C1 Genome Mutations Reprograming en

Universal Genetic Code
DNA Strider Program
Murine Erythropoietin Receptor Sequence

1/1 atg gac aaa M D K 121/41 ctg ctg gca L L A 241/81 cag ctc gag Q L E 361/121 ctg cag gtg L Q V 481/161 gtg gtg ctg V V L 601/201 cgc act gag R T E 721/241 cta ctg acc L L T 841/281 atc tgg cct I W P 961/321 tcc ttc cct S F P 1081/361 gag cat gcc E H A 1201/401 gaa aca tct E T S 1321/441 cct ccc gag P P E 1441/481 gat ggc ccc D G P 1561/521 agc cat act S H T 1681/561 cct tga aaa P * K 31/11 ctc agg gtg ccc ctc tgg cct cgg gta ggc L R V P L W P R V G 151/51 tcc cgg ggc tcc gaa gaa ctt ctg tgc ttc S R G S E E L L C F 271/91 ggt gag tca cga aag tca tgt agc ctg cac G E S R K S C S L H 391/131 acg gag gcg tcc ggt tct cct cgc tat cac T E A S G S P R Y H 511/171 cgc tgg ctg cca cct cct gga gca cct atg R W L P P P G A P M 631/211 tgt gtt ctg agc aac ctg cgg ggc ggg acg C V L S N L R G G T 751/251 gct agc gac ctg gac cct ctc atc ttg acg A S D L D P L I L T 871/291 ggc atc cca agc cca gag agc gag ttt gag G I P S P E S E F E 991/331 gag gat cca cct gcc cac cta gag gtc ctc E D P P A H L E V L 1111/371 cag gac acc tac ttg gta ttg gat aag tgg Q D T Y L V L D K W 1231/411 tcc tgc ccg tct gac ttg gcc tca aag ccc S C P S D L A S K P 1351/451 cta cct ccc act cca cct cac ttg aag tac L P P T P P H L K Y 1471/491 tac tcc cac ccc tat gag aac agc ctt gtc Y S H P Y E N S L V 1591/531 taa agt cag agc tga cct tgg ccc tct gag * S Q S * P W P S E 1711/571 aaa aaa aaa aaa aaa aaa aaa aaa a K K K K K K K K 61/21 ccc ctc tgt ctc cta ctt gct ggg gca gcc P L C L L L A G A A 181/61 acc caa cgc ttg gaa gac ttg gtg tgt ttc T Q R L E D L V C F 301/101 cag gct ccc acc gtc cgc ggc tcc gtg cgt Q A P T V R G S V R 421/141 cgc atc atc cat atc aat gaa gta gtg ctc R I I H I N E V V L 541/181 acc acc cac atc cga tat gaa gtg gac gtg T T H I R Y E V D V 661/221 cgc tac acc ttc gct gtt cga gcg cgc atg R Y T F A V R A R M 781/261 ctg tct ctc att ctg gtc ctc atc tcg ctg L S L I L V L I S L 901/301 ggt ctc ttc acc acc cac aag ggt aac ttc G L F T T H K G N F 1021/341 tca gag cca cgc tgg gca gtg act cag gct S E P R W A V T Q A 1141/381 ttg ctg ccc cgg acc cca tgc agt gag aac L L P R T P C S E N 1261/421 agg cca gag ggc acc tca cct tcc agc ttt R P E G T S P S S F 1381/461 cta tac ctt gtg gtg tcc gat tct ggc atc L Y L V V S D S G I 1501/501 cca gac tca gag cct ctg cat ccc ggc tat P D S E P L H P G Y 1621/541 cag gaa gag aca gcc ttg caa tgt taa gat Q E E T A L Q C * D 91/31 tgg gca cct tca ccc agc ctc ccg gac ccc aag ttt gag agc aaa gcg gcc W A P S P S L P D P K F E S K A A 211/71 tgg gag gaa gcg gcg agc tcc ggg atg gac ttc aac tac agc ttc tca tac W E E A A S S G M D F N Y S F S Y 331/111 ttc tgg tgt tca ctg cca aca gcg gac aca tcg agt ttt gtg ccg ctg gag F W C S L P T A D T S S F V P L E 451/151 ctg gac gcc ccc gcg ggg ctg ctg gcg cgc cgg gca gaa gag ggc agc cac L D A P A G L L A R R A E E G S H 571/191 tcg gca ggc aac cgg gca gga ggg aca caa agg gtg gag gtc ctg gaa ggc S A G N R A G G T Q R V E V L E G 691/231 gcc gag ccg agc ttc agc gga ttc tgg agt gcc tgg tct gag ccc gcg tca A E P S F S G F W S A W S E P A S 811/271 ttg ctg acg gtt ctg gcc ctg ctg tcc cac cgc cgg act ctg cag cag aag L L T V L A L L S H R R T L Q Q K 931/311 cag ctg tgg ctg ctg cag cgt gat ggt tgt ctg tgg tgg agc ccg ggc agc Q L W L L Q R D G C L W W S P G S 1051/351 ggg gac cca ggg gca gat gat gag ggg ccc tta ctg gag ccg gtg ggc agt G D P G A D D E G P L L E P V G S 1171/391 ctc tca ggg cct ggg ggc agt gtg gac cct gtg act atg gat gaa gct tca L S G P G G S V D P V T M D E A S 1291/431 gag tac acc atc ctg gac ccc agc tct cag ctc ctg tgc cct cgg gca ctg E Y T I L D P S S Q L L C P R A L 1411/471 tca aca gat tac agt tcg ggg ggc tct cag gga gtc cac ggg gac tca tct S T D Y S S G G S Q G V H G D S S 1531/511 gtg gcc tgc tcc tag gac tcc agc cta caa cgt ctt gaa cgg gat tgg tga V A C S * D S S L Q R L E R D W * 1651/551 taa gag tta tct gtc tgt ata tag aaa tat ata tat ata tcg att ttt cta * E L S V C I * K Y I Y I S I F L
DNA Strider Program
Non-Coding DNA Frame

