Xist A
X
1. ... 3 2. ... 11 3. ... 22 hnRNP U Xist RNA ... 22 3 Xist RNA ... 28 950 Xist RNA hnRNP U .... 35 A ... 41 950-nt ... 44 4. ... 48 5. ... 52 6. SUMMARY ... 55 7. ... 57 8. ... 58 9. ... 65
3 1. X XX XY Y X 1000 X 2 X 2 ( 1) 2 X X X X (Lyon, 1961) X X
inactive specific transcript (Xist) X 23 kb
18 kb RNA
(lncRNA) Xist RNA ( 2) (Borsani et al., 1991; Brockdorff et al., 1991; Brockdorff et al., 1992; Penny et al., 1996) Xist
(ES ) Xist
X Xist RNA X
(Marahrens et al., 1997; Penny et al., 1996)
X Xist
Xist RNA X RNA FISH
Xist cloud Xist cloud Xist RNA
1 PRC2 X H3 27 (H3K27me3) X (Marks et al., 2009; Plath et al., 2003) ( 3)
Xist RNA mRNA RNA
II
mRNA X (Brockdorff et al., 1992) Xist RNA X
RNA
heterogeneous nuclear ribonucleoprotein U (hnRNP U) (Hasegawa et al., 2010) X hnRNP U
Xist RNA X ES
hnRNP U Xist RNA X
X
lncRNA
(Chu et al., 2011) Xist RNA
hnRNP U (Chu et al., 2015; McHugh et al., 2015; Minajigi et al., 2015) 3D structured illumination (3D-SIM)
hnRNP U Xist RNA
(Smeets et al., 2014) hnRNP U Xist RNA
Xist RNA X X
structural maintenance of chromosomes flexible hinge domain containing 1 (SmcHD1)
HP1-binding protein enriched in inactive X
chromosome 1 (HBiX1) X
(Blewitt et al., 2008; Nozawa et al., 2013) SMCHD1 HBiX1
X XIST
RNA X (Nozawa et al., 2013)
XIST / Xist RNA X X
X SMCHD1 / SmcHD1 HBiX1
Xist RNA ES
5
(Wutz et al., 2002) Xist RNA A F
( 2) (Nesterova et al., 2001)
Wutz A Xist RNA 5' X
A
7.5
( 4 ) A
X ( 4 )
(Duszczyk et al., 2011; Duszczyk et al., 2008; Wutz et al., 2002) Wutz
X Xist RNA
Xist RNA X
X Xist RNA
Hasegawa hnRNP U X
Xist RNA (Hasegawa et al., 2010)
Xist RNA hnRNP U X
hnRNP U Xist RNA
Xist RNA
(iMEFs)
hnRNP U Xist RNA RNA
NIH3T3 6-kb 3 Xist RNA
hnRNP U cross-linking immunoprecipitation combined with high-throughput sequencing (CLIP-seq)
(Huelga et al., 2012) Xist RNA hnRNP U
(950-nt) 950-nt 950-nt A (950-nt mut) 1 Col1a1 ES Xist RNA A hnRNP U
7
1.
( ) X ( ) 2 XX ( ) X ( ) Y
8
2. Xist
Xist 23 kb 7 (ex1-ex7) 18 kb Xist RNA Xist RNA
6 (A-F) A X (Wutz et al., 2002)
A F B C
D
E
Xist
X
22.5-1
X-inactive specific transcript (Xist)
Xist
exon1 ex2 ex3 ex5 ex6 exon7 ex4AAAAAA
Xist
遺伝子
ex2
ex3
ex4
ex5
ex6
ex7
ex1
5 kb
F
B
C
A
D
E
9
3. X
X Xist RNA ( ) PRC2
H3K27me3 ( ) X
X Xist RNA ( ) H3K27me3 ( ) immuno-FISH
Xist cloud H3K27me3 (Leeb and Wutz,
2007)
X
染色体不活性化の確立過程
Xist RNA H3K27me3
PRC2 Xist RNAの集積 抑制型ヒストン修飾の導入 X染色体の凝縮 ヘテロクロマチン化 遺伝子の不活性化 サイレンシングドメインの形成 Leeb et al., 2007
10 4. A ( ) 7.5 ( ) ( ) A ( ) (Wutz et al., 2002)
Xist A
リピート領域は不活性化に必須である
‣ A
リピート領域
- X
染色体不活性化を引き起こすのに重要
(Wutz et al., 2002)
-
種間で配列が高度に保存されている
-
ステムループ構造をとる
(Wutz et al., 2002, Duszczyk et al., 2011)
CTGTTATTATTTTTTTTTCTTTTTCTTTTCGGGATCGGGGCTGTGGATACCTG
TGTGTCCTCCCCGCCATTCCATGCCCAACGGGGTTTTGGATACTTA
CCTGCCTTTTCATTCTTTTTTTTTCTTATTATTTTTTTTTCTAAACTTGCCCATCTGGGCTGTGGATACCTG
CTTTTATTCTTTTTTTCTTCTCCTTAGCCCATCGGGGCCATGGATACCTG
CTTTTTGTAAAAAAAAAAAAAAAAAAAAAAACCTTTCTCGGTCCATCGGGACCTCGGATACCTG
CGTTTAGTCTTTTTTTCCCATGCCCAACGGGGCCTCGGATACCTG
CCTTTCTTTCATTGTTTATATAATCTTGCCCATCGGGGCCACGGATACCTG
CTTTAAATTTTTTTTTTACAGGCCCAACGGGGCTTGGTGGA
GCCCAWCGGGGCYNYGGATACCTG
GCCCATCGGGGYNNYGGATANCTG
Mouse consensus sequence Human consensus sequence5'—
-3’
--GCCCAUCGGGGCC-CGGAUACCUG--
Wutz et al., 200211 2.
