cell cycle regulation of dnmt1 and cancer. m m m m m m m m dnmt1 m m m m m m m m g0/g1sg2m dnmt1...
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Cell cycle regulation of DNMT1 and cancer
MM
M
MM
M
MM
DNMT1
MM
MM
MM
MM
G0/G1 S G2 M
DNMT1 transcription
Enzymatic activity
Steady state mRNA
The Genome and Epigenome Must be Faithfully Replicated with The Genome and Epigenome Must be Faithfully Replicated with Each Cell CycleEach Cell Cycle
aaaa
0 3 6 8 12 16 20 24 4832
serum induction (h)
18S
DNMT1
0
10
20
30
40
50
60
70
80
90
DNMT1mRNA
% G0/G1% S% G2/M
serum induction(h)0 3 6 8 12 16 20 24 32 48
Expression of DNMT1 mRNA is regulated with the cell cycle
- - - - - - - - - - - - - CT T T GT GCAT T CT Grattusutr(5072-5093)(5123-5211)
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - daniorarioutr(2342-2364)- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - chickenutr(4936-4991)AAT T T T AA- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -mouseutr(5076-5106)(5194-5233)GAT T T T AAAAGT T T T T T AT T AT GCAT T AT AT CAAAT CT AChumanutr(5089-5408)AAT T T T AA- - - T T T T T T AT T CT GT AT T CT AT CAGAT CT GCrattusutr(5072-5093)(5123-5211)
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GT T T T T ATdaniorarioutr(2342-2364)- - - - - - - - - - - - - - - - - - - - - - ACT T T AT GT AGT T T T T AT chickenutr(4936-4991)- - - - - - - - - - - - - - - - - - - - AGACT T GAT GT AGT TNT T ATmouseutr(5076-5106)(5194-5233)CACT GT AT GAGT GGAAAT T AAGACT T T AT GT AGT T T T T AT humanutr(5089-5408)CACT GT GT GGGT GGCAAGT GAGACT T GAT GT AGT TNT T ATrattusutr(5072-5093)(5123-5211)
AT GT T GT AAT AT T T C daniorarioutr(2342-2364)AT GT T GT AAT AT T T CT T CAAAT AAAT CT CT CCT AT AAA chickenutr(4936-4991)AT GT T GT AAT AT T T CT T CAAA mouseutr(5076-5106)(5194-5233)AT GT T GT AAT AT T T CT T CAAAT AAAT CT CT CCT AT AAACChumanutr(5089-5408)AT GT T GT AAT AT T T CT T CAAA rattusutr(5072-5093)(5123-5211)
daniorarioutr(2342-2364)chickenutr(4936-4991)
mouseutr(5076-5106)(5194-5233)AAAAAAAAAAAAAAAAAAAAAAAAAA humanutr(5089-5408)
rattusutr(5072-5093)(5123-5211)
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - daniorarioutr(2342-2364)- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - chickenutr(4936-4991)- - - - - - - - - - - - - - - - - - GT GT AGT ACT T T GT GCAT T CT Gmouseutr(5076-5106)(5194-5233)T GAT T T AGT GAT CAAAT T GT GCAGT ACT T T GT GCAT T CT Ghumanutr(5089-5408)
The DNMT1 3’UTR Contains A Highly Conserved ElementThe DNMT1 3’UTR Contains A Highly Conserved Element
5’ 3’
3’UTR probes / competitors
UTR
UTR 3’- 158
UTR 3’- 56
UTR 5’- 259
5090-5408
5349-5405
5090-5350
5090-5248
0h 20h serum induced
84
41
32
kDa
0 168 20 24 36
serum induction (h)
competitor:
13085
43
32
kDa no
ne
UT
R
UT
R
3 ’5
6 U
TR
3’ 1
58 U
TR
5’ 2
59
A Specific Protein (p40) Binds the Conserved Region of A Specific Protein (p40) Binds the Conserved Region of the DNMT1 3’UTR in Arrested Cellsthe DNMT1 3’UTR in Arrested Cells
0 .5 1 2 40 .5 1 2 4
DNMT1 3’UTR destabilizes mRNA in arrested cells
t1/2=105 min
t1/2=58 min
0.01
0.1
1
0 100 200 300
+serum arrested
56 nt
growth arrested serum induced (20h)
DNMT1
GFP
DNMT1 UTR DNMT1
DNMT1UTR
56
0
5
10
15
20
25
30
DNMT1 UTR
DNMT1UTR 56
DNMT1
growth arrestedserum induced (20h)
No
rma
lize
d D
NM
T1
mR
NA
pAD-DNMT-1
Kan
5350
5408
0
5085
DNMT1
DNMT1-UTR 56
DNMT1 UTR
CMV
poly
AG
FP
The Highly Conserved Region of the 3’UTR is Required The Highly Conserved Region of the 3’UTR is Required to Downregulate the DNMT1 mRNA in Growth Arrested Cellsto Downregulate the DNMT1 mRNA in Growth Arrested Cells
DNMT1 UTR DNMT1
DNMT1UTR
56
h-DNMT1
h-DNMT1-3’UTR
NIH 3T3
Deregulated cell cycle expression of DNMT1 leads to cellular transformation
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A + serumarrested
DNMT1 UTRDNMT1Nih-3T3
- serum + serum0
5
10
15
20
25
30C
3579
111315
2 4 6 8 10 12
time (days)
B
hDNMT1 UTRhDNMT1 hDNMT1 UTRhDNMT1
The 3’UTR inhibits ectopic expression of DNMT1 at the G0 phase of the cell cycle
Ectopic expression of DNMT1 induces entry into the S phase of the cell cycle
Mitogenic signals
DNMT1
DNA replication
Fig. 2
DNA replication DNA methylation
Protection of the DNA methylation pattern
Inhibition of DNMT1 by antisense oligonucleotides inhibits Y1 tumor growth in vivo
Tumor suppressor genes
Oncogenic signals
MeTase
Tumor suppressor genes
M M M M MM
Cell arrest
Tumor suppresors
Cell growth
Nancy DetichQiangLi ZhuangNadia Cervoni
Ian WeaverMichael Meany
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