Articles in PresS. Am J Physiol Renal Physiol (January 11, 2012). doi:10.1152/ajprenal.00642.

2011

1 Mechanisms of p53 Activation and Physiological Relevance in the

2 Developing Kidney
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4 Karam Aboudehen†¶, Sylvia Hilliard†, Zubaida Saifudeen†, and Samir El-Dahr†¶1
5 Departments of †Pediatrics and ¶Genetics, Tulane University School of Medicine, New Orleans,
6 Louisiana 70112
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19 Abbreviated Title: Regulation of tumor suppressor p53 in nephrogenesis
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30 Corresponding Author:
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32 Samir S. El-Dahr, M.D.
33 Tulane University School of Medicine
34 Department of Pediatrics
35 1430 Tulane Avenue, SL-37
36 New Orleans, LA 70112.
37 Tel: 504-988-6692
38 Fax: 504-988-1771
39 E-mail: seldahr@tulane.edu
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Copyright © 2012 by the American Physiological Society.

45 ABSTRACT

46 The tumor suppressor protein, p53, is a short-lived transcription factor due to Mdm2-

47 mediated proteosomal degradation. In response to genotoxic stress, p53 is stabilized via post-

48 translational modifications which prevent Mdm2 binding. P53 activation results in cell cycle

49 arrest and apoptosis. We previously reported that tight regulation of p53 activity is an

50 absolute requirement for normal nephron differentiation. However, the mechanisms of p53

51 activation in the developing kidney are unknown. We show here that metanephric p53 is

52 phosphorylated and acetylated on key serine and lysine residues, respectively, in a temporal

53 profile which correlates with the maturational changes in total p53 levels and DNA-binding

54 activity. Site-directed mutagenesis revealed a differential role for these post-translational

55 modifications in mediating p53 stability and transcriptional regulation of renal function genes

56 (RFGs). Section immunofluorescence also revealed that p53 modifications confer the protein

57 with specific spatio-temporal expression patterns. For example, phos-p53S392 is enriched in

58 maturing proximal tubular epithelial cells, whereas acetyl-p53K373/K382/K386 are expressed in

59 nephron progenitors. Functionally, p53 occupancy of RFG promoters is enhanced at the onset

60 of tubular differentiation, and p53 loss- or gain-of function indicates that p53 is necessary but

61 not sufficient for RFG expression. We conclude that post-translational modifications are

62 important determinants of p53 stability and physiological functions in the developing kidney.

63 We speculate that the stress/hypoxia of the embryonic microenvironment may provide the

64 stimulus for p53 activation in the developing kidney.

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66 Key words: Nephrogenesis; Post-Translational Modifications; Mdm2; nephron differentiation

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67 INTRODUCTION

68 The TP53 gene encodes a transcription factor that maintains genomic integrity via its

69 ability to induce cell cycle arrest, senescence or apoptosis (4, 46). The p53 protein is

70 composed of 393 amino acids in humans (390 amino acids in mouse), and consists of several

71 functional domains: N-terminus transactivation and proline rich domains, a central core DNA

72 binding domain, and a C-terminus regulatory domain (38). Hot spot mutations found in 50% of

73 human cancer are for the most part located in the DNA-binding domain (12).

74 Regulation of cellular p53 expression is mainly controlled at the protein level via post-

75 translational modifications and by interactions with the E3 ubiquitin ligase, Mdm2 (21, 28).

76 Under normal conditions, cellular p53 levels and activity are kept low via Mdm2-mediated

77 ubiquitination and proteosomal degradation. Mdm2 gene expression is positively regulated by

78 p53, thus defining a negative feedback loop that controls p53 activity (23). Also, Mdm2

79 associates with chromatin-bound p53 at target promoters (24); in that capacity, Mdm2 recruits

80 histone modifiers that remodel local chromatin structure as well as modify p53 itself.

81 p53 activity and stability are regulated through a multitude of post-translational

82 modifications, such as phosphorylation, acetylation, methylation, sumoylation, and

83 ubiquitination (3, 6, 21, 38, 50). In response to stress, p53 is phosphorylated by a number of

84 kinases on serine residues, mainly clustered within the N-terminus region (e.g., S6,S9,S15,S20)

85 (7). Phosphorylation of p53 prevents Mdm2 binding and leads to p53 stabilization and

86 transactivation (34). In addition, several histone acetyl transferases are known to acetylate

87 p53 at different lysine residues, including CBP/p300 (K370,K372,K373,K381,K382,K386), PCAF

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 88 (K320) and Tip60/hMOF (K120,K164) (5, 6, 15, 18, 21). Acetylated p53 has a higher DNA-

89 binding affinity, is protected from ubiquitination, and modulates transcription through

90 recruitment of co-activators/repressors (5, 24, 26, 32). It has been proposed that different p53

91 acetylation cassettes serve as a code providing p53 with DNA binding specificity and selective

92 gene activation potential (26). Three different methyltransferases have been shown to

93 methylate C-terminal lysine residues of p53. Set7/9-mediated mono-methylation of K372

94 promotes p53 activity, whereas mono-methylation at K370 and K382 by Smyd2 and Set8/PR-

95 Set7, respectively, represses p53 activity (13, 21, 22, 48). Modification of p53 by the ubiquitin-

96 like modifiers, SUMO and Nedd8, further add to the competition for the C-terminus Lysines.