2.1
2.1.1 pEFrtTAIB
EF1α pCSII-EF-RfA ( )
internal ribosome entry site (IRES) pSA-IRES-EGFP
PstI BamHI S (BSD)
pcDNA6TR (Thermo) SpeI NotI PCR
pBluescript II SK (+) pEFIB
(rtTA)
ptTA-bleo ( ) tTA ClaI SpeI
pBluescript II SK (+) (Gossen et al., 1995) PCR
D95N,L101S,G102D E71K
rtTA SpeI NotI pEFIB
EF1α mRNA rtTA BSD 2 S rtTA pEFrtTAIB 2.1.2 pPBTight RNA CAG IRES-Puromycin (PuroR) Piggybac pPBCAG-BstXI-IP ( ) pTRE-Tight (Clonteh) (pTight) RNA pPBTight 6-kb
TSS 6-kb Mid 6-kb End 950-nt 950-nt as 3’ NotI
PCR pPBTight
(pPBTight6-kbTSS pPBTight6-kb Mid pPBTight6-kb End pPBTight950-nt pPBTight950-nt as) EGFP pTRE-Tight
pTight-EGFP pPBCAG-BstXI-IP (pPBTightEGFP) 950-nt mut 950-nt pBluescript II SK (+) pBSii950-nt PCR 469-bp A (IDT) In-Fusion (Clontech) 3' PCR PCR 950-nt mut NotI 950-nt
mut (pPBTight950-nt mut) Xist
cDNA pPBTight6-kbTSS SpeI SalI
pXistcDNASS12.9 Xist cDNA SpeI-XhoI (pPBTightXistfull)
2.1.3 pColpuroTight Col1a1
Col1a1 5' DNA PCR
XbaI SacI pBluescript
II SK (+) In-Fusion pBSiiColLA pPGK-neo-pA PGK
PCR pBSiiColLA In-Fusion
pBSiiColLAneo pBSiiColLAneo KpnI
DNA Col1a1 3' DNA
PCR KpnI
pBSiiColLAneoSA pTRE-Tight pTight
XhoI SalI pBSiiColLAneoSA
pColLAneoTightSA pColLAneoTightSA
PCR PCR
PuroR In-Fusion pColpuroTight
13
(pColpuroTight950-nt pColpuroTight950-nt mut pColpuroTight950-nt as)
2.1.4 CRISPR-Cas9
CRISPR-Cas9 gRNA Cas9 1
pX330 (Cong et al., 2013) gRNA CRISPRdirect (https://crispr.dbcls.jp) (Naito et al.,
2015) 2 1 10mM 95 5 BbsI 2 BbsI px330 Xist pXPR-Xist(-20) pXPR-Xist(0.9k) Col1a1 pCRISPR-Col1a1 2.1 1
15 2.2 (iMEFs) NIH3T3 (DMEM SIGMA-ALDRICH) 10% (FBS) 1% / (nacalai) EF
2.2.1 rtTA NIH3T3 rtTAIB
1.1µg pEFrtTAIB 8.8 µL Fugene HD (Promega) 130 µL
Opti-MEM (Thermo) 15 35mm 1x105 NIH3T3 24 6 µg/mL Blasticidin S HCl (Thermo) 2 1 µg/mL Blasticidin S HCl 0.9 /well 96 well rtTAIB 2.2.2 pPBTight RNA 1.1 µg Piggybac ( ) 1.1 µg 130 µL Opti-MEM 15 35mm 1x105 rtTAIB 24 2 µg/mL Puromycin (Thermo) 1 PBTightEGFP pPBTightXistfull
PBTight6-kbTSS PBTight6-kb Mid PBTight6-kb End PBTight950-nt PBTight950-nt as PBTight950-nt mut
1 µg/mL
Puromycin 1 µg/mL
ROSA26 nls-rtTA ES (J1rtTA/N Anton Wutz ) (Wutz and Jaenisch, 2000) DMEM 15 %
KnockOut Serum Replacement (KSR Thermo) 1%
(nacalai) 1% / 0.1mM
(sigma) 500U Leukemia Inhibitory Factor (nacalai) 2i (1 µM PD0325901 3 µM CHIR99021)
2.2.3 Xist J1rtT/N ∆Xist ES
1.1µg pXPR-Xist(-20) pXPR-Xist(0.9k) pEFIB 8.8 µL Fugene
HD 130 µL Opti-MEM 10 1x105 J1rtTA/N 5 35 mm 24 4 µg/mL Blasticidin S HCl 60mm DNA 2 PCR J1rtT/N ∆Xist ES 2.2.4 Col1a1 1 ES
