研究

出芽酵母細胞周期関連遺伝子の制御

30遺伝子

Gene NameTranscriptionTranslationDegradationPhosphorylationOthers
BUB2
CDC14
CDC15
CDH1NDCDK*1, CDC5*2
CLB2CDC20,CDH1*3
PDS1CDC20, CDH1*4CDK*5,pic
CLB5CDC20*6
CDC20CDH1*7
SWE1CDK,CLA4,CDC5*8, HSL1
SWI5SFFCDC4*9SRB10*10, CDK*11

その他

Gene NameTranscriptionTranslationDegradationPhosphorylationOthers
ACM1MCB, SCBCDC20, CDH1*12CDK*13
ALK1SFFCDH1?(200-KEN,224-Dbox,16855400?)
ALK2CDH1?(116-KEN,150-Dbox,16855400?)
CDC5?SFFCDH1*14CDK*15
CIN8HCM1(16912276?)CDH1*16
FIN1CDH1*17CDK*18
HSL1MCB, SCBHOG1*19CDC20,CDH1*20
MPS1CDC20,CDH1

細胞周期用語集

  • R-X-X-L-X-X-X-X-[N/D/E] Destruction (D) box APC_Cdc20の標的モチーフ
  • K-E-N (KEN box) APC_Cdh1の標的モチーフ*21 KEN → AAA
  • [S/T]-P-X-[K/R] CDKのより厳密なコンセンサス
  • [S/T]-P CDKの緩いコンセンサス
  • [D/E]-X-[S/T]-Phi-X-[D/E] Polo kinaseのコンセンサス(where 'Phi' denotes a hydrophobic residue)
  • S-[pT/pS]-[P/X] Polo Box binding (PBB) site
  • SCF_Cdc4の標的
    • Sic1, Swi5
  • APC_Cdc20の標的
    • Clb5, Clb2, DBF4*22,Pds1 (Securin)
  • APC_Cdh1の標的
    • BUB1*23,Clb1, Clb2, Cdc5, Cin8, Kip1, Hsl1, Cdc20, Ase1, Fin1, Pds1p
    • A simple database search of budding yeast sequences reveals 92 proteins containing matches to both a basic D box (RxxLxxxx?(NDEQ)) and a basic KEN box (KENxxx(NDEQ)), in 32 of which the two motifs are separated by fewer than 100 amino acids.(11562348?)
  • Skaar JR, Pagano M., Cdh1: a master G0/G1 regulator., Nat Cell Biol. 2008 Jul;10(7):755-7.
    • The current study verifies previous models of Cdh1 function in the cell cycle, strengthens the case for Cdh1 as a tumour suppressor and validates a role for Cdh1 in the nervous system.
    • It is apparent that Cdh1 loss causes genomic instability, but the precise cause of this instability is unclear.
 

Selected binding sites for cell-cycle transcription factors (TFs)

Budding yeastFission yeast
NamePhaseBinding site*1TFBinding site*1TF
MCBG1/SACGCGTMBFACGCGTMBF
SCBG1/SCRCGAAA*2SBFNot describedNot described
SFF/ForkheadG2/MMcm1*3 + GTAAACAA*2Mcm1 + Fkh2GTAAACAAbSep1? Fkh2?
PCBG2/MNot describedNot describedGNAACRPBF, Mbx1?
ECBM/G1YAATTA + Mcm1*3Yox1/Yhp1 + Mcm1Not describedNot described
Swi5/Ace2M/G1ACCAGCR*2Swi5, Ace2ACCAGCCNT*2Ace2
  1. Different versions of binding sites have been described, and typical sequence patterns were selected for this table to represent each binding site.
  2. These sites are also present in reverse orientation.
  3. A typical Mcm1 site is: TTWCCYAAWNNGGWAA.

N is any base; R is A or G; Y is C or T;Wis A or T.

Bähler J., Cell-cycle control of gene expression in budding and fission yeast., Annu Rev Genet. 2005;39:69-94.

細胞周期で制御されている遺伝子はいくつあるのか?

これの答えは意外に難しい。

制御の方法は?

  1. 転写
  2. 翻訳
  3. リン酸化
  4. 分解

調べる方法は?

  • マイクロアレイ解析
  • ChIP-chip解析
  • ゲノム塩基配列から保存された配列を抽出
  • プロテオーム解析

細胞周期を同調させる方法は?

