HP1のリン酸化修飾制御とその機能の解明

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Nagoya City University Academic Repository

学位の種類博士 (生体情報) 報告番号甲第 1500 号学位記番号第 13 号氏名西淵剛平授与年月日平成 27 年 3 月 25 日学位論文の題名 HP1 のリン酸化修飾制御とその機能の解明論文審査担当者主査：中山潤一副査：湯川泰, 田上英明, 大隅圭太

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HP1

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1 3 1-1 4 1-2 6 1-3 8 1-4 Heterochromatin Protein 1 11 1-4-1 12 1-4-2 13 1-4-3 N 14 1-4-4 HP1 14 1-4-5 HP1 16 1-5 17

HP1α

18 2-1 19 2-2 20 2-2-1 20 2-2-2 20 2-2-3 20 2-2-4 21 2-2-5 22 2-2-6 in vitro 23 2-2-7 FLAG 23 2-2-8 24 2-2-9 25 2-3 26 2-3-1 HP1α 26 2-3-2 HP1α 27 2-3-3 29 2-3-4 HP1α N 31

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HP1α

39 3.1 40 3.2 41 3-2-1 41 3-2-2 41 3-2-3 41 3-2-4 42 3-2-5 FLAG 42 3-2-6 in vitro 42 3-2-7 42 3-2-8 43 3-2-9 43 3.3 44 3-3-1 HP1 44 3-3-2 HP1α 45 3-3-3 HP1α 46 3-3-4 51 3-3-5 HP1α 52 3.4 54 56 59

64

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DNA DNA DNA H3 9 (H3K9me) H3 4 (H3K4me) . H3K9me Heterochromatin Protein 1 (HP1) . HP1 α β γ HP1 H3K9me HP1 HP1α

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H3K9me . HP1α . HP1 . HP1α HP1α DNA H3K9me HP1α HP1α N HP1α DNA α H3K9 H3K9me . HP1α . α . . HP1 . HP1 . H3K9 10 . HP1α HP1 HP1 HP1α .

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1-1

DNA DNA H2A H2B H3 H4 146 bp DNA ( 1-1) DNA . DNA C . N . ( 1-2) . . H3 9 (H3K9me) Heterochromatin Protein 1 (HP1) .

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HP1 2 3 HP1 5 . 1-1 H2A H2B H3 H4 2 . 8 DNA 146bp ( 1 α) 1-2 リン酸化アセチル化メチル化 (アルギニン) メチル化 (活性化リジン) メチル化 (不活性化リジン) ユビキチン化

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1-2

1879 W. Flemming α ( ) (3) 1928 E. Heitz (3,4) ( DAPI DNA 1-3 ) (constitutive heterochromatin) X

(facultative heterochromatin) DNA

. (4) . 1930 H.J. Muller X (3,4) White . PEV (position effect variegation) (3,4) . PEV 50 . . % ( ) DNA DNA DNA bp M bp M bp

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% (6) . 1-3 NIH3T3 DAPI ( 5 )

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1-3

. 1884 A. Kossel DNA . 10 1964 V. Allfrey (7) . . α (8) .

(histone acetyl transferase: HAT) 1990

. 1996 D. Allis α HAT (9) HAT Gcn5 Gcn5 HAT DNA DNA 1994 G. Reuter PEV 1 Su(var)3-9 C . (10) HOX

Enhancer of zeste: E(z)

Trithorax .

SET 2000

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SET . (11) . SET H3K9 . E(z) H3K27 Trithorax H3K4 (12) SET SET

− (me) − (me2) (me3) ( 1-4) (13) 1-4 (A) (B) !"# $!_%& !'( $ ! ) ) "*+*,-./012 3145+. 3145+. $!_'& $ ! ) ) !$ !_%$ $ ! ) ) 6785+5+. $!%& "*+*,-./012 6785+5+. (!_' !$ !$ $ ! ) ) $!_%& !"# 91+--./75:;2<=5,-./012 6785+5+. (!' !$ !$ $ ! ) ) $!& (!' *7 641--./75:;2<=5,-./012 6785+5+. (!_' !$ $ $ ! ) $!% & !_'( ) !"# $!& !'( $ ! ) ) >5,-./012 3145+. (!_' !"# !"# $& !'( $ ! ) ) #75,-./012 3145+. (!_' (!' A B

