Role of P63 and the Notch Pathway in Cochlea Development and Sensorineural Deafness

Corrections

CELL BIOLOGY Correction for “Role of p63 and the Notch pathway in cochlea ously duplicated a p53-RE-III panel from an earlier paper development and sensorineural deafness,” by Alessandro Terrinoni, (1). We thank the reader for bringing this issue to our at- Valeria Serra, Ernesto Bruno, Andreas Strasser, Elizabeth Valente, tention, and we deeply apologize to the scientific community for Elsa R. Flores, Hans van Bokhoven, Xin Lu, Richard A. Knight, the error.” and Gerry Melino, which appeared in issue 18, April 30, 2013, of The corrected figure and its corrected legend appear below. Proc Natl Acad Sci USA (110:7300–7305; first published April 15, 2013; 10.1073/pnas.1214498110). “ 1. Tucci P, et al. (2012) Loss of p63 and its microRNA-205 target results in enhanced The authors note that Thanks to an alert reader, we no- cell migration and metastasis in prostate cancer. Proc Natl Acad Sci USA 109(38): ticedthatinFig.3D, the control ChIP for MDM2 errone- 15312–15317.

A Promoter RE Hes5 gene

200 1000 2000 2200 2400 2600 2800 3000 3200 3400 45 B Luciferase activity TID 40

35 30 SaOs-2 TAp63α C SaOs-2 HA-TAp63α D 25 Input HA IgG SAM DBD Doxy - + 20 HA Hes5 Fold over control 15 p21 10 Actin 5 MDM2 0

I537T G530V Q536LR280CR304WS272NR279H Q634X E pcDNA-HATAp63a Wt Q566fsX94 RE I 5’ Promoter RE II 3’

Atoh1 mRNA transcript Enhancer A Enhancer B 3000 4200 7200 F G H 7 Luciferase activity TID SaOs-2 TAp63α SaOs-2 HA-TAp63α 6 SAM DBD IgG Doxy - + Input HA 5 HA 4

3 Atoh1 2 Fold over control

p21 1

0 Tub I537T G530V R279HR280CR304WS272N Q634X Ap63a Wt pcDNA-HAT Q566fsX94

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2854–2855 | PNAS | February 18, 2014 | vol. 111 | no. 7 www.pnas.org Downloaded by guest on September 23, 2021 CELL BIOLOGY Correction for “Loss of p63 and its microRNA-205 target results Melino, which appeared in issue 38, September 18, 2012, of in enhanced cell migration and metastasis in prostate cancer,” by Proc Natl Acad Sci USA (109:15312–15317; first published Paola Tucci, Massimiliano Agostini, Francesca Grespi, Elke K. September 4, 2012; 10.1073/pnas.1110977109). Markert, Alessandro Terrinoni, Karen H. Vousden, Patricia A. J. The authors note that “The y-axis label of Fig. 5C is incorrect. Muller, Volker Dötsch, Sebastian Kehrloesser, Berna S. Sayan, Instead of ‘Biochemical Recurrence,’ it should read ‘Biochemical- Giuseppe Giaccone, Scott W. Lowe, Nozomi Takahashi, Peter Free Recurrence.’ We apologize to readers for the erroneous la- Vandenabeele, Richard A. Knight, Arnold J. Levine, and Gerry beling.” The corrected figure and its corrected legend appear below.

