CATATGGTTCA) located in osteopontin promoter minus2245to minus2259 nt can be regulated by vitamin D3 [27] This DNAregulation region can easily be affected by the change ofcalcium concentration The promoter region of porcine OPNwas analyzed in transcription factor binding sites exceptthe region minus2615 to minus2239 Interestingly this region ofporcine OPN promoter is rich in CpG sites compared tohuman mouse and bovine genome Sakata also proved that

0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10

Figure 17 Phase space analysis of comparing the first two eigen-vectors (PC1 and PC2) for principle component analysis Theeigenvalues of the two eigenvectors are projected into one phasespace small motion of protein structure could be grouped intoclusters

OPN promoter transcription activity is regulated by somespecific DNA modification mechanism of rearrangement ofchromatin structure [22]

In the present study four cloned pigs were surroundedby many defects For example Copy1 pig had a retardation oflimb bone growth Copy2 heart organ showed a pericarditisand copy3 heart had valvular heart disease This physiologydefects appeared aberrant development especially in boneor heart may involved in the initially fetus stage with inap-propriate organ differentiate Thus our data suggested thatthe consequent result in aberrant OPN expression or incom-pletely epigenetic modification in OPN promoter (Figure 5)These aberrant molecular data of OPN are correlated withthe defects of bone and heart in cloned pigs SemiquantitativePCR of OPN mRNA showed that discrepant expression pat-tern was identified in several cloned pig tissues especially inbrain (9975 up-regulation) heart (115 downregulation)and ear (1803 downregulation) (Figure 2) OPN mRNAhas different expression in brain development in differentembryonic stages [28] The overexpression of OPN in braintissue may cause some unexpected brain damage or neurondevelopmentOPN can inducemyocardial fibrosis and repairtissue after inflammation Lacking OPN will cause faultywound healing after myocardial infarction [29 30] Silentexpression ofOPN in cloned pigrsquo heart tissue may also be themain cause of heart disease

Recent studies indicated that OPN gene expression maybe affected by treatment of TSA (a histone deacetylaseinhibitor) The results applied that OPN promoter could beregulated by epigenetic mechanism [22] In this study weinvestigated OPN methylation profile after 5-aza-dc treat-ment The results indicated that mRNA expression of OPN is


0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster

Figure 18 Cluster analysis of all MD frames of c-Jun and TFIIB complex during simulation time of 5 ns for identifying representativestructure All MD frames were grouped into fourteen clusters by linkage method the RMSD cut-off distance between each neighbor frameis 014 nm The most predominant group is the cluster 14 which is displaced in the region of simulation time from 4000 to 5000 ps and theMD frames in cluster 14 are the most number among all clusters The middle frame of cluster 14 is displaced in simulation time of 4260 ps

Close form

Open form

Chain A of c-Jun Chain B of c-Jun

TFIIB

Time = 0ps

Time = 4260ps

Figure 19 The structural comparison between the first frame(0 ps) and representative structure (4260 ps) The structures of c-Jun and TFIIB are represented by blue ribbon and red solid phaserespectively The chain A of c-Jun was surrounded more compactlyby TFIIB at 4260 ps by the structural inward rotation to make morecompactness between TFIIB and c-Jun through 0 ps to 4260 ps

Chain A of c-JunChain B of c-Jun

Channel 2

Channel 1TFIIB

Figure 20 The possible space prediction between c-Jun and TFIIBamong all simulation times The predicted channels are colored inred and green The structures of c-Jun and TFIIB are colored inblue and orange respectively Each possible space is represented bychannels each channel was generated by Caver 30 program

directly affected by addingmethyltransferase inhibitor 5-aza-dc (Figure 4) COBRA was performed to study the methy-lation of OPN front end promoter in wild-type and clonedpig different tissues Sodium bisulfite sequencing analysisalso revealed that the methylation of CpG sites concentratedin front of the 20 CpG sites in front of OPN promoter(Figure 5) Discrepancy methylation in this promoter region


also happened in brain heart ear and liver tissues betweenwild-type and cloned pigs (Figure 5) It revealed that DNAmethylation of OPN promoter may be involved with regula-tion of expression of OPN mRNA In order to characterisewhich promoter DNA element is important four constructsof OPN promoter (minus2615-luc minus2239-luc minus1505-luc andminus495-luc) were used for analysis Obvious downregulationin methylated minus2615-luc (HpaII andHhaI methyltransferase)was observed Compared to the deletion of this controlregion (sim390 nt) in front of OPN promoter the decreasinglevel of promoter activity is not as obvious as minus2615M-luc construct (Figure 7) It means that methylation in thefront of OPN promoter caused some silent mechanism thatmake chromosome structure more compact or block somepromotion transcription factors The EMSA data indicatedthat 13th CpG site of our analyzed region could bind toAP1 transcription factor and binding activity is affected bymethylation in this CpG site (Figure 7) Taken together allthese findings correlated with DNA methylation in tissue-or cell-specific gene expression OPN promoter region wasdensely methylated in some low expression (Figures 2 4 and5)