  1. Induced synchrony, which forces cells to synchronize by some intervention such as pheromone arrest or the use of temperature-sensitive cdc mutants
  2. Selection synchrony, which selects a cohort of cells at the same cell cycle stage, as in the elutriation method
  3. Yeasat Metabolic Cycle (YMC) synchrony, which is a naturally synchronized continuous yeast culture proceeds through 5-h metabolic cycles
 

どんな制御因子があるのか?

  1. 転写制御
    • SWI4
    • SWI6
    • MBP1
    • HCM1
    • MCM1
    • FKH2
    • NDD1
    • SWI5
  2. リン酸化制御
 

マイクロアレイ解析?によるゲノムワイドな細胞周期転写制御の解析

マイクロアレイによる細胞周期によって制御される転写産物の同定の始まり

  • Cho RJ, Campbell MJ, Winzeler EA, Steinmetz L, Conway A, Wodicka L, Wolfsberg TG, Gabrielian AE, Landsman D, Lockhart DJ, Davis RW., A genome-wide transcriptional analysis of the mitotic cell cycle., Mol Cell. 1998 Jul;2(1):65-73.
    ゲノムワイドなマイクロアレイと温度感受性変異による同調をつかって、細胞周期で制御される416の転写産物を手動データ解析により同定。この論文は(こちらの方が先に発表されたにもかかわらず)、下のSpellmanの論文ほど引用されない(私も今回調べてみて始めてこの論文の存在を知った)。
  • Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Eisen MB, Brown PO, Botstein D, Futcher B., Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization., Mol Biol Cell. 1998 Dec;9(12):3273-97.
    上の論文とやっていることは同じなのだが、クラスタリングやフーリエ解析を利用したデータ解析により800の遺伝子を同定。2番煎じになってしまったため、MCBになってしまったがデータ解析に優れているためこちらの方がより引用されている。ちなみにこちらの解析では、上の論文の416の遺伝子のうち304のみが細胞周期で変動するとされた。
     

最近の解析結果

  • Rowicka M, Kudlicki A, Tu BP, Otwinowski Z., High-resolution timing of cell cycle-regulated gene expression., Proc Natl Acad Sci U S A. 2007 Oct 23;104(43):16892-7.
    • 酵母の代謝周期(Yeast Metabolic Cycle:YMC)によって同調した細胞のマイクロアレイ解析と、最大エントロピーデコンボリューション法をつかってかなり高解像度に細胞周期によって変化する転写産物(Cell Cycle Regulated Transcript: CCRT)を同定している。おそらく現在一番高解像度な転写マップ。
    • 同定できたCCRT(Rowicka's CCRT)は、1,129個!
       
      F4.large.jpg
       

  • Orlando DA, Lin CY, Bernard A, Wang JY, Socolar JE, Iversen ES, Hartemink AJ, Haase SB., Global control of cell-cycle transcription by coupled CDK and network oscillators., Nature. 2008 Jun 12;453(7197):944-7.
  • Simmons Kovacs LA, Orlando DA, Haase SB., Transcription networks and cyclin/CDKs: the yin and yang of cell cycle oscillators., Cell Cycle. 2008 Sep 1;7(17):2626-9.
    • clb1,2,3,4,5,6, GAL-CLB1株を用いてサイクリンの発現を止めてしまっても、転写の周期は起き続ける!ーCDKによる生化学振動子のほかに転写の振動子があるという発見。
      データを見ると、振動してはいない。順番に転写活性化が起きているだけ。「振動子がある」というのは言い過ぎ。
       
      nature06955-f4.2.jpg
       

 

出芽酵母のゲノムを細胞周期制御モチーフからみる

SGDのPattern Matchingを使って、細胞周期制御に関わる蛋白質のモチーフを検索する(Sep.30 2008)

MotifUnique hitTotal hit
KEN box (KEN)8711025
D-box (RxxLxxxx?[DEN])16862246
D-box (RxxLxxxx?[DENQ])19512741
CDK ([ST]Px[KR])14402108
Moc1 (LFP)498529
Moc2 ([ST]Pxx[KR])16392254
Total ORF5884
 

Mocにあるように適当な配列を用いて検索をかけても、多くの蛋白質がヒットする。単純にモチーフからだけではどの遺伝子が制御されているのか結論を出すのは難しい。そこで、これらの組み合わせ(Rowicka's CCRT, CDK, D_hit, KEN_hit)で見てみる。

