1

Supplementary Data

Figure S1. The structure of the p73 gene promoters. The P1 promoter (X denotes non-consensus nts; X preferred nts; X mutated nts)

The P1 promoter p53RE RRRCWWGYYY N 0-16 RRRCWWGYYY (consensus) TAp73RE AGTCTAGCCT gcgaggagacgcg GGTCCTGCCC (+294, +316) TAp73REm AGTATATCCT gcgaggagacgcg GGTACTTCCC The P2 promoter ΔNp73RE GGGCAAGCTG aggcctgcccc GGACTTGGAT (+380062,+380032) Two fragments one including the P1 promoter, another the P2 promoter were amplified with primers TA1/TA2 (TA1, GGtacctgcc gcctggccct tgcctcacgc tccag; TA2, gaagctTagg cctcgggatt ggcacagccc tgcag) and DN1/DN2 (DN1, ggtacctgga atcgtccttt cgtcctcagc ccg ; DN2, AAGcttagtt ctgtcaactg gctgaatcca acaac) from genomic DNA, respectively, and cloned into a promoterless luciferase construct, pGL3-Basic vector. The highest-affinity binding sites with the consensus motif GNG(T/C/G)GGG(T/C)G for Egr1 in the p73 regulatory regions are marked EBS . The numbers indicate the position relative to the start of P1 transcription. Egr1 binding sites in TAp73 promoter (P1): EBSa, -1728 GCGCGGGCG –1710; EBSb, -1676 CACCCCCAC –1668; EBSc, -321 GCGTGGGCG -313; EBSd, -37 CGCCCGCGC –29; EBSe, +490 CGCCCGCGC +498. Egr1 binding site in � N promoter (P2): EBsf, +37031 GTGGGGGTG +37039. The positions and sequence of the two p53/p73 responsive element (RE) in the intron 1 (P1 promoter ) and intron3 (P2 promoter) are shown , with deviations from the consensus sequence consists of two copies of the 10 base pair (bp) motif 5’-PuPuPuC(A/T)(T/A)GpyPyPy-3’ separated by 0 to 13 bp. The consensus p53/p73 binding site was mutated by replacing the critical C/G residues with A/T in the construction of luciferase reporters.

TAp73 promoter ( P1)

!Np73 promoter ( P2)

-1919(TA1) -1600 -390 +1 55 77 251 539(TA2)EBSa EBSb EBSc EBSd EBSe exon1

A (320 bp) B (445 bp) C (288 bp)

36907(DN1) 38131 (DN2)ATG(38342)

exon3’(38107~381)EBSf

D(300 bp) E(180 bp)

exon2(29769~866)

ATG(29802)

37206 37952

TAp73 promoter ( P1)

!Np73 promoter ( P2)

-1919(TA1) -1600 -390 +1 55 77 251 539(TA2)EBSa EBSb EBSc EBSd EBSe exon1

A (320 bp) B (445 bp) C (288 bp)

36907(DN1) 38131 (DN2)ATG(38342)

exon3’(38107~381)EBSf

D(300 bp) E(180 bp)

exon2(29769~866)

ATG(29802)

37206 37952

2

Figure S2

Transfection of pGL3-TAp73 and pGL3-ΔNp73 into H4 cells together with expression plasmids for Egr1, WT1 and the dominant negative WT1/Egr1

Figure S3. Structure of the Egr1 promoter (X denotes non-consensus nts; X preferred nts; X mutated nts)

RRRCWWGYYY RRRCWWGYYY p53A1 CGGCAGGCTC cgagac GAGGGAGTCC (-1906,–1881) (80%) p53A2 CAACCAGCTG cgaccc GGAAATGCCA (-455,–430) (75%) p53A2m CAAACATCTG cgaccc GGATATTCCA p53A3 GGTCCTGCCA tatta GGGCTTCCTG (-126,-102) (75%) p53A3m GGTACTTCCA tatta GGGATTTCTG p53A4 CAGCCAGCTT ccgccgccgca GGACCGGCCC (+39,+69) (80%) The potential p53/p73 binding sites in the non-coding sequences from –2 kb to +142 bp of the promoter and the 5’-UTR of the Egr1 gene. The numbers indicate the position relative to the start of the transcription.