3/1 gga caa act G Q T 123/41 gct ggc atc A G I 243/81 gct cga ggg A R G 363/121 gca ggt gac A G D 483/161 ggt gct gcg G A A 603/201 cac tga gtg H * V 723/241 act gac cgc T D R 843/281 ctg gcc tgg L A W 963/321 ctt ccc tga L P * 1083/361 gca tgc cca A C P 1203/401 aac atc ttc N I F 1323/441 tcc cga gct S R A 1443/481 tgg ccc cta W P L 1563/521 cca tac tta P Y L 1683/561 ttg aaa aaa L K K 33/11 cag ggt gcc cct ctg gcc tcg ggt agg ccc Q G A P L A S G R P 153/51 ccg ggg ctc cga aga act tct gtg ctt cac P G L R R T S V L H 273/91 tga gtc acg aaa gtc atg tag cct gca cca * V T K V M * P A P 393/131 gga ggc gtc cgg ttc tcc tcg cta tca ccg G G V R F S S L S P 513/171 ctg gct gcc acc tcc tgg agc acc tat gac L A A T S W S T Y D 633/211 tgt tct gag caa cct gcg ggg cgg gac gcg C S E Q P A G R D A 753/251 tag cga cct gga ccc tct cat ctt gac gct * R P G P S H L D A 873/291 cat ccc aag ccc aga gag cga gtt tga ggg H P K P R E R V * G 993/331 gga tcc acc tgc cca cct aga ggt cct ctc G S T C P P R G P L 1113/371 gga cac cta ctt ggt att gga taa gtg gtt G H L L G I G * V V 1233/411 ctg ccc gtc tga ctt ggc ctc aaa gcc cag L P V * L G L K A Q 1353/451 acc tcc cac tcc acc tca ctt gaa gta cct T S H S T S L E V P 1473/491 ctc cca ccc cta tga gaa cag cct tgt ccc L P P L * E Q P C P 1593/531 aag tca gag ctg acc ttg gcc ctc tga gca K S E L T L A L * A aaa aaa aaa aaa aaa aaa aaa aa K K K K K K K 63/21 cct ctg tct cct act tgc tgg ggc agc ctg P L S P T C W G S L 183/61 cca acg ctt gga aga ctt ggt gtg ttt ctg P T L G R L G V F L 303/101 ggc tcc cac cgt ccg cgg ctc cgt gcg ttt G S H R P R L R A F 423/141 cat cat cca tat caa tga agt agt gct cct H H P Y Q * S S A P 543/181 cac cca cat ccg ata tga agt gga cgt gtc H P H P I * S G R V 663/221 cta cac ctt cgc tgt tcg agc gcg cat ggc L H L R C S S A H G 783/261 gtc tct cat tct ggt cct cat ctc gct gtt V S H S G P H L A V 903/301 tct ctt cac cac cca caa ggg taa ctt cca S L H H P Q G * L P 1023/341 aga gcc acg ctg ggc agt gac tca ggc tgg R A T L G S D S G W 1143/381 gct gcc ccg gac ccc atg cag tga gaa cct A A P D P M Q * E P 1263/421 gcc aga ggg cac ctc acc ttc cag ctt tga A R G H L T F Q L * 1383/461 ata cct tgt ggt gtc cga ttc tgg cat ctc I P C G V R F W H L 1503/501 aga ctc aga gcc tct gca tcc cgg cta tgt R L R A S A S R L C 1623/541 gga aga gac agc ctt gca atg tta aga tta G R D S L A M L R L 93/31 ggc acc ttc acc cag cct ccc gga ccc caa gtt tga gag caa agc ggc cct G T F T Q P P G P Q V * E Q S G P 213/71 gga gga agc ggc gag ctc cgg gat gga ctt caa cta cag ctt ctc ata cca G G S G E L R D G L Q L Q L L I P 333/111 ctg gtg ttc act gcc aac agc gga cac atc gag ttt tgt gcc gct gga gct L V F T A N S G H I E F C A A G A 453/151 gga cgc ccc cgc ggg gct gct ggc gcg ccg ggc aga aga ggg cag cca cgt G R P R G A A G A P G R R G Q P R 573/191 ggc agg caa ccg ggc agg agg gac aca aag ggt gga ggt cct gga agg ccg G R Q P G R R D T K G G G P G R P 693/231 cga gcc gag ctt cag cgg att ctg gag tgc ctg gtc tga gcc cgc gtc act R A E L Q R I L E C L V * A R V T 813/271 gct gac ggt tct ggc cct gct gtc cca ccg ccg gac tct gca gca gaa gat A D G S G P A V P P P D S A A E D 933/311 gct gtg gct gct gca gcg tga tgg ttg tct gtg gtg gag ccc ggg cag ctc A V A A A A * W L S V V E P G Q L 1053/351 gga ccc agg ggc aga tga tga ggg gcc ctt act gga gcc ggt ggg cag tga G P R G R * * G A L T G A G G Q * 1173/391 ctc agg gcc tgg ggg cag tgt gga ccc tgt gac tat gga tga agc ttc aga L R A W G Q C G P C D Y G * S F R 1293/431 gta cac cat cct gga ccc cag ctc tca gct cct gtg ccc tcg ggc act gcc V H H P G P Q L S A P V P S G T A 1413/471 aac aga tta cag ttc ggg ggg ctc tca ggg agt cca cgg gga ctc atc tga N R L Q F G G L S G S P R G L I * 1533/511 ggc ctg ctc cta gga ctc cag cct aca acg tct tga acg gga ttg gtg aag G L L L G L Q P T T S * T G L V K 1653/551 aga gtt atc tgt ctg tat ata gaa ata tat ata tat atc gat ttt tct acc R V I C L Y I E I Y I Y I D F S T
Frequent Stop Codons
DNA Strider Program
AatII AccI Acc65I AceII AceIII AciI AclI AflII AflIII AgeI AhdI AluI AlwI AlwNI ApaI ApaBI ApaLI ApoI AscI AseI AvaI AvaII AvrII BaeI BamHI BanI BanII BbeI BbsI BbvI BbvCI Bce83I BcefI BcgI BciVI BclI BfaI BfiI BfrBI BglI BglII BlpI BplI BpmI Bpu10I BsaI BsaAI BsaBI BsaHI BsaJI BsaWI BscAI BseMII BseRI BsgI BsiEI BsiHKAI BsiWI BslI
gacgt/c gt/mkac g/gtacc gctag/c cagctc ccgc aa/cgtt c/ttaag a/crygt a/ccggt gacnnn/nngtc ag/ct ggatc cagnnn/ctg gggcc/c gcannnnn/tgc g/tgcac r/aatty gg/cgcgcc at/taat c/ycgrg g/gwcc c/ctagg acnnnngtayc g/gatcc g/gyrcc grgcy/c ggcgc/c gaagac gcagc cctcagc cttgag acggc cgannnnnntgc gtatcc t/gatca c/tag actggg atg/cat gccnnnn/nggc a/gatct gc/tnagc gagnnnnnctc ctggag cctnagc ggtctc yac/gtr gatnn/nnatc gr/cgyc c/cnngg w/ccggw gcatc ctcag gaggag gtgcag cgry/cg gwgcw/c c/gtacg ccnnnnn/nngg
7/11 -3/-1
4/5
12/7
2/6 8/12 -5/-2 16/14 12/13 12/10 6/5
5/4
13/8 16/14 -5/-2 1/5
4/6 10/8 10/8 16/14
BsmI BsmAI BsmBI BsmFI Bsp24I Bsp120I Bsp1286I BspEI BspHI BspKT6I BspLU11I BspMI BsrI BsrBI BsrDI BsrFI BsrGI BssHII BssKI BssSI BstAPI BstBI Bst4CI BstDSI BstEII BstF5I BstNI BstUI BstXI BstYI BstZ17I Bsu36I Cac8I ChaI CjeI CjePI ClaI Csp6I CviAII CviJI CviRI DdeI DpnI DraI DraIII DrdI EaeI EagI EarI Ecl136II Eco47III Eco57I EcoHI EcoNI EcoO109I EcoRI EcoRII EcoRV
gaatgc 1/-1 gtctc 1/5 cgtctc 1/5 gggac 10/14 gacnnnnnntgg 12/7 g/ggccc gdgch/c t/ccgga t/catga gat/c a/catgt acctgc 4/8 actgg 1/-1 ccgctc -3/-3 gcaatg 2/0 r/ccggy t/gtaca g/cgcgc /ccngg cacgag -5/-1 gcannnn/ntgc tt/cgaa acn/gt c/crygg g/gtnacc ggatg 2/0 cc/wgg cg/cg ccannnnn/ntgg r/gatcy gta/tac cc/tnagg gcn/ngc gatc/ ccannnnnngt 15/9 ccannnnnnntc 14/8 at/cgat g/tac c/atg rg/cy tg/ca c/tnag ga/tc ttt/aaa cacnnn/gtg gacnnnn/nngtc y/ggccr c/ggccg ctcttc 1/4 gag/ctc agc/gct ctgaag 16/14 /ccsgg cctnn/nnnagg rg/gnccy g/aattc /ccwgg gat/atc
FauI FmuI Fnu4HI FokI FseI FspI GdiII HaeI HaeII HaeIII HgaI HhaI HinP1I HincII HindIII HinfI HpaI HpaII HphI KasI KpnI LpnI MaeII MaeIII MboI MboII MfeI MluI Mlu113I MlyI MmeI MnlI MscI MseI MslI MspA1I MwoI NaeI NarI NciI NcoI NdeI NgoMIV NheI NlaIII NlaIV Nli387/7I NotI NruI NsiI NspI PacI PflMI PleI PmeI PmlI Ppu10I PpuMI
cccgc 4/6 ggnc/c gc/ngc ggatg 9/13 ggccgg/cc tgc/gca cggccr -5/-1 wgg/ccw rgcgc/y gg/cc gacgc 5/10 gcg/c g/cgc gty/rac a/agctt g/antc gtt/aac c/cgg ggtga 8/7 g/gcgcc ggtac/c rgc/gcy a/cgt /gtnac /gatc gaaga 8/7 c/aattg a/cgcgt cc/gcgg gactc 5/5 tccrac 20/18 cctc 7/6 tgg/cca t/taa caynn/nnrtg cmg/ckg gcnnnnn/nngc gcc/ggc gg/cgcc cc/sgg c/catgg ca/tatg g/ccggc g/ctagc catg/ ggn/ncc cycgr/g gc/ggccgc tcg/cga atgca/t rcatg/y ttaat/taa ccannnn/ntgg gagtc 4/5 gttt/aaac cac/gtg a/tgcat rg/gwccy
PshAI PssI PstI PvuI PvuII RleAI RsaI RsrII SacI SacII SalI SanDI SapI Sau96I SbfI ScaI SchI SciI ScrFI SelI SexAI SfaNI SfcI SfiI SfoI SgfI SgrAI SimI SmaI SmlI SnaBI SpeI SphI SrfI Sse8647I SspI Sth132I StsI StuI StyI SwaI TaiI TaqI TaqII TatI TfiI TseI Tsp45I Tsp509I TspRI Tth111I Tth111II VpaK11AI XbaI XcmI XhoI XmaI XmnI
gacnn/nngtc rggnc/cy ctgca/g cgat/cg cag/ctg cccaca 12/9 gt/ac cg/gwccg gagct/c ccgc/gg g/tcgac gg/gwccc gctcttc 1/4 g/gncc cctgca/gg agt/act gagtc 5/5 ctc/gag cc/ngg /cgcg a/ccwggt gcatc 5/9 c/tryag ggccnnnn/nggcc ggc/gcc gcgat/cgc cr/ccggyg gggtc -3/0 ccc/ggg c/tyrag tac/gta a/ctagt gcatg/c gccc/gggc ag/gwcct aat/att cccg 4/8 ggatg 10/14 agg/cct c/cwwgg attt/aaat acgt/ t/cga gaccga 11/9 w/gtacw g/awtc g/cwgc /gtsac /aatt castg 2/-7 gacn/nngtc caarca 11/9 /ggwcc t/ctaga ccannnnn/nnnntgg c/tcgag c/ccggg gaann/nnttc
DNA Strider Program
Universal Genetic Code
DNA Strider Program
Plasmids-4 Kb Cosmids-40 Kb BAC, YAC 100-500 Kb Bacterial genome-2Mb
E. Coli F plasmid- BAC allows stable cloning of up to 1 million bp