J1rtT/N ∆Xist ES pColpuroTight Col1a1 30µg 10µg pCRISPR-Col1a1 24 1 2 µg/mL Puromycin DNA 2 PCR 2µg/mL
17
2. J1rtT/N Xist J1rtT/N ∆Xist
2.3 hnRNP U
hnRNP U (Hasegawa et al., 2010)
siRNA 1x105 iMEFs 35-mm 24
50 µL Opti-MEM 2µM siRNA 50 µL
Opti-MEM 1µL Lipofectamine RNAiMAX (Invitrogen)
iMEFs 1 siRNA 1
siRNA 24 4 1 35-mm
24 1 2 siRNA
2 siRNA 24 RNA-FISH siRNA
24
2.4 hnRNP U
RIPA (25 mM Tris-Cl pH7.4 150 mM NaCl 1% NP-40 1% Sodium Deoxycholate 0.1% SDS 1x
(nacalai)) 30 (62.5 mM Tris-Cl pH6.8 25% 2% SDS 0.01% BPB 5% ) 100 5 SDS-PAGE (8% 375mM Tris-Cl pH8.8 0.1% SDS) 200V 400mA (Amarsham) hnRNP U (Abcam 20666) HRP IgG
(Jackson Immuno Research 111-035-144)
(nacalai) ImageQuant LAS 500
tubulin (CALBIOCHEM CP06) HRP IgG (Amersham NA931V) tubulin 2.5 RNA NP-40 (10 mM Tris-Cl pH 7.5 10 mM NaCl 3 mM MgCl2 0.5% Nonidet P-40) (8000 rpm 4 5
19
RNA TRIzol (Ambion)
0.1 M Tris–Cl pH 7.5, 0.22 M NaCl, 1% SDS, 12.5 mM EDTA
200 µg/mL K 37°C 60
/ RNA
(Pandya-Jones
and Black, 2009) (20
mM Tris-Cl 75 mM NaCl 0.5 mM EDTA 0.85 mM DTT 1x Proteinase
inhibitor (nacalai) 50% ) (10 mM
HEPES 1 mM DTT 7.5 mM MgCl2 0.2 mM EDTA 0.3 M NaCl 1 M
1% NP-40) (14000rpm 4 2 )
RNA
2.6 RT-PCR RT-qPCR cDNA 1 µg RNA
Superscript III (Invitrogen)
cDNA 50 1 RT-PCR qPCR qPCR
KAPA SYBR FAST qPCR kit (Kapa Biosystems) 103 106
qPCR RT-qPCR
21
2.7 RNA fluorescent in situ hybridization (RNA-FISH)
RNA-FISH (Sado et al., 2001) Nick translation Reagent
Kit (Abbott) iMEF NIH3T3
PFA ES 2x Hybridization Buffer 37 16 42 2x SSC/0.05% tween20 50% /2x SSC 2 vectashield (VECTOR) 2.8 HNRNP U XIST HEK293
HNRNP U CLIP-seq (Huelga et al., 2012) hg19 TopHat v2.0.12 (Kim et al., 2013)
RNA-seq XIST 5'
Integrative Genomics Viewer (IGV) v2.3.47 (Robinson et al., 2011) (Thorvaldsdóttir et al., 2013)
3.
hnRNP U Xist RNA
hnRNP U Xist RNA X
(Hasegawa et al., 2010) Xist RNA X hnRNP U
RNA-FISH Xist RNA
X
Xist RNA
Xist RNA hnRNP U X
iMEFs
siRNA hnRNP U Xist RNA
hnRNP U mRNA siRNA (sihnRNP U) sihnRNP U
siRNA 2
hnRNP U sihnRNP U
hnRNP U
2 sihnRNP U iMEFs hnRNP U
( 5-A) siRNA iMEFs
RNA-FISH Xist RNA
Xist cloud sihnRNP U
(Hasegawa et al., 2010) Xist RNA
( 5-B) Xist RNA DAPI
hnRNP U Xist RNA
siRNA iMEFs
RNA RT-qPCR
23
Malat1 β-Actin
Xist RNA 1 7
2 (Xist1 Xist7)
sihnRNP U Xist RNA
( 5-C) hnRNP U
X Xist RNA
RNA-FISH
hnRNP U Xist RNA RNA-FISH
siRNA
(Pandya-Jones and Black, 2009)
RNA RT-PCR
Gapdh RNA (pre-mRNA)
Gapdh RNA
Gapd pre-mRNA Gapd mRNA
RT-PCR Xist
RNA RT-PCR sihnRNP U
Xist RNA
( 5-D) hnRNP U X
Xist RNA
Xist RNA iMEFs hnRNP U X
Xist RNA
24 5. siRNA hnRNP U (A) iMEF hnRNP U siRNA (si hnRNP U) siRNA (Scr.) hnRNP U tubulin
Scr.