Egr1

-2820 -2078 -1030 0 142 270

Egr1F

ATG

Egr1P1Egr1P2

Egr1P3

A1 A2 A3 A4

Egr1

-2820 -2078 -1030 0 142 270

Egr1F

ATG

Egr1P1Egr1P2

Egr1P3

A1 A2 A3 A4

H4

0

5

10

15

20

25

30

pGL3-basic pGL3-TAp73 pGL3-!Np73

Ind

uc

tio

n F

old

Vector

Egr1

WT1

WT1/Egr1

3

Figure S4. Induction of transfected pGL3-Egr1 and pGL3-Egr1F was tested also in

H4 cells.

Figure S5. Immunoblot to show the induction of Egr1 protein after transfection of

expression vectors for ΔNp73α , TAp73α or p53 in MCF7 cells. MCF7 cells were transfected with HA-tagged expression vectors for ΔNp73α, TAp73α or p53 and 24 h later the cells were lysed for immunoblotting. All the proteins induced Egr1 (last row) but TAp73α was the most effective judged by the intensity of the Egr1 bands shown below the image.

H4

0

5

10

15

20

25

30

35

pGL3-

Basic

pGL3-

Egr1

pGL3-

Egr1F

Ind

uc

tio

n F

old

cDNA

p53

TAp73!

"Np73!

4

Figure S6A. Three potential p53 responsive elements in the p53 promoter and one

Egr1 binding site ebs (X denotes non-consensus nts; X much-preferred nts; X mutated nts)

RRRCWWGYYY RRRCWWGYYY

p53 F CAGCAGGTCT t GCACCTCTTC (-577–557) (75%) p53Fm CAGAAGTTCT t GCAACTTTTC p53 M GATCCAGCTG agagcaaac GCAAAAGCTT (-236–208) (80%); p53Mm GATACATCTG agagcaaac GCATAATCTT p53Z GAGCGTGCTT tccacgacgg TGACACGCTT (+183 +212 (85%). p53Zm GAGAGTTCTT tccacgacgg TGAAACTCTT ebs -651 CCCCCCCGC –593;

Figure S6B

293T

0

20

40

60

80

100

120

pGL3-P-p53F pGL3-P-p53Fm pGL3P-p53M pGL3P-p53Mm

Ind

ucti

on

Fo

ld

cDNA

p53

TAp73a

TAp73b

!Np73a

!Np73b

One copy p53F or p53M (See Fig S6A) of the putative non-consensus p53RE (the length of the oligonucleotide is about 80 bp) was cloned into pGL3-promoter (Promega Co.) by KpnI/XhoI. Seeding 12,000 cells into 96-well plate, total DNA=90 ng. 24h after transfection, cells were lysed and assayed.

p53L(-764)kpnI p53R(+229)

0 223exon1

ebs F M Z

p53L(-764)kpnI p53R(+229)

0 223exon1

ebs F M Z

5

Figure S6C. The binding sites of F and M in the p53 promoter contain (CTG).(CAG)

trinucleotide repeats that form CTG/CAG hairpin structures. The hairpin structures were analyzed on the F or M-central 200-bp sequences with DNASIS (V2.5). The p53F-80bp contains 5 CTG/CAG repeats and the p53M-80bp contains 3 CTG/CAG repeats. The following structures were minimal on the total secondary structure energy among structures to form. Consistent with experimental results, the non-consensus p53 binding sites F and M fall into the category of hairpin structures based on (CTG).(CAG) repeats (located on the loops) that are known to provide strong binding sites for p53 and related proteins. p53F-80bp TCTTAAAGCACCTGCACCGCCCCCCCGCCGCCTGCAGAGGGCGCAGCAGGTCTTGCACCTCTTC

TGCATCTCATTCTCCA

p53M ACATTTTAACTGATGAGAAGAAAGGATCCAGCTGAGAGCAAACGCAAAAGCTTTCTTCCTTCCAC

CCTTCATATTTGACA

6

Figure S7

In H4 cells, neither transiently-expressed Egr1 nor WT1 has any effect on the p53 promoter; in Saos2 cells Egr1, WT1 and the dominant negative WT1/Egr1 all weakly induce.