 
NameCCRTCDKD_hitKEN
ACC1G1411
AFR1M111
ALK1*G2111
ALK2*S211
ATF1G2/M111
BFR1S111
BNR1G1/S211
BUD3G2521
CDC20*M111
CDC5*G2121
CSR1G1(P)111
ERM6G2111
FAA3G2/M111
FKH2G1(P)311
GPT2G2/M111
HPC2S411
HSL1*S243
IQG1*M432
ISW1G1(P)133
KAR3S221
KEL1S212
KEL2G1/S111
KIP1S111
LTE1G1(P)811
MMS4G1/S311
MPS1*M111
MSC7G1(P)111
MYO2S111
NTE1S111
NUM1G12213
PDS1*S311
SGS1S112
SIR4G2511
SKM1G1/S111
SPC72G1111
SPT16M/G1112
STH1S113
STU1G1/S111
STU2S111
SUM1G1(P)111
SVL3S121
SWI4M211
SWI5G2/M811
TOS2G1/S111
TRA1G1(P)454
UBP7G1131
ULS1M/G1212
VAC17G2211
YCS4S111
YHP1S211
YLR057WG1(P)111
 *CDH1のターゲットであることがわかっている遺伝子。

Morgan's CDK substrate

ProteinClb5/Clb2Biological Process
Ace21.3Required for G1-specific transcription
Ady33.4Protein of unknown function
Arg10.7Argininosuccinate synthetase
Ase115.8Microtubule binding protein required for spindle stability
Ash11.7Daughter cell-localized transcription factor
Atg200.5Protein of unknown function
Axl22.0Required for axial budding pattern
Bbp13.1Spindle pole body component
Bck20.4Protein involved in the SIT4 pathway for CLN activation
Bem10.9Required for cell polarization and bud formation
Bem31.5GTPase-activating protein Cdc42p and Rho1p
Bna31.7Arylformamidase
Bni14.3Involved in cytoskeletal control
Bsp12.2Organization of actin cytoskeleton
Bud40.4Required for axial budding
Bud60.4Required for bipolar budding
Caf1201.2Transcriptional regulation
Cdc202.3APC-activating factor in anaphase
Cdc51.1Protein kinase required for mitotic exit
Cdc6758.6pre-replicative complex formation and maintenance
Cdh15.6APC activating factor in M/G1
Chs20.4Responsible for primary septum disk during cytokinesis
Cnn145.7Protein of unknown function
Csr20.6Protein of unknown function
Cst91.4Required for synaptonemal complex formation
Cyc20.8Cytochrome-c mitochondrial import factor
Dbf22.2Protein kinase required for mitotic exit
Dbf202.4Protein kinase required for mitotic exit
Dbf41.9Regulatory subunit for Cdc7p protein kinase
Ddc113.1DNA damage checkpoint protein
Dna20.6DNA helicase
Dna431.5DNA replication initiation / DNA strand elongation
Dnf21.2Protein of unknown function
Dpb210.5DNA polymerase epsilon subunit
Eco12.5Required for establishment of sister chromatid cohesion
Elm10.7Protein kinase regulating pseudohyphal growth
Far13.3Cln-specific Cdk1 inhibitor
Fin113.8Intermediate filament protein
Fkh23.5Transcription factor regulating G2/M gene expression
Fmt11.6Mitochondrial methionyl-tRNA transformylase
Frt220.3ER protein of unknown function
Fun210.7Protein of unknown function
Fun300.9Involved in resistance to UV radiation
Fus13.0Required for cell fusion during mating
Gin41.9Protein kinase required for septin organization
Hcm12.0Member of the forkhead family of DNA-binding proteins
Hdr10.8Protein involved in meiotic segregation
Hos41.0Protein of unknown function
Icy20.8Protein of unknown function
Ifh10.9Involved in regulation of ribosomal RNA expression
Ino21.3Transcription factor required for derepression of phospholipid synthetic genes
Ipl11.8Protein kinase involved in chromosome segregation
Jsn11.7RNA-binding protein
Kar35.9Kinesin-like protein required for proper spindle assembly
Kar94.8Involved in mitotic spindle orientation
Kel15.1Involved in cell fusion and morphology
Kel20.4Protein involved in cell fusion and morphology
Kip20.7Kinesin-like protein required for proper spindle assembly
Kip32.1Kinesin-like protein required for proper spindle assembly
Lre11.5Protein of unknown function
Lrs42.5Involved in rDNA silencing
Lte10.3GDP exchange factor involved in mitotic exit
Luv11.6Involved in protein sorting in the late Golgi
Mcm20.8Subunit of MCM complex at origins of DNA replication
Mcm316.2Subunit of MCM complex at origins of DNA replication
Mcm60.7Subunit of MCM complex at origins of DNA replication
Mih16.1Phosphatase that dephosphorylates Y19 on Cdk1