Our data revealed that DNA methylation of CpG sites inOPN promoter was the main mechanism through specifictranscription factor that makes the tissue-specific expres-sion In previous study AP1-like binding site (TGAGCGA)was identified as a methylated insulator region in humanblastoma cell line [31] Analyzed region in front of porcineOPN promoter showed that CpG 1st binding site containedthe specific binding site sequence While in our interestingDNA regulation region range from minus2615 to minus2239 nt ofOPNpromoter also exhibited little block access in the competitionof probe It is suggested that CpG 1st and CpG 13th playan important role in methylation controlled mechanism toregulate gene expression We finally utilize Z-dock program[32] to analyze the interaction between AP1 (PDB code1JNM) and RNA polymerase II initial transcription factor(TFIIB) (PDB code 1VOL) [33] to see if they had stablebinding From the docking result of Z-dock (Figure 8) weproved theAP1 is significantly bound to TFIIBWe also foundtheAP1 can autoregulate theHDAC-1 in promoter region andlead to significant higher degrees of hypermethylation in theOPN promoter region and cause AP1 to be hypermethylatedconsequently ceasing the OPN mRNA expression [34]

Further mechanical studies by the computational biologyalso pointed out that theDNAsequence for hypermethylationof OPN promoter binding sites is c-Jun The chain A of c-Jun could be encompassed more tightly by inward rotationalstructure change of TFIIB during theMDprocess (Figure 19)Therefore we found c-Jun had crucial role for interaction ofinitiating transcription by RNA polymerase II The methy-lation of c-Jun leads to of hyper-condense helix structuralchange and makes transcription termination which stopsOPN mRNA production Therefore the MD docking resultsreconfirm the c-Jun partake the crucial roles in consequentOPN transcription that matches our wet laboratory studiesWe suppose this will cause the problems in the embryonic

development and lead to threatened conditions Thereforeadjusting OPN promoter c-Jun (AP1) methylation will affecttranscription binding and could be the treatment for geneticdeveloping errors in the future

In conclusion aberrantmethylation of porcineOPN genewas frequently found in different tissues of somatic nucleartransferred cloning pigs and bisulfite sequence data suggestedthat the OPN promoter region of minus2615 to minus2239 nt maybe a crucial regulation DNA element In pig ear fibroblastcell culture study the demethylation of OPN promoter wasfound in dose-dependent response of 5-aza-dc treatment andfollowed the OPN mRNA reexpression In cloned pig studydiscrepant expression pattern was identified in several clonedpig tissues especially in brain (9975 up-regulation) heart(115 down-regulation) and ear (1803 downregulation)Promoter assay data revealed that four methylated CpG sitespresenting in the minus2615 to minus2239 nt region cause signifi-cant downregulation (approximately 75) of OPN promoteractivity (119875 lt 0001) EMSA data also suggested that CpG13th and 1st sites showed methylation noncompetition phe-nomenon which had influence on binding with transcriptionfactor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Chih-Jie Shen Yung-An Tsou and Hsiao-Ling Chen con-tributed equally to this work

Acknowledgments

This research was supported by Grants NSC-95-2313-B-005-030-MY3 NSC-101-2313-B-005-014-MY3 101-2314-B-039-013-MY3 NSC102-2325-B039-001 and NSC102-2221-E-468-027- from the National Science Council and was partlysupported by the Ministry of Education Taiwan under theaiming top university plan (ATU-101-s0508) The authorswould like to thank their colleagues (Drs Tung-Chou TsaiCheng-Wei Lai and Zi-Lun Lai) in the Molecular Embry-ology amp DNA Methylation Laboratory for their help withdiscussions and technical issues This study is also supportedin part by Taiwan Department of Health Clinical Trialand Research Center of Excellence (DOH102-TD-B-111-004)Taiwan Department of Health Cancer Research Center ofExcellence (MOHW103-TD-B-111-03) and CMU under theAim for Top University Plan of the Ministry of EducationTaiwan

References

[1] Y M Leung Y M Wong S W Chen et al ldquoDown-regulationof voltage-gated Ca2+ channels in Ca2+ store-depleted ratinsulinoma RINm5F cellsrdquo BioMedicine vol 3 no 3 pp 130ndash139 2013


[2] M-C Lin S-Y Tsai F-Y Wang et al ldquoLeptin induces cellinvasion and the upregulation of matrilysin in human coloncancer cellsrdquo BioMedicine vol 3 no 4 pp 174ndash180 2013

[3] Y-M Chang B K Velmurugan W W Kuo and et alldquoInhibitory effect of alpinate Oxyphyllae fructus extracts onAng IIinduced cardiac pathological remodeling-related path-ways in H9c2 cardiomyoblast cellsrdquo BioMedicine vol 3 no 4pp 148ndash152 2013

[4] T H Bestor ldquoThe DNA methyltransferases of mammalsrdquoHuman Molecular Genetics vol 9 no 16 pp 2395ndash2402 2000

[5] P H Tate and A P Bird ldquoEffects of DNA methylation onDNA-binding proteins and gene expressionrdquo Current Opinionin Genetics and Development vol 3 no 2 pp 226ndash231 1993

[6] W H Eyestone and K H Campbell ldquoNuclear transfer fromsomatic cells applications in farm animal speciesrdquo Journal ofReproduction and Fertility vol 54 pp 489ndash497 1999

[7] T Wakayama A C F Perry M Zuccotti K R Johnson and RYanagimachi ldquoFull-term development of mice from enucleatedoocytes injected with cumulus cell nucleirdquo Nature vol 394 no6691 pp 369ndash374 1998

[8] TWakayama andR Yanagimachi ldquoMouse cloningwith nucleusdonor cells of different age and typerdquo Molecular Reproductionand Development vol 58 no 4 pp 376ndash383 2001

[9] J Ohgane T Wakayama Y Kogo et al ldquoDNA methylationvariation in clonedmicerdquoGenesis vol 30 no 2 pp 45ndash50 2001

[10] G A Johnson R C Burghardt F W Bazer and T E SpencerldquoOsteopontin roles in implantation and placentationrdquo Biologyof Reproduction vol 69 no 5 pp 1458ndash1471 2003

