Crosstalk between site-specific modifications on p53 and histone H3

Oncogene (2008) 27, 1639–1644

& 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc

SHORT COMMUNICATION

Crosstalk between site-specific modifications on p53 and histone H3 LJ Warnock, R Adamson, CJ Lynch and J Milner YCR p53 Research Group, Department of Biology, University of York, York, UK

Previously, we have observed a link between p53 expression and histone H3 post-translational modifications. Here, we ask if specific post-translational modifications of p53 impact upon histone H3 modifications in a selective manner. We have also screened for internal co-operative effects within the repertoire of p53 modifications. Exogenous p53 constructs were expressed in HCT116 p53/ cells. Four mutant p53 constructs were used, with single ‘phosphorylation’ mutations at serines 15 and 37 (S15A, S15D, S37A and S37D) and compared with exogenously expressed wild-type p53. The results showed that the replacement of serine 15 with either alanine (S15A) or aspartic acid (S15D) induced phosphorylation at S33P, S37P and S46P. In contrast, phosphorylation mutants p53(S37A) and p53(S37D) were not phosphorylated on S33. S46 phosphorylation appeared specifically enhanced by p53(S37D) relative to p53(S37A). Distal induction of S392 phosphorylation was observed for each of the p53 N-terminal phosphorylation mutants. Analysis of endogenous histone H3 (from the transfected cells) revealed loss of di-methylated K9 following expression of wild type and mutant p53 constructs. Expression of p53 (S15A), (S15D) and (S37A) selectively induced acetylation at K9 and K14. In contrast, wt p53 and p53(S37D) had no effect upon K9 or K14 acetylation. K18 acetylation status was unaffected throughout. Oncogene (2008) 27, 1639–1644; doi:10.1038/sj.onc.1210787; published online 24 September 2007 Keywords: p53 protein; histone H3; phosphorylation; acetylation; methylation

Results and discussion In the event of genotoxic stress the tumor suppressor p53 is transiently stabilized and transported to the nucleus where it acts as a transcription factor (Ljungman, 2000; Zhao et al., 2000). During this period, p53 may undergo a cascade of post-translational modifications including N- and C-terminal phosphorylations, C-terminal acetylCorrespondence: Dr LJ Warnock, YCR p53 Research Group, Department of Biology, Zone 2, University of York, Heslington, York, York YO10 5DD, UK. E-mail: [email protected] Received 6 February 2007; revised 13 August 2007; accepted 13 August 2007; published online 24 September 2007

ation, sumoylation, methylation, ubiquitination and neddylation (Appella and Anderson, 2001; Brooks and Gu, 2003; Sykes et al., 2006). In particular, serines 15 and 37 act as substrates for DNA-PK and ATM following genotoxic stress (Vousden, 2002). These posttranslational events stabilize and activate p53 for DNA repair, apoptosis, cell cycle arrest at G1/S or G2/M or senescence (Barlev et al., 2001; Okorokov et al., 2002; Vousden, 2006). p53 is thought to function as the ‘molecular node’ connecting upstream signaling cascades and downstream DNA-repair–recombination pathways. The role of p53 in genomic global repair is now well established (see Sengupta and Harris, 2005, for review). DNA repair and gene transactivation also require histone acetylation (Zhang and Dent, 2005). We have previously shown p53-dependent UV-induced global chromatin relaxation achieved by p53-mediated acetylation of histone H3 and recruitment of the histone acetylase p300 to sites of NER (Rubbi and Milner, 2003), as well as specific p53-dependent K9 and K14 acetylation of histone H3 following UV irradiation (Allison and Milner, 2003). In the present study, we examine whether such sitespecific histone H3 modifications are influenced by the modification status of p53: that is, does modification of p53 at a given residue, for example phosphorylation at serine 15, have a knock-on effect on p53-dependent sitespecific modifications of histone H3? For this purpose, we expressed phosphorylation mutants of p53 in HCT116 p53/ cells and analysed the effects on expressed proteins for additional p53 post-translational modifications at intact sites (Figure 1a—serines 15, 33, 37, 46 and 392 and lysine 382) and histone H3 modifications (Figure 1b— serine 10 and lysines 4, 9, 14 and 18). Phosphorylation events at the N terminus of p53 have been reported as crucial in the transactivation of p53responsive genes (Saito et al., 2002). Upregulation of p21 was observed in HCT116 p53/ cells transfected with wild-type p53 and mutant constructs (Figure 2), thus demonstrating that the expressed p53 proteins are transcriptionally active. Interestingly, wt p53 failed to upregulate hdm2 (Figure 2) and this correlated with the lack of phosphorylation at serine 392 (Figure 2). S392 is known to be phosphorylated in response to UV radiation and has been implicated in site-specific DNAbinding (Keller et al., 2001). N-terminal p53 phosphorylation site interdependencies have been determined following UV radiation in H1299 lung carcinoma cells (deleted for both p53 alleles)