A C D Np63/miR205 function Np63/miR205 intermediate Np63/miR205 loss PROBABILITY PROBABILITY PROBABILITY PROBABILITY (Overall Follow-up time)

TIME (months) TIME (months) (Biochemical-Free Recurrence, censored) B E 60

20 *** *** Number of metastasis 0 Ctrl Scr miR-205 Np63

Fig. 5. In human prostate cancer, loss of ΔNp63 and miR-205 associates with invasive phenotype and poor clinical outcome. Tumor and normal prostate samples clustered into three groups reflect the activity of the ΔNp63–miR-205 complex. Groups were indicative of metastatic and invasive behavior as well as clinical prognosis. (C) Time to biochemical-free recurrence for the three groups. (D) Clinical follow-up in the cohort was recorded over 5 y; all data are censored for survival. Kaplan–Meier analysis with suppressed censoring shows a significant trend within this follow-up time toward poor survival in the ΔNp63–miR-205 loss group. (A) miR-205/ΔNp63 relationship. Average expression of ΔNp63 was calculated from ΔNp63-specific probes and compared with miR- 205 expression. Metastatic tumor samples are indicated in red diamonds, primary tumors in black squares, and normal samples in blue circles. Pearson correlation was calculated together with the significance of the correlation. Notable is the clean separation between metastatic and normal samples. (B) Heatmap illustrating miR-205 and ΔNp63 expression in prostate cancer. Data show (i) clustering of samples by their ΔNp63/miR-205 expression, Middle bars; (ii) association between normal and ΔNp63/miR-205 expression as well as metastasis and ΔNp63/miR-205 loss, Top bars; and (iii) association between the EMT signature and the expression of the ΔNp63/miR-205 axis, Bottom bars. Middle bars (clustering): Samples were clustered by their miR-205/ΔNp63 expression (red indicates significant overexpression and blue, significant underexpression of the gene/miR, true color: P ≤ 1.0E-05). This determined one group of samples with an active miR-205/ΔNp63 axis (“function”), one group with a clear loss of expression (“loss”), and one intermediate group (“intermediate”) as indicated in the clustering bar. Top bars (primary, metastasis, normal): The group exhibiting loss of miR-205/ΔNp63 expression was enriched in metastatic samples, CORRECTIONS whereas the miR-205/ΔNp63 function group was enriched in normal tissue samples (P < 1.0E-06; compare also Fig. S8 A–C). Bottom bars: The miR-205/ΔNp63- loss group was also associated with an EMT transcriptional profile (P < 0.01, compare also Fig. S8D). Signature scores (Sarrio bars) for experimentally derived signatures according to Sarrio et al. (27) characterizing EMT are shown. Red indicates significant positive, and blue indicates significant negative association of a sample with a signature. P value for enrichment of the cluster loss with the signature EMT_up is 0.0014. Full statistics are in Fig. S8D.(E) Lung metastasis in nude mice. A total of 1.5 × 106 scrambled control–PC3–Tet–On (12 mice), ΔNp63α–PC3–Tet–On (10 mice), or miR-205–PC3–Tet–On (10 mice) cells were injected through the tail vein of BALB/c nude male mice. ΔNp63α and miR-205 expression was induced with doxycycline through their drinking water. Animals were killed after 3 wk and total number of lung metastases was counted using a stereomicroscope. ***P < 0.001. See also Fig. S9.

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PNAS | February 18, 2014 | vol. 111 | no. 7 | 2855 Downloaded by guest on September 23, 2021 Role of p63 and the Notch pathway in cochlea development and sensorineural deafness

Alessandro Terrinonia,1,2, Valeria Serraa,1, Ernesto Brunob, Andreas Strasserc,d, Elizabeth Valentec,d, Elsa R. Florese, Hans van Bokhovenf, Xin Lug, Richard A. Knighth, and Gerry Melinoa,h,2 aBiochemistry Laboratory Istituto Dermopatico Dell’Immacolata, c/o Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata,” 00133 Rome, Italy; bDepartment of Clinical Sciences and Translational Medicine, University of Rome “Tor Vergata,” 00133 Rome, Italy; cThe Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; dDepartment of Medical Biology, Melbourne University, Parkville, VIC 3052, Australia; eDivision of Basic Science Research, Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; fDepartment of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6500 HB, Nijmegen, The Netherlands; gNufﬁeld Department of Clinical Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford OX3 7DQ, United Kingdom; and hToxicology Unit, Medical Research Council, Leicester University, Leicester LE1 9HN, United Kingdom