[11] G FWeber S Zawaideh S Hikita V A Kumar H Cantor andS Ashkar ldquoPhosphorylation-dependent interaction of osteo-pontin with its receptors regulates macrophage migration andactivationrdquo Journal of Leukocyte Biology vol 72 no 4 pp 752ndash761 2002

[12] M Mazzali T Kipari V Ophascharoensuk J A WessonR Johnson and J Hughes ldquoOsteopontinmdasha molecule for allseasonsrdquo QJM vol 95 no 1 pp 3ndash13 2002

[13] N S Fedarko A Jain A Karadag M R van Eman and LW Fisher ldquoElevated serum bone sialoprotein and osteopontinin colon breast prostate and lung cancerrdquo Clinical CancerResearch vol 7 no 12 pp 4060ndash4066 2001

[14] G Chakraborty S Jain and G C Kundu ldquoOsteopontinpromotes vascular endothelial growth factor-dependent breasttumor growth and angiogenesis via autocrine and paracrinemechanismsrdquo Cancer Research vol 68 no 1 pp 152ndash161 2008

[15] R Agnihotri H C Crawford H Haro L M MatrisianM C Havrda and L Liaw ldquoOsteopontin a novel substratefor matrix metalloproteinase-3 (stromelysin-1) and matrixmetalloproteinase-7 (matrilysin)rdquo The Journal of BiologicalChemistry vol 276 no 30 pp 28261ndash28267 2001

[16] D Wang S Yamamoto N Hijiya E N Benveniste and C LGladson ldquoTranscriptional regulation of the human osteopontinpromoter Functional analysis and DNA-protein interactionsrdquoOncogene vol 19 no 50 pp 5801ndash5809 2000

[17] H Rangaswami A Bulbule and G C Kundu ldquoOsteopontinrole in cell signaling and cancer progressionrdquo Trends in CellBiology vol 16 no 2 pp 79ndash87 2006

[18] H Rangaswami A Bulbule and G C Kundu ldquoNuclear factor-inducing kinase plays a crucial role in osteopontin-inducedMAPKI120581B120572 kinase-dependent nuclear factor 120581B-mediatedpromatrix metalloproteinase-9 activationrdquo Journal of BiologicalChemistry vol 279 no 37 pp 38921ndash38935 2004

[19] A B Tuck D M Arsenault F P OrsquoMalley et al ldquoOsteopon-tin induces increased invasiveness and plasminogen activatorexpression of human mammary epithelial cellsrdquo Oncogene vol18 no 29 pp 4237ndash4246 1999

[20] H Okamoto ldquoOsteopontin and cardiovascular systemrdquoMolec-ular and Cellular Biochemistry vol 300 no 1-2 pp 1ndash7 2007

[21] F Schoensiegel R Bekeredjian A Schrewe et al ldquoAtrial natri-uretic peptide and osteopontin are useful markers of cardiacdisorders inmicerdquoComparativeMedicine vol 57 no 6 pp 546ndash553 2007

[22] R Sakata S Minami Y Sowa M Yoshida and T Tamaki ldquoTri-chostatin A activates the osteopontin gene promoter throughAP1 siterdquo Biochemical and Biophysical Research Communica-tions vol 315 no 4 pp 959ndash963 2004

[23] K H Choi H Basma J Singh and P W Cheng ldquoActiva-tion of CMV promoter-controlled glycosyltransferase and 120573-galactosidase glycogenes by butyrate tricostatin A and 5-aza-21015840-deoxycytidinerdquo Glycoconjugate Journal vol 22 no 1-2 pp63ndash69 2005

[24] B M Kumar H-F Jin J-G Kim et al ldquoDNA methylationlevels in porcine fetal fibroblasts induced by an inhibitor ofmethylation 5-azacytidinerdquo Cell and Tissue Research vol 325no 3 pp 445ndash454 2006

[25] S Pronk S Pall R Schulz et al ldquoGROMACS 45 a high-throughput and highly parallel open source molecular simula-tion toolkitrdquo Bioinformatics vol 29 no 7 pp 845ndash854 2013

[26] E Chovancova A Pavelka P Benes et al ldquoCAVER 30 atool for the analysis of transport pathways in dynamic proteinstructuresrdquo PLoS Computational Biology vol 8 no 10 ArticleID e1002708 2012

[27] Q Zhang J L Wrana and J Sodek ldquoCharacterization of thepromoter region of the porcine opn (osteopontin secretedphosphoprotein 1) gene Identification of positive and negativeregulatory elements and a ldquosilentrdquo second promoterrdquo EuropeanJournal of Biochemistry vol 207 no 2 pp 649ndash659 1992

[28] M Y Lee J S Choi S W Lim J H Cha M H Chun and JW Chung ldquoExpression of osteopontinmRNA in developing ratbrainstem and cerebellumrdquo Cell and Tissue Research vol 306no 2 pp 179ndash185 2001

[29] L Liaw D E Birk C B Ballas J S Whitsitt J M Davidsonand B L Hogan ldquoAltered wound healing in mice lacking afunctional osteopontin gene (spp1)rdquo The Journal of ClinicalInvestigation vol 101 no 7 pp 1468ndash1478 1998

[30] N A Trueblood Z Xie C Communal et al ldquoExaggeratedleft ventricular dilation and reduced collagen deposition aftermyocardial infarction in mice lacking osteopontinrdquo CirculationResearch vol 88 no 10 pp 1080ndash1087 2001

[31] M Fujimoto R Kitazawa SMaeda and S Kitazawa ldquoMethyla-tion adjacent to negatively regulating AP-1 site reactivates TrkAgene expression during cancer progressionrdquo Oncogene vol 24no 32 pp 5108ndash5118 2005