Crosstalk between p53 and histone H3 modifications LJ Warnock et al

1640 S15 S37 P P

N

TA1

TA2

DBD

NLS TET

REG Ac P

PP P P S15 S33 S37 S46 Ac Me P

Me

C

K382 S392

Ac

Ac

N-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKK..QKS…FKT….-C 4 9 10 14 18 P

P

P

P P

P

P

P

N-MEEPQSDPSVEPPLSQQTFSDLTKLLPENNVLSPLPSQAMDDLMLSPDD…….. -C 6 9 15 18 20 33 37 46 Figure 1 (a) Schematic indicating the position of p53 phosphorylation mutations p53S15A, p53S15D, p53S37A and p53S37D (above the linear protein) and the endogenous sites of post-translational modifications investigated (below the linear protein, modified from Appella and Anderson, 2001). (b) Schematic indicating the position of the sites of endogenous human histone H3 post-translational modifications investigated in this study. (c) Schematic demonstrating the juxtaposition of known exogenous N-terminal sites of phosphorylation in human p53 proteins.

transfected with p53 in which each of the N-terminal threonine and serine residues were mutated to alanine (Saito et al., 2003). These phosphorylation interdependencies were divided into four groups: S6 and S9; S9, S15, T18 and S20; S33 and S37; and S46. Here, we identify site interdependencies between the group: S15, S33, S37 and S46 in the absence of applied stress such as treatment with IR or UV. The juxtaposition of known N-terminal serine phosphorylation sites is shown in Figure 1c. In the present study, we found that wt p53 and phosphorylation mutant proteins p53S37A and p53S37D were constitutively phosphorylated at S15 when expressed in HCT116 p53/ cells (Figure 2). Phosphorylation at S33 was greatly increased in those cells expressing p53S15A or p53S15D, but was absent on expression of wt p53 and p53S37A and p53S37D (Figure 2) illustrating that wt p53 is not constitutively phosphorylated at S33 and efficient phosphorylation/de-phosphorylation at S33 is influenced by alterations of a serine at residue 15. Nonetheless, p53 negative for S33 phosphorylation appeared to be able to upregulate p21 and hdm2 in HCT116 p53/ cells indicating that S33 phosphorylation may be dispensable for transactivating activity (Figure 2). Exogenously expressed wt p53 is constitutively phosphorylated at S15 but not at S37 or S46. Phosphorylation at S37 and S46 is clearly increased by the expression of p53 S15 phosphorylation mutants suggesting that efficient phosphorylation/de-phosphorylation at S37 and S46 is influenced by the substitution of a serine residue at S15 (Figure 2). However, the increase in S46 phosphorylation seen following the expression of S37A appears to be lower than that for S37D p53, indicating that S37 phosphorylation in vivo may facilitate phosphorylation at S46. This is important as it has been suggested that phosphorylation at S46 may influence the activation of Oncogene