Mlp10.7Myosin-like protein
Mmr12.3Protein of unknown function
Mms40.8DNA damage repair protein
Mob10.8Regulatory subunit of Dbf2, required for mitotic exit
Mpc542.0Component of the meiotic plaque
Mps218.3Required for spindle pole body assembly and normal chromosome segregation
Mpt11.4Component of RNA polymerase II transcription factor TFIID
Msb11.5Required for establishment of cell polarity
Msh61.0DNA repair protein
Nbp11.7Essential protein required for G2/M transition
Ndd12.6Interacts with Fkh2 to control G2/M gene expression
Net10.4Control of the Cdc14 phosphatase
Nth13.6Neutral trehalase
Nup10.9Nuclear pore protein (nucleoporin)
Nup602.7Nuclear pore protein (nucleoporin)
Orc12.4Required in pre-replicative complex formation
Orc226.9Required in pre-replicative complex formation
Orc667.9Required in pre-replicative complex formation
Pak11.7Kinase that suppresses DNA pol. alpha mutations
Pcl71.0Pho85 cyclin
Pin21.2Protein of unknown function
Pkh21.2protein kinase with similarity to 3-phosphoinositide-dependent protein kinase
Plm21.5Required for plasmid maintenance
Pol11.0DNA polymerase I alpha subunit
Pom1521.0Nuclear pore membrane glycoprotein
Ptk20.6Protein kinase required for polyamine transport
Pxl10.9Involved in polarized growth
Rad90.5DNA damage checkpoint protein
Rav11.9Involved in regulation of (H+)-ATPase in vacuolar membrane
Rbs11.3Protein of unknown function
Rfc10.4Subunit of RFC complex required for DNA replication
Rlf21.1Involved in nucleosome assembly
Rrp61.7Involved in 5.8S ribosomal RNA 3' end processing
Rts12.0PP2A regulatory subunit
Sac71.3GTPase-activating protein for Rho1p
Sec31.7Component of exocyst complex
Sfb30.9Involved in transport of protein from the ER to the Golgi
Sld214.2Required for initiation of DNA replication
Sld32.1Involved in initiation of DNA replication
Slk191.8Kinetochore protein required for spindle assembly
Smc42.7Subunit of the condensin protein complex
Spa20.8Involved in cell polarity and cell fusion during mating
Spc11054.2Spindle pole body component
Src10.2Protein of unknown function
Srm11.5GDP/GTP exchange factor for Gsp1p and Gsp2p
Ssk10.6Involved in osmosensory signal transduction
Stb12.5Involved in transcription regulation at Start
Stu20.9SPB component regulatin microtubule dynamics
Swe11.7Protein kinase that phosphorylates Cdk1 on Y19
Swi52.4G1-specific transcription factor
Swi61.0G1-specific transcription factor
Tfg13.3RNA polymerase II transcription initiation factor TFIIF (factor g)
Tus10.6Protein with similarity to GDP-GTP exchange factors
Ufe11.3t-SNARE homolog of the endoplasmic reticulum
Ulp25.3Degrades conjugated ubiquitin-like protein Smt3p
Whi51.6Regulation of progression through Start
Xbp11.9Stress-induced transcriptional repressor
YDL025C0.9Protein kinase of unknown function
YDL089W0.6Protein of unknown function
YDR348C1.8Protein of unknown function
Yen1364.4Protein of unknown function
YER158C1.2Protein of unknown function
YGR035C0.5Protein of unknown function
YHL035C1.2Protein of unknown function
YHR149C1.0Protein of unknown function
YJL084C1.3Protein of unknown function
YJR054W4.8Protein of unknown function
YKR078W0.3Protein of unknown function
YLR187W1.5Protein of unknown function
YLR278C0.9Protein of unknown function
YML119W0.5Protein of unknown function
YNL058C1.2Protein of unknown function
YNL321W3.1Member of the calcium permease family of membrane transporters
YNR047W4.3Protein kinase of unknown function
YOL036W2.6Protein of unknown function
YOL070C0.6Protein of unknown function
YOR066W1.9Protein of unknown function
YOR315W0.5Protein of unknown function
YPL267W1.6Protein of unknown function
Yrf1-31.4Protein of unknown function
Yrf1-60.7Protein of unknown function
Yta71.4Protein of unknown function
Zip13.4Structural protein of the synaptonemal complex