[32] Accelerys Discovery Studio Client v25 Accelrys San DiegoCalif USA 2009

[33] D B Nikolov H Chen E D Halay et al ldquoCrystal structure ofa TFIIB-TBP-TATA-element ternary complexrdquo Nature vol 377no 6545 pp 119ndash128 1995

[34] P Sharma S Kumar and G C Kundu ldquoTranscriptional regu-lation of human osteopontin promoter by histone deacetylaseinhibitor trichostatin A in cervical cancer cellsrdquo MolecularCancer vol 9 article 178 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


process [8] This dilemma of SCNT animal may be caused bymethylation controlled genes such as imprinting genes [9]

OPN is an extracellular matrix protein and hydrophilicglycoprotein identified firstly in the bone as a sialoprotein Itcontains a thrombin and transglutaminase cutting site andthe molecular weight is about 25 kDa to 75 kDa in pig themolecular is about 67 kDa it contains numerous isoforms[10] OPN has a hydrophobic N terminal thus it can besecreted out of cell membrane the amino sequence ofOPN isfull of Asp Thr and Ser that can elevate the binding activitywith calcium glycosylation and phosphorylation respec-tively [11] Thus OPN plays numerous roles in many aspectssuch as bone remodeling cell migration iNOS regulationrepairment and leucocyte recruitment [12] And acquiredOPN expression has been found in a variety of cancer celltypes especially in the liver lung breast prostate colonbrain and spleen [13 14] OPN is cleaved by MMPs proteinto generate functional OPN that can bind to 120572v1205733 [15] Thisintegrin binding with OPN has influence on NF120581B signalingtransduction [16 17] Therefore overexpressed OPN is asso-ciated with tumorigenesis tumor invasion and metastasis[18 19] Previous study suggested that overexpressed OPNinduces the serious cardiac fibrosis [20 21] Thus our clonedpigs were also surrounded by various defects in heart fibrosisand retardation of growth of bones Therefore this studyfocuses on the methylation change of OPN promoter thatmay be disrupted by inappropriate reprogramming processConsequently aberrant methylation of promoter could leadto aberrant expression of OPN In the previous studies OPNexpression was induced with TSA (trichostatin A) in mouseundifferentiated mesenchymal cell line by AP1 site [22]The TSA is a histone deacetylase inhibitor It can lose thechromatin structure in order to let gene restore its expression5-aza-dc is also an analog with the same structure of cytosinewithout methyl group adding in the 51015840C end [23]

Thus 5-aza-dc addition leads to low methylation per-centage in the CpG sites rich region The hypomethylationstatus in the promoter may contribute its gene transcriptionactivity Porcine fetal fibroblasts in 5th passage cultures weretreated with 05 10 20 and 30 120583M 5-aza-dc for 96 h 5-aza-dc inhibited the growth of cell at all concentrations 5-aza-dc induced a reduction of transcripts level in DNMT1 andincreasing expression in imprinted gene IGF2 [24] Further-more human OPN promoter sequence is similar to porcinein the front 400 nt of the porcine promoter Therefore weinvestigatedOPN RNA and promotermethylation changes inthe porcine ear fibroblast cell Data showed that the elevatedOPN expression and in 5-aza-dc treated fibroblast cell is dueto the decreased methylation of OPN promoter Cloned pigssamples had found extremelymethylation changes especiallyin the brain (9975 upregulation) heart (1150 down-regulation) and ear (1803 down-regulation) Deletionanalysis of the promoter region revealed 5-aza-dc inducedluciferase response that was regulated by minus2615 to minus2239 ofthe OPN promoter These data suggested that methylation inthe OPN promoter plays a crucial role in the regulation ofOPN expression Methylation of OPN promoter may be anepigenetic marker of diagnosis of cancer

2 Materials and Methods

21 CpG Island Prediction The sequence of a putative CpGisland in OPN promoter was analysed by using MethPrimersoftware (httpwwwurogeneorgmethprimerindex1html)

22 Cell Culture The porcine fibroblast cell line was grownin Dulbeccorsquos modified Eaglersquos medium (DMEM Gibco-BRL Gaithersburg MD USA) supplemented with 10 fetalbovine serum (FBS Gibco BRL) and containing 100UmLpenicillin and streptomycinThe cells were incubated at 37∘Cin humidified incubator with 5 CO

2

23 5-aza-dc Demethylation Drug Treatment For 5-aza-dctreatment porcine fibroblast cell in 5th passage cultures wastreated with 5-aza-dc (sigma) at various concentrations thatis 0 (control) 05 15 and 20120583M for 72 h Medium waschanged every 24 h and then cells were collected for RNA andDNA extraction and stored at minus80∘C [24]

24 Quantitative Real Time-PCR 2 120583g RNA of ear fibroblastcell was used to be transformed to cDNA 05 120583L of cDNAwas performed for quantitative real time-PCR with Rotor-Gene 6000 (Corbett) 120573-actin was the internal control fornormalize target gene OPN The calculated gene expressionfold from CT value was according to the previous study119875 value less than 05 exhibited the obviously significantdifference

25 Methylation Analysis by Combined Bisulfite Restric-tion Analysis (COBRA) For amplification of porcine OPNpromoter methylation analysis site PCR was performedusing 2120583L of bisulfite-converted genomic DNA as tem-plate The primer sets of COBRA were OPN-C sense 51015840-TTTTTTGAGGGAGATTAGTTTTTG-31015840 and antisense 51015840-ATTCTACTAAAATCCAACCACCC-31015840 The COBRA-PCRproducts were purified by phenolchloroform followed byethanol precipitationThe DNAwas resuspended in 85 120583L ofdistilled deionized water Purified PCR products were thendigested with 10U BstUI restriction enzyme (New EnglandBiolabs MA USA) at 65∘C Products were electrophoresedon 6 native acrylamide gel stained with 200 gmL ethidiumbromide and visualized using a Kodak 1D software