97 Sumoylation of p53 at K386 by Summo-1 and neddylation by Mdm2 at K370, 372,373 inhibit

98 p53-mediated transcriptional activation (9, 11, 21). Although the nature and role of p53 post-

99 translational modifications in the p53 tumorigenic and genotoxic responses have been

100 extensively investigated, much less attention has been paid to the nature and potential role of

101 these modifications during normal development.

102 Although previous studies have shown that tight regulation of basal p53 levels/activity

103 is essential for proper nephron differentiation (20, 42), the developmental mechanisms

104 responsible for p53 activation and stability remain largely unknown. The present study was

105 designed to determine whether embryonic p53 is post-translationally modified and how might

106 these modifications affect the developmental expression, transcriptional activity and spatial

107 localization of p53 in the developing kidney. We also examined the effect of loss- and gain-of-

108 function of p53 on nephron differentiation gene expression.

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109 MATERIALS AND METHODS

110 Animals, tissues and organ culture - All animal protocols utilized were in strict adherence to

111 guidelines established by the Institutional Animal Care and Use Committee at Tulane

112 University. Wild-type CD1 mice were purchased from Charles Rivers Laboratories. TPp53-/-

113 mice on C57Bl6 background were purchased from the Jackson Laboratory. Mdm2 deletion

114 from the ureteric bud lineage was accomplished by crossing Mdm2LoxP/LoxP mice with Hoxb7-Cre

115 mice (20). For the organ culture studies, E13.5 metanephroi were cultured on Transwell filters

116 in DMEM/F12 medium with 10% FCS (fetal calf serum) at 37oC/5% CO2, as described (20, 42).

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118 Semi-Quantitative and Quantitative Reverse Transcriptase-PCR - Total RNA was isolated from

119 kidney samples using the RNAqueous-96 Automated Kit (Ambion, Austin, TX). For semi

120 quantitative RT-PCR, the SuperscriptTM First-Strand Synthesis System for RT-PCR (Invitrogen)

121 was used. Generally, between 1 and 2μg of RNA was used for reverse transcription and 1 or

122 2μl of cDNA was used for PCR. Sequences of PCR primers are listed in Table 1. PCR products

123 were visualized by electrophoresis on a 1% agarose gel pre-stained with Ethidium bromide and

124 analyzed using Alpha Innotech FluorChem© FC2 imaging system (Alpha Innotech, San Leandro,

125 CA). Gene expression was normalized to Gapdh. Quantitative real-time PCR was performed in

126 quadruplicate using the Applied Biosystems 7500 Fast Real-time PCR system and TaqMan

127 GenExpression Assays (Applied Biosystems) for aquaporin-2 (AQP2; Mm00437575_m1),

128 Atp6v1v1 V1-H-ATPase β1 (Mm00460309_m1), carbonic anhydrase II (Mm00501572_m1),

129 Bradykinin B2 receptor, Bdkrb2 (Mm00437788_m1) and GAPDH (endogenous control,

130 FAM/MGB probe, non-primer limited; no. 4352932E). The setup of reaction consisted of 1μl of

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131 cDNA (100 ng), 1μl of TaqMan primer set, 10μl Taq [TaqMan Fast Universal PCR master mix,

132 No AmpErase UNG; no. 4366072], and 8μl of H2O under the following PCR conditions: step 1,

133 95°C for 20 s; step 2, 95°C for 3 s; and step 3, 60°C for 30 s; steps 2 and 3 were repeated 40

134 times.

135

136 Cell Culture, DNA constructs and Reporter Assays - p53-null human lung carcinoma cells

137 (H1299) were maintained in minimum essential medium supplemented with 10% fetal bovine

138 serum (Invitrogen) at 37 °C in a humidified incubator with 5% CO2. The rat BdkrB2 (pBK2 -

139 94/+55-CAT), rat Agtr1a (pAT1a-1.2/LUC), mouse AQP-2 (-10 kb/CAT), and pG13-LUC promoter

140 reporter constructs have been described previously (35, 40). p53 mutant constructs (SA-15,

141 TA-18, SA-20, 6KR) were generously provided by Dr. T-P Yao (Duke University). Additional

142 mutant p53 constructs were generated in our laboratory using the QuikChange site directed

143 mutagenesis kit (Stratagene) according to manufacturer protocol. Co-transfection of reporter

144 constructs along with wild-type(pCMV-p53, from G. Morris, Tulane University) or mutant p53

145 constructs was performed using the Lipofectamine Plus reagent (Invitrogen) according to the

146 manufacturer’s recommendations as described (43). A control β-galactosidase-encoding

147 vector, pSVZ (Promega), was co-transfected to correct for transfection efficiency. Additional

148 controls included transfections with pCMV-CAT (pCAT3Basic) or pCMV-empty (pCDNA3.1)

149 vectors. Aliquots of cell lysate were analyzed for CAT or LUC activity after normalization for

150 protein content or β-galactosidase activity.