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H3K9 H4K20 H3K9 (H3K9me) . HOX H3K27 H3K27 H3K27 H3K4 H3K36 H3K4 H3K36 (14) ( ) H3K9me HP1 (Heterochromatin protein 1)

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1-4 Heterochromatin protein 1 (HP1)

HP1 α (15) HP1 PEV Su(ver)2-5 . (16) HP1 . HP1α,-β,-γ ( CBX5,-1,-3 ) 3 HP1 ( 1-5) (CD) (CSD) CD N CD CSD HP1 HP1 HP1 HP1 1-5 HP1 ( ) HP1 ( ) HP1

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1-4-1 (CD)

(Chromodomain) Chromatin organization modifier domain

HOX Pc (Polycomb) HP1 (17) CD HP PEV Su(var)3-9 H3K9 HP1 CD H3K9 . (18-20) . CD 3 (aromatic cage) ( 1-6A) (21) HP1 H3K9me HP1 CD 60% Pc CD H3K27me . (22) HP1 HP1 CD H3K9me (Kd ) µM (23) GFP HP1 α FRAP ( ) HP1 . (24) HP1 . . (25) H3K9me H3S10 CD α . (H3S10phos) HP1 H3K9me (23, 26) . HP1α α HP1α H3K9me, S10phos (27,28) HP1α (25). HP1

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1-4-2 (CSD) HP1 CSD CD (29) . CSD . ( 1-6B) (30) 1 CSD PXVXL (X ) HP1 . (31) 1-6 HP1 CD CSD (A) HP1α H3K9me3 (PDB:3FDT) CD K9me3 (B) HP1β CSD CAF1 (PDB:1S4Z) CSD PXVXL CAF1 32 α

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1-4-3 N CD N CD CSD . N ( 1-5) CD α CD (35) HP1a Swi6 HP1α N (33-35) HP1 (36-40) HP1 RNA DNA . . HP1 (38) 1-4-4 HP1 3 HP1 N SUMO SUMO ( 1) (41,43) (35) HP1α 11 14 H3K9me HP1α

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1 H P 1 ( 32 )

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1-4-5 HP1 α DNA α HP1 α . (40,48-50) H3K9 D. Canzio Swi6 α (48) α Swi6 H3K9me . α Swi6 . α Swi6 H3K9me . α HP1α α . (49) HP1β α H3K9me (50) α HP1 HP1

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1-5

in vitro α α α α . HP1α . (35) HP1 .

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2-1

HP1α α CD N S11-14 HP1α . (35) . . HP1a Swi6 2

(Casein Kinase 2: CK2) HP1α in vitro

CK2 (33-35) . HP1α . α HP1 HP1α HP1α α

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2-2

2-2-1 α

HeLa . RNA SuperScript Reverse Transcriptase (Invitrogen) α

cDNA HP1α (NM_012117) HP1β (NM_006807)

HP1γ (BAA_83340) cDNA PfuTurbo (Agilent) α TOPO-TA

cloning kit (Invitrogen) α pCRII His

HP1 cDNA pCold I

(TaKaRa) FLAG HEK293T

HP1α cDNA pFLAG-C1 (35)

CK2 pRSFDuet (Novagen) Ck2a Ck2b

α (35)

α PCR site-directed mutagenesis α

2-2-2 α

HeLa HEK293T U2OS IMR90 10% FBS (GIBCO)

( ) DMEM ( )

QIAGEN midiprep kit α Lipofectamine 2000 (Invitrogen) α

. 24 48

2-2-3

RIPA buffer (50 mM Tris-HCl[pH8.0], 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, Protease inhibitor cocktail [Complete EDTA-free; Roche], 10 mM NaF) 4 12,000 rpm 10