a specific subset of p53-dependent genes, in particular apoptotic target genes (Vousden, 2006). Acetylation at p53 C-terminal lysines activates p53 sequence-specific DNA-binding. Following genotoxic stress, phosphorylation at S15 enhances the binding of p53 to CBP/p300, thereby increasing acetylation at K320 and K382 (Appella and Anderson, 2001). In the present study, we found that expressed wt p53 was constitutively acetylated at K382. Levels of acetylation were enhanced in cells expressing the phosphorylation mutant proteins p53S15D and p53S37D (Figure 2), suggesting that phosphorylation at S15 and S37 at the N terminus of p53 enhances K382 acetylation at the C terminus. Acetylation at K382 has been shown to specifically determine the PAb421 epitope in murine p53 (Warnock et al., 2005) and here acetylation at K382 correlates with PAb421 reactivity following exogenous expression in human HCT116 p53/ cells (Figure 2). The above results suggest that modifications at S15 influence the levels of p53 post-translational modification at other sites, including C-terminal S392 phosphorylation and K382 acetylation (Figure 2). Multisite modification of proteins is thought to constitute a complex regulatory program described as a dynamic ‘molecular barcode’ containing intermolecular and intramolecular signaling for the in vivo qualitative and quantitative control of protein function (Yang, 2005). The importance of N- and C-terminus interdependencies has recently been highlighted, following the resolution of a novel structure for the full-length p53 protein (Okorokov et al., 2006). This revolutionary structure predicts that the N- and C-termini interact directly in dimeric and tetrameric p53. The relationship between N- and C-termini post-translational modifications would therefore not only be relevant for the formation of dimeric and tetrameric p53 but may be crucial for the recruitment of specific proteins and DNA


p53S37D

p53S37A

p53S15D

p53S15A

p53hwt

Vector

Lipofectamine

Untreated

1641

D0-1

S15P

S33P

S37P

S46P

S392P

K382Ac

PAb421

p21

hdm2

Actin Figure 2 Phosphorylation mutation of p53 has site-specific effects on N- and C-terminus phosphorylation and acetylation of human p53. HCT116 p53/ cells were grown in DMEM with 10% FCS, 2 mM glutamine, 100 U ml1 penicillin and 100 mg ml1 streptomycin at 37 1C in 5% CO2 in air. BamH1/EcoR1 restriction sites were used for cloning human wt p53 and phosphorylation mutants into pCMV-Script. All constructs were verified by sequencing. 24 h after transfection, cells were harvested and whole cell lysates prepared. Western blotting was performed as previously described (Warnock et al., 2005) using actin as reference control for total protein (Antiactin antibody, Chemicon, Hampshire, UK, #MAB 1501). Human p53 was detected using anti-DO-1 (Santa Cruz, Santa Cruz, CA, USA, #sc-126). p21 and hdm2 were detected by anti-p21 and SMP 14, respectively (Santa Cruz, #sc-817 and #sc-965). S15, S33, S37 and S46 phosphorylated p53 proteins were detected by antiphospho-p53 (S15), (S33), (S37) and (S46) antibodies, respectively (Cell Signaling Technology, Danvers, MA, USA, #9284, #2526, #9289 and #2521). Phosphorylated S392 p53 was detected by antip-p53(Ser392) antibody (Santa Cruz, #sc-7997). Acetylation at K382 was detected using anti-acetyl-p53 (K382) Ig (Cell Signaling Technology, #2525). PAb421 was obtained from hybridoma supernatant. Peroxidase chemiluminescence was performed in accordance with manufacturer’s instructions (Roche Diagnostics Ltd, Sussex, UK).