Figure S8

Transfection of expression plasmids for the 5 members of the p53 family each induced the p53 promoter to different extents depending on the cell lines.

Figure S9. The p53 promoter is dose-dependently transactivated by p53.

Transfection of a gradient of the p53 expression plasmid into 293T and Saos2 cells, followed by measurement of the luciferase induction ratio was plotted as a function of the amount of plasmid DNA. This showed that increasing amounts of DNA correlate with increased transactivation up to a maximum of 20 to 25 ng DNA, and then declined at higher levels.

293T

0

50

100

150

200

250

300

350

cDNA p53

05

p53

10

p53

15

p53

20

p53

25

p53

30

p53

40 ng

Ind

ucti

on

Fo

ld

pGL3-p53

pGL3-Basic

Saos2

0

2

4

6

8

10

12

14

16

18

20

cDNA p53 5 p53

10

p53

15

p53

20

p53

25

p53

30

p53

40 ng

Ind

ucti

on

Fo

ld

pGL3-Basic

pGL3-p53

293T

0

50

100

150

200

250

300

350

cDNA p53

05

p53

10

p53

15

p53

20

p53

25

p53

30

p53

40 ng

Ind

ucti

on

Fo

ld

pGL3-p53

pGL3-Basic

Saos2

0

2

4

6

8

10

12

14

16

18

20

cDNA p53 5 p53

10

p53

15

p53

20

p53

25

p53

30

p53

40 ng

Ind

ucti

on

Fo

ld

pGL3-Basic

pGL3-p53

H4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

pGL3-Basic pGL3-p53

Ind

uc

tio

n F

old

Saos2

0

1

2

3

4

5

pGL3-Basic pGL3-p53

cDNA

Egr1

WT1

WT1/Egr1

H4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

pGL3-Basic pGL3-p53

Ind

uc

tio

n F

old

Saos2

0

1

2

3

4

5

pGL3-Basic pGL3-p53

cDNA

Egr1

WT1

WT1/Egr1

H4

0

20

40

60

80

100

120

pGL3-Basic pGL3-p53

Ind

ucti

on

Fo

ld

cDNA

p53

TAp73á

TAp73â

!Np73á

!Np73â

Saos2

0

2

4

6

8

10

12

14

16

18

pGL3-Basic pGL3-p53

H4

0

20

40

60

80

100

120

pGL3-Basic pGL3-p53

Ind

ucti

on

Fo

ld

cDNA

p53

TAp73á

TAp73â

!Np73á

!Np73â

Saos2

0

2

4

6

8

10

12

14

16

18

pGL3-Basic pGL3-p53

7

Figure S10. Time course of the expression levels of the p53 family and Egr1 genes

after Etoposide (ETO) treatment in U2OS cells.

A

B C

A, Egr1, p53 and p73 mRNA expression in a time course from 0 to 11 h after treatment of cells with 20 ug/ml ETO; B, Western blot to show Egr1, p73α and p53 protein levels in a time course, with effects of RNAi to p53 in the lower panel; C, ChIP analysis to show the time course of p53 binding to the Egr1 and p53 promoters. Input means total DNA sample collected from the 6h samples + or - ETO; Mock indicates that non-specific IgG was used + or - ETO