26 Methylation Specific-PCR Genomic DNA (05 120583g) wastreated with sodium bisulfite according to the manufacturersquosrecommendations (EZDNAMethylationKit Zymo researchCA USA) and amplified with specific primers for methylatedor unmethylated DNA The primer sets of MS-PCRwereOPN-M sense 51015840-AAGCGGGGAAGGAGTTATTACGT-31015840 antisense 51015840-TCCGACAAAACGAAACGATCATACA-31015840OPN-U sense 51015840-GAAGTGGGGAAGGAGTTTATTATGT-31015840 and antisense 51015840-CAATAACTCCAACAAAACAAAACAATC-31015840 All PCR reactions were performed on PTC 200thermocyclers (MJ Research MA USA) and in 25 120583Lvolume using the PlatiumTaq DNA polymerase system(Invitrogen CA USA) PCR products were separated on15 agarose gels The M-set primers contained at least three







WT pig Cloned pig


OPN

120573-Actin

(a)

Liver

Heart

Ear

Muscle

Brain

Cloned pigWT pig

0

10

20

30

40

90

100

OPN

mRN

A ex

pres

sion

leve

l


(b)


2O

TSS


minus2615

minus2500

minus2000

minus1500

minus1000

minus500

+1

(a)

M-set

U-set



214 bp

222 bp

(b)


2O


1 SB8765432

Mock Control

287

Methylation ()9084512413350759775296

35

30

25

20

15

10

5

0

0 05 15 20

Met

hyla

tion

()

243bp

144bp

387bp

05 120583M 15 120583M 20 120583M

lowast

5-aza-dc (120583M)

(a)

35

30

25

20

15

10

5

0

0 05 15 20

Met

hyla

tion

()

15 35302520

Cycles

The fl

uro

amou

nt o

f syb

er g

reen

05 120583M15 120583M20 120583M10 120583M

lowast

5-aza-dc (120583M)

10minus05

10minus07

10minus09

10minus11

10minus13

10minus15

(b)





119889

2119903

119889119905

= 119872

minus1119865

(1)





Total 57400 = 143CpG1-10 49200 = 245

Total 57400 = 29CpG1-10 49200 = 47

Total 57400 = 117CpG1-10 49200 = 233

Total 57400 = 85CpG1-10 49200 = 115


Total 57400 = 133CpG1-10 49200 = 222

Total 57400 = 133CpG1-10 49200 = 261

Total 57400 = 181CpG1-10 49200 = 35

Total 57400 = 50CpG1-10 49200 = 94

Total 57400 = 181CpG1-10 49200 = 328

Total 57400 = 53CpG1-10 49200 = 94


Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc

++

minus2615-luc

minus2239-luc

minus1505-luc

minus495-luc


(a)

Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc


minus2239-luc

minus1505-luc

minus495-luc


(b)











1 2 3

4 5

6 7 8 9

10 11 12 13 14

15 16 17

18 19 20

HhaI

HhaI

HpaII

HpaII

AP1-like

AP1-like



1st CpG

100()

75353466526693912

++++++minus minus

(a)

13th CpG

100()

157239589676844561

(b)

20th CpG

100()

352493400820837800

(c)


c-Junc-fos


Binding ()

758

816

51448100

963

994

555

627

100

++ +

+ ++ + +

++

+ +


minusminus

minusminus

minus

minus

minusminusminus

minusminus

minusminus

minus





Chain A of c-Jun

TFIIB

Chain B of c-Jun

181

180

179

178

177

176

175

174

1730 1 2 3 4 5 6 7 8 910 11

Docking pose

Zdoc

k sc

ore


0 50 100 150 200 250 30000

02

04

06

08

10

12TFIIB

Diso

rder

disp

ositi

on

Residue index

c-Jun




3 Results




0 1000 2000 3000 4000 500000

02

04

06

08Pr

otei

n RM

SD (n

m)

Time (ps)

(a)

0 1000 2000 3000 4000 500019

20

21

22

Radi

us o

f gyr

atio

n (n

m)

Time (ps)

(b)

0 1000 2000 3000 4000 5000

Tota

l ene

rgy

(kJm

ol)

Time (ps)

minus910

minus912

minus914

minus916

minus918

minus920

times105

(c)






250 255 260 265 270 275 28000

01

02

03

04

05

c-Jun chain A

RMSF

(nm

)

Residue

(a)

250 255 260 265 270 275 28000

01

02

03

04

05

06

07

c-Jun chain B

RMSF

(nm

)

Residue

(b)

100 120 140 160 180 200 220 240 260 280 300 32000

01

02

03

04

05

06

TFIIB

RMSF

(nm

)

Residue

(c)





0 1000 2000 3000 4000 5000

10

20

30

40

50

Resid

ue

Time (ps)

CoilBendTurn


(a)

0 1000 2000 3000 4000 5000

40

80

120

160

200

Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)










5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







WT pig Cloned pig


OPN

120573-Actin

(a)

Liver

Heart

Ear

Muscle

Brain

Cloned pigWT pig

0

10

20

30

40

90

100

OPN

mRN

A ex

pres

sion

leve

l


(b)


2O

TSS


minus2615

minus2500

minus2000

minus1500

minus1000

minus500

+1

(a)