FrontPage

2010-01-24 (日) 00:00:00

*1 localization: S-16,42,176,227,239
*2 18500339?~ activity: S-215,259
*3 Wäsch R, Cross FR., APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit., Nature. 2002 Aug 1;418(6897):556-62. 25-Dbox,85-,100-KEN
*4 8-KEN, 85-Dbox
*5 ., , . ;:.
*6 34-Dbox
*7 tow-Dbox,585-KEN
*8 Asano S, Park JE, Sakchaisri K, Yu LR, Song S, Supavilai P, Veenstra TD, Lee KS., Concerted mechanism of Swe1/Wee1 regulation by multiple kinases in budding yeast., EMBO J. 2005 Jun 15;24(12):2194-204.
*9 Kishi T, Ikeda A, Koyama N, Fukada J, Nagao R., A refined two-hybrid system reveals that SCF(Cdc4)-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry., Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14497-502.
*10 Kishi T, Ikeda A, Koyama N, Fukada J, Nagao R., A refined two-hybrid system reveals that SCF(Cdc4)-dependent degradation of Swi5 contributes to the regulatory mechanism of S-phase entry., Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14497-502. CDC4 interaction: S-225,231,246,250,261,300,T-320,323
*11 NLS: S-552,646,664
*12 Enquist-Newman M, Sullivan M, Morgan DO., Modulation of the mitotic regulatory network by APC-dependent destruction of the Cdh1 inhibitor Acm1., Mol Cell. 2008 May 23;30(4):437-46.
*13 Ostapenko D, Burton JL, Wang R, Solomon MJ., Pseudosubstrate inhibition of the anaphase-promoting complex by Acm1: regulation by proteolysis and Cdc28 phosphorylation., Mol Cell Biol. 2008 Aug;28(15):4653-64.~Enquist-Newman M, Sullivan M, Morgan DO., Modulation of the mitotic regulatory network by APC-dependent destruction of the Cdh1 inhibitor Acm1., Mol Cell. 2008 May 23;30(4):437-46.~ Stability&localization: S-3,31,48,161,T-161
*14 Schwab M, Neutzner M, Möcker D, Seufert W., Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC., EMBO J. 2001 Sep 17;20(18):5165-75.~Visintin C, Tomson BN, Rahal R, Paulson J, Cohen M, Taunton J, Amon A, Visintin R., APC/C-Cdh1-mediated degradation of the Polo kinase Cdc5 promotes the return of Cdc14 into the nucleolus., Genes Dev. 2008 Jan 1;22(1):79-90.~ N70, 35-KEN
*15 Mortensen EM, Haas W, Gygi M, Gygi SP, Kellogg DR., Cdc28-dependent regulation of the Cdc5/Polo kinase., Curr Biol. 2005 Nov 22;15(22):2033-7.~ Activation: S-242
*16 Hildebrandt ER, Hoyt MA., Cell cycle-dependent degradation of the Saccharomyces cerevisiae spindle motor Cin8p requires APC(Cdh1) and a bipartite destruction sequence., Mol Biol Cell. 2001 Nov;12(11):3402-16.~ 35-Dbox,970-KEN
*17 Woodbury EL, Morgan DO., Cdk and APC activities limit the spindle-stabilizing function of Fin1 to anaphase., Nat Cell Biol. 2007 Jan;9(1):106-12.~ 8-Dbox
*18 Woodbury EL, Morgan DO., Cdk and APC activities limit the spindle-stabilizing function of Fin1 to anaphase., Nat Cell Biol. 2007 Jan;9(1):106-12.~ S-36,54,117,148,T-68
*19 Clotet J, Escoté X, Adrover MA, Yaakov G, Garí E, Aldea M, de Nadal E, Posas F., Phosphorylation of Hsl1 by Hog1 leads to a G2 arrest essential for cell survival at high osmolarity., EMBO J. 2006 Jun 7;25(11):2338-46.
*20 Burton JL, Solomon MJ., D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p., Genes Dev. 2001 Sep 15;15(18):2381-95.~ 94-Dbox,775-,812-KEN,
*21 Pfleger CM, Kirschner MW., The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1., Genes Dev. 2000 Mar 15;14(6):655-65.
*22 Sullivan M, Holt L, Morgan DO., Cyclin-specific control of ribosomal DNA segregation., Mol Cell Biol. 2008 Sep;28(17):5328-36.
*23 Mammal,Qi W, Yu H., KEN-box-dependent degradation of the Bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome., J Biol Chem. 2007 Feb 9;282(6):3672-9.