U2OS cells Egr1-mRNA

0

5

10

15

20

25

30

No RNAi p53+p73 RNAi

Ind

ucti

on

Fo

ld

UN

ETO 1h

ETO 3h

ETO 5h

ETO 7h

ETO 11h

U2OS cells p53-mRNA

0

1

2

3

4

5

6

7

8

No RNAi p53+p73 RNAiIn

du

cti

on

Fo

ld

UN

ETO 1h

ETO 3h

ETO 5h

ETO 7h

ETO 11h

U2OS cells p73-mRNA

0

5

10

15

20

25

30

35

No RNAi p53+p73 RNAi

Ind

uc

tio

n F

old

UN

ETO 1h

ETO 3h

ETO 5h

ETO 7h

ETO 11h

U2OS cells Egr1-mRNA

0

5

10

15

20

25

30

No RNAi p53+p73 RNAi

Ind

ucti

on

Fo

ld

UN

ETO 1h

ETO 3h

ETO 5h

ETO 7h

ETO 11h

U2OS cells p53-mRNA

0

1

2

3

4

5

6

7

8

No RNAi p53+p73 RNAiIn

du

cti

on

Fo

ld

UN

ETO 1h

ETO 3h

ETO 5h

ETO 7h

ETO 11h

U2OS cells p73-mRNA

0

5

10

15

20

25

30

35

No RNAi p53+p73 RNAi

Ind

uc

tio

n F

old

UN

ETO 1h

ETO 3h

ETO 5h

ETO 7h

ETO 11h

0 1 2 3 4 5 6 8 12 ( h)p73αEgr1p53s15p-p53s20p-p53s46p-p53actin

No RNAi

0 2 4 6 8 12 Egr1p73αp53actin

p53+p73 RNAi

total p53s46p-p53s15p-p53total p53s46p-p53s15p-p53

Input +Ab Mock + - 0 1 2 3 4 5 6 8 12 + - Abp53RE

p53A2

p53A3

p53F

p53M

Egr1 promotertotal p53s15p-p53total p53s15p-p53

p53 promoter

0 1 2 3 4 5 6 8 12 ( h)p73αEgr1p53s15p-p53s20p-p53s46p-p53actin

No RNAi

0 2 4 6 8 12 Egr1p73αp53actin

p53+p73 RNAi

0 1 2 3 4 5 6 8 12 ( h)p73αEgr1p53s15p-p53s20p-p53s46p-p53actin

No RNAi

0 2 4 6 8 12 Egr1p73αp53actin

p53+p73 RNAi

total p53s46p-p53s15p-p53total p53s46p-p53s15p-p53

Input +Ab Mock + - 0 1 2 3 4 5 6 8 12 + - Abp53RE

p53A2

p53A3

p53F

p53M

Egr1 promotertotal p53s15p-p53total p53s15p-p53

p53 promoter

total p53s46p-p53s15p-p53total p53s46p-p53s15p-p53

Input +Ab Mock + - 0 1 2 3 4 5 6 8 12 + -

Input +Ab Mock + - 0 1 2 3 4 5 6 8 12 + - Abp53RE

p53A2

p53A3

p53F

p53M

Egr1 promotertotal p53s15p-p53total p53s15p-p53

p53 promoter

8

S11-Primer sequences For cloning and mutation TAp73 promoter (Fig.S1) TA1: GGtacctgccgcctggcccttgcctcacgctccag, KpnI TA2: gaagctTaggcctcgggattggcacagccctgcag, Hind III ΔNp73 promoter (Fig.S1) DN1: ggtacctggaatcgtcctttcgtcctcagcccg,KpnI DN2: AAGcttagttctgtcaactggctgaatccaacaac, HindIII Mutation of Egr1 binding sites (EBS) in p73 promoters (Fig.S1) EBSam: gagggagcgagctcctgcTcgAgcgctcggtccgcaggtttcgc, XhoI, EBSbm: ggcgtgcctcgttctcacGTGcactccggaccccggtcctcttc, PmlI EBScm: ccggacctgcttcggccctgcgtCgAcggcctcgccgggctctg, SalI EBSdm: agcccgccctgcctccccgcTAgcgcacccgcccggaggctcgc, NheI EBSem: atcgccagcaagctcctccccgcAcgcgTgctccctccgacctg, MluI