M-set

U-set



214 bp

222 bp

(b)


2O


1 SB8765432

Mock Control

287

Methylation ()9084512413350759775296

35

30

25

20

15

10

5

0

0 05 15 20

Met

hyla

tion

()

243bp

144bp

387bp

05 120583M 15 120583M 20 120583M

lowast

5-aza-dc (120583M)

(a)

35

30

25

20

15

10

5

0

0 05 15 20

Met

hyla

tion

()

15 35302520

Cycles

The fl

uro

amou

nt o

f syb

er g

reen

05 120583M15 120583M20 120583M10 120583M

lowast

5-aza-dc (120583M)

10minus05

10minus07

10minus09

10minus11

10minus13

10minus15

(b)





119889

2119903

119889119905

= 119872

minus1119865

(1)





Total 57400 = 143CpG1-10 49200 = 245

Total 57400 = 29CpG1-10 49200 = 47

Total 57400 = 117CpG1-10 49200 = 233

Total 57400 = 85CpG1-10 49200 = 115


Total 57400 = 133CpG1-10 49200 = 222

Total 57400 = 133CpG1-10 49200 = 261

Total 57400 = 181CpG1-10 49200 = 35

Total 57400 = 50CpG1-10 49200 = 94

Total 57400 = 181CpG1-10 49200 = 328

Total 57400 = 53CpG1-10 49200 = 94


Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc

++

minus2615-luc

minus2239-luc

minus1505-luc

minus495-luc


(a)

Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc


minus2239-luc

minus1505-luc

minus495-luc


(b)











1 2 3

4 5

6 7 8 9

10 11 12 13 14

15 16 17

18 19 20

HhaI

HhaI

HpaII

HpaII

AP1-like

AP1-like



1st CpG

100()

75353466526693912

++++++minus minus

(a)

13th CpG

100()

157239589676844561

(b)

20th CpG

100()

352493400820837800

(c)


c-Junc-fos


Binding ()

758

816

51448100

963

994

555

627

100

++ +

+ ++ + +

++

+ +


minusminus

minusminus

minus

minus

minusminusminus

minusminus

minusminus

minus





Chain A of c-Jun

TFIIB

Chain B of c-Jun

181

180

179

178

177

176

175

174

1730 1 2 3 4 5 6 7 8 910 11

Docking pose

Zdoc

k sc

ore


0 50 100 150 200 250 30000

02

04

06

08

10

12TFIIB

Diso

rder

disp

ositi

on

Residue index

c-Jun




3 Results




0 1000 2000 3000 4000 500000

02

04

06

08Pr

otei

n RM

SD (n

m)

Time (ps)

(a)

0 1000 2000 3000 4000 500019

20

21

22

Radi

us o

f gyr

atio

n (n

m)

Time (ps)

(b)

0 1000 2000 3000 4000 5000

Tota

l ene

rgy

(kJm

ol)

Time (ps)

minus910

minus912

minus914

minus916

minus918

minus920

times105

(c)






250 255 260 265 270 275 28000

01

02

03

04

05

c-Jun chain A

RMSF

(nm

)

Residue

(a)

250 255 260 265 270 275 28000

01

02

03

04

05

06

07

c-Jun chain B

RMSF

(nm

)

Residue

(b)

100 120 140 160 180 200 220 240 260 280 300 32000

01

02

03

04

05

06

TFIIB

RMSF

(nm

)

Residue

(c)





0 1000 2000 3000 4000 5000

10

20

30

40

50

Resid

ue

Time (ps)

CoilBendTurn


(a)

0 1000 2000 3000 4000 5000

40

80

120

160

200

Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)










5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 SB8765432

Mock Control

287

Methylation ()9084512413350759775296

35

30

25

20

15

10

5

0

0 05 15 20

Met

hyla

tion

()

243bp

144bp

387bp

05 120583M 15 120583M 20 120583M

lowast

5-aza-dc (120583M)

(a)

35

30

25

20

15

10

5

0

0 05 15 20

Met

hyla

tion

()

15 35302520

Cycles

The fl

uro

amou

nt o

f syb

er g

reen

05 120583M15 120583M20 120583M10 120583M

lowast

5-aza-dc (120583M)

10minus05

10minus07

10minus09

10minus11

10minus13

10minus15

(b)





119889

2119903

119889119905

= 119872

minus1119865

(1)





Total 57400 = 143CpG1-10 49200 = 245

Total 57400 = 29CpG1-10 49200 = 47

Total 57400 = 117CpG1-10 49200 = 233

Total 57400 = 85CpG1-10 49200 = 115


Total 57400 = 133CpG1-10 49200 = 222

Total 57400 = 133CpG1-10 49200 = 261

Total 57400 = 181CpG1-10 49200 = 35

Total 57400 = 50CpG1-10 49200 = 94

Total 57400 = 181CpG1-10 49200 = 328

Total 57400 = 53CpG1-10 49200 = 94


Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc

++

minus2615-luc

minus2239-luc

minus1505-luc

minus495-luc


(a)

Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc


minus2239-luc

minus1505-luc

minus495-luc


(b)











1 2 3

4 5

6 7 8 9

10 11 12 13 14

15 16 17

18 19 20

HhaI

HhaI

HpaII

HpaII

AP1-like

AP1-like



1st CpG

100()

75353466526693912

++++++minus minus

(a)

13th CpG

100()

157239589676844561

(b)

20th CpG

100()

352493400820837800

(c)


c-Junc-fos


Binding ()