Edited by Michael Karin, University of California, San Diego School of Medicine, La Jolla, CA, and approved March 20, 2013 (received for review August 22, 2012) The ectodermal dysplasias are a group of inherited autosomal abnormalities, urogenital problems, facial dysmorphism, and hearing dominant syndromes associated with heterozygous mutations in loss. Nucleotide sequence analyses have provided evidence for the Tumor Protein p63 (TRP63) gene. Here we show that, in addition a striking genotype–phenotype correlation with mutations in in- to their epidermal pathology, a proportion of these patients have dividual domains of p63 in ED patients (8, 9) The SAM domain distinct levels of deafness. Accordingly, p63 null mouse embryos seems to be particularly important for skin development, whereas show marked cochlea abnormalities, and the transactivating isoform the DBD and TID are crucial for limb development. In general, of p63 (TAp63) protein is normally found in the organ of Corti. TAp63 mutations in EEC patients show substitutions in the DBD that transactivates hairy and enhancer of split 5 (Hes5) and atonal homo- impair p63’s ability to bind to specific target sequences in DNA. log 1 (Atoh1), components of the Notch pathway, known to be in- MicelackingbothcopiesoftheTrp63 gene are born lacking volved in cochlear neuroepithelial development. Strikingly, p63 null limbs, skin, and skin appendages, such as hair shafts, follicles, and mice show morphological defects of the organ of Corti, with super- sebaceous glands, and consequently die from dehydration shortly CELL BIOLOGY numerary hair cells, as also reported for Hes5 null mice. This pheno- after birth (10, 11). ΔNp63 is the predominant isoform in the basal type is related to loss of a differentiation property of TAp63 and not layer of the epidermis (12, 13) and is crucial for the maintenance of to loss of its proapoptotic function, because cochleas in mice lacking epithelial stem cells (10, 11). Accordingly, reconstitution with − − the critical Bcl-2 homology domain (BH-3) inducers of p53- and p63- ΔNp63 at least partially rescues the epidermal defects seen in p63 / mediated apoptosis—Puma, Noxa, or both—are normal. Collectively, mice (12). In EEC patients, ΔNp63 mutant proteins accumulate, these data demonstrate that TAp63, acting via the Notch pathway, is and these proteins generally show reduced transcriptional activity crucial for the development of the organ of Corti, providing a molec- on skin-specific promoters (compared with TAp63 proteins), al- ular explanation for the sensorineural deafness in ectodermal dys- though in other ED syndromes ΔNp63 mutant proteins are tran- plasia patients with TRP63 mutations. scriptionally more active than the WT protein. TAp63 also exerts critical functions in the development and epidermis | cell death | p53 family function of the heart (14) and oocytes (15, 16). The latter suggests a role for this gene in female infertility (17). Only limited infor- umor protein p63 (TRP63) is the most ancient member of the mation is available for the role of p63 in development and main- Tp53 family of transcription factors (1) and acts as a key molecule tenance of other tissues/organs. Although the relationship between in embryonic development. The structural organization of p63 (2, 3) ΔNp63 mutations and ED is consistent with the epidermal defects −/− is similar to p53, containing a transactivation domain (TA), DNA seen in the p63 mice, the role of mutant p63 isoforms in the binding domain (DBD), and an oligomerization domain. The ex- conductive and sensorineural deafness seen in some patients (8, 18) pression of p63 is regulated by two distinct promoters giving rise to is unclear. Because a deaf EEC patient presented with abnormal- proteins that either contain (TAp63) or do not contain (ΔNp63) the ities in the morphology of the cochlea (18), we have investigated the N-terminal TA domain, which is critical for the transcriptional in- role of p63 in the development of the inner ear and, in particular, duction of particular subsets of p63 target genes (2). Both p63 the organ of Corti. Our studies, using gene expression analysis and isoforms give rise to differentially spliced mRNAs and proteins, morphological examination of gene-targeted mice, demonstrate with at least six p63 isoforms (α, β, γ)recognized(1).p63α also that TAp63, through activation of the Notch signaling pathway, is contains a sterile α motif (SAM) and a carboxy terminal-inhibitory important for normal development of the cochlea. domain (TID) (4), both absent in p53 (5). The TID binds and Results inhibits the N-terminal TA domain by masking important N-ter- Deafness in ED Patients and Cochlea Morphology in p63-Null Mice. To minal residues. The importance of this regulatory domain is evident evaluate the incidence of deafness among ED patients with p63 from the identification of mutations in the TAp63α-C terminus in human patients (6). The ectodermal dysplasia (ED) syndromes are a large and het- Author contributions: A.T. and G.M. designed research; A.T., V.S., E.B., and H.v.B. per- erogeneous group of inherited human diseases that are character- formed research; A.S., E.V., E.R.F., and X.L. contributed new reagents/analytic tools; A.T., ized by developmental abnormalities of ectodermally derived A.S., H.v.B., R.A.K., and G.M. analyzed data; and A.T., R.A.K., and G.M. wrote the paper. structures. A large subset of autosomal dominant ED syndromes The authors declare no conflict of interest. are caused by heterozygous mutations in the TRP63 gene (7). The This article is a PNAS Direct Submission. prototypic Ectrodactyly, Ectodermal dysplasia, and Cleft lip/palate 1A.T. and V.S. contributed equally to this work. (EEC) syndrome (Online Mendelian Inheritance in Man: OMIM 2 To whom correspondence may be addressed. E-mail: [email protected] 604292) has highly variable expression and penetrance. Clinically, or [email protected]. EEC patients show ectodermal dysplasia affecting skin, hair, nails This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and teeth, and facial clefts, as well as frequent lacrimal duct 1073/pnas.1214498110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1214498110 PNAS Early Edition | 1of6 Table 1. Incidence of deafness in EEC-like patients p63-associated syndrome Patients affected, % (n) Trp63 mutations