Lander et al., Nature 2001, 409, 877
-Human genome = 30,000 genes, 3x109 bp -Hundreds of genes acquired by horizontal transfer from bacteria -Dozens of genes acquired from transposons -50% of the genome is derived from transposable elements of which DNA and LTR transposons are inactive
-Alu transposable elements predominate in GC rich regions while GC-poor regions are associated with dark G- bands in karyotypes
Lander et. al., Nature 2001, 409, 877
-LINE1 and Alu constitute 60% of all interspersed repeat sequence -LINE1 and Alu are vertically transmitted -Genomes of the worm, y, and mustard weed have many more types of recent active transposons of which LINE and SINE elements are 5-6% -3 million transposable elements remain in the human genome -Presently these elements are responsible for new mutations, like for 2/1000 mutations -Hypothesis: 170 million years ago : critical speciation events leading to mammalian radiation for a common ancestor may have involved a burst in transposition activity
Lander et. al., Nature 2001, 409, 877
Transposons: -LINES (LINE1 is still active !) are most ancient, 6kb in length, encode 2 orfs and have a polymerase II promoter, move to the nucleus a complex of proteins and the RNA; an endonuclease makes a ss nick and the RT uses the nicked RNA to prime RT from the 3 end- imperferct with unnished 5 ends; new insertions are anked by 7-20bp target site Duplications; LINES target AT rich gene-poor regions due to TTTT/A endonuclease preferred cleavage site Long interspersed nuclear elements- can insert into the gene for Factor VIII and produce hemophilia
-SINES are short 100-400 bp and use LINES to function -Promoter regions are shared with tRNA sequences or with the 7SL RNA of the signal recognition particle- this subfamily of SINES is the Alu repeat which accumulate in GC-rich DNA via a yet to be understood mechanism ! -Alu elements in the human genome: the active Alu, the inactive MIR and Ther2/MIR3 -LTR transposons are anked by LTRs and contain gag, pol coding for RT and RNAseH; transposition occurs via the RT in a cytoplasmic virus-like structure, primed by a tRNA as opposed to chromosomal priming for SINES. They generated extracellular retroviruses by acquiring an envelope protein -DNA transposons resemble bacterial transposons having terminal Inverted repeats and using cut and paste mechanisms- they are short lived elements
-The human genome contains much longer intron sequences -Only 5% of the 28% that is transcribed into RNA actually codes for proteins -Repetitive eements: 45% the transposons; 3% repeats of CA type and 5% recent duplications -Only 94 of the 1278 protein families are specic to vetrebrates
-SNP= Single Nucleotide Polymorphism -Two human genomes compared will differ at 2.5 million places corresponding to a frequency of 1 per 1300 nucleotide pairs -Rate of nucleotide change/genome 5 nt/ 1000 are changed in 1 million years due to the acuracy of replication -Human and chimpanzee chromosomes are separated by 5 million years of evolution- very similar: human and mouse chromosomes separated by 100 million years of evolution are much more different Lander et al., Nature 2001, 409, 877
-Homologs of human genes can be found in nematode worms, fruit y, yeasts and bacteria -Hundreds of bacterial genes were transferred horizontally to humans -Gene sequences are more conserved than genome structures -Number of genes: 6000 for yeast Saccharomyces cerevisiae; 18,000 for the nematode C. elegans; 13,000 for Drosophila melanogaster; 30,000 for humans -There is no machinery dedicated to evolution except mistakes: substitutions, deletions, inversions, translocations, transposons -A total of 3 billion years of evolution -Human-chimpanzee divergence of 1.2%, human-to-human 0.12%
Chimpanzee and human chromosomes are almost identical except for human chromosome 2 99% of Alu repeats) are in the same place in the human and chimpanzee genomes The 1% repeats that differ contain human-specic Alu, still active which can induce genetic diseases. Human Alu seqs (1 million) and mouse B1 (400,000) evolved from the 7SL RNA which encodes the SRP RNA Alu restriction site = ag/ct
Methionine (Met)- rich p54 binds hydrophobic signal sequences
RNA component
Lodish et al., Molecular Cell Biology, 2000, Freeman Press
-Why are Alu elements accumulating in GC rich regions? -Higher loss in AT-rich regions -Negative selection for Alu elements in AT-rich regions -Positive selection for GC rich regions -Rapid transcription of SINE elements into RNA can only occur near genes in opened chromatin; SINE RNA can appear in massive amounts, inhibit PKR, stimulating translation. -Stress induced SINE transcription which leads to massive increases in protein translation- mechanism of evolution? -Y chromosome has lower levels of somatic genes transcribed and shows lower than expected numbers of Alu elements; the reverse is true for chromosome 19
Mutations
Mutation (CpG, slipped mispairing, hot spot, splicing) or/ and Epigenetic change (methylation) (Chromatin state differs in each cell type) SELECTION PRESSURES AMPLIFIES MUTATED CLONE (cell type-specic mechanisms) Proliferation, clonal dominance SECOND, THIRD HIT (p53, etc) ANGIOGENESIS= regulated by bone-marrow derived and stroma-derived cells HIGHLY MALIGNANT AND METASTATIC CANCER
1) Mutation occuring in CpG regions are most prevalent (30% of all mutations) 2) Slipped mispairing and single nucleotide substitutions 3) Hotspots independent of CpG 4) Single-Pair Substitutions affected by neighboring residues 5) Strand difference in mutation and repair 6) Single base pair substitutions in human mRNA splice junctions
Nature and Mechanisms of Human Gene Mutations Antonarkis SE, Krawczak M and Cooper DN
Mutation mechanisms Single base substitutions account for the majority of gene defects Gene deletion, insertions, duplications, inversions Retrotransposition of LINE and Alu or of pseudogenes can induce disease Expansion of trinucleotide repeats (in 5 or 3 UTRs, in coding genes or in introns); expansion of 12-mer repeat induces progressive myoclonus epilepsy
SINGLE PAIR SUBSTITUTIONS A major mechanism is misincorporation of bases during replication Transition: purine to purine or pyrimidone to pyrimidine Transversion: purine to pyrimidine or pyrimidine to purine 1) Mutation occuring in CpG regions are most prevalent (30% of all mutations) Most frequent mutations CG to TG and CG to CA due to methylation of C at 5 position (5m-C) and deamination resulting in T CG to CA is due to the 5m-C to T mutation on the anti-sense strand C is also deaminated but U is eliminated from DNA due to the action of DNA glycosylase
Different distribution of mutations in chromosomes CG to TG and CG to CA are more frequent in autosomal genes; X-chromosome genes are less affected by these types of mutations due to more pronounced CpG suppression (lower CpG content) Silent versus non silent mutations mutations will be discovered as a function of phenotype and will depend on codon usage
CpG dinucleotides= hotspots for nucleotide substitutions 5mC occurs predominantly in CpG dinucleotides; the majority of which are methylated CpG distribution is non-random 1% of genome fraction rich in CpG that are unmethylated = CpG islands coincide with transcribed regions Excess C to T in CpG was intially reported for Hb mutations CG to TG and CG to CA occur with different frequency in different genes Methylation in germ line of cytosine promotes mutations; male germ cells are much more methylated than female germ cells
As a consequence: Codons for Arg and Gly are mutated to the highest frequency (see codon usage: GGA, GGC, GGT, GGG: G frequently in 5 of mutated G; Arg is more mutated tha that of Gly-four codons for Arg contain CG
R>G>L=Q=E=C>S=A>W>Y=V=P>K=D>N=H=T=I=M=F>>> STOP FGFR= mutation of R380 to glycine in the transmembrane domain leads to the most common form of genetic dwarsm
Mutation rates are lower for genes on chromosome X, Arg is still the most mutated codon
2) Slipped mispairing and single nucleotide substitutions Transient mis-alignment of the primer template caused by looping out of one base of the templete: 5-G T C G G T T T T A G C-3 5-G T C G G T T T A G C-3 T 3-C A G C C A A A T C G-5 5-G T C G G T T T A G C-3 T
Misalignment Misincorporation
3- C A G C C A A A T C G-5 Realignment 5-G T C G G T T T T A G C-3 3-C A G C C C A A A T C G-5 Correction in 5-G T C G G G T T T A G C-3 favor of C
In slipped mispairing the nt at 5 is replaced by the nt at 3 and the nucleaotide at 3 is replaced by the nt at 5= a mechanism to avoid introduction of STOP codons Slipped mispairing depends on surrounding sequence
3) Hotspots independent of CpG Either a single base affected by several two non-identical substitutions Mutations affecting two or three bases of one codon Motifs of TTT, CTT, TGA TTG, CTTT, TCTT and TTTG within 10bp of the codon CTT= site for topoisomerase-I cleavage TGGA= deletion hotspot consensus - putative arrest site for DNA polymerase !
4) Single-Pair Substitutions affected by neighboring residues Differences have been noted between adjacent positions of mutated bases: G is overrepresented as 3 anking nucleotides when T is mutated a mutated G is often anked by another G in 5 nucleotides at 2bp distance inuence non CpG mutations non-sense mutations are more likely to become detected due to major damage of truncating proteins 5) Strand difference in mutation and repair relative rates of CT to CC and AG to GG differ by > 2-fold; these transitions are complimentary and so strands differ with respect to correction & mutation rates
6) Single base pair substitutions in human mRNA splice junctions -a. mutations in 5 and 3 splice sites: exon skipping and expression of additional intronic sequences (cryptic splice site utilization) -b. mutations in intron or exon leading to activation of cryptic splice sites -c. mutations in the introns that may regulate efciency of splicing -d. mutations within a branch point sequence- (an intermediate point in eukaryotiec RNA splicing is the formation of a lariat structure utilizing an Adenosine residue 10-50 nt past the 3 ss. After lariat structure formation the rst AG site is used as acceptor site 5 splice site 3 splice site A/C AG GT A/GAGT...........CTRAY...(10-50 nt) C/TAG G..... exon intron exon.
1) Mutation occuring in CpG regions are most prevalent (30% of all mutations) 2) Slipped mispairing and single nucleotide substitutions 3) Hotspots independent of CpG 4) Single-Pair Substitutions affected by neighboring residues 5) Strand difference in mutation and repair 6) Single base pair substitutions in human mRNA splice junctions
Mutations are permanent, epigenetic changes are reversible
Phil. Trans. R. Soc. B (2008) 363, 20792087 doi:10.1098/rstb.2008.2261 Published online 28 March 2008
Pluripotency and nuclear reprogramming