151

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152 Western blot analysis - Nuclear and cytosolic protein fractions were prepared using

153 Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, CA) according to manufacturer

154 protocol. Immunoblotting was performed using 20-50 µg of protein samples. The following

155 primary antibodies were used: total p53, FL-393X (sc-6243, Santa Cruz), acetyl-p53K373/K382 (06-

156 758, Upstate Biotechnologies), acetyl-p53K379 (2570S, Cell Signaling), phospho-p53S392 (9281S,

157 Cell Signaling), phospho-p53S6 (9285, Cell Signaling), phospho-p53S9 (9288, Cell signaling),

158 phospho-p53S15 (9284S, Cell Signaling), and mouse anti-β-actin (610153, BD Transduction

159 Laboratories).

160

161 Electrophoretic Mobility Shift Assays (EMSA) - EMSA was performed as previously described

162 (40). Briefly, [32P] Labeled duplex oligonucleotides (~50,000 cpm) were incubated for 20 min at

163 room temperature with 5 µg of nuclear extracts. Reactions containing an antibody against ac-

164 p53K373/K382 (Upstate Biotechnologies) or p-p53S392 (Cell signaling) were pre-incubated with the

165 nuclear extract on ice for 30 minutes before adding the radiolabeled probe. The binding

166 reaction was loaded onto a 6% acrylamide gel electrophoresed at 200 volts for 2 hrs in 0.25X

167 Tris Borate EDTA (TBE) solution. Following electrophoresis, the acrylamide gel was soaked in a

168 10% glycerol solution for 10 minutes, dried for 1.5 hours at 80°C using a gel dryer, and exposed

169 overnight to capture autoradiography.

170

171 Chromatin Immunoprecipitation (ChIP) - ChIP assays were performed using the EZ-ChIPTM kit

172 (Upstate Biotechnology) with modifications (41). Kidney tissue was minced and cross-linked in

173 PBS/1% formaldehyde solution for 15 min, and quenched by addition of 0.125 M glycine for 5

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174 min. Tissue was rinsed in 1X PBS, homogenized and lysed in SDS-lysis buffer. DNA was sheared

175 by sonication to produce an average DNA fragment size between 500 and 1,000 bp and diluted

176 10-fold in ChIP dilution buffer. Immunoprecipitation was performed with anti-p53 (FL-393X)

177 antibody or control normal immunoglobulin (IgG) overnight at 4°C. Precipitated complexes

178 were captured on protein G Dynabeads (Invitrogen), washed and eluted from the beads and

179 cross-links were reversed at 65°C overnight. Immunoprecipitated DNA was purified and used

180 for semi quantitative PCR. Sequences for the primers used for PCR are as follows (all mouse):

181 Bdkrb2 - forward, 5’-TTGAAAGATGAGCTTGTTC-3’, and reverse, 5’-AAACTCACCTTTTCTCAA;

182 Atp6V0A2 - forward, 5’-GAGAGAAGAGAGAAGAGA, and reverse, 5’-CTGCTTTTAGAGTTTTGTT;

183 Nkcc1 - forward, 5’-GTTAATCTGGGAGGAATG, and reverse, 5’-GCATCAATGTTATCTTCAA; Enac-γ

184 - forward, 5’-CCAAAGAATAAATGATCTGT, and reverse, 5’-TCTCTTATCTCAGTTCCA; p21 -

185 forward, 5’-CCTTTCTATCAGCCCCAGAGGATACC-3’, and reverse, 5’-

186 GACCCCAAAATGACAAAGTGACAA-3’; DDX-17 - forward, 5’-GATTAAAACTTGAGCATCC, and

187 reverse, 5’-AGATCTTTATCCGTTTGG; and BAD - forward, 5’-CATTTTACAGGAGGGAAT, and

188 reverse, 5’-CTGACCCAATCAGTTTTC.

189

190 Immunohistochemistry - Kidneys were fixed in 10% formalin at 4°C overnight, processed for

191 paraffin embedding, sectioning (5 μm) and immunostaining as described (20). The primary

192 antibodies used are: acetyl-p53K373/K382, 1:100 dilution (06-758, Upstate Biotechnologies),

193 acetyl-p53K386 , 1:100 dilution (ab52172, Abcam), phospho-p53S392, 1:100 (sc-56173, Santa

194 Cruz), PCNA, 1:200 dilution (M0879, Dako Cytomation), phospho-histone H3 (Ser10), 1:300

195 dilution (9701, Cell Signaling), E-Cadherin, 1:300 (610181, BD Transduction Laboratories),

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196 AQP2, 1:500 dilution (sc-9882, Santa Cruz), NCAM, 1:200 dilution (C9672, Sigma), FITC-

197 conjugated lotus tetragonolobus lectin agglutinin (LTA), 1:300 dilution (FL-1321, Vector

198 Laboratories, Inc.) and DAPI, 1:500 dilution (D1306, Invitrogen). For Immunofluorescence

199 detection, we used donkey anti-mouse or donkey anti-rabbit secondary IgG antibodies with

200 Alexafluor 555 or 488 conjugates (Invitrogen). The immunofluorescent images were captured

201 using a 3D or deconvolution microscope (Leica DMRXA2).

202

203 Statistical analysis

204 The results are presented as mean ± SEM. Comparisons between the groups were

205 performed by unpaired t-test or ANOVA using GraphPad Prism software (5.01 Version).

206 Differences were considered significant if the p value was <0.05.

207

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208 RESULTS

209 p53 protein is post-translationally modified in a developmentally regulated manner – We have

210 previously demonstrated that kidney p53 mRNA levels decline by approximately 50% from

211 embryonic to adult life (20, 42). Here, we show that p53 protein expression is highly abundant

212 in the embryonic kidney but declines dramatically postnatally to almost undetectable levels in

213 the adult kidney (Fig. 1A). To determine whether p53 modifications known to regulate p53

214 stability in response to DNA damage also occur in the embryo, we analyzed the temporal

215 changes in p53 phosphorylation and acetylation, schematically depicted in Fig. 1B. Western

216 blot analysis of nuclear protein extracts showed that the ratio of ac-p53K373,K382/total p53 is

217 significantly higher in the embryonic than postnatal kidney (Fig. 1C). Adult kidneys express

218 very low to undetectable levels of ac-p53K373,K382 (data not shown). Similarly, the relative

219 levels of p-p53S6,S9,S15,S392/total p53 declined significantly during transition from embryonic to

220 adulthood (Fig. 1D). Adult kidneys expressed very low levels of total or modified p53 (Fig. 1

221 A,D). The parallel developmental changes in total and modified p53 suggest a possible role for

222 p53 phosphorylation/acetylation in mediating p53 protein stability during kidney maturation.