si hn
RNP
U
C
op
y
nu
mb
er
ra
tio
(%
)
Scr.
si hnRNP U
hnRNP U
tubulin
DAPI
Xist
Scr.
si hnRNP U
Figure 1
A
B
C
0%
20%
40%
60%
80%
100%
Xist1
Xist7 Malat1
Actb
Xist1
Xist7
Malat1
Actb
Cytoplasm
Nucleus
Scr.
si hnRNP U
Cyt
op
lasm
Nu
cle
op
lasm
Ch
ro
ma
tin
Cyt
op
lasm
Nu
cle
op
lasm
Ch
ro
ma
tin
Xist
D
Xist
25
(B) siRNA RNA-FISH Xist RNA ( ) DAPI
( ) Scr. Xist cloud 2 Xist
cloud 4
hnRNP U si hnRNP U
Xist RNA (Hasegawa et al., 2010)
10 µm
Scr.
si hn
RNP
U
C
op
y
nu
mb
er
ra
tio
(%
)
Scr.
si hnRNP U
hnRNP U
tubulin
DAPI
Xist
Scr.
si hnRNP U
Figure 1
A
B
C
0% 20% 40% 60% 80% 100%Xist1
Xist7 Malat1
Actb
Xist1
Xist7
Malat1
Actb
Cytoplasm
Nucleus
Scr.
si hnRNP U
Cyt
op
lasm
Nu
cle
op
lasm
Ch
ro
ma
tin
Cyt
op
lasm
Nu
cle
op
lasm
Ch
ro
ma
tin
Gapd mRNA Gapd pre-mRNA XistD
Gapd mRNA Gapd pre-mRNA Xist(C) siRNA iMEFs
RNA qPCR Xist RNA Xist RNA
Xist RNA 1 7 2
(Xist1 Xist7) lncRNA Malat1
mRNA β-Actin
Xist siRNA Malat1
Actb RNA
Scr.
si hn
RNP
U
C
op
y
nu
mb
er
ra
tio
(%
)
Scr.
si hnRNP U
hnRNP U
tubulin
DAPI
Xist
Scr. si hnRNP UFigure 1
A
B
C
0% 20% 40% 60% 80% 100%Xist1 Xist7 Malat1 Actb Xist1 Xist7 Malat1 Actb
Cytoplasm Nucleus Scr. si hnRNP U Cytop lasm Nucle oplasm Chro matin Cytop lasm Nucle oplasm Chro matin Gapd mRNA Gapd pre-mRNA Xist
D
Gapd mRNA Gapd pre-mRNA Xist27
(D) siRNA iMEFs RT-PCR
Xist RNA hnRNP U (B)
Xist RNA Scr.
Gapd mRNA (Gapd pre-mRNA) Gapd mRNA
Scr.
si hn
RNP
U
C
op
y
nu
mb
er
ra
tio
(%
)
Scr.
si hnRNP U
hnRNP U
tubulin
DAPI
Xist
Scr.
si hnRNP U
Figure 1
A
B
C
0% 20% 40% 60% 80% 100%Xist1 Xist7 Malat1 Actb Xist1 Xist7 Malat1 Actb
Cytoplasm Nucleus
Scr.
