Supplemental data

Poly(ADP-ribosyl)ation of p53induces

gene-specific transcriptional repression of MTA1

Min-Ho Lee, Hyelin Na, Eun-Jin Kim, Han-Woong Lee, and Mi-Ock Lee

Supplementary Table

The sequences of oligonucleotides used for RT-PCR, ChIP and EMSA.

oligonucleotide name / Nucleotide sequence
RT-PCR
p53 / Sense
Antisense / 5-CATCTACAAGCAGTCACAGCA-3
5-TTCCGTCCCAGTAGATTACCA-3
MTA1 / Sense
Antisense / 5'-CACGCACATCAGGGGCAA-3'
5'-GTGCGAAGGTGCCCACA-3'
p21
(Madison and Lundblad, 2010) / Sense
Antisense / 5'-ATGTCAGAACCGGCTGGGGATGTC-3
5'-GGGCTTCCTCTTGGAGAAGATC-3
Bax
(Liu et al., 2008) / Sense
Antisense / 5'-CCCTTTTGCTTCAGGGTTTC-3
5'-TGTTACTGTCCAGTTCGTCC-3
survivin
(Cheung et al., 2009) / Sense
Antisense / 5'-ATGGGTGCCCCGACGTT-3
5'-TCAATCCATGGCAGCCAG-3
-actin / Sense
Antisense / 5'-CGTGGGCCGCCCTAGGCACCA-3'
5'-TTGGCTTAGGGTTCAGGGGGG-3'
ChIP
ChIP1 / Sense
Antisense / 5'-AAAGAGCACGGCCGCTCCTGGA-3'
5'-GGTCCTCCGGCATTCCTCCCTGA-3'
ChIP2 / Sense
Antisense / 5'-ATCGCGCCTCCATTTTCC-3'
5'-ATTTTGGGTTGGGGTGAG-3'
p21
(Saramäki et al., 2006) / Sense
Antisense / 5'-GAAATGCCTGAAAGCAGAGG-3'
5'-GCTCAGAGTCTGGAAATCTC-3'
BAX
(Sinha et al., 2010) / Sense
Antisense / 5'-TCAGCACAGATTAGTTTCTG-3'
5'-GGGATTACAGGCATGAGCTA-3'
survivin / Sense
Antisense / 5-TTGAACTCCAGGACTCAAGTGA-3
5-GATGCGGTGGTCCTTGAGA-3
EMSA
p53RE1 / Sense
Antisense / 5'-GCACGACCACCTGTCCAGAGATGCCACGCG-3
5'-CGCGTGGCATCTCTGGACAGGTGGTCGTGC-3
p53RE2 / Sense
Antisense / 5'-CGAAGGCCAAGGACACGCCCTGCCTGGTGT-3
5'-ACACCAGGCAGGGCGTGTCCTTGGCCTTCG-3
p53RE3 / Sense
Antisense / 5'-CGCCGCCGCCGGCCCGGACATGGCCGCCAA-3
5'-TTGGCGGCCATGTCCGGGCCGGCGGCGGCG-3
Mut 2,3 / Sense
Antisense / 5'-CGAAGGCCGGTGACGGTCCCTGCCTGGTGT-3
5'-ACACCAGGCAGGGACCGTCACCGGCCTTCG-3
Mut 4 / Sense
Antisense / 5'-CGCCGCCGCCGGCCCGGACGGGGCCGCCAA-3
5'-TTGGCGGCCCCGTCCGGGCCGGCGGCGGCG-3
p21 promoter p53RE
(Yin et al., 2004) / Sense
Antisense / 5'-GGAAGAAGACTGGGCATGTCTGGGCAGAGA-3
5'-TCTCTGCCCAGACATGCCCAGTCTTCTTCC-3

Supplementary Figure Legends

Supplementary Figure 1p53 represses the expression of MTA1 at transcription level.(A) MCF7 or HepG2 cells were treated with 50 μM 5-FU for the indicated periods. The mRNA level of MTA1 was analyzed by RT-PCR. (B)Chang liver or WRL-68 cells were transfected with empty vector (EV) or pCMV-Myc-p53. The mRNA level of MTA1 was analyzed by RT-PCR. (C) HepG2 cells were transfected with si-GFP or si-p53, and then treated with 50 μM 5-FU for 24 h. The mRNA level of MTA1 was analyzed by RT-PCR. The density of RT-PCR bands was determined by an image analysis system and normalized to the corresponding β-actin band. The fold changes in expression levels are shown at the bottom of the gel illustrations.

Supplementary Figure 2Treatment of 5-FU represses the expression level of MTA1 mRNA.Cells with differential p53 status were treated with 5-FU at the indicated concentrations. The mRNA expression of MTA1 was analyzed by RT-PCR. The density of RT-PCR bands was determined by an image analysis system and normalized to the corresponding β-actin band. The fold changes in expression levels are shown at the bottom of the gel illustrations.

Supplementary Figure 3Effects of PHEN on the 5-FU-induced regulation of gene expression for survivin, p21 and Bax.HepG2 cells were treated with 100 μM PHEN with or without 50 μM 5-FU for 24 h as indicated. The mRNA expression of MTA1 was analyzed by RT-PCR. The density of PCR bands was determined by an image analysis system and normalized to the corresponding β-actin band. The fold changes in expression levels are shown at the bottom of the gel illustrations.

Supplementary Figure 4 Repression of the MTA1-mediated VEGF expression by p53 and PARP-1. HepG2 cells were transfected with si-GFP, si-p53, or si-PARP-1. After 48 h of transfection, cells were treated with 50 μM 5-FU for 24 h. RT-PCR analysis was performed to monitor the expression of mRNA.The density of bands was determined by an image analysis system and normalized to the corresponding β-actin band. The fold changes in expression levels are shown at the bottom of the gel illustrations.

Supplementary Figure 5PARylation of mutant p53 presentin MDA-MB-231 cells. (A), (B) Whole cell lysates were immunoprecipitated (IP) with normal IgG or anti-p53 antibodies, and precipitates were probed by western blotting (WB) using anti-PAR antibodies. Arrow indicates p53 that was verified by WB.The membrane was strippedand reprobed with an anti-p53 antibody used as anIP control. (C) MDA-MB-231 cells were treated with 50 M 5-FU for 24h. DNA fragments that immunoprecipitated by an anti-p53 antibodywere amplified by PCR using primers for ChIP1 and ChIP2.

SupplementaryReferences

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Liu FT, Agrawal SG, Gribben JG, Ye H, Du MQ, Newland AC et al. (2008). Bortezomib blocks Bax degradation in malignant B cells during treatment with TRAIL.Blood111:2797-2805.

MadisonDL, Lundblad JR.(2010). C-terminal binding protein and poly(ADP)ribose polymerase 1 contribute to repression of the p21(waf1/cip1) promoterOncogene29:6027-6039.

Saramäki A, Banwell CM, Campbell MJ, Carlberg C. (2006). Regulation of the human p21(waf1/cip1) gene promoter via multiple binding sites for p53 and the vitamin D3 receptor.Nucleic Acids Res34:543-554.

Sinha S, Malonia SK, Mittal SP, Singh K, Kadreppa S, Kamat R et al.(2010). Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element.EMBO J29:830-842.

Yin Y, Zhu A, Jin YJ, Liu YX, Zhang X, Hopkins KM et al. (2004). Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21.Proc Natl Acad Sci U S A101:8864-8869.