50 µl (0.6 mg/ml) 2x alkaline-phosphatase reaction buffer (100 mM Tris-HCl [pH 9.0], 10 mM MgCl2, and 60 units/ml shrimp alkaline phosphatase [SAP; TaKaRa]) 3

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SDS-PAGE 5 mM EDTA Transfer buffer (25 mM Tris, 190 mM Glycine, 20% Methanol) 20 EDTA Transfer buffer

15 PVDF (Immobilon-P ; Millipore)

(BIO-RAD) α 5%

/TBS-T (20 mM Tris-HCl [pH7.5], 500 mM NaCl, 0.05% Tween 20)

1 TBS-T 3

ECL (GE Healthcare) LAS300mini α α

anti-HP1α (BMP001 ; MBL) anti-HP1β (1MOD-1A9; Millipore) anti-HP1γ (2MOD-1G6; Millipore) anti-CK2α (1AD9; Calbiochem) anti-α-tubulin (T5168; Sigma)

peroxidase-conjugated anti-histone H3 (ab21054; Abcam) peroxidase-conjugated anti-FLAG M2 (A8592; Sigma) peroxidase-conjugated anti-rabbit IgG (A6667; Sigma)

peroxidase-conjugated anti-mouse IgG (112-035- 072; Jackson ImmunoResearch) 2-2-4 2-3A 2-2-4a BL21(DE3) 100 µg/ml xYT 5 ml 2xYT 37 1 L 37 OD595 0.5 0.6 30 200 µM IPTG 15 24

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2-2-4b His

Extraction buffer (50 mM Sodium phosphate [pH7.0], 300 mM NaCl, 0.5% NP-40, 1 mM Phenylmethylsulfonyl fluoride [PMSF])

TALON (Clontech) 1 4

Wash buffer (50 mM Sodium phosphate [pH7.0], 300 mM NaCl, 5 mM Imidazole) α . Elution buffer (50 mM Sodium phosphate [pH7.0], 300 mM NaCl, 150 mM Imidazole)

SOURCE 15Q (GE Healthcare) α 50 µg/ml 25 µg/ml 2-2-5 2-2-5a (S100) (NE) Dignam (51) 5 Buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.5 mM Dithiothreitol [DTT]) 4 2,000 rpm 10 2 10 20 15 ml 2,000 rpm 10 MLS50 (Beckman) α 4 14,000 rpm 20 ( ) 0.11 Buffer B (0.3M HEPES [pH 7.9], 1.4 M KCl, 30 mM MgCl2) 4 36,000 rpm 1 -80 3 Buffer C (20

mM HEPES [pH 7.9], 0.42 M NaCl, 25% Glycerol, 1.5 mM MgCl2, 0.2 mM EDTA,

0.5 mM PMSF, 0.5 mM DTT) 10

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16,000 rpm 30 (SPECTRUMLABS, MWCO: 6-8,000)

50 Buffer D (20 mM HEPES [pH 7.9], 0.15 M KCl, 10% Glycerol, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) 1

2 1.5 ml 15,000

rpm 20 -80

2-2-5b

HeLa . S100 NE Buffer Q (20 mM HEPES [pH 7.9], 10% glycerol, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT)

HiTrap Q HP (GE Healthcare) 0 1 M (KCl)

Superose 6 Column (GE Healthcare) α

2-2-6 in vitro

in vitro 1 µg HP1α (N . CD [1-80aa])

5 µl 25 µl Kinase reaction buffer (20 mM Tris-HCl [pH 7.5], 50 mM KCl, 10 mM MgCl2, 200 µM ATP) 3 30 50 units CK2 (NEB) α CK2 50 µM 4,5,6,7,-tetrabromobenzotriazole (TBB; Chemicon) 2-2-7 FLAG α 2-2-7a FLAG HP1α HEK293T (10 cm2 _{1 )} 24 PBS 1 ml IP buffer (50

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rpm 10 IP buffer 200 µl Sepharose 4B (GE