Shinya Yamanaka1,2,*
1
Center for iPS Cell Research & Application, Kyoto University, 53 Kawahara-machi, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan 2 CREST, JST, Kawaguchi 332-0012, Japan
Embryonic stem cells are promising donor cell sources for cell transplantation therapy, which may in the future be used to treat various diseases and injuries. However, as is the case for organ transplantation, immune rejection after transplantation is a potential problem with this type of therapy. Moreover, the use of human embryos presents serious ethical difculties. These issues may be overcome if pluripotent stem cells are generated from patients somatic cells. Here, we review the molecular mechanisms underlying pluripotency and the currently known methods of inducing pluripotency in somatic cells. Keywords: embryonic stem cell; induced pluripotent stem cell; transcription factor; retrovirus
1. INTRODUCTION Embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and have the unique capacity to proliferate extensively while maintaining pluripotency. The isolation of the rst ES cells from mouse embryos (Evans & Kaufman 1981; Martin 1981) led to the development of the revolutionary knockout mouse technology (Doetschman et al. 1987; Hooper et al. 1987), which is widely used today. Since ES cells theoretically have the capacity to develop into any cell type, the generation of ES cell lines from human blastocyst embryos ( Thomson et al. 1998) has offered the possibility of using these cells as a donor source for cell transplantation therapies. Potential clinical applications include treatment of degenerative diseases such as juvenile diabetes, Parkinsons disease and heart failure as well as spinal cord injury and burns. However, as is the case for organ transplantation, tissue rejection is a concern for ES cell transplantation. One possible means to avoid immune rejection is the reprogramming of the nuclei of differentiated cells to an ES cell-like, pluripotent state, and using these cells to generate appropriate donor cells for transplantation. Here, we will review the following three methods of inducing nuclear reprogramming and pluripotency in somatic cells: nuclear transfer, fusion with ES cells and reprogramming using dened factors (gure 1).
2. REPROGRAMMING BY NUCLEAR TRANSFER Much progress has been made in the last 50 years in understanding how differentiation and pluripotency are controlled. First, in order for the somatic cells to regain pluripotency, it is essential that they maintain complete genomes. While it is now widely accepted that all cells in an organism possess the same genome, historically this has not always been the case. For instance, in his
* yamanaka@frontier.kyoto-u.ac.jp One contribution of 17 to a Theme Issue Japan: its tradition and hot topics in biological sciences.
1893 book The germ-plasma: a theory of heredity, Weismann proposed that somatic cells lose or irreversibly inactivate unnecessary genes in the course of differentiation. It took more than 50 years to disprove this theory (Gurdon & Byrne 2003). In 1952, Briggs & King showed that nuclear transfer was possible in frogs by transplanting nuclei from blastula stage embryos into enucleated Rana pipiens eggs. The resulting embryos developed into normal hatched tadpoles (Briggs & King 1952). However, their later work showed that transplantation of nuclei from tail-bud stage somatic cells into enucleated eggs resulted in abnormal embryonic development, suggesting that differences in nuclear state affect the capacity of somatic cells to be reprogrammed (King & Briggs 1955, 1956). Gurdon and colleagues then succeeded in producing adult frogs by transferring tadpole intestine cell nuclei into enucleated Xenopus laevis eggs (Gurdon 1962; Gurdon & Uehlinger 1966), showing that the genes required for normal development can be activated even in differentiated cells. However, when they transferred nuclei from adult somatic cells, animals developed to tadpole, but not to adult stage (Laskey & Gurdon 1970; Gurdon et al. 1975; DiBerardino & Hoffner 1983). This raised the possibility that terminally differentiated cells are unable to activate genes required for full development to adulthood. Nuclear transfer in mammals is more difcult than in frogs due to the small size of eggs. Nonetheless, in 1975, Bromhall reported development to the morula stage following nuclear transfer of rabbit morula cell nuclei into enucleated rabbit eggs, albeit with low efciency (Bromhall 1975). In 1983, McGrath & Solter obtained live mice by transferring a zygotic donor nucleus into an enucleated zygote (McGrath & Solter 1983). In 1986, Willadsen fused cells of 8- or 16-cell embryos with enucleated sheep eggs and was able to obtain healthy cloned animals (Willadsen 1986). Successful nuclear transfer of embryonic donor cell nuclei was subsequently reported in rabbits (Stice & Robl 1988),
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Pluripotency and nuclear reprogramming cells of patients suffering from spinal cord injury and juvenile diabetes (Hwang et al. 2004, 2005). However, in a much-publicized fraud scandal, their data were shown to be a fabrication; indeed, they were unable to generate a single cloned ES cell line from more than 2000 human eggs. 3. REPROGRAMMING BY FUSION WITH ES CELLS Another strategy for reprogramming the somatic cell nuclei is fusion with ES, embryonic germ (EG) or embryonic carcinoma (EC) cells. In 1965, Harris showed that fusion of HeLa cells with Sendai virus was able to activate erythrocytes (Harris 1965; Harris & Watkins 1965; Harris et al. 1965). Their work facilitated studies in cell fate conversion by fusion with other cell types. In 1976, Miller & Ruddle demonstrated that thymocytes acquired pluripotency upon fusion with EC cells (Miller & Ruddle 1976), and similar results were later obtained by electrofusion with EG cells ( Tada et al. 1997) and mouse ES cells (Tada et al. 2001). Reprogramming by spontaneous fusion with mouse ES cells was also demonstrated (Terada et al. 2002; Ying et al. 2002). In tetraploid cells generated by fusion of somatic cells with ES or EG cells, ES cell marker genes such as Oct3/4 acquire an ES cell-like epigenetic state, adopting the DNA methylation and histone modication patterns normally seen in ES cells (Kimura et al. 2004). The transplantation of these cells into nude mice results in formation of teratomas consisting of various tissues from all three germ layers, conrming the pluripotency of these cells. Finally, reprogramming by fusion with human ES cells was reported in 2005 (Cowan et al. 2005; Yu et al. 2005). Thus, it appears that ES cells contain factors that induce pluripotency in somatic cells, though it is still controversial whether these factors reside in the nucleus (Do & Scholer 2004) or cytoplasm (Strelchenko et al. 2006) of ES cells. However, pluripotent cells generated by fusion contain both somatic cell and ES cell-derived chromosomes. As a result, rejection upon implantation remains an issue. In an effort to circumvent this problem, Tada and colleagues recently developed a system to remove whole chromosomes from tetraploid cells (Matsumura et al. 2007). In addition, other groups have been attempting to reprogramme the somatic cells with ES cell extracts ( Taranger et al. 2005). 4. REPROGRAMMING WITH DEFINED FACTORS The fact that somatic cell nuclei can be reprogrammed by transfer into oocytes or fusion with ES cells indicates that oocytes and ES cells contain reprogramming factors. It is probable that reprogramming factors largely overlap with those maintaining pluripotency in ES cells. Some of the candidate factors are described below. (a) Oct3/4 (POU5F1) POU5F1 is a POU family transcription factor specically expressed in ES cells, early embryos and germ cells, and was originally designated as Oct3 (Okamoto et al. 1990) or Oct4 (Scholer et al. 1989). Oct3/4-null embryos die in utero at peri-implantation stages of
nuclear transfer
somatic cells fusion
eggs ES cells pluripotent stem cells defined factors
Figure 1. Three methods of inducing pluripotency in somatic cells.
mice (Cheong et al. 1993), pigs (Prather et al. 1989), cows (Sims & First 1994) and monkeys (Meng et al. 1997). However, it proved difcult to generate cloned animals by nuclear transfer from differentiated cells into eggs. A breakthrough came in 1996, when Campbell & Wilmut performed nuclear transfer using day 9 embryo-derived differentiated cells and successfully obtained healthy cloned sheep. Their success was probably due to the fact that they induced their cells into a quiescent state prior to nuclear transfer (Wilmut et al. 2002). Using similar techniques, Wilmut was able to produce an adult sheep, famously known as Dolly, using nuclei derived from follicle cells ( Wilmut et al. 1997). Subsequently, somatic cloning was successfully performed in other species, including the cow (Kato et al. 1998), mouse (Wakayama et al. 1998), goat (Baguisi et al. 1999), pig (Polejaeva et al. 2000), cat (Shin et al. 2002), rabbit (Chesne et al. 2002) and dog (Lee et al. 2005). Furthermore, Jaenisch and colleagues generated cloned mice from B lymphocytes that had undergone immunoglobulin rearrangement ( Hochedlinger & Jaenisch 2002). These studies demonstrated that terminally differentiated cells are in fact able to reactivate all of the genes required for normal development, and that egg cytoplasm contains factors that are able to reprogramme somatic cell nuclei. Given that embryos can now be cloned using somatic cell nuclei, derivation of ES cells from these cloned embryos might provide a means of avoiding immune rejection after transplantation therapy. However, many countries have laws prohibiting cloning of humans for ethical reasons. Cloned ES cells face even stronger ethical objections than ordinary human ES cells, since they involve generation of human embryos exclusively for the production of ES cells. Obtaining a sufcient number of human oocytes presents another challenging issue. As a solution to overcome this issue, Eggan and associates showed that reprogramming can be achieved by fertilized eggs, when mitosis is arrested (Egli et al. 2007). In 2005, a group in Korea reported that they had successfully generated cloned ES cells from skin
Phil. Trans. R. Soc. B (2008)