758

816

51448100

963

994

555

627

100

++ +

+ ++ + +

++

+ +


minusminus

minusminus

minus

minus

minusminusminus

minusminus

minusminus

minus





Chain A of c-Jun

TFIIB

Chain B of c-Jun

181

180

179

178

177

176

175

174

1730 1 2 3 4 5 6 7 8 910 11

Docking pose

Zdoc

k sc

ore


0 50 100 150 200 250 30000

02

04

06

08

10

12TFIIB

Diso

rder

disp

ositi

on

Residue index

c-Jun




3 Results




0 1000 2000 3000 4000 500000

02

04

06

08Pr

otei

n RM

SD (n

m)

Time (ps)

(a)

0 1000 2000 3000 4000 500019

20

21

22

Radi

us o

f gyr

atio

n (n

m)

Time (ps)

(b)

0 1000 2000 3000 4000 5000

Tota

l ene

rgy

(kJm

ol)

Time (ps)

minus910

minus912

minus914

minus916

minus918

minus920

times105

(c)






250 255 260 265 270 275 28000

01

02

03

04

05

c-Jun chain A

RMSF

(nm

)

Residue

(a)

250 255 260 265 270 275 28000

01

02

03

04

05

06

07

c-Jun chain B

RMSF

(nm

)

Residue

(b)

100 120 140 160 180 200 220 240 260 280 300 32000

01

02

03

04

05

06

TFIIB

RMSF

(nm

)

Residue

(c)





0 1000 2000 3000 4000 5000

10

20

30

40

50

Resid

ue

Time (ps)

CoilBendTurn


(a)

0 1000 2000 3000 4000 5000

40

80

120

160

200

Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)










5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Total 57400 = 143CpG1-10 49200 = 245

Total 57400 = 29CpG1-10 49200 = 47

Total 57400 = 117CpG1-10 49200 = 233

Total 57400 = 85CpG1-10 49200 = 115


Total 57400 = 133CpG1-10 49200 = 222

Total 57400 = 133CpG1-10 49200 = 261

Total 57400 = 181CpG1-10 49200 = 35

Total 57400 = 50CpG1-10 49200 = 94

Total 57400 = 181CpG1-10 49200 = 328

Total 57400 = 53CpG1-10 49200 = 94


Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc

++

minus2615-luc

minus2239-luc

minus1505-luc

minus495-luc


(a)

Mock

0 45403530252015105

Luc

Luc

Luc

Luc

Luc


minus2239-luc

minus1505-luc

minus495-luc


(b)











1 2 3

4 5

6 7 8 9

10 11 12 13 14

15 16 17

18 19 20

HhaI

HhaI

HpaII

HpaII

AP1-like

AP1-like



1st CpG

100()

75353466526693912

++++++minus minus

(a)

13th CpG

100()

157239589676844561

(b)

20th CpG

100()

352493400820837800

(c)


c-Junc-fos


Binding ()

758

816

51448100

963

994

555

627

100

++ +

+ ++ + +

++

+ +


minusminus

minusminus

minus

minus

minusminusminus

minusminus

minusminus

minus





Chain A of c-Jun

TFIIB

Chain B of c-Jun

181

180

179

178

177

176

175

174

1730 1 2 3 4 5 6 7 8 910 11

Docking pose

Zdoc

k sc

ore


0 50 100 150 200 250 30000

02

04

06

08

10

12TFIIB

Diso

rder

disp

ositi

on

Residue index

c-Jun




3 Results




0 1000 2000 3000 4000 500000

02

04

06

08Pr

otei

n RM

SD (n

m)

Time (ps)

(a)

0 1000 2000 3000 4000 500019

20

21

22

Radi

us o

f gyr

atio

n (n

m)

Time (ps)

(b)

0 1000 2000 3000 4000 5000

Tota

l ene

rgy

(kJm

ol)

Time (ps)

minus910

minus912

minus914

minus916

minus918

minus920

times105

(c)






250 255 260 265 270 275 28000

01

02

03

04

05

c-Jun chain A

RMSF

(nm

)

Residue

(a)

250 255 260 265 270 275 28000

01

02

03

04

05

06

07

c-Jun chain B

RMSF

(nm

)

Residue

(b)

100 120 140 160 180 200 220 240 260 280 300 32000

01

02

03

04

05

06

TFIIB

RMSF

(nm

)

Residue

(c)





0 1000 2000 3000 4000 5000

10

20

30

40

50

Resid

ue

Time (ps)

CoilBendTurn


(a)

0 1000 2000 3000 4000 5000

40

80

120

160

200

Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)










5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










1 2 3

4 5

6 7 8 9

10 11 12 13 14

15 16 17

18 19 20

HhaI

HhaI

HpaII

HpaII

AP1-like

AP1-like



1st CpG

100()

75353466526693912

++++++minus minus

(a)

13th CpG

100()

157239589676844561

(b)

20th CpG

100()

352493400820837800

(c)


c-Junc-fos


Binding ()

758

816

51448100

963

994

555

627

100

++ +

+ ++ + +

++

+ +


minusminus

minusminus

minus

minus

minusminusminus

minusminus

minusminus

minus





Chain A of c-Jun

TFIIB

Chain B of c-Jun

181

180

179

178

177

176

175

174

1730 1 2 3 4 5 6 7 8 910 11

Docking pose

Zdoc

k sc

ore


0 50 100 150 200 250 30000

02

04

06

08

10

12TFIIB

Diso

rder

disp

ositi

on

Residue index

c-Jun




3 Results




0 1000 2000 3000 4000 500000

02

04

06

08Pr

otei

n RM

SD (n

m)

Time (ps)

(a)

0 1000 2000 3000 4000 500019

20

21

22

Radi

us o

f gyr

atio

n (n

m)