with the p53 protein to elicit an appropriate biological response. Core histone proteins undergo complex post-translational events resulting in a ‘molecular barcode’ similar to that described for the tumor suppressor p53 protein (Yang, 2005). Indeed, the ‘histone code’ hypothesis has been put forward identifying the interplay between various post-translational histone tail modifications that serve as markers for the recruitment of proteins which, when in complex with one another, regulate diverse chromatin functions, including DNA replication and gene expression (Jenuwein and Allis, 2001). Phosphorylation of the core histone H3 at S10 in particular, is a highly conserved modification which occurs at G2-M of the cell cycle and is important for normal chromosome condensation, segregation and overall chromosome dynamics. p53 has been shown directly or indirectly to perturb the normal regulation of S10 phosphorylation and is thought to contribute toward the maintenance of normal ploidy and the prevention of malignant transformation (Allison and Milner, 2003). In the present study, the controls were phosphorylated at S10 compared to cells expressing human p53 proteins in which S10 phosphorylation was barely detectable (Figure 3). This is consistent with the observations of Allison and Milner (2003) who reported lower levels of S10 histone H3 phosphorylation in HCT116 p53 þ / þ compared with HCT116 p53/ isogenic cell lines. Interestingly, S10 phosphorylation levels remain barely detectable despite the expression of phosphorylation mutant proteins which produce marked changes in histone H3 acetylation and methylation. This raises the possibility that changes in histone H3 S10 phosphorylation (Allison and Milner, 2003) additionally require signals generated in response to applied stress. We have previously demonstrated that p53 acts as a chromatin accessibility factor mediating p53-dependent UV-induced global chromatin relaxation achieved by the p53-dependent acetylation of histone H3 and recruitment of p300 to sites of NER (Rubbi and Milner, 2003). Other workers have since described mechanisms that involve direct interactions of p300 with p53 and obligatory modifications of the corresponding histone substrates (An et al., 2004). We also have evidence for p53 directly or indirectly downregulating UV-induced acetyl transferase activity at histone H3 K9 thereby preventing abnormally high levels of globally acetylated K9 enabling precise regulation of histone modification (Allison and Milner, 2003). In the present study, in the absence of applied UV stress, there is clearly a marked increase in acetylation at K9 and K14 (Figure 3) in cells expressing p53S15A or p53S15D. We, therefore, suggest that the presence of a serine at residue 15 may be required to elicit an efficient acetylation/de-acetylation response at these sites. Phosphorylation at S15 is known to enhance the binding of p53–p300 (Appella and Anderson, 2001) and alteration of serine 15 may affect the interaction with p300 and thereby indirectly affect histone acetylation. In contrast, S37 phosphorylation status however exerted a specific influence on the acetylation of histone H3 at K9, since K9 acetylation Oncogene


p53S37D

p53S37A

p53S15D

p53S15A

p53hwt

Vector

Untreated

1642

D0-1

S10 P

K9Ac Histone H3 K14Ac

K18Ac

Mono-Me K4

Tri-Me K4

Histone H3

Di-Me K9

Set 9

Histone H3

Actin

Figure 3 Effect of p53 and site-specific phosphorylation mutations on histone H3 S10 phosphorylation, K4 mono-methylation, K9 acetylation and di-methylation and K14 acetylation. Western blotting identified total histone H3 and p53 proteins using antihistone H3 C-16, (Santa Cruz, #sc-8654) and anti-DO-1. Phosphorylation at S10 was detected by anti-phospho-histone H3 (Ser 10), (Cell Signaling Technology, #9701). Acetylation modifications were detected by anti-acetyl-histone H3 (Lys 9), (Upstate, #06-942), anti-acetyl-histone H3 (Lys 14), (Cell Signaling Technology, #9675) and anti-acetyl-histone H3 (Lys 18), (Upstate, #06-911). Histone H3 methylation modifications were detected using anti-mono-methylhistone H3 (Lys 4), anti-tri-methyl-histone H3 (Lys 4) and anti-dimethyl-histone H3 (Lys 9), respectively (Upstate, #07-476, #07-473 and #07-521). Set 9 methyltransferase was detected by anti-SET9 antibody (Upstate, #07-314). Peroxidase chemiluminescence was performed in accordance with manufacturer’s instructions (Roche Diagnostics).