223

224 Developmental changes in p53 DNA binding activity - We performed EMSA on kidney nuclear
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225 extracts to determine the maturational changes in p53 DNA binding activity. A P-labeled

226 oligonucleotide duplex corresponding to the conserved high affinity p53-binding site in the

227 Bkdr2 promoter (43) was used as a probe. Fig. 2A shows that multiple DNA-protein complexes

228 form when the BK2-P1 probe is incubated with E17.5 nuclear kidney extracts. As expected, no

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229 binding is detected in control p53-deficient H1299 cell extracts. Addition of ac-p53K373/K382

230 antibody to the reaction mixture supershifts the DNA-protein complex, while addition of anti-

231 p53-pS392 antibody inhibits complex formation (Fig. 2A). Furthermore, when we compared

232 probe binding to protein extracts at different stages, we found a progressive decrease in

233 binding from E13.5 to adulthood, especially postnatally (Fig. 2 B,C).

234

235 Effects of post-translational modifications on p53 protein stability - We next examined the

236 effects of phosphorylation and acetylation on p53 protein stability under normal cellular

237 conditions. A series of p53 mutant constructs were generated by site directed mutagenesis

238 (Fig. 3A) and transiently transfected into p53-deficient H1299 cells followed by p53

239 immunoblotting. Mutagenesis of p53S15/T18/S20, but not p53S392, dramatically reduces p53

240 protein stability (Fig. 3 B,D). Whereas mutations of individual C-terminus lysines

241 p53K373/K382/K386 had no effect on p53 stability, combined elimination of the six acetylation sites

242 (p536KR) enhanced p53 stability relative to the wild-type protein (Fig. 3 C,D). Together, these

243 findings indicate that post-translational modifications exert differential effects on p53 stability.

244

245 Effects of p53 modifications on activity of RFG promoters - We previously demonstrated that

246 p53 transactivates the promoters of BdkrB2, AQP2, and Na-K-ATPase α, and represses the

247 Agtr1a promoter (40). Co-transfection experiments performed in H1299 cells revealed that

248 relative to wild type p53, mutant p53S/A15 displayed significantly reduced transcriptional effects

249 on the AQP2, BdkrB2, and Agtr1a promoter reporter constructs even after correction for p53

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250 protein levels determined by Western blotting and densitometric analysis (Fig. 4). However,

251 p53S/A15 was equally capable of activating PG13-Luc, a synthetic reporter construct driven by

252 13 tandem copies of a p53 consensus sequence (Fig. 4). p53T/A18 and p53S/A20 mutant

253 constructs displayed lower transcriptional activity of AQP2 but not BdkrB2 promoter

254 constructs; however, both mutants exhibited lower repression of Agtr1a (Fig. 4). Compared to

255 WT-p53, p53S/A392 and p53K/R382 constructs showed only modest activation of the PG13-Luc

256 construct but not others (Fig. 4). Although p536KR is a more stable protein (Fig. 3 C,D), this did

257 not translate into enhanced transcriptional activity (Fig. 4). We conclude that post-

258 translational modifications of p53 have differential effects on its transcriptional activity of RFG

259 promoters.

260

261 Spatial expression of ac-p53K386 and ac-p53K373/K382 - In the developing kidney, two distinct

262 zones can be distinguished histologically: an outer cortical nephrogenic zone (NZ) and a sub-

263 cortical differentiating zone (DZ). The NZ houses renal progenitors and nascent nephrons

264 which express proliferating cell nuclear antigen (PCNA) and Neural Cell-Associated Marker

265 (NCAM), whereas the DZ contains maturing tubular structures which express differentiation

266 markers. We sought to determine whether p53 modifications are associated with specific

267 spatial expression patterns. Acetylation of p53 at K373/382/386 by CBP/P300 correlates with

268 enhanced transcriptional activity and DNA binding affinity (5). Immunofluorescence using

269 antibodies specific to ac-p53K373/K382 or ac-p53K386 showed that ac-p53K373,K382,K386 are

270 predominantly expressed in the NZ in an overlapping manner with PCNA and NCAM (Fig. 5 A-E,

271 and Fig. 6 A,B). However, there seems to be more co-localization of ac-p53K386 with PCNA than

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272 there is between ac-p53K373/K382 and PCNA (Fig 5 A-D). Moreover, ac-p53K373/K382/K386 are

273 expressed within epithelial, E-cadherin-positive, tubules (Fig. 5F and Fig. 6E). Co-staining with

274 AQP-2, a marker of collecting ducts, or LTA, a marker of proximal tubules, revealed that ac-

275 p53K373/K382/K386 are mostly (but not exclusively) expressed in the collecting duct/distal nephron

276 segments (Fig. 5 G,H and Fig. 6 C,D,F).