Pluripotency and nuclear reprogramming development (Nichols et al. 1998). Although these embryos are able to reach blastocyst stage, in vitro culture of the inner cell mass of homozygous mutant blastocyst produces only trophoblast lineages, and Oct3/4 mutant ES cells differentiate only into trophoblast cells, while cells expressing a single copy of Oct3/4 maintain their pluripotential ES cell state ( Niwa et al. 2000). Likewise, knock-down of Oct3/4 by siRNA in human ES cells caused these cells to differentiate into trophoblast lineages (Zaehres et al. 2005). By contrast, twofold overexpression of Oct3/4 in ES cells causes differentiation into primitive endoderm and mesoderm ( Niwa et al. 2000). Hence, the level of Oct3/4 expression is an important determinant of cell fate in mouse ES cells. (b) Sox2 Sox2 is a Sox (SRY-related HMG-box) family transcription factor expressed in ES cells, early embryos, germ cells and neural stem cells (Koopman et al. 2004). The Sox2-null embryos die around implantation due to a failure in epiblast (primitive ectoderm) development (Avilion et al. 2003). Homozygous mutant blastocysts appear morphologically normal, but undifferentiated cells fail to proliferate when blastocysts are cultured in vitro, and only trophectoderm and primitive endoderm-like cells are produced. Disruption of Sox2 in mouse ES cells by either conditional knockout or RNAi resulted in rapid differentiation (Ivanova et al. 2006; Masui et al. 2007). These data demonstrate that Sox2 is indispensable for maintaining pluripotency in both early embryos and ES cells. Another Sox family protein, Sox15, is also highly expressed in ES cells, but its role remains unclear (Maruyama et al. 2005). (c) Nanog Nanog is a homeobox protein specically expressed in pluripotent cells and the inner cell mass of the blastocyst stage embryo (Chambers et al. 2003; Mitsui et al. 2003). Nanog-null embryos show disorganization of extraembryonic tissues at E5.5, with no discernible epiblast or primitive ectoderm (Mitsui et al. 2003). Nanog-decient blastocysts appear to be morphologically normal, but the inner cell mass produces only parietal endoderm-like cells and not epiblastderived cells when blastocysts are cultured in vitro. ES cells lacking Nanog can be derived, but they tend to differentiate into extraembryonic endoderm lineages even in the presence of LIF. Another group reported that even heterozygous Nanog mutant ES cells were unstable and susceptible to spontaneous differentiation (Hatano et al. 2005). RNAi-mediated knockdown of Nanog led to differentiation in both the mouse (Hough et al. 2006; Ivanova et al. 2006) and the human (Hyslop et al. 2005; Zaehres et al. 2005) ES cells. Importantly, overexpression of Nanog in mouse ES cells permits cells to self-renew in the absence of LIF. Likewise, overexpression of Nanog in human ES cells enabled growth without feeder cells (Darr et al. 2006). In addition, Nanog-overexpressing ES cells showed markedly increased reprogramming activity after fusion with somatic cells (Silva et al. 2006). Nanog expression is upregulated by Oct3/4 and Sox2 (Kuroda et al. 2005; Rodda et al. 2005; Wu da & Yao 2005) and suppressed
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by p53 (Lin et al. 2005), GCNF (Gu et al. 2005) and TCF3 (Pereira et al. 2006). Genome-wide chromatin immunoprecipitation analyses demonstrated that Oct3/4, Sox2 and Nanog share many target genes in both the mouse and the human ES cells (Boyer et al. 2005; Loh et al. 2006). (d) STAT3 Both the mouse and the human ES cells are usually derived and maintained on a feeder layer of mouse embryonic broblasts (MEFs) in order to promote selfrenewal. Conditioned media from MEFs can also support self-renewal of mouse ES cells, indicating that MEFs secrete soluble factor(s) that inhibit differentiation (Smith & Hooper 1987). Indeed, MEFs are known to inhibit ES cell differentiation via production of the IL-6 family cytokine, leukaemia inhibitory factor (LIF; Smith et al. 1988; Williams et al. 1988). With the addition of recombinant LIF protein into the culture medium, mouse ES cells can be cultured without MEF feeder cells. The LIF receptor is a heteromeric complex consisting of gp130 and the LIF receptor (LIFR, also known as LIFRb; Ernst & Jenkins 2004). The tyrosine kinase Janus kinase ( JAK) binds constitutively to the intracellular domain of this receptor complex in its inactive form. Upon LIF binding, JAK kinase phosphorylates tyrosine residues on both gp130 and LIFR. Phosphorylation of Y765/812/904/914 of the intracellular domain of gp130 and Y976/996/1023 of LIFR recruits signal transducers and activators of transcription (STAT ) 1 and STAT3 through their SH2 domains (Stahl et al. 1995). The STAT proteins are then activated by JAK-mediated tyrosine phosphorylation to form homodimers and/or heterodimers and translocate into the nucleus, where they function as transcription factors (Auernhammer & Melmed 2000). Among the many STAT proteins, STAT3 has been shown to be essential for the maintenance of pluripotency in mouse ES cells (Boeuf et al. 1997; Niwa et al. 1998; Matsuda et al. 1999; Raz et al. 1999). Using a tamoxifen-inducible form of STAT3 generated by fusion with the ligand-binding domain of the oestrogen receptor, it was shown that STAT3 activation is sufcient for self-renewal in the presence of foetal bovine serum (Matsuda et al. 1999). STAT3 also cooperates with Smad and Id proteins to support clonal expansion of mouse ES cells in the absence of serum (Ying et al. 2003). The role of LIF appears to be different among different species. For example, LIF cannot promote self-renewal of human or monkey ES cells (Humphrey et al. 2004; Sumi et al. 2004). Human ES cells express relatively low levels of LIF signalling components (LIFR, JAK and STAT3), and high levels of suppressor of cytokine signalling (SOCS), which negatively regulates LIF signalling (Wei et al. 2005a). In monkey ES cells, suppression of LIF signalling by a dominant negative form of STAT3 does not cause them to differentiate (Sumi et al. 2004). Thus, human and monkey ES cells appear to maintain pluripotency using a LIF/STAT3-independent mechanism. Since contamination from feeder cells represents a potential problem for transplantation therapy, it will be useful
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Pluripotency and nuclear reprogramming expression of the mesoderm marker brachyury (Yamaguchi et al. 1999). Additional experiments are required to clarify the function of Wnt/b-catenin signalling in ES cells and other stem cells. (f ) c-Myc c-Myc is a helix-loop-helix/leucine zipper transcription factor that associates with its partner protein, Max (Adhikary & Eilers 2005). c-Myc is regulated by STAT3 (Kiuchi et al. 1999) and plays important roles in self-renewal and maintenance of pluripotency in mouse ES cells (Cartwright et al. 2005): forced expression of stable c-Myc causes mouse ES cells to self-renew without LIF, whereas the dominant negative form of c-Myc induces differentiation even in the presence of LIF. GSK3b negatively regulates c-Myc activity by phosphorylation-dependent degradation by the proteasome (Sears et al. 2000). Thus, c-Myc is a common target for both the LIF and Wnt signalling pathways. c-Myc has a large number of binding sites in the genome ( Fernandez et al. 2003; Li et al. 2003; Cawley et al. 2004) and is thought to modify chromatin structure ( Knoeper et al. 2006) and activate expression of some miRNAs (ODonnell et al. 2005). (g) Klf4 Klf4 is a Kruppel-like transcription factor (also known as gut-enriched Kruppel-like factor, GKLF) (Rowland et al. 2005) originally identied as a tumour suppressor that is deleted in gastrointestinal cancers (Zhao et al. 2004; Wei et al. 2005b). A conditional knockout mouse model supports a role for Klf4 in gastrointestinal cancers (Katz et al. 2005). Klf4, however, is overexpressed in squamous cell carcinoma and breast cancers (Foster et al. 1999, 2000). Moreover, induction of KLF4 in basal keratinocytes blocks the proliferationdifferentiation switch and initiates squamous epithelial dysplasia (Foster et al. 2005). Thus, Klf4 is associated with both tumour suppression and oncogenesis. Ectopic expression of Klf4 suppresses cell proliferation, but ablation of only one of its target genes, p21, is sufcient to neutralize the cytostatic effect of Klf4 (Rowland et al. 2005). In p21-null cells, Klf4 promotes cell proliferation by downregulating p53 (Rowland et al. 2005). This may in part account for the dual function of Klf4 in cancers. In addition to its well-known expression in gastrointestinal tract, testis and skin, we found that Klf4 is highly expressed in undifferentiated mouse ES cells (Tokuzawa et al. 2004, unpublished data). We also found that inactivation of STAT3 in mouse ES cells dramatically decreases Klf4 expression and that forced expression of Klf4 enables LIF-independent selfrenewal (Tokuzawa et al. 2004, unpublished data). Another group also reported a positive effect of Klf4 in self-renewal of mouse ES cells (Li et al. 2005), and Klf4 is known to cooperate with Oct3/4 and Sox2 to activate the Lefty1 core promoter ( Nakatake et al. 2006). (h) Induction of pluripotent stem cells by four factors To test these candidates for their pluripotencyinducing activity, we have developed a system in which induction of pluripotency can be detected as
to determine the mechanisms by which feeder cells maintain pluripotency of human ES cells. (e) APC/b-catenin b-catenin is a dual-function protein that participates in cellcell adhesion by linking cadherins to the actin cytoskeleton and also acts as an intracellular signalling molecule of the canonical Wnt signalling pathway (Reya & Clevers 2005). In the absence of Wnt activation, b-catenin is phosphorylated by a complex consisting of adenomatous polyposis coli gene (APC), Axin and glycogen synthase kinase (GSK) 3b. Phosphorylated b-catenin is degraded by the ubiquitin proteasome system, thereby keeping the level of cytoplasmic b-catenin low. Upon binding of Wnt to its receptors, Frizzled and LRP5/6, GSK3b is inactivated through a poorly understood mechanism involving the direct interaction of Axin with LRP5/6, and/or the action of an Axin-binding molecule, dishevelled. As a result, b-catenin accumulates in the cytoplasm and travels to the nucleus, where it regulates transcription through association with lymphoid enhancer factor (LEF)/T-cell factor (TCF) transcription factors. Neural differentiation of mouse ES cells was attenuated by the activation of Wnt signalling, either by overexpression of Wnt1 or treatment with lithium chloride, an inhibitor of GSK3b (Aubert et al. 2002). Moreover, Wnt3a mutant mice display ectopic neural tube formation at gastrulation stage ( Yoshikawa et al. 1997). In addition, ES cells with a mutant form of APC were impaired in their ability to differentiate into the three germ layers (Kielman et al. 2002). Wnt also accelerates the proliferation of stem cells in the intestinal, epidermal and haematopoietic systems, and may be a common factor controlling stem cell proliferation (Reya et al. 2003). These data suggest that Wnt signalling promotes self-renewal of ES cells and other stem cells. Comparisons of the gene expression prole of undifferentiated and differentiated human ES cells revealed that frizzled 5 is enriched in undifferentiated ES cells (Sato et al. 2003). In addition, 6-bromoindirubin-3 0 -oxime (BIO), a newly identied pharmacological inhibitor of GSK3b, was able to maintain mouse ES cell pluripotency in the absence of LIF (Sato et al. 2004). Most notably, BIO was also able to maintain human ES cells in an undifferentiated state, and to maintain the expression of Oct3/4, Rex1 and Nanog. Given that LIF has little effect on human ES cells and culture on feeder cells may result in crossspecies contamination upon transplantation, BIO may provide a useful means of improving the culture techniques of human ES cells. How Wnt/b-catenin signalling promotes selfrenewal of mouse ES cells remains to be resolved. b-catenin is reported to upregulate the expression of STAT3 (Hao et al. 2006), and there appears to be a synergistic interaction between Wnt and LIF (Ogawa et al. 2006). On the other hand, BIO treatment permits LIF-independent self-renewal of mouse ES cells (Sato et al. 2004). This suggests that BIO may have additional functions besides GSK inhibition. In addition, it has been reported that Wnt facilitates neural differentiation of ES cells and induces the

