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

aaaa

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