si hnRNP U
Cytop lasm Nucle oplasm Chro matin Cytop lasm Nucle oplasm Chro matin Gapd mRNA Gapd pre-mRNA XistD
Gapd mRNA Gapd pre-mRNA Xist3 Xist RNA hnRNP U Xist RNA EF1α (rtTA) NIH3T3 ( 6-A ) CAG Puromycin (PuroR) piggybac piggybac ( 6-A )
EGFP Xist RNA
RNA
RNA-FISH
RNA EGFP mRNA RNA-FISH qPCR
mRNA ( 6-B,D) Xist RNA
RNA-FISH Xist cloud
qPCR ( 6-C,E)
RNA RNA
hnRNP U
Xist RNA Xist cDNA 6-kb 3
( 7-A 6-kb TSS, 6-kb Mid, 6-kb End)
6-kb
TSS 6-kb Mid 2 RNA-FISH PuroR mRNA
Xist cloud 6-kb End
29 3 ( 7-E-G) 3 TSS Mid End ( 7-H) End 2 End RNA 2 6-kb TSS
6-kb Mid 2 Xist RNA Xist cloud
6. RNA NIH3T3
(A) EF1α rtTA rtTA
IRES-BSD Blasticidin S rtTA (pTight) NotI (Cloning site) CAG PuroR puromycin 2 Piggybac (PBIR)
Figure S1
A
B
C
PBIR PuroR CAG pTight pA PBIR
Cloning site pA
EF1α rtTA IRES BSD
DAPI Xist
Puro
R0% 20% 40% 60% 80% 100% GFP Malat1 Cytoplasm Nucleus C op y nu mb er ra tio (% ) C op y nu mb er ra tio (% ) 0% 20% 40% 60% 80% 100%
Xist full Malat1 Cytoplasm Nucleus
D
E
31
(B) (D) EGFP (B) RNA-FISH
EGFP mRNA ( ) (D)
RNA qPCR EGFP mRNA
(C) (E) Xist RNA (C)
RNA-FISH PuroR ( ) Xist
cloud ( ) (E) RNA qPCR
Xist RNA
10 µm
Figure S1
A
B
C
PBIR Puro
RCAG pTight
pA PBIR
Cloning site
pA
EF1α rtTA IRES BSD
DAPI
Xist
Puro
R0% 20% 40% 60% 80% 100% GFP Malat1 Cytoplasm Nucleus
C
op
y
nu
mb
er
ra
tio
(%
)
C
op
y
nu
mb
er
ra
tio
(%
)
0% 20% 40% 60% 80% 100%Xist full Malat1 Cytoplasm Nucleus
D
E
32
7. 6-kb Xist RNA
(A) Xist cDNA 6-kb 3 (6-kb TSS 6-kb Mid 6-kb End) NIH3T3
Figure 2
A
B
C
D
Xist cDNA
6-kb TSS
6-kb Mid
6-kb End
1
1
18102
6119
6079
12097
12096
18102
E
F
G
DAPI
6-kb TSS
Puro
RDAPI
6-kb Mid
Puro
RDAPI
6-kb End
Puro
RC
op
y
nu
mb
er
ra
tio
(%
)
6-kb TSS
6-kb Mid
6-kb End
20%
40%
60%
80%
100%
Cytoplasm
Nucleus
20%
40%
60%
80%
100%
Cytoplasm
Nucleus
20%
40%
60%
80%
100%
Cytoplasm
Nucleus
33
(B-D) 6-kb Xist RNA NIH3T3 RNA-FISH
6-kb TSS (B) 6-kb Mid (C) PuroR ( )
Xist cloud ( ) 6-kb End PuroR ( )
10 µm
(E-G) 6-kb Xist RNA NIH3T3
RNA RT-qPCR
6-kb TSS (E) 6-kb Mid (F) 6-kb End (G)
10 µm
Figure 2
A
B
C
D
Xist cDNA 6-kb TSS 6-kb Mid 6-kb End 1 1 18102 6119 6079 12097 12096 18102E
F
G
DAPI 6-kb TSS PuroR DAPI 6-kb Mid PuroR DAPI 6-kb End PuroR
C op y nu mb er ra tio (% ) 6-kb TSS 6-kb Mid 6-kb End 0% 20% 40% 60% 80% 100% 6-kb TSS Malat1 Actb Cytoplasm Nucleus 0% 20% 40% 60% 80% 100%
6-kb Mid Malat1 Actb Cytoplasm Nucleus 0% 20% 40% 60% 80% 100%
6-kb End Malat1 Actb Cytoplasm Nucleus
(H) 6-kb TSS 6-kb Mid 6-kb End 6-kb TSS 1 6-kb TSS 6-kb Mid 6-kb End
Figure S2
0%
20%
40%
60%
80%
100%
120%
6-kb TSS 6-kb Mid 6-kb End
Relative ratio vs 6k-b TSS (%)
H
35
950 Xist RNA hnRNP U
6-kb Xist RNA Xist cloud ( 7-B,C) RIP-qPCR Xist
RNA hnRNP U
(Yamada et al., 2015) hnRNP U Xist RNA X
HEK293 hnRNP U RNA
CLIP-seq (Huelga et al., 2012) hnRNP U XIST RNA
5' A-repeat hnRNP U
( 8-A) Huelga et al. (2012)
RNA-seq HEK293 XIST RNA
( 8-A)
hnRNP U CLIP-seq A Xist RNA
5' hnRNP U (Hendrickson et
al., 2016) A
X (Sakata et
al., 2017; Wutz et al., 2002) hnRNP U Xist RNA NIH3T3 Xist RNA 5' 950 (950-nt) ( 8-B) 950-nt RNA-FISH ( 8-C ) hnRNP U Xist RNA ( 5-B ) 950-nt NIH3T3 RNase RNA-FISH 950-nt RNA ( 8-C ) 950-nt 950-nt NIH3T3 RNA qRT-PCR ( 8-D) 950-nt RNA
RNA RT-PCR PCR ( 8-E) RNA-FISH 950-nt 950-nt RT-PCR 950-nt 950-nt hnRNP U ” ” Xist RNA hnRNP U A hnRNP U 5' 950
37 8. XIST hnRNP U
(A) HEK293 hnRNP U CLIP-seq RNA-seq
(Huelga et al., 2012) A ( ) 5' hnRNP U RNA-seq
Figure 3