EEC syndrome 7 (11/86) DBD: R204, S272, R279, R304, D312, and ins 3′ end. RHS syndrome 20 (2/10) TID: S541 and frameshift. AEC syndrome 38 (6/16) SAM: L514, C522, G530, I537, and frameshift Total EEC-Like 17 (19/112)

AEC, Ankyloblepharon Ectodermal dysplasia Cleft lip/palate; RHS, Rapp-Hodgkin Syndrome. mutations, we analyzed a cohort of 112 patients: 17% of these were scala tympani,andscala vestibuli (Fig. 1 A and B)inE18.5 − − affected by deafness, albeit to different extents. Table 1 summa- p63 / embryos. rizes the relative incidence of deafness for each ED subgroup. Development of the cochlea during embryogenesis occurs from Additional analysis on two new patients revealed by audiometric prosensory cells at E14.5, which leads to the formation of both tests a mild hearing loss, both conductive and sensorineural (Fig. supporting cells as well as inner (IHC) and outer hair cells (OHC). S1 A and B). Because the p63-deficient mice phenocopy to a sub- At the molecular level, Sex determining region Y-box 2 (Sox2) is expressed in supporting cells, whereas Myo-VIIa is found in IHC stantial extent the symptoms observed in ED patients, we in- − − and OHC. In p63 / E14.5 embryos, we identified Sox2 (Fig. S2 C vestigated whether these mice show cochlear abnormalities, and F), Myosin-VIIa (Fig. S2 I and K), and Connexin 26 (Cx26), consistent with the human deafness phenotype. To this end, we − − a cell-specific marker of the stria vascularis(Fig. S2 D and G)in compared p63 / and WT embryos from embryonic day (E)14.5 −/− germinative prosensory cells and in the organ of Kolliker. Whereas and E18.5 (adult p63 mice could not be investigated, because Sox2 and Myo-VIIa appeared to be normally expressed at this the pups die a few hours after birth). H&E staining of sections stage, Cx26 showed a slightly altered distribution compared with from embryos showed no structural abnormalities in the cochlea of E14.5 WT embryos (Fig. S2 D and G). − − p63 / E14.5 embryos (Fig. S2 A and B). However, morphological At E18.5, Sox2 expression was detected in supporting cells, at defects were clearly detected in the cochlear duct (scala media), the base of the organ of Corti (Fig. S3 A and D)inbothWTand