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278 p-p53ser392 is enriched in proximal tubules - At E15.5, most tubular structures have not

279 differentiated yet into mature proximal tubules as indicated by paucity of LTA staining as

280 compared to older age groups (Fig. 7 A). At this stage, occasional p-p53S392-expressing cells are

281 localized in newly formed tubular structures expressing LTA (Fig. 7 A-C). At E17.5 and PN1,

282 when the cortex has demarcated into NZ and DZ, p-p53S392 expression is clearly enriched

283 within LTA-positive proximal tubules (Fig. 7 D-I). Also, p-p53S392 displayed minimal if any co-

284 localization in proliferating cells (Fig. 7 J,K) or AQP2+ collecting ducts (Fig. 7L). Collectively,

285 these results demonstrate a stage- and cell-type specific expression of p-p53S392 in maturing

286 proximal nephron segments.

287

288 Ontogeny of p53 and RFGs - We next examined the ontogeny of a subset of nephron

289 differentiation genes, i.e., RFGs, in relation to the temporal expression of p53. RFG mRNA

290 levels were quantified by RT-PCR using freshly harvested kidneys (in vivo samples, E13.5-E17.5)

291 and compared to kidneys harvested and cultured (ex vivo samples, E13.5+96hr). p53 mRNA

292 levels were maintained in the developing kidney from E13.5-E17.5 (Fig. 8). On the other hand,

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293 most RFGs were significantly up-regulated between E13.5 and E17.5 (Fig. 8). This discrepant

294 expression pattern suggests that RFG expression is not primarily driven by ambient p53 levels.

295 Furthermore, we found that RFG expression pattern from in vivo samples was similar in ex vivo

296 samples (Fig. 8), suggesting that blood flow and glomerular filtration are not required for the

297 inductive expression of RFGs.

298

299 Developmental changes in p53 binding to RFGs targets – Since tissue levels may not reflect the

300 actual functional importance of a transcription factor, we performed ChIP-PCR assays to

301 examine p53 occupancy on RFG promoters in developing kidneys; as a control, we included

302 known p53 targets such as p21, DDX-17 and BAD. The results demonstrate that p53 binding to

303 RFGs peaks at the onset of tubular differentiation on E15.5 (Fig. 9 A,B). Although p53 was also

304 bound to non-RFGs (DDX and BAD), a developmental pattern was not observed (Fig. 9 A,B).

305 These data suggest that onset of nephron differentiation is accompanied by enhanced p53

306 binding to RFGs promoters.

307

308 Effect of altered p53 activity on RFG expression - To assess the biological effects of altered p53

309 activity on the expression of RFGs in vivo, we performed quantitative RT-PCR on RNA extracted

310 from embryonic p53+/+ and p53-/- kidneys, and from mice with conditional ureteric bud-specific

311 deletion of Mdm2 (UBMdm2-/-). The latter mice lack Mdm2 from the collecting duct system. We

312 also performed ex vivo studies in E13.5 embryonic kidneys cultured in the presence of

313 Pifithrin-α (PFT-α), a small molecule p53 inhibitor, or Nutlin-3, a chemical compound which

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314 inhibits Mdm2-p53 interactions and thus stabilizes p53. Fig. 10 shows that genetic or chemical

315 inactivation of p53 downregulates Bdkr2, whereas carbonic anhydrase 2, CA2 (a marker of

316 intercalated cells), was downregulated by PFT-α. Other markers, such as H+-ATPase and AQP2

317 were not affected. AQP2 is known to be regulated by the p53 family member, p73 (39);

318 therefore, compensation by other p53 family members might explain the modest effects of

319 p53 inhibition.

320 Excessive p53 activity in vivo (UBmdm2-/-) and ex vivo (Nutlin) up-regulates Bdkr2, CA2,

321 and H+-ATPase (Fig. 10). Total kidney AQP2 mRNA levels are up-regulated in response to

322 Nutlin, but not in UBmdm2-/- mice; however, in situ hybridization did show upregulation of AQP2

323 mRNA in collecting ducts of UBmdm2-/- mouse (data not shown). These data are consistent with

324 our overall hypothesis that p53 modulates RFG expression in vivo.

325

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326 DISCUSSION

327 In the present study, we examined the mechanisms of p53 regulation during murine

328 kidney development. The findings indicate that embryonic kidney p53 is phosphorylated and

329 acetylated, and its endogenous activity is regulated by Mdm2. These two factors may

330 contribute to its stability, cell type-specific expression, and target gene activation during

331 nephrogenesis. We also report that p53-chromatin interactions on RFG target promoters are

332 developmentally regulated.

333 We have previously reported that p53 is enriched in renal epithelial cells during

334 nephron differentiation (16, 40, 42). We also identified several RFGs (Bdkrb2, AQP-2, Na-K-

335 ATPase-α1, and Agtr1) as a novel group of p53 target genes (35, 40, 41, 43, 44). Here, we

336 report that the p53 protein is subject to developmental regulation, whereby expression is

337 highest during nephrogenesis and is downregulated in the mature kidney. Importantly, we

338 found that the p53 modification cassette during normal kidney development resembles the

339 one elicited during the p53 response to DNA damage (10, 19, 21, 29, 37). This finding implies

340 that the hyperproliferative state of embryogenesis may provide a stress signal leading to p53

341 activation (33, 45). The nature of the stress signal is unclear; we speculate that it may be

342 related to a greater need for DNA repair during active DNA replication, cellular hypoxia,

343 increased metabolic needs and competition of rapidly dividing cells for nutrients, or handling

344 of accumulating reactive oxygen species.