Healthcare) 30

IP 50 µl (50% ) ANTI-FLAG M2

Affinity Gel (SIGMA) 3

IP buffer

FLAG elution buffer (250 µg/ml 3xFLAG peptide / IP buffer)200 ul 1 α 2-2-8 α H2A, H2B, H4 (52) H3 9 (K9C) 110 (C110A) K9C (H3Kc9me3) (53) C110A α (H3K9me0) 193 bp-601 DNA (52) 1 µg streptavidin T1 Dynabeads 80 µg (Invitrogen) Binding buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 5% glycerol, 0.05 mM EDTA, 0.1 mg/ml BSA, 1 mM DTT)

4 Binding buffer

100 pmol HP1 .

FLAG-HP1α 4 1 3

Wash buffer (10 mM Tris-HCl [pH 7.5], 300 mM NaCl, 0.1% Triton X-100, 5% glycerol, and 0.05 mM EDTA) SDS

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2-2-9

10 µl Gelshift buffer (20 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM DTT, 0.1

mg/ml BSA) HP1 0.2 pmol 193 bp-601

DNA 37 15 1 µl 30%

0.5 x Tris-borate-EDTA (TBE) 5% native polyacrylamide gels 100 V 1.5 SYBR Gold (Invitrogen) LAS3000 mini (GE Healthcare) α ImageQuant software α

fraction bound = 1 - fraction unbound α Igor Pro software α Curve-fitting

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2-3

2-3-1 HP1α

HP1α

. (35) HP1α .

. .

HeLa HEK293T U2OS IMR90 .

SDS-PAGE Phos-tag SDS-PAGE HP1α α ( 2-1A) Phos-tag . HP1α . HP1α 11 14 HP1α (S11-14A) ( 2-1B) (WT) S11-14A . ( 2-1C) 2-1 HP1α (A) HP1α SAP HP1α (B) FLAG HP1α N α (C) HP1α

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2-3-2 HP1α HP1a Swi6 HP1α 2 (Casein Kinase 2: CK2) . (33,34) HP1α in vitro CK2 . (35) CK2 α,β . CK2 100 CK2 (54) HP1 CK2 HP1α HP1α N . CD (1-80aa) α HP1α HeLa . (S100) (NE) ( 2-2A,B) S100 200 500 kDa . ( 2-2C) CK2 . . CK2α α CK2 ( 2-A,B,C) CK2 TBB ( 2-2D) . HP1α CK2 .

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2-2 HP1α N

(A,B) HeLa . (A) (B)

in vitro CK2

α WB (C) A 12-17

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2-3-3 α HP1α HP1α 2-3-2 CK2 HP1α HP1α His ( 2-3A) SDS-PAGE CBB WB CK2 . . ( 2-3B) HP1α . A

B 2-3 (A) (B) CK2 CK2 CK2

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Phos-tag SDS-PAGE HP1α . . ( 2-4A) HP1α α in vitro . 97 (35) S11-14 S97A ( 2-4A) HP1α,-β,-γ ( 2-4B) HP1α-HM (KRK89-91AAA, KKK104-106AAA) ( 2-4C,D) 2-4 HP1 (A) 2-5,6 α WT S97A (B) 2-5,6 α HP1α,-β,-γ (C) HP1α Hinge mutant ; HM (D) 2-7 α HP1α-HM

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2-3-4 HP1α N HP1 CD α H3K9me . Swi6 α H3K9me (48) HP1α N . DNA α H3K9me3 HP1α,-β,-γ α ( 2-5A,B) HP1α H3K9me3 . Swi6 (48) HP1β H3K9me3 . HP1β (50) HP1α α HP1α H3K9me3 . ( 2-5C,F) S97A α H3K9me3 . ( 2-5C) . HP1α H3K9me3 . ( 2-5E-G) CK2 HP1α N H3K9me3 ( 2-5E,F,G)