2000 10 kb 2000 2000 0 0 0 3200 0 hnRNP U CLIP-seq RNA-seq hnRNP U CLIP-seq RNA-seq XIST XIST A-repeat 950-nt Xist exon1 A-repeat DAPI 950-nt PuroRA
B
C
950-nt Cytop lasm Nucle oplasm Chro matin Gapd mRNA Gapd pre-mRNAE
RNase 950-nt(B) (A) hnRNP U 5' 950-nt NIH3T3 (C) 950-nt RNA-FISH ( ) 950-nt DAPI ( ) RNase 950-nt 10 µm
Figure 3
2000
10 kb
2000
2000
0
0
0
3200
0
hnRNP U
CLIP-seq
RNA-seq
hnRNP U
CLIP-seq
RNA-seq
XIST
XIST
A-repeat
950-nt
Xist
exon1
A-repeat
DAPI
950-nt
Puro
RA
B
C
D
950-nt
Cyt
op
lasm
Nu
cle
op
lasm
Ch
ro
ma
tin
Gapd mRNA
Gapd pre-mRNA
E
RNase
950-nt
C
op
y
nu
mb
er
ra
tio
(%
)
0%
20%
40%
60%
80%
100%
950-nt
Malat1
Actb
Cytoplasm
Nucleus
Figure 3
2000
10 kb
2000
2000
0
0
0
3200
0
hnRNP U
CLIP-seq
RNA-seq
hnRNP U
CLIP-seq
RNA-seq
XIST
XIST
A-repeat
950-nt
Xist
exon1
A-repeat
DAPI
950-nt
Puro
RA
B
C
D
950-nt
Cyt
op
lasm
Nu
cle
op
lasm
Ch
ro
ma
tin
Gapd mRNA
Gapd pre-mRNA
E
RNase
950-nt
C
op
y
nu
mb
er
ra
tio
(%
)
0%
20%
40%
60%
80%
100%
950-nt
Malat1
Actb
Cytoplasm
Nucleus
39 (D) 950-nt NIH3T3 RNA RT-qPCR 950-nt
Figure 3
2000
10 kb
2000
2000
0
0
0
3200
0
hnRNP U
CLIP-seq
RNA-seq
hnRNP U
CLIP-seq
RNA-seq
XIST
XIST
A-repeat
950-nt
Xist
exon1
A-repeat
DAPI
950-nt
Puro
RA
B
C
D
950-nt
Cyt
op
lasm
Nu
cle
op
lasm
Ch
ro
ma
tin
Gapd mRNA
Gapd pre-mRNA
E
RNase
950-nt
C
op
y
nu
mb
er
ra
tio
(%
)
0%
20%
40%
60%
80%
100%
950-nt
Malat1
Actb
Cytoplasm
Nucleus
(E) 950-nt NIH3T3 RT-PCR 950-nt 950-nt
Figure 3
2000
10 kb
2000
2000
0
0
0
3200
0
hnRNP U
CLIP-seq
RNA-seq
hnRNP U
CLIP-seq
RNA-seq
XIST
XIST
A-repeat
950-nt
Xist
exon1
A-repeat
DAPI
950-nt
Puro
RA
B
C
D
950-nt
Cyt
op
lasm
Nu
cle
op
lasm
Ch
ro
ma
tin
Gapd mRNA
Gapd pre-mRNA
E
RNase
950-nt
C
op
y
nu
mb
er
ra
tio
(%
)
0%
20%
40%
60%
80%
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950-nt
Malat1
Actb
Cytoplasm
Nucleus
41 A
Xist RNA5' 950-nt
950-nt Xist RNA X A
(Sakata et al., 2017; Wutz et al., 2002) A
A ( )
RNA ( 4)
Xist RNA X
(Duszczyk et al., 2011; Wutz et al., 2002) A 950-nt Wutz et al. (2002) 7.5 2 (C G G A) (950-nt mut) 950-nt (950-nt as) NIH3T3 ( 9-A,B) RNA-FISH 950-nt mut 950-nt as PuroR RNA 950-nt ( 9-C,D) hnRNP U A A X
42
9. A 950-nt
(A) (950-nt mut 950-nt as) A
(B) A
( ) (Wutz et al., 2002) Wutz et al. (2002) 950-nt
A 2 (950-nt mut)
Figure 4
DAPI
950-nt as
Puro
RDAPI
950-nt mut
Puro
R950nt
A-repeat
950-nt as
950-nt mut
C
C
C
G
C
C
G
G
G
G
A
U
C
G
C
G
C
C
G
G
G
A
A
U
A-repeat
Mutant
C
D
A
B
950-nt mut
950-nt as
950-nt as
950-nt mut
Figure 4
DAPI
950-nt as
Puro
RDAPI
950-nt mut
Puro
R950nt
A-repeat
950-nt as
950-nt mut
C
C
C
G
C
C
G
G
G
G
A
U
C
G
C
G
C
C
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G
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A-repeat
Mutant
C
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A
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950-nt mut
950-nt as
950-nt as
950-nt mut
43
(C,D) 950-nt mut 950-nt as NIH3T3 RNA-FISH
950-nt mut (C) 950-nt as (D) ( )
10 µm
Figure 4
DAPI
950-nt as
Puro
RDAPI
950-nt mut
Puro
R950nt A-repeat 950-nt as 950-nt mut
C
C
C
G
C
C
G
G
G
G
A
U
C
G
C
G
C
C
G
G
G
A
A
U
A-repeat
Mutant
C
D
A
B
950-nt mut
950-nt as
Puro
RPuro
R950-nt as
950-nt mut
Figure 4
DAPI
950-nt as
Puro
RDAPI
950-nt mut
Puro
R950nt A-repeat 950-nt as 950-nt mut
C
C
C
G
C
C
G
G
G
G
A
U
C
G
C
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G
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Mutant