345

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346 The results of the present study revealed that p53 phosphorylation on various Serine

347 residues is differentially acquired during nephron differentiation. Thus, whereas p53 N-

348 terminus phosphorylation levels on Ser6, 9, 15 are abundant during embryonic life,

349 phosphorylation of p53 on Ser392 is relatively low in the embryonic kidney, but is induced

350 postnatally. The physiological relevance of p53 phosphorylation in the developing kidney is

351 unknown. This study offers some clues. Phosphorylation is required for p53 protein

352 stabilization in response to DNA damage. Indeed, our in vitro data support the notion that

353 phosphorylation of Ser15, 20 and Thr18 is required for full stabilization of p53. We therefore

354 surmise that p53 N-terminus phosphorylation plays a role in p53 stability perhaps by limiting

355 its interaction with Mdm2.

356 Ser392 (mouse Ser389) is one of the target residues phosphorylated in p53. Kinases

357 that target this site in vitro are casein kinase II, the double-stranded-RNA-activated protein

358 kinase (PKR), and p38 MAP kinase (25). Although homozygous mutant p53S389A mice (serine

359 392 in human) are viable, p53S389A mutant cells are compromised in transcriptional activation

360 of p53 target genes and apoptosis after UV irradiation (8). Moreover, p53S389A mice show

361 increased sensitivity to UV-induced skin tumor development (8). Thus, serine 392(389)

362 phosphorylation is required for the tumor-suppressive function of p53. The surge in p-

363 p53Ser392 during later stages of kidney development suggests that this modification may be

364 involved in programming of terminal differentiation gene expression. Further, p-p53Ser392 is

365 nephron segment-specific (proximal tubule) and thus may be important in cell fate

366 maintenance. Future studies are needed to test these hypotheses.

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367 Unlike Ser392 phosphorylation, acetylation of p53 on Lys373,382,386 is more active at

368 embryonic stages than after birth. Physiologically, p53K373/K82 acetylation correlates with

369 enhanced DNA binding affinity and transcriptional activity (5), implying that p53 is

370 transcriptionally more active in the embryonic than postnatal kidney. However, knock-in

371 mouse studies in which C-terminus lysines were mutated did not reveal a specific phenotype

372 (27). We speculate that the distinct p53 post-translational cassettes in the embryo may

373 empower p53 with selective access to different target genes or recruitment of different co-

374 factors, although their in vivo relevance may not become apparent under unstressed

375 conditions.

376 Differential spatial expression patterns of p53 post-translational modifications have not

377 previously been associated with tissue differentiation. Given the roles of p53 in patterning of

378 the intermediate mesoderm and development of kidneys, liver, and neural tube (1, 2, 17, 30,

379 31, 47, 49), it would be important to determine whether key morphogenetic pathways (PI3

380 kinase, TGF-β, Wnt) target differential modifications on the p53 protein to affect

381 developmental fate switches (14). It has been suggested that post-translational modifications

382 provide p53 with target gene specificity. For example, phosphorylation of p53 Ser46 and

383 acetylation of Lys120 are proposed to influence the induction of specific apoptotic target

384 genes, whereas acetylation of p53 Lys320 favors DNA binding to the p21 gene, promoting cell

385 cycle arrest (36). Our promoter-reporter assays indicate that interference with N-terminus

386 phosphorylation, but not acetylation, impacts p53-mediated activation of select RFGs.

387 Although these results may not completely represent the in vivo conditions, they do confirm

388 that p53 modifications confer transcriptional specificity.

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389

390 We examined using ChIP p53 binding to RFGs and other known targets during kidney

391 development. p53 occupancy of RFG promoters is highly enriched at the onset of tubular

392 differentiation at E15.5 as compared to the postnatal kidney. Maintenance of RFG expression

393 (with the exception of Bdkrb2) in p53-null mice may be related to compensation by other p53

394 family members (39). In addition, p53 did not display a similar developmental binding to the

395 DEAD Box gene, DDX17, or the proapoptosis gene, BAD. It is tempting to speculate that in

396 response to a differentiation signal, combinatorial patterns of p53 modifications allow

397 distinguishing between different biological responses via chromatin association and RFG

398 transactivation.

399 The ubiquitin ligase Mdm2 mediates p53 proteosomal degradation (34). Moreover, the

400 Mdm2 gene is positively regulated by p53, thus establishing a negative feedback loop which

401 controls p53 levels physiologically. In order to examine how Mdm2-p53 interactions modulate

402 RFG expression during nephron differentiation, we employed two complementary approaches:

403 treatment of embryonic kidney cultures with chemical inhibitors of Mdm2 or p53, and

404 targeted disruption of the Mdm2 or p53 genes. When analyzing the effect of altered p53

405 activity, we found changes in a subset of RFGs (AQP2, Bdkrb2, CA2) that are mainly expressed

406 in UB derivatives; this is not surprising given that p53 is enriched within these structures (20,

407 42). Collectively, our data suggests that changes in RFG expression in vivo are more

408 pronounced following p53 stabilization than p53 inactivation. We speculate that this may be

409 due to redundancy by the p53 family members, p63 and p73.

19
410 In summary, the present study demonstrates that the p53 protein in the embryonic

411 kidney is subject to post-translational modifications. These modifications impart p53 with

412 nephron segment-specific expression patterns and are able to regulate p53 stability and target

413 gene activation. The onset of nephron differentiation is accompanied by enhanced occupancy

414 of p53 on promoters of RFGs. Future studies should elucidate the upstream kinases and

415 signaling pathways which mediate these developmental changes in the Mdm2-p53 module

416 and whether p53 modifications act as a fate switch during tissue patterning and

417 organogenesis.