was increased in cells expressing the non-phosphorylated protein p53S37A, whereas in cells expressing p53S37D, the levels of histone H3 acetylation remained as low as in those expressing wt p53. This data suggests that acetylation at histone H3, K9 is associated with de-phosphorylation at S37. Our overall findings are consistent with and confirm the hypothesis that stress-induced changes in p53 phosphorylation may alter patterns of histone post-translational modification (Allison and Milner, 2003; Sengupta and Harris, 2005). Use of site-specific Oncogene

p53 phosphorylation mutants has allowed us to pinpoint p53 phosphorylation sites, such as S37, that appear crucial for histone H3 modification. Moreover, we further show that the histone H3 modifications are in themselves site-specific and governed by site-specific p53 modifications, indicating a functional link between the p53 ‘code’ and the histone ‘code’. Similar to p53, histones play a role in transcriptional control, enhancement and repression of transcription being determined by the precise position and extent of lysine methylation in histone tails (Morgunkova and Barlev, 2006). Histones have been shown to be essential substrates for p53-dependent methyl transferase activity, in particular CARM1 and PRMT1 are known to bind directly to p53. The work of An et al. (2004) indicates a p53-dependent accumulation of distinct histone modifications in response to the activity of CARM1 and PRMT1. In the present study, mono-methylation at histone H3 K4 was elevated in cells expressing p53 while di-methylation at histone H3 K9 was abrogated (Figure 3), suggesting a role for p53 in histone H3 methylation at K4 and K9 which is not influenced by modifications of p53 at S15 or S37. We initially envisaged an indirect role for p53 as recent studies describe histone H3 K4 as a major substrate specifically for mono-methylation by the novel histone methyltransferase Set 9 (Sims and Reinberg, 2006). We expected to find high levels of Set 9 in cells expressing p53 accounting for the elevated levels of histone H3 K4 methylation as RNA silencing of Set 9 has been reported to decrease total levels of p53 (Chuikov et al., 2004). We were surprised to see Set 9 expressed at a high level in all cell samples (Figure 3). These findings were supported by Set 9 mRNA expression levels detected by quantitative RT–PCR (data not shown). On this basis, we suggest that p53 may have a direct role in the increase in histone H3 K4 mono-methylation. Our findings demonstrate high levels of histone K9 dimethylation in the controls and reduced levels in cells expressing p53. As one would expect decreased K9 methylation was associated with elevated K9 acetylation (Figure 3) as these modifications are thought to be mutually exclusive (Zhang and Reinberg, 2001). This study points to a role for p53 in site-specific histone H3 methylation as reciprocity of methylation at K9 and K4 while independent of post-translational modifications of p53 remains nonetheless p53-dependent (Figure 3). The ‘histone code’ suggests that an increase in histone acetylation is a signal to transcribe open chromatin (Jenuwein and Allis, 2001). We performed quantitative chromatin immunoprecipitation (ChIP) analysis in HCT116 p53/ cells expressing exogenous wild type and phosphorylation mutants p53 S15A, S15D, S37A and S37D. Our results demonstrate the presence of wild type and mutant p53 proteins at the p21 promoter, consistent with previous reports of endogenous p53 at the promoter of p21 in unstressed cells (Kaeser and Iggo, 2001). Interestingly, there was an eight fold increase in recruitment of p53 S15A to the p21 promoter compared with wild-type p53 (Figure 4b), consistent with the observation that phosphorylation of


1643 -2292 to -1850

p21waf1 gene

(443 bp) p53

(13.0 Kb shown)