Time (ps)

(b)

0 1000 2000 3000 4000 5000

Tota

l ene

rgy

(kJm

ol)

Time (ps)

minus910

minus912

minus914

minus916

minus918

minus920

times105

(c)






250 255 260 265 270 275 28000

01

02

03

04

05

c-Jun chain A

RMSF

(nm

)

Residue

(a)

250 255 260 265 270 275 28000

01

02

03

04

05

06

07

c-Jun chain B

RMSF

(nm

)

Residue

(b)

100 120 140 160 180 200 220 240 260 280 300 32000

01

02

03

04

05

06

TFIIB

RMSF

(nm

)

Residue

(c)





0 1000 2000 3000 4000 5000

10

20

30

40

50

Resid

ue

Time (ps)

CoilBendTurn


(a)

0 1000 2000 3000 4000 5000

40

80

120

160

200

Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)










5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Chain A of c-Jun

TFIIB

Chain B of c-Jun

181

180

179

178

177

176

175

174

1730 1 2 3 4 5 6 7 8 910 11

Docking pose

Zdoc

k sc

ore


0 50 100 150 200 250 30000

02

04

06

08

10

12TFIIB

Diso

rder

disp

ositi

on

Residue index

c-Jun




3 Results




0 1000 2000 3000 4000 500000

02

04

06

08Pr

otei

n RM

SD (n

m)

Time (ps)

(a)

0 1000 2000 3000 4000 500019

20

21

22

Radi

us o

f gyr

atio

n (n

m)

Time (ps)

(b)

0 1000 2000 3000 4000 5000

Tota

l ene

rgy

(kJm

ol)

Time (ps)

minus910

minus912

minus914

minus916

minus918

minus920

times105

(c)






250 255 260 265 270 275 28000

01

02

03

04

05

c-Jun chain A

RMSF

(nm

)

Residue

(a)

250 255 260 265 270 275 28000

01

02

03

04

05

06

07

c-Jun chain B

RMSF

(nm

)

Residue

(b)

100 120 140 160 180 200 220 240 260 280 300 32000

01

02

03

04

05

06

TFIIB

RMSF

(nm

)

Residue

(c)





0 1000 2000 3000 4000 5000

10

20

30

40

50

Resid

ue

Time (ps)

CoilBendTurn


(a)

0 1000 2000 3000 4000 5000

40

80

120

160

200

Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)










5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 1000 2000 3000 4000 500000

02

04

06

08Pr

otei

n RM

SD (n

m)

Time (ps)

(a)

0 1000 2000 3000 4000 500019

20

21

22

Radi

us o

f gyr

atio

n (n

m)

Time (ps)

(b)

0 1000 2000 3000 4000 5000

Tota

l ene

rgy

(kJm

ol)

Time (ps)

minus910

minus912

minus914

minus916

minus918

minus920

times105

(c)






250 255 260 265 270 275 28000

01

02

03

04

05

c-Jun chain A

RMSF

(nm

)

Residue

(a)

250 255 260 265 270 275 28000

01

02

03

04

05

06

07

c-Jun chain B

RMSF

(nm

)

Residue

(b)

100 120 140 160 180 200 220 240 260 280 300 32000

01

02

03

04

05

06

TFIIB

RMSF

(nm

)

Residue

(c)





0 1000 2000 3000 4000 5000

10

20

30

40

50

Resid

ue

Time (ps)

CoilBendTurn


(a)

0 1000 2000 3000 4000 5000

40

80

120

160

200

Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)










5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


250 255 260 265 270 275 28000

01

02

03

04

05

c-Jun chain A

RMSF

(nm

)

Residue

(a)

250 255 260 265 270 275 28000

01

02

03

04

05

06

07

c-Jun chain B

RMSF

(nm

)

Residue

(b)

100 120 140 160 180 200 220 240 260 280 300 32000

01

02

03

04

05

06

TFIIB

RMSF

(nm

)

Residue

(c)





0 1000 2000 3000 4000 5000

10

20

30

40

50

Resid

ue

Time (ps)

CoilBendTurn


(a)

0 1000 2000 3000 4000 5000

40

80

120

160

200

Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)










5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 1000 2000 3000 4000 5000

10

20

30

40

50

Resid

ue

Time (ps)

CoilBendTurn


(a)

0 1000 2000 3000 4000 5000

40

80

120

160

200

Resid

ue

Time (ps)

CoilBendTurn

A-helix3-helix

(b)










5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)

15

(a)

5 10 15 20 25

5

10

15

20

25

Resid

ue in

dex

Residue index

0

Dist

ance

(nm

)15

(b)









0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 1000 2000 3000 4000 5000145

150

155

160

165

170

175

SASA

(nm

S2

N)

Time (ps)

(a)

0 1000 2000 3000 4000 500012

13

14

15

16

17

18

Dist

ance

(nm

)

Time (ps)

(b)

0 1000 2000 3000 4000 500000

01

02

03

04

c-JunTFIIB

MSD

(nm

-S2-

N)

Time (ps)

(c)





0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 5 10 150

5

10

15

20

25

Num

ber


(a)

0 5 10 150

5

10

15

20

25

30

35

40

45

50

55

Num

ber


(b)



4 Discussion


0 10 20

0

10

20

PC2

PC1minus20

minus20minus10

minus10






0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 1000 2000 3000 4000 50000123456789

101112131415

1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70Cl

uste

r

Time (ps)

Fram

e num

ber

Cluster


Close form

Open form


TFIIB

Time = 0ps

Time = 4260ps



Channel 2

Channel 1TFIIB













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

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