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950-nt mut
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RPuro
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950-nt mut
950-nt Xist RNA 5' 950-nt hnRNP U A NIH3T3 RNA ROSA26 rtTA ES (
10-A) (Wutz and Jaenisch, 2000) Xist
950-nt 5' CRISPR-Cas9
( 10-B,C)
Col1a1 (Beard et al., 2006) 950-nt
950-nt mut 950-nt as 3 1 ( 10-D ) 2 Col1a1 950-nt 950-nt mut 950-nt as PCR ( 10-D ) RNA-FISH 950-nt mut 950-nt as 2 950-nt NIH3T3 ( 10-E-G) A 950-nt NIH3T3 ES 950-nt RNA
45
10. Col1a1 1 ES
RNA
(A) ROSA26 rtTA ES (J1rtTA/N
(Wutz and Jaenisch, 2000))
(B) Xist 5' 950-nt Xist
Tsix 4 ES
Tsix RNA 950-nt RNA-FISH
J1rtTA/N 950-nt CRISPR-Cas9 (J1rtTA/N ∆Xist) (C) CRISPR-Cas9 (B) PCR J1rtTA/N J1rtTA/N ∆Xist PCR pA nlsrtTA SA ROSA26 locus Xist A repeat CRISPR gRNA 2 CRISPR gRNA 1 Tsix 950-nt exon1
Figure S4
J1rtT A/N ∆Xist #7 Primer1 Primer2C
A
B
46 (D) J1rtTA/N ∆Xist Col1a1
1 (950-nt 950-nt mut 950-nt as) (pTight) Col1a1 PCR 950-nt 950-nt mut 950-nt as 1 WT PCR (TA)
Figure 5
D
pTight
Puro
RTargeting
vector
Col1a1 gene
CRISPR gRNA
Test fragments
Targeted allele
Puro
RpTight
Test fragments
DAPI
950-nt
Puro
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Puro
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Puro
RWT
TA
95
0-n
t #
11
95
0-n
t #
8
95
0-n
t a
s
#5
95
0-n
t mu
t #
1
95
0-n
t a
s
#1
95
0-n
t mu
t #
4
F
G
E
950-nt
950-nt mut
950-nt as
∆Xi
st
#
7
Primer1 Primer291.8%
0.0%
0.0%
950-nt mut
Puro
R950-nt as
Puro
R950-nt
Puro
RFigure 5
A
pTight
Puro
RTargeting
vector
Col1a1 gene
CRISPR gRNA
Test fragments
Targeted allele
Puro
RpTight
Test fragments
DAPI
950-nt
Puro
RDAPI
950-nt mut
Puro
RDAPI
950-nt as
Puro
RWT
TA
95
0-n
t #
11
95
0-n
t #
8
95
0-n
t a
s
#5
95
0-n
t mu
t #
1
95
0-n
t a
s
#1
95
0-n
t mu
t #
4
C
D
B
950-nt
950-nt mut
950-nt as
∆Xi
st
#
7
Primer1
Primer2
91.8%
0.0%
0.0%
950-nt mut
950-nt as
950-nt
47
(E-G) Col1a1 950-nt 950-nt mut 950-nt as
RNA-FISH PuroR mRNA
DAPI 950-nt RNA % 10 µm
Figure 5
D
pTight PuroR Targeting vectorCol1a1 gene CRISPR gRNA
Test fragments Targeted allele PuroR pTight
Test fragments
DAPI 950-nt PuroR DAPI 950-nt mut PuroR DAPI 950-nt as PuroR WT TA 950-n t #11 950-n t #8 950-n t as #5 950-n t mu t #1 950-n t as #1 950-n t mu t #4
F
G
E
950-nt 950-nt mut 950-nt as ∆Xi st # 7 Primer1 Primer2 91.8% 0.0% 0.0% 950-nt mut PuroR 950-nt as PuroR 950-nt PuroR4. 5' 950-nt Xist RNA hnRNP U 950-nt A A Xist RNA A Xist RNA X X
(Sakata et al., 2017; Wutz et al., 2002) A
X A
X A
X PRC2
H3K27me3 X
(Maenner et al., 2010; Zhao et al., 2008)
A Xist RNA
A Xist
RNA X H3K27me3
(Kohlmaier et al., 2004; Sakata et al., 2017) A B
C Xist RNA X
H3K27me3 (Almeida et al., 2017)
PRC2 A
PRC2 A
A
A Xist RNA Xist cloud
(Chaumeil et al., 2006) X
A X Xist cloud
A
Xist RNA lncRNA (Chu et al., 2011)
49
Xist RNA Xist X Xist
(Engreitz et al., 2013) A Xist RNA
Xist RNA A Xist RNA
(Engreitz et al., 2013) hnRNP
U A Xist
RNA A Xist RNA
A Xist RNA Xist
hnRNP U X Xist RNA hnRNP U A Xist cloud ( 11 ) A Xist RNA hnRNP U X A A A ( 11 ) A Spen
Lamin B receptor (Chu et al., 2015; McHugh et al., 2015) (Chen et al., 2016) A
RAP-seq Xist RNA A
Xist RNA X
(Engreitz et al., 2013) Xist RNA A
Xist RNA X RNA
950-nt 950-nt Xist RNA A X A Xist RNA
(Sakata et al., 2017) Xist A
51
11. Xist RNA A
Xist RNA hnRNP U X Xist cloud
( ) A
本研究から導かれたAリピートの作用モデル
Xist
∆AXist
WT Xist
A-repeat
A-repeat
Xist
∆AXist cloud
の作る
5.