Table 1. Pros and cons of three methods that induce pluripotency in somatic cells. use of human embryos nuclear transfer ES cell fusion iPS cell yes yes no application to human cells unknown yes unknown tumour formation yes yes yes
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chromosomal abnormalities normal tetraploid retroviral insertion c-Myc expression
marker gene expression. Fbx15 is specically expressed in ES cells and early embryos, but is dispensable for self-renewal of ES cells and development (Tokuzawa et al. 2003). We inserted a b-geo cassette (a fusion of b-galactosidase and the neomycin-resistant gene) into the mouse Fbx15 locus by homologous recombination. ES cells homozygous for the b-geo knock-in (Fbx15bgeo/bgeo) were resistant to an extremely high concentration of G418 (up to 12 mg mlK1), whereas somatic cells derived from Fbx15bgeo/bgeo mice were sensitive to the selection. Thus, it is probable that even partial induction of pluripotency would render somatic cells resistant to G418 at normal concentrations (0.3 mg mlK1). We introduced candidate genes into Fbx15bgeo/bgeo MEFs by retrovirus-mediated transfection and cultured them in ES cell medium containing G418. With any single factor, we did not obtain G418-resistant colonies. However, by combining four factors (Oct3/4, Sox2, c-Myc and Klf4), we obtained multiple G418resistant colonies. These cells resembled ES cells in both their morphology and proliferation capacity. Furthermore, when transplanted into nude mice, these induced pluripotent stem (iPS) cells produced teratomas containing tissues of all three germ layers. Thus, pluripotent cells can be generated from broblasts by introducing just a few dened factors (Takahashi & Yamanaka 2006). Fbx15-selected iPS cells do not contribute adult or germ line chimeras, indicating that their reprogramming is partial. Recently, we and two other groups reported signicant improvement in quality of iPS cells (Maherali et al. 2007; Okita et al. 2007; Wernig et al. 2007). The three groups used the same four factors, but used Nanog as a selection marker. Nanog-selected iPS cells showed more similar gene expression pattern to ES cells than did Fbx15-selected ones. These improved iPS cells also contributed to adult and germ line chimeras. These data showed that the combination of the four factors and proper selection can generate pluripotent cells that are indistinguishable from ES cells. How the four factors induce pluripotency remains elusive. Our model is that c-Myc plays crucial roles. As a potent proto-oncogene, c-Myc should contribute to rapid proliferation of iPS cells. In addition, by binding numerous positions of genome and recruiting histone acetylase complexes, c-Myc should open up chromatins (Adhikary & Eilers 2005). However, overexpression of c-Myc alone in broblasts would induce p53-dependent apoptosis or senescence. We postulate that it is Klf4 that neutralizes the adverse effect of c-Myc by suppressing p53. Oct-3/4 and Sox2 then bind to their target sites and induce pluripotency by enhancing stemness genes and suppressing
differentiation-associated genes. Niwa and associates recently showed that Klf4 also functions as a cofactor of Oct-3/4 and Sox2 ( Nakatake et al. 2006). At present, however, we do not know whether the same four factors can induce iPS cells from human somatic cells. It is possible that additional factors are required to induce human iPS cells, but more work is necessary to determine whether or not this is the case. In addition, the use of retroviral vectors and the protooncogene c-Myc poses serious safety concerns that must be resolved prior to application of human iPS cells in regenerative medicine. In fact, we showed that approximately 20% of mice derived from Nanogselected iPS cells developed tumours (Okita et al. 2007). Thus, the retroviral introduction of c-Myc should be avoided prior to clinical application. Once established, human iPS cells should also be useful in the elds of drug discovery and toxicology.
5. CONCLUSION In this review, an overview of the three currently proposed methods of inducing pluripotency in somatic cells is given. These technologies each have their own pros and cons for future clinical applications (table 1). However, rather than discussing which approach is best at present, basic research should push ahead on all fronts so that the best possible technologies may be developed in the future ( Yamanaka 2007).
6. NOTE ADDED IN PROOF After we submitted this manuscript, we and others succeeded in producing iPS cells from human broblasts with the same four factors (Takahashi et al. 2007; Yu et al. 2007; Park et al. 2008). In addition, the c-Myc retrovirus was shown to be dispensable for iPS cell generation (Nakagawa et al. 2008). Furthermore, another group reported generation of human iPS cells with a partially different combination consisting of Oct3/4, Sox2, Nanog and Lin28 ( Yu et al. 2007). These data collectively showed that nuclear reprogramming in human cells can be achieved by combinations of small numbers of factors.
The author would like to thank the members of the laboratory for their scientic, technical or administrative help.
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Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J. & Campbell, K. H. 1997 Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810813. (doi:10. 1038/385810a0) Wilmut, I., Beaujean, N., De Sousa, P. A., Dinnyes, A., King, T. J., Paterson, L. A., Wells, D. N. & Young, L. E. 2002 Somatic cell nuclear transfer. Nature 419, 583 587. (doi:10.1038/nature01079) Wu da, Y. & Yao, Z. 2005 Isolation and characterization of the murine Nanog gene promoter. Cell Res. 15, 317 324. (doi:10.1038/sj.cr.7290300) Yamaguchi, T. P., Takada, S., Yoshikawa, Y., Wu, N. & McMahon, A. P. 1999 T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specication. Genes Dev. 13, 3185 3190. (doi:10.1101/gad.13.24.3185) Yamanaka, S. 2007 Strategies and new developments in the generation of patient-specic pluripotent stem cells. Cell Stem Cell 1, 39 49. (doi:10.1016/j.stem.2007.05.012) Ying, Q. L., Nichols, J., Evans, E. P. & Smith, A. G. 2002 Changing potency by spontaneous fusion. Nature 416, 545 548. (doi:10.1038/nature729) Ying, Q. L., Nichols, J., Chambers, I. & Smith, A. 2003 BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281292. (doi:10.1016/S00928674(03)00847-X) Yoshikawa, Y., Fujimori, T., McMahon, A. P. & Takada, S. 1997 Evidence that absence of Wnt-3a signaling promotes neuralization instead of paraxial mesoderm development in the mouse. Dev. Biol. 183, 234242. (doi:10.1006/dbio. 1997.8502) Yu, J., Vodyanik, M. A., He, P., Slukvin, I. I. & Thomson, J. A. 2005 Human embryonic stem cells reprogram myeloid precursors following cellcell fusion. Stem Cells 24, 168 176. (doi:10.1634/stemcells.2005-0292) Yu, J. et al. 2007 Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 19171920. (doi:10.1126/science.1151526) Zaehres, H., Lensch, M. W., Daheron, L., Stewart, S. A., Itskovitz-Eldor, J. & Daley, G. Q. 2005 High-efciency RNA interference in human embryonic stem cells. Stem Cells 23, 299 305. (doi:10.1634/stemcells.2004-0252) Zhao, W., Hisamuddin, I. M., Nandan, M. O., Babbin, B. A., Lamb, N. E. & Yang, V. W. 2004 Identication of Kruppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene 23, 395 402. (doi:10. 1038/sj.onc.1207067)
Length of nuclear DNA in linear form=105xcell diameter Histones pack DNA to microscope dimensions One chromosome:one linear DNA molecule 2.8x108bp Linear chromosome dimensions= 10cm Even packed DNA remains available for transcription & replication Histones: small basic proteins H1, H2A, H2B, H3, H4 Low salt extraction, no Mg= beads on a string, DNA linker and nucleosomes (basic unit) linker DNA sensitive to DnaseI Physiologic salt extraction: condensed form, 30 nm diameter Nucleosome=composed of (H2A, H2B, H3, H4)2=octamer Nucleosome 147 bp, linker 10-90 bp
Lodish et al. Molecular Cell Biology 5th Edition Freeman
Isotonic buffer extraction 0.15M NaCl, 0.004 M MgCl2= structure 30 nm fiber, two start helix, left handed, H1 is also present Untranscribed chromatin adopts 30 nm fiber structure Transcribed chromatin: extended beads on a string His conservation: H3 extremely conserved, H1 more variable Variants of His: H2AX in ds DNA breaks is replacing H2, gets phosphorylated and attracts DNA repair enzymes
Modification of histone tails Each His contains a flexible N-terminus, H2A and H2B contain also flexible C-termini His tails are required to support the transition between beads-onstring to the 30 nm fiber structure The N tail of H4 contains Lys K16 which is required for contact with the next nucleosome in the fiber structure; the positive charge of K16 epsilon NH2 must interact with a negative patch at the interface between H2A and H2B Aceylation of K16 prevents this interaction and prevents fiber formation, thus allowing a structure more amenable for activation of transcription
His acetylation on other sites of H4 (Ks or Rs) increase sensitivity to DNAseI >His acetylases HAC are required for transcription of many genes Methylation Mono, di and thri-methylation of epsilon NH2 of K residues Methylation prevents change in charge by acetylation His code=reading of the modification of His residues by proteins that regulate transcription Heterochromatin protein 1= HP1 maintains heterochr. Chromodomain domain of HP1 binds to H3K9Me3 present in hetero-chromatin; these proteins have chromoshadow domains, which oligomerize with similar domains in other proteins and which methylate as HMT=histone methyl transferase H3K9
hp1 spreads heterochromatin structure due to HMT activity and interaction with chromoshadow domains of other proteins, this spreading stops at boundary regions during mitosis H3 K9 Me3 is distributed to daughter cells and there by attracting HP1 heterochromatin structure is spread in heterochr H3K9Me4, H3K27Me3 For EUCHROMATIN- bromodomain proteins bind AcetylHis TFIID has two such domains- transcription is promoted TFIID is also an acetylase which reinforces the effect K9Ac, K14Ac, in H3 K14Ac in H3
HDAC= histone deacetylase, reverses acetylation marks
HDAC inhibitors are utilized in cancer therapy
Several cancers appear to exhibit decreased acetylation
Methylation at CG- direct DNA methylation results in 5-methylcytidine at sequences CG CG methylation occurs both in promoters and in other DNA sequences Promoters of transcriptionally active genes contain a lower number of methylated CG sequences CG methylation leads to recruitment of Histone deacetylases which will diminish transcription
Chromatin remodeling complexes regulate availability of DNA to the transcriptional machinery SWI/SNF complex has an ATP-dependent helicase in the center and modifies nucleosome conformation to regulate access of transcription factors to chromatin SWI/SNF complex causes changes in position/structure of nucleosomes Gene induction by certain stimuli require SWI/SNF remodeling SWI/SNF plays a role in repressing E2F-induced genes, slowing down cell cycle, SWI/SNF cooperate with Rb SNF5 mutations/loss of function is involved in human cancer Brg1 mutations (catalytic subunit of SWI/SNF) are involved in prostate, lung and breast tumors
Embryonic Stem Cells