EBSfm: cgctctcccgggggtgCACgtggggagcgatgaggcccctgccg, ApaLI p53 promoter (Fig.S6A) p53L: gcgacccacggTacccgcggagccgggacgtgaa, KpnI p53R: caagttcactcTCgagcttacccaatccagggaagc, XhoI One copy of p53F or p53M in the p53 promoter (Fig.S6B) p53F-K: gtcccgcggtaCCtcttaaagcacctgcaccgccc, KpnI p53F-X: tgaatgCTCgagacaggtctgaagcctggagaatg, XhoI p53M-K: gaaagGGtAcctaaaaaatgacattttaactgatg, KpnI p53M-X: cctgcCTCgAgtcaaatatgaagggtggaaggaag, XhoI For ChIP Fragment A in TAp73 promoter (Fig. 3C, 7F & S1) TA1: ggtacctgccgcctggcccttgcctcacgctccag TR1: ctactagcggcctgcgggagccaggggtgg Fragment B in TAp73 promoter (Fig. 3C, 7F & S1) TL2: gctctgccgaagatcgcggtcgggtctggc TR2: catcggcgtccgtcccggctggcctggcgc Fragment C in TAp73 promoter (Fig. 3C, 4C, 7F & S1) TA2: gacttggcgc tcgcgagagg ctggagcggc TR3: gaagcttaggcctcgggattggcacagccctgcag Fragment D in ΔNp73 promoter (Fig.3C & S1) DN1: ggtacctggaatcgtcctttcgtcctcagcccg DR1: atccatggggagctgagaaatccatgcgga Fragment E in ΔNp73 promoter (Fig. 4C & S1) DL1: gggtttaattatggagccggcgctgaccgg DN2: aagcttagttctgtcaactggctgaatccaacaac p53A1 in Egr1 promoter (Fig.5C & S3) 268-L1: atttggctaaggtaccctaccagggagtgg p53A1R: ggagggtgtaactttcttggacttaatccc p53A2 in Egr1 promoter (Fig.5C, 7C, 7F, S3 & S10C) p53A2L: tgacagcgatagaaccccggcccgactcgc p53A2R: cgttgcccctccctccgccttcttccctcc p53A3 in Egr1 promoter (Fig.5C, 7C, 7F, S3 & S10C)

9

p53A3L: ggccccggcggcggctagagctctaggctt p53A3R: gcggctccccaagttctgcg p53A4 in Egr1 promoter (Fig.5C & S3) p53A4L: cgcagaacttggggagccgc 268-R0: gaacactgagaagcttgcaggcggcgaccc p53F in p53 promoter binding to p53/p73 (Fig.6C, 7C, S6 & S10C) p53L: gcgacccacggtacccgcggagccgggacgtgaa p53FR: agggagacaggtctgaagcctggagaatga p53M in p53 promoter binding to p53/p73 (Fig.6C, 7C, S6 & S10C) p53ML: tgggagaaaacgttagggtgtgatattacg p53MR: gcagaattggtggaaatcattttggaggaa p53Z in p53 promoter binding to p53/p73 (Fig.6C & S6) p53ZL: caggcggattacttgcccttacttgtcatg p53R: caagttcagtctcgagcttacccaatccagggaagc For semi-quantitative RT-PCR and LightCycler quantitative RT-PCR cyclophilin A (CPH, as a control, Fig 1B, 7A, 7D & S10A) CPH-L: ctcctttgagctgtttgcag CPH-R: caccacatgcttgccatcc p53 (Fig 7A & S10A) TP53LC-L: catttgcacctacctcacag TP53LC-R: gacttgacaactccctctacc Human p73 (Fig 7D & S10A) p73-L: agtaccgcatgaccatctg p73-R: cgtgaactcctccttgatg Mouse p73 (Fig 1B) p73m-L: accatcacaatccccaac p73m-R: ttccaagagcagtttctcac Egr1 (Fig 1B, 7A, 7D & S10A) Egr1N-L: cagtcccagctcatcaaac Egr1N-R: tctgctttcttgtccttctg