418
419

20
420 Acknowledgments

421 These studies were performed as part of a pre-doctoral dissertation thesis (K.A.). We thank
422 the members of the El-Dahr and Saifudeen laboratories for insightful discussions and technical
423 assistance.

424

425 Grants

426 This work was supported by NIH grant RO1DK62550 (S.E.D). Z.S. is supported by Center of
427 Biomedical Research Excellence 1P20 RR017659.

428

429 Disclosures

430 The Authors have no conflict of interest, financial or otherwise.

431

432 Author Contributions

433 - Karam Aboudehen: is a graduate student in the Biomedical Sciences Program and has
434 performed all of the experiments and results presented in the manuscript. He also wrote
435 the manuscript and prepared the figures.
436 - Sylvia Hilliard, Ph.D.: Dr. Hilliard provided tissues from the conditional UB-targeted
437 MDM2-null mice.
438 - Zubaida Saifudeen, Ph.D.: Dr. Saifudeen provided tissue and reagents related to the p53-
439 null mice, and played a key role in the overall supervision and troubleshooting of the
440 project and in editing the manuscripts and figures.
441 - Samir S. El-Dahr, M.D.: Dr. El-Dahr is the doctoral supervisor of Mr. Aboudehen. He
442 played a key role in the overall design, interpretation, troubleshooting, and editing of the
443 manuscript and figures.

21
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573
574

575

24
576 FIGURE LEGENDS

577 FIGURE 1. Developmental changes in total and modified p53 in mouse kidneys. (A) Western

578 blot analysis of total p53 in nuclear extracts from embryonic (E), postnatal (PN) and adult

579 kidneys. (B) Schematic of p53 post-translational modifications investigated in this study. (C,D)

580 Western blot analysis of acetyl p53 and phosphorylated p53 in nuclear kidney extracts.

581 Specificity of antibodies was tested in PN1 p53+/+ and p53-/- whole kidney extracts. Equal

582 protein loading was monitored by Ponceau S staining (not shown) and anti-ß-actin antibody.

583 Acetylated and phosphorylated p53 densitometric values were normalized to total p53 values

584 shown in A and expressed relative to the values on E13.5.

585 FIGURE 2. Developmental changes in kidney p53 DNA binding activity. (A) EMSA with a 32P-

586 labeled probe corresponding to the consensus p53 binding site in the Bdkrb2 promoter

587 incubated with nuclear extract (NE) from E17.5 or H1299 cell lysate in the presence or absence

588 of antibodies to ac-p53K373/K382 and p-p53Ser392. (B,C) EMSA showing DNA binding activity of

589 labeled probe to kidney extracts at E13.5, E17.5 and PN1. For competition assays, 10 to 50-

590 fold excess cold p53 oligonucleotide duplex was used. Lanes: 1, free probe; 2, probe in the

591 presence of NE from the indicated stage; 3, probe in the presence of NE and competitor; 4,

592 probe in the presence of NE and the indicated antibodies.

593 FIGURE 3. Post-translational modifications are important for p53 protein stability. (A)

594 Schematic of the p53 mutations tested in this study. (B, C) Western blots of p53 protein in

595 H1299 cells transfected with p53-mutant constructs (see text for details). Bottom panels

596 represent immunoblots for β-actin. (D) Densitometric analysis of p53 protein expression of

25
597 mutant constructs relative to wild-type p53. Data in the graph represent mean ± S.E. of at

598 least three experiments. * p<0.05 vs. WTp53.

599 FIGURE 4. Effects of p53 modifications on RFG gene expression. Wild-type and mutant p53

600 constructs were co-transfected along with promoter-reporter constructs of BdkrB2, AQP2,

601 Agtr1a (AT1a) or PG13 into H1299 cells and lysates were assayed for luciferase or CAT activity

602 24 h post transfection (see text for details). The graphs show the change in reporter activity

603 for the indicated constructs adjusted to wild-type p53 which was arbitrarily set to 1.00. Data

604 in each graph represent mean ±S.E. of at least three experiments (* p<0.05).

605 FIGURE 5. Spatial expression of ac-p53K373/382 in the developing kidney. Immunofluorescence

606 staining in kidney sections using antibodies to ac-p53K373/K382, PCNA, NCAM, LTA, E-cadherin,

607 AQP-2 and PH3. (A-D) Ac-p53K373/K382 is expressed in the nephrogenic zone (NZ) in embryonic

608 (E) and postnatal day 1 (PN1) kidneys. (E) Ac-p53K373/K382 is enriched in NCAM-positive cap

609 mesenchyme (CM). (F) Ac-p53K373/K382 is expressed in E-cadherin-positive epithelial tubules.

610 (G, H) Ac-p53K373/K382 is expressed in maturing collecting ducts (CD) but not in proximal tubules

611 (PT).

612 FIGURE 6. Spatial expression of ac-p53K386 in the developing kidney. Immunofluorescence

613 staining of kidney sections on E15.5 (A,B) or E17.5 (C,D) using antibodies to ac-p53K386, PCNA

614 E-cadherin, or AQP-2. Ac-P53K386 is expressed in proliferating cells of nephrogenic zone (NZ)

615 (A,B), ureteric bud branches (C) and collecting duct (D).