+1 E1

p53

E2

p53

E3

Scale (bp) -3000

-2000

-1000

+1

1000

2000

3000

4000

5000

6000

S15D

S37A

S37D

7000

8000

10000

x-fold relative to wtp53

10

8

6

4

2

Vector

wtp53

S15A

Figure 4 p53 enrichment at the p21 promoter according to wt or mutant p53 expression. ChIP analysis of in vivo recruitment of p53 to a region of the p53-dependent promoter p21 which contains a p53 binding site (a) was performed using the EZ ChIP Chromatin Immunoprecipitation kit in accordance with manufacturer’s instructions (Upstate). Briefly, HCT116 p53/ cells were transfected with the wild type and mutant p53 constructs indicated above and harvested after 24 h by crosslinking with 1% para-formaldehyde. Cells were lysed and sonicated to shear genomic DNA into fragments of 200–1200 bp in length. 2 106 cells and 1 mg of antibody for either p53 (clone DO-1, Santa Cruz), positive control RNA pol II (clone CTD4H8, Upstate) or negative control non-specific Mouse IgG (Upstate) were used per ChIP. Immuno-complexes were collected and washed using Protein G-agarose beads and prepared for PCR analysis. Real-time quantitative PCR to examine p53 enrichment in the 50 -region of the p21 promoter was performed for 45 cycles with a Qiagen SYBERgreen Gigakit (Cat no. 204145, Qiagen, Crawley, Sussex, UK) using an MJ Research Opticon II cycler (MJ Research, Boston, MA, USA). The p21 promoter-specific primer sequences were forward primer 50 -CTGTGGCTCTGATTG GCTTTC-30 ; reverse primer 50 -CACCACCACCACGACATTC-30 . (b) Background negative control IgG signals were subtracted from the values obtained by chromatin immuno-precipitation with DO-1 (specific for total p53). Data were then corrected according to total input chromatin signals and expressed as fold-difference with respect to wild-type p53-transfected cells set at 1.0. Thus, the graph illustrates relative enrichment of total p53 protein at the target site of the p21 promoter according to wild type or site-specific mutant p53 expression.

endogenous p53 at serine 15 is not required for recruitment to the p21 promoter in HCT116 p53 þ / þ cells or primary human keratinocytes under non-stressed conditions (Lynch CJ and Milner J, unpublished; Schavolt and Pietenpol, 2007). The p53 mutants S15D, S37A and S37D exhibited a 2-fold increase in p21 promoter occupancy relative to wild-type p53 (Figure 4b). These findings parallel the observed variations in p21 protein expression levels in HCT116 p53/ cells (Figure 2). We demonstrate interdependencies between p53 Nterminal phosphorylation sites and N-terminal phosphorylation and C-terminal phosphorylation/acetylation sites in mammalian cells. The newly proposed structure for full-length p53 suggests that such post-translational modifications within the N- and C-termini may fine-tune the flexibility of the p53 molecule and directly affect the efficiency of p53-protein and p53-DNA interactions (Okorokov et al., 2006). Our findings also clearly reveal a knock-on effect of p53-dependent site-specific modification on histone

H3 at S10, K4, K9 and K14. We also define a more direct role for p53 in mono-methylation at histone H3 K4. Furthermore, we identify modifications that are influenced by the post-translational modification status of p53 proteins since K9 acetylation was associated with de-phosphorylation at S37. Thus, we propose the integration of a ‘histone code’ and ‘p53 code’ (Espinosa and Emerson, 2001). Such a histone-p53 code may provide an important epigenetic mechanism to maintain the exquisite control of DNA repair, cell cycle arrest and apoptosis required to avoid malignant cellular transformation. Acknowledgements We are grateful to Dr Karen Vousden for kindly providing the p53 phosphorylation mutant cDNAs and Dr Bert Vogelstein for providing the HCT116 p53 null cell line. This work was supported by a Yorkshire Cancer Research program grant awarded to Professor Jo Milner. Oncogene