Mouse ES renew in Leukemia Inhibitory Factor medium Renewal requires: Nanog (homeodomein protein, Tir Na Nog:= land of ever young) Oct 4 (POU transcription factor) Sox2 and FoxD3 are Oct4 partners Nanog allows mouse ES cells to renew without LIF Nanog is a 305 amino acid protein that binds to DNA promoter at the consensus sequence C/GG/AC/GCG/CATTANG/C
Lodish et al. Molecular Cell Biology 2000, Freeman Press
In the absence of LIF, ES cells can differentiate into many cell types -embryoid bodies which lack any pattern or tissue structure ES renewal in LIF medium
When injected into adult organisms ES cells form teratocarcinomas When injected into blastocysts, ES cells can contribute to the formation of all tissues- chimerism Alberts et al., Molecular Biology of the Cell, 2002, Garland Science
Lodish et al. Molecular Cell Biology 2000, Freeman Press
Nuclei from specialized cells retain the potential to direct development of an animal - nuclear transfer in amphibians
3% of transferred nuclei result in viable clones Sheep, cow, rabbit, mouse, cat, pig, goat clones
Nature March 3, 2003 Dolly, the Sheep, died on February 14
T and B cell nuclei from peripheral node- 1,000 transplanted oocytes, 41 blastocysts, 2 ES cell lines
Hochedlinger and Jaenisch Nature 2002, 415, 1035
Human Embryo at Blastocyst Stage
Inner Cell Mass
HuES cell line
15 human ES cell lines, NIH Culture on mitotically inactivated MEFs FGF, LIF, human plasma Initially 150 h doubling time After extended passages: trisomy, chromosome 12 additions to chromosome 2
ES-derived blood progenitors are poorly functional; phenotype and not function; such progenitors resemble more yolk sac cells HOXB4 or BCR-ABL transduced ES cells generate functional blood progenitors Functional HOXB4 transduced ES-derived blood progenitors are rejected by NK cells due to low MHC-I expression
Adult Stem Cells (Tissue Derived) Plasticity ?
Hematopoietic Stem Cells
Socolovsky et al., Proc. Natl. Acad. Sci. USA 1998, 95, 6574
Expansion of HSCs on stromal cells & cytokines
CFU-S = short term blood reconstitution
LTCIC = long term blood reconstitution