616 FIGURE 7. Spatial expression of p-p53Ser392 in the developing kidney. Immunofluorescence

617 staining of kidney sections using antibodies to ac-p53Ser392, LTA, phospho-histone H3, PCNA or

26
618 AQP-2. (A-I) The nephrogenic zone (NZ) is devoid of ac-p53Ser392 but is enriched in

619 differentiated proximal tubules (PT). (J,K) Ac-p53Ser392 is not expressed in dividing cells or

620 collecting ducts (L).

621 FIGURE 8. Developmental expression of RFGs in vivo and ex vivo organ culture. RFGs

622 transcript levels in RNA samples from embryonic (E13.5, E14.5, E15.5, E16.5, E17.5) and organ

623 cultured kidneys (E13.5 + 24 h, 48 h, 72 h or 96 h) were determined by RT-PCR. Gene

624 expression levels were normalized to Gapdh. The expression level at E13.5 was arbitrarily set

625 to 1.0. Data in each graph represent mean ± S.E. of at least three experiments. A

626 representative ethidium bromide-stained gel is shown for each gene.

627 FIGURE 9. Occupancy of RFG promoters by p53 during nephron differentiation. (A)

628 Representative gel images of ChIP experiments performed in embryonic (E15.5) and postnatal

629 (PN1) kidneys using p53 (FL-393) antibody or control IgG. The precipitated chromatin was

630 analyzed for p53 binding using primers for differentiation RFGs (Bdkrb2, NKCC1, ATPV0A2,

631 ENAC-g, p21) and other p53 target genes (DDX-17, BAD). (B) Quantitative analysis of percent

632 of promoter-bound p53 for different target genes. Data is expressed in percent change

633 relative to the amount of input DNA. Data in each graph represent mean ± S.D. of at least

634 three experiments (* p<0.05).

635 FIGURE 10. Effect of altered p53 activity on RFGs expression. p53-/- and UBMdm2-/- and

636 littermate wild-type kidneys were harvested at E17.5 and extracted RNA was analyzed by qRT-

637 PCR. For organ culture studies, metanephroi were dissected at E13.5 and incubated in the

638 presence of PFT-α (10 μM) or Nutlin-3 (10µM) for 96 h. Gene expression in PFT-α or Nutlin-

27
639 treated kidneys was compared to control DMSO-treated kidneys, while expression in p53-/- and

640 Ubmdm2-/- kidneys was compared to wild-type littermates. Data is expressed in relative fold

641 change from control/wild-type normalized at 1.0 (* p<0.05).

642

28
643

Table 1. List of primers used in semi-quantitative RT-PCR.

29
 A
Total p53
E13.5 E17.5 PN1 Adult PN1p53+/+ PN1p53-/-

50kd

actin

B phosphorylation acetylation
S6 S9 S15 K373 K382 S392

Transactivation Proline DNA-Binding Domain Oligomerization Regulatory

Domain Domain Domain Domain
(1-61) (64-101) (102-300) (307-355) (356-393)

C
E13 5
E13.5 E17 5
E17.5 PN1 p53+/+
p / p53-/-
p /

acK382

acK373,382
actin

D
E13.5 E17.5 PN1 Adult

pSer6

pSer9

pSer15 p53+/+ p53-/-

pSer392
actin

Figure 1
 B
E13.5 E17.5 PN1
acp53K373/382 - - - + - - - + - - - +
Comp - - + - - - + - - - + -
1 2 3 4 1 2 3 4 1 2 3 4

Bound

Free

C
E13.5 E17.5 PN1
PSer392 - - - + - - - + - - - +
Comp - - + - - - + - - - + -
1 2 3 4 1 2 3 4 1 2 3 4

Bound

Free

Figure 2
A

WT-p53 SA-15 TA-18 SA-20 SA-392

B

actin

WT-p53
p KR-372 KR-382 KR-386 6-KR
C

actin

Figure 3
A
Rat Bdkrb2 reporter construct

+1
-1184 bp
CAT

B
Mouse AQP2 reporter construct

+1
-10000 bp
CAT

C
Human p21 reporter construct

+1
-2183 bp
LUC

Rat AT1a reporter construct

+1
-3231 bp
LUC

Figure 4
 Ac-p53K373,K382/PCNA/Dapi
10x 20x
20x
10x

CM

UB
E15.5

UB
AA BB
5

10x 20x

PT

CD

C D

p53K373,K382/ NCAM/Dapi
Ac-p53
Ac Ac-p53K373,K382
, / E-Cad/Dapi
20x 20x
CM

UB

UB

E F
E17.5

Ac-p53K373,K382
, / AQP2/Dapi Ac-p53K373,382
, / LTA/pH3
5

20x 10x

PT

CD

G H

Figure 5
 p53K386/PCNA
ac-p53
ac
A B

10x10x 20x
10x
10x ac-p53K386/LTA/pH3
20x
C D
E17.5

10x 20x

ac-p53K386/E-cad/Dapi ac-p53K386/AQP-2/Dapi
E F

20x 20x

Figure 6
 p-Ser392/LTA/Dapi
10x 20x 40x

NZ
E15.5

PT

A B C

10x 20x 40x

E17.5

PT

D E F
10x 20x 40x
PN1

G H I

p-Ser392/pH3/Dapi p-Ser392/LTA/Cyto p-Ser392/AQP2/Dapi

40x 20x 20x
E17.5

J K L

Figure 7
Figure 8
A

F ig u r e 9
Figure 10