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2-5 HP1 H3K9 K9me3 ( H3unmod H3K9Cme3) α HP1 (A) HP1α HP1β HP1γ HP1 (input HP1 α WB (B) A (C,D) HP1α (C; WT, D; S97A) α (E,F) FLAG-HP1α (G) C,F

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HP1α N H3K9me3 . (35) . α H3K9me3 ( 2-5C) DNA HP1α HP1 . α . . α DNA α HP1 HP1 HP1α HP1 DNA α ( 2-6A ,C-F) HP1α DNA α . ( 2-6A ) S97A DNA . ( 2-6B) N . HP1α HP1 ( ) ( 2-6F) HP1α CD H3K9me3 µM (23,35) HP1α HP1α H3K9me

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2−6 HP DNA

(A-D) α DNA HP1

HP1α-WT (A) -S97A (B) HP1β

(C) HP1γ (D) HP1 α (E) DNA

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HP1α . (40) HP1α . ( 2-4C) (39,40) (hinge mutant ; HM) α (HP1α-HM) DNA DNA ( 2-7A,C) ( 2-7A,B) ( 2-7B,C) HP1α-HM . ( 2-7B,D) HP1α N HP1α-HM α HP1α-HM . ( 2-7F . 3 5 ) (40) CK2 HP1α-HM H3K9 H3K9me3 . ( 2-7F . 6 ) CK2 HP1α H3K9me3 DNA H3K9me3 HP1α H3K9me3

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2−7 α

(A-D) DNA HP1 HP1α-WT (A)

HP1α-WT (B) HP1α-HM (C) HP1α-HM (D) α

2-6 (E, F)

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2-4

HP1α HP1α HP1α . HP1α N ES HP1α ( ) CK2 N HP1α HP1β N HP1α . Swi6 HP1a CK2 . α H3K9me3 HP α CK2 in vitro 97 N α PP2 N HP α . HP1 DNA RNA H3K9me3 HP α DNA . . RNase HP1α

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RNA (37,38) HP1 DNA RNA N CD . N . HP1β α NMR DNA (50) . . HP1α HP1α N H3K9me3 DNA . HP1α (35,54) .

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3-1

HP1 . HP H3S10 HP1 (23,26) . HP1α . (25) HP1 1999 E. Minc HP1α,-β,-γ α HP1α γ . (55) HP1α 93 . . HP1α HP1α α α HP1α

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3-2

3-2-1 α HP1α α pcDNA4/TO_3xFLAG_puro HP1α Fspl α 1 Aurora B

Aurora B (NM_001284526) pACYCDuet (Novagen)

PP2Cβ (NM_002706) .

pColdI α 3-2-2 α

HeLa HEK293T

Invitrogen Tet-ON α TRex-HeLa 10 % FBS

1 mM Sodium pyruvate ( ) - MEM

( ) 3xFLAG HP1α PolyFect (QIAGEN) α 2 µg/ml 48 15 cm 50 5 ug/ml 48 5% CO2 37 3-2-3 G2/M 200 ng/ml α α anti-HP1α_Sp_{92 (} ₎

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3-2-4 HP1 α pACYCDuet_AuroraB 25 µg/ml 3-2-5 FLAG α HP1α . 10 mg 1 mg/ml IP buffer Sepharose 4B 1 ml 1 4 IP buffer M2 agarose 100 µl over night 4 IP buffer 8 ml 8 1.5 ml 250 µlm −30 over night 15,000rpm 4 30 SDS-PAGE LC/MS/MS (MS CDB ) IP buffer freeze-thaw 3-2-6 in vitro

HP1α α in vitro 50 units Aurora A

(Invitrogen) Aurora B (Invitrogen) α

20 units PP2A (Invitrogen)

PP2Cβ (2 µg) α RIPA buffer PP2 buffer

(80 mM Tris-HCl pH8.4, 60 mM MgCl2) PP2 30 3 3-2-7

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3-2-8

HP1α DNA α

3-2-9

PBS 200 µl

Chromatin fractionation buffer (20 mM Tris-HCl [pH7.5], 50-150 mM NaCl, 5mM MgCl2, 0.2% NP-40, 10% Glycerol, 1 mM NaF) 10