Alberts et al., Molecular Biology of the Cell, 2002, Garland Science
Donor HSC-derived blood cells must be functional
Phenotype Function
Klf4+Sox2+Myc+Oct4= iPS or ES-like
Use of tet-inducible lentiviruses shows that expression of the four factors can be transient for reprogramming
Brambrink et al.
Brambrink et al.
Brambrink et al.

C1 Genome Mutations Reprograming en

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C1 Genome Mutations Reprograming en

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Universal Genetic Code

DNA Strider Program

Murine Erythropoietin Receptor Sequence

DNA Strider Program

Non-Coding DNA Frame

Frequent Stop Codons

DNA Strider Program

2/6 8/12 -5/-2 16/14 12/13 12/10 6/5

13/8 16/14 -5/-2 1/5

4/6 10/8 10/8 16/14

DNA Strider Program

Universal Genetic Code

DNA Strider Program

Plasmids-4 Kb Cosmids-40 Kb BAC, YAC 100-500 Kb Bacterial genome-2Mb

E. Coli F plasmid- BAC allows stable cloning of up to 1 million bp

Lander et al., Nature 2001, 409, 877

Lander et al., Nature 2001, 409, 877

Lander et al., Nature 2001, 409, 877

Lander et. al., Nature 2001, 409, 877

Lander et al., Nature 2001, 409, 877

Lander et al., Nature 2001, 409, 877

Methionine (Met)- rich p54 binds hydrophobic signal sequences

Lodish et al., Molecular Cell Biology, 2000, Freeman Press

Mutations are permanent, epigenetic changes are reversible

Pluripotency and nuclear reprogramming

somatic cells fusion

eggs ES cells pluripotent stem cells defined factors

Figure 1. Three methods of inducing pluripotency in somatic cells.

Pluripotency and nuclear reprogramming

chromosomal abnormalities normal tetraploid retroviral insertion c-Myc expression

Pluripotency and nuclear reprogramming

Pluripotency and nuclear reprogramming

Pluripotency and nuclear reprogramming

Pluripotency and nuclear reprogramming

Phil. Trans. R. Soc. B (2008)

Lodish et al. Molecular Cell Biology 5th Edition Freeman

Lodish et al. Molecular Cell Biology 5th Edition Freeman

Lodish et al. Molecular Cell Biology 5th Edition Freeman

HDAC= histone deacetylase, reverses acetylation marks

HDAC inhibitors are utilized in cancer therapy

Several cancers appear to exhibit decreased acetylation

Lodish et al. Molecular Cell Biology 5th Edition Freeman

Lodish et al. Molecular Cell Biology 5th Edition Freeman

Lodish et al. Molecular Cell Biology 5th Edition Freeman

Embryonic Stem Cells

Lodish et al. Molecular Cell Biology 2000, Freeman Press

Lodish et al. Molecular Cell Biology 2000, Freeman Press

Nature March 3, 2003 Dolly, the Sheep, died on February 14

Hochedlinger and Jaenisch Nature 2002, 415, 1035

Human Embryo at Blastocyst Stage

Inner Cell Mass

HuES cell line

Adult Stem Cells (Tissue Derived) Plasticity ?

Hematopoietic Stem Cells

Expansion of HSCs on stromal cells & cytokines

CFU-S = short term blood reconstitution

LTCIC = long term blood reconstitution

Donor HSC-derived blood cells must be functional

Alberts et al., Molecular Biology of the Cell, 2002, Garland Science

Alberts et al., Molecular Biology of the Cell, 2002, Garland Science

Klf4+Sox2+Myc+Oct4= iPS or ES-like

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