(XX) (XY) X
2 X
X Xist RNA X
X
Xist RNA RNA II
mRNA X Xist RNA X Xist RNA X hnRNP U hnRNP U X Xist RNA Xist RNA X X SmcHD1 HBiX1 X
Xist RNA Xist RNA X
X hnRNP U Xist RNA hnRNP U Xist RNA X Xist RNA hnRNP U Xist RNA X
Xist RNA Xist RNA
NIH3T3 Xist RNA
53 hnRNP U CLIP-seq hnRNP U hnRNP U XIST RNA 5' A A XIST RNA (950-nt) hnRNP U NIH3T3 950-nt RNA 950-nt hnRNP U Xist RNA Xist RNA A X 950-nt A A X 950-nt RNA A
(950-nt mut) 950-nt (950-nt as) NHI3T3 950-nt mut 950-as RNA-FISH 950-nt 950-nt mut 950-nt as Col1a1 1 ES NIH3T3 Xist RNA hnRNP U X Xist RNA X A A 950-nt RNA Xist RNA X
A X PRC2
H3K27me3 X
A Xist RNA X H3K27me3
X A Xist RNA X Xist RNA 5' hnRNP U Xist RNA hnRNP U X A Xist RNA X X
55 6. SUMMARY
X chromosome inactivation (XCI) is a mechanism to compensate for the dose difference of X-linked genes between XX females and XY males. X-linked X-inactive specific transcript (Xist) is a long noncoding RNA that plays an essential role in XCI in the mouse. Although Xist RNA, like common protein-coding mRNAs, is transcribed by RNA polymerase II, spliced and polyadenylated, it is retained in the nucleus and associates with the X chromosome it originates from. One of the major proteins constituting the nuclear matrix, hnRNP U, has been shown to be required for the association of Xist RNA with the inactive X chromosome (Xi). It has also been suggested that in human cultured cells, structural maintenance of chromosomes hinges domain containing 1 (SMCHD1), and its binding partner, HBiX1, are involved in compaction of Xi. However, the sequences in Xist RNA that are involved in compaction of Xi have not yet been identified.
In this study, I found that the first 950-nt sequence of Xist RNA had the potential to associate with chromatin in a manner independent of hnRNP U. Furthermore, its chromatin association is apparently dependent on the presence of an intact A-repeat sequence, which is one of the repeats in Xist/XIST RNA conserved among many mammalian species, and has been shown to be essential for Xist RNA-mediated chromosome silencing. This raises an interesting possibility that the ability of chromatin association of the 950-nt RNA might be functionally involved in the mechanisms of XCI.
It has been proposed that the role of the A-repeat in XCI is to mediate the interaction between Xist RNA coating the X chromosome and polycomb repressive complex 2 (PRC2) which catalyzes trimethylation of the lysine residue located at the twenty-seventh position from the N-terminus of histone H3 (H3K27me3), which is one of histone modifications typical for facultative heterochromatin such as Xi. However previous studies showed that H3K27me3 was not sufficient for chromosome silencing as it still accumulated on the X chromosome coated with the mutated Xist RNA
lacking the A-repeat defective in chromosome silencing. Subsequent study further demonstrated that X-linked genes, which failed to be silenced by the mutated Xist RNA were located outside or at the periphery of the domain formed by the mutated Xist RNA in the nucleus. Taking all these findings and those in this study, I suggest a new hypothesis that the A-repeat captures chromatin near the initial loading site of Xist RNA and relocates it into the core of the Xist RNA domain consisting of heterochromatin.
57 7.
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9.
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