100 µl (Whole cell lysate; WCL) 12,000 rpm 4

10 (Supernatant; Sup) WCL Sup 100 µl 2x

SDS (Ppt) 200

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3-3

3-3-1 HP1 HP1α 2 α (55) Phos-tag α HeLa HP1α ( 3-1 ) 3 . 2 α HP1β HP1γ . . HP1β . ( 3-1 ) HP1γ 24 HP1γ . ( 3-1 ) α HP1γ . Phos-tag WB HP1γ HP1α 3-1 HP1 Phos-tag WB 3 6 12 24 .

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3-3-2 HP1α HP1α α . 93 (35) . ( 3-2 ) HP1α HP1α N HP1α FLAG α . HP1α HP1α . . . S92A ( 3-3) S85A S87A . . . S97A S92 HP1α S92 . 3-2 HP1α HP1α ( )

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3-3 HP1 FLAG HP1α 293T 3-3-3 HP1α 3-3-1 HP1α HP1γ . HP1α HP1γ HP1α HP1α α HP1α α . . HP1α Aurora B (56) Aurora B H3S10 Aurora B

Chromosome passenger complex HP1 α . (26,56)

HP1 α ( ) PP2A

(56) PP2A

Shugoshin HP1 α

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(51)

. Aurora B PP2A . . . HP1α α PP2Cβ ( 3-4) PP2C 16 (58) Mass . PP2A PP2Cβ (IP-WB) α Mass PP2Cβ HP1 α . PP2A ( 3-5)

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3-4 FLAG α HP1

FLAG HP1α .

α (A) Mass

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3-5 WB α FLAG HP1α HEK293T . HP1α HP1α . . in vitro Aurora HP1γ Aurora A (44) HP1α Aurora B . ( 3-6A) HP1α S92 S92A . Aurora B S92 . ( 3-6A) Aurora B ZM-447439 . S92 Aurora B . ( 3-6B) S85 S87 . . PP2Cβ PP2A PP2Cβ PP2A . ( 3-C)

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3-5 HP1α

(A) in vitro HP1α Aurora kinase Phos-tag WB

(B) HEK293T 5 µM ZM-447439 Phos-tag WB

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3-3-4 α α HP1α HP1α Aurora B Aurora B HP1α ( 3-6A) CK2 S97 S97A α ( 3-6C ) 3-7B α S92 HP1α 70~80% S92 HP1α . 3-6

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3-3-5 HP1α N N Aurora B S92 HP1α α CK2 Aurora B HP1α CK2 HP1α H3K9me3 ( 3-7A) DNA α CK2 Aurora B HP1α CK2 HP1α DNA . ( 3-7B) CK2 S97 ( 2-6) HP1α N DNA . . in vitro α HP1α . . (Sup) (Ppt) HP1α HP1α . . ( 3-7C) HP1α .

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3-7 HP1α

(A,B) HP1α α (A) DNA

(B) (C) HeLa α (Tubulin) H3

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3-4

HP1α . S92 Aurora B S85, S87 . in vitro S85 S87 Aurora B Aurora B . S85 S87 Aurora B α . NDR HP1α S95 (45) . HP1α α S95 . ( 3-3) Phos-tag α . HP1α S95 HP1α . HP1 . PP2Cβ (59) HP1α HP1α . (25) HP1 Shugoshin (Sgo1) Sgo1 α PP2A (58,60) HP1α PP2A H3S10 HP α HP1α PP2A Sgo1 . ( 3-8)

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3-8 HP1α

(Interphase) HP1α N (NTD)

H3K9me (Mitotic phase)

HP1α .

