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2011
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
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
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
65
2
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
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.
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
3
88 (K320) and Tip60/hMOF (K120,K164) (5, 6, 15, 18, 21). Acetylated p53 has a higher DNA-
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
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
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-
4
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).
117
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
130 FAM/MGB probe, non-primer limited; no. 4352932E). The setup of reaction consisted of 1μl of
5
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
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
147 vector, pSVZ (Promega), was co-transfected to correct for transfection efficiency. Additional
149 vectors. Aliquots of cell lysate were analyzed for CAT or LUC activity after normalization for
151
6
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
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
7
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):
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
202
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).
207
9
208 RESULTS
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
32
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
10
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
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
11
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
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
12
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
277
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
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,
13
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
298
299 Developmental changes in p53 binding to RFGs targets – Since tissue levels may not reflect the
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
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
14
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
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
15
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
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
345
16
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
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
365 nephron segment-specific (proximal tubule) and thus may be important in cell fate
17
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
18
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
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
431
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
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,
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
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).
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
610 (G, H) Ac-p53K373/K382 is expressed in maturing collecting ducts (CD) but not in proximal tubules
611 (PT).
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).
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
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
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
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
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
642
28
643
29
A
Total p53
E13.5 E17.5 PN1 Adult PN1p53+/+ PN1p53-/-
50kd
actin
B phosphorylation acetylation
S6 S9 S15 K373 K382 S392
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
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
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
+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
PT
D E F
10x 20x 40x
PN1
G H I
J K L
Figure 7
Figure 8
A
F ig u r e 9
Figure 10