1644 References Allison SJ, Milner J. (2003). Loss of p53 has site-specific effects on histone H3 modification, including serine 10 phosphorylation important for maintenance of ploidy. Cancer Res 63: 6674–6679. An W, Kim J, Roeder RG. (2004). Cooperative ordered functions of p300, PRMT1 and CARM1 in transcriptional activation by p53. Cell 117: 735–748. Appella E, Anderson CW. (2001). Post-translational activation modifications and activation of p53 by genotoxic stresses. Eur J Biochem 268: 2764–2772. Barlev NA, Liu L, Chehab NH, Masfield K, Harris KG, Halazonetis TD et al. (2001). Acetylation of p53 activates through recruitment of coactivators/histone acetyltransferases. Mol Cell 8: 1243–1254. Brooks CL, Gu W. (2003). Ubiquitination, phosphorylation and acetylation: the molecular basis for p53 regulation. Curr Opin Cell Biol 15: 164–171. Chuikov S, Kurash JK, Wilson JR, Xiao B, Justin N, Ivanov GS et al. (2004). Regulation of p53 activity through lysine methylation. Nature 432: 353–360. Espinosa JM, Emerson BM. (2001). Transcriptional regulation by p53 through intrinsic DNA/chromatin binding and site-directed cofactor recruitment. Mol Cell 8: 57–69. Jenuwein T, Allis CD. (2001). Translating the histone code. Science 293: 1074–1080. Kaeser MD, Iggo RD. (2001). Chromatin immunoprecipitation analysis fails to support the latency model for regulation of p53 DNA binding activity in vivo. Proc Natl Acad Sci 99: 95–100. Keller DM, Zeng X, Wang Y, Zhang QH, Kapoor M, Shu H et al. (2001). A DNA damage-induced p53 serine 392 kinase complex contains CK2, hSptl6, and SSRP1. Mol Cell 7: 283–292. Ljungman M. (2000). Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress. Neoplasia 2: 208–225. Morgunkova A, Barlev NA. (2006). Lysine methylation goes global. Cell Cycle 5: 1308–1312. Okorokov AL, Sherman MB, Plisson C, Grinkevich V, Sigmundsson K, Selivanova G et al. (2006). The structure of p53 tumour suppressor protein reveals the basis for its functional plasticity. EMBO J 25: 5191–5200. Okorokov AL, Warnock L, Milner J. (2002). Effect of wild-type, S15D and R175H p53 proteins on DNA end joining in vitro: potential

Oncogene

mechanism of DNA double-strand break repair modulation. Carcinogenesis 23: 549–557. Rubbi CP, Milner J. (2003). P53 is a chromatin accessibility factor for nucleotide excision repair of DNA damage. EMBO J 22: 975–986. Saito S, Goodarzi AA, Higashimoto Y, Noda Y, Lees-Miller SP, Appella E et al. (2002). ATM mediates phosphorylation in multiple p53 sites, including Ser(46), in response to ionizing radiation. J Biol Chem 277: 12491–12494. Saito S, Yamagichi H, Higashimoto Y, Chao C, Xu Y, Fornace Jr AJ et al. (2003). Phosphorylation site interdependence of human p53 post-translational modifications in response to stress. J Biol Chem 278: 37536–37544. Schavolt KL, Pietenpol JA. (2007). p53 and DNp63a differentially bind and regulate target genes involved in cell cycle arrest, DNA repair and apoptosis. Oncogene; e-pub ahead of print 2 April 2007. Sengupta S, Harris CC. (2005). p53: traffic cop at the crossroads of DNA repair and recombination. Nat Rev Mol Cell Biol 6: 44–55. Sims RJ, Reinberg D. (2006). Histone H3 Lys 4 methylation: caught in a bind? Genes Dev 20: 2779–2786. Sykes SM, Mellert HS, Holbert MA, Marmorstein R, Lane WS, McMahon SB. (2006). Acetylation of the pS3 DNA-binding domain regulates apoptosis induction. Mol Cell 24: 841–851. Vousden KH. (2002). Activation of the p53 tumour suppressor protein. Biochim Biophys Acta 1602: 47–59. Vousden KH. (2006). Outcome of p53 activation—spoilt for choice. J Cell Sci 119: 5015–5027. Warnock LJ, Raines SA, Mee TR, Milner J. (2005). Role of phosphorylation in p53 acetylation and PAb421 epitope recognition in baculoviral and mammalian expressed proteins. FEBS J 272: 1669–1675. Yang X-Y. (2005). Multisite protein modification and intramolecular signaling. Oncogene 24: 1653–1662. Zhang K, Dent SYR. (2005). Histone modifying enzymes and cancer: going beyond histones. J Cell Biochem 96: 1137–1148. Zhang Y, Reinberg D. (2001). Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev 15: 2343–2360. Zhao R, Gish K, Murphy M, Yin Y, Notterman D, Hoffman WH et al. (2000). Analysis of p53-regulated gene expression patterns using oligonucleotide arrays. Genes Dev 14: 981–993.