(60)

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4

H3K9me HP1 H3K9me CD 2001 (18-20) 10 . CD H3K9me . . HP1 (48,49) . H3K9me3 (50) α HP1 Swi6 HP1α HP1β CK2 HP α N . DNA . SUMO HP1 RNA α (46) HP1 . α α

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α . ( ) CSD HP1 HP1 HP1α HP1α HP1α α α . H3K9me HP1 Aurora HP1 . . DNA : 12-301, 13-302

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(2012

4 2015

3 )

1. RBP2 α 6 2012 5 2. RBP2 α 35 2012 12 3. HP1 30 2012 12 4. HP1 2013 5 5. HP1 31 2013 11 6. HP1 36 2013

7. G. Nishibuchi, Y. Shibata, T. Hayakawa, K. Shinmyozu and J. Nakayama. Physical and functional interaction between histone H3K4 demethylase JARID1A and NuRD complex Keystone Symposia Conference [Chromatin Mechanisms and Cell Physiology], Oberstdorf (Germany), March 2014.

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8. G. Nishibuchi, Y. Shibata, T. Hayakawa, K. Shinmyozu, H. Tagami and J. Nakayama.

Physical and functional interaction between histone H3K4 demethylase JARID1A and NuRD complex IIAS Research Conference [Chromatin Decoding], Kyoto, May 2014.

9. Fischle Wolfgang HP1 H3K9me3 nucleosome 87 2014 10 10. HP1 32 2014 12

1. Nishibuchi G, Shibata Y, Hayakawa T, Hayakawa N, Ohtani Y, Shinmyozu K, Tagami H and Nakayama JI.

「Physical and Functional Interactions between the Histone H3K4 Demethylase KDM5A and the Nucleosome Remodeling and Deacetylase (NuRD) Complex.」

The Journal of Biological Chemistry. 289(42):28956-70 (2014)

2. Nishibuchi G, Machida S, Osakabe A, Murakoshi H, Hiragami-Hamada K, Nakagawa R, Fischle W, Nishimura Y, Kurumizaka H, Tagami H and Nakayama JI.

「N-terminal phosphorylation of HP1α increases its nucleosome-binding specificity._」

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Nishibuchi G and Nakayama JI.

「Biochemical and structural properties of Heterochromatin Protein 1: understanding its role in chromatin assembly_」

The Journal of Biochemistry. 156(1):11-20 (2014)

1. 4 p49-65

2013

2. 5

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6 CDB

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DNA ( ) DNA ( ) DNA ( ) DNA H3 9 (H3K9me) H3 4 H3K9me Heterochromatin Protein 1 (HP1) HP1 α β γ HP1

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HP1 HP1 HP1α N HP1 HP1α HP1 HP1α HP1 DNA HP1 N

(Casein Kinase 2; CK2) Aurora

B HP1α HP1α N HP1α DNA H3K9 H3K9me ( ) HP1α DNA ( ) HP1 HP1 DNA

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HP1α

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CSD CD CD CSD Me _P -+ -+ CSD CD CD CSD -+ - +

CD

CSD

クロモドメイン _{クロモシャドウドメイン}

HP1α

NTD (N-terminal domain) Hinge Casein Kinase 2 によるリン酸化 H3K9 未修飾ヌクレオソーム ( ヘテロクロマチン存在量：少量） _{( ヘテロクロマチン存在量：多量）}H3K9me ヌクレオソーム Aurora B などによるリン酸化 PP2 ホスファターゼによる脱リン酸化 HP1α NTD-phos Hinge-phos H3K9me H3S10phos

恒常的なリン酸化による H3K9me ヌクレオソームへの結合促進

HP1αのリン酸化修飾による

ダイナミックな機能制御メカニズム

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: Epigenetics DNA 1942 C. Waddington DNA DNA : Chromatin 1879 W. Flemming Chroma- DNA : Chromosome H2A H2B H3 H4 H1 H4 : Nucleosome DNA 1.6

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: Histone tail C N : Euchromatin H3K4, H3K36 : Heterochromatin X H3K9, H3K27, H4K20 : Centromere ( ) H3 CENP-A H3K9me Heterochromatin protein 1; HP1 H3K9me

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: Chromodomain (CD)

Polycomb Pc HP1

Chromatin organization modifier domain

PHD Phos-tag Wako Phos-tag SDS-PAGE