AB18.5 E WT 18.5 E p63 -/- CM Cochlea Skin

TAp63α

ΔNp63α

Actin

MyoVIIa D E F Cx26

18.5 E WT 18.5 E p63 -/- 18.5 E TAp63 -/-

G AEPs H I 10 2,5 TAp63-/- mice Wt-L 70 8 Wt-w TAp63-/- -L 50 6 2 TAp63-/- -w 4 30 1,5 2 10 Milliseconds 8KHz

0 Cochlea dimension mm 3/6 4/6 4/6 6/1 1 3/5 6/11 1 Wt TAp63-/- % of Supernumerary I/OHC Sup. cells ratio

Fig. 1. Morphological and functional defects in cochlea of p63-deficient mice. (A and B) H&E-stained sections of murine WT and p63−/− E18.5 embryos. The − − p63 / embryo shows an abnormal structure of the scalae tympani and media compared with the corresponding WT samples. (Scale bars, 350 μm.) (C) TAp63α and ΔNp63α expression in cochlea and epidermis of WT embryos. Semiquantitative PCR analysis shows much higher expression of TAp63α in cochlear tissue compared with the expression of ΔNp63α. In contrast, expression of only ΔNp63α was seen in the skin; expression was normalized to the expression of actin. − − − − (D–F) Immunofluorescence staining of cochlear sections of mouse embryos (E18.5): WT (D), p63 / (E), and TAp63 / (F). MyoVIIa (green), Cx26 (red), and DAPI − − − − nuclei (blue) staining. (Scale bars, 20 μm.) E and F show supernumerary inner and outer hair cells in both p63 / and TAp63 / embryos. The staining for Cx26 protein suggests that the p63−/− embryos lack intercellular junctions compared with the WT embryos in which connections were correctly formed; the same effect was observed in TAp63−/− (F). (G) Auditory evoked potential analysis shows a variable delay of the fifth wave cerebral cortex potentials in response to 8-KHz stimulation. (H) Comparative study of the width (w) and length (L) of the mouse Cochlea by CT images (Fig. S4). (I) Histological analysis of at least three − − sections (base, middle, apex) from each cochlea TAp63 / mice revealed the presence of supernumerary IHC/OHC cells. The supernumerary cells were present − − at least in 50% of section (n= 6 mice analyzed for each group), ratio shows defective sections over total sections analyzed in TAp63 / mice.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1214498110 Terrinoni et al. −/− p63 mice. Cx26 was expressed in WT mice, as previously re- ABCMyoVIIa ported (19), in supporting and stria vascularis cells, with modest Cx26 − − overexpression seen in the p63 / embryos (Fig. S3 B and E), where it was abnormally localized below the stria. The distribution of − − supporting cells was similar in WT and p63 / mice. Staining of hair cells with Myo-VIIa in E18.5 embryos showed an abnormal number − − of hair cells in p63 / mice (Fig. 1 D and E): four OHC and two IHC − − were identiﬁed in p63 / mice instead of the regular three OHC 18.5 E Noxa -/- 18.5 E Puma -/- 18.5 E Puma-Noxa -/- and one IHC seen in the WT. Moreover, both IHC and OHC in −/− DE35 SaOs-2 HA-TAp63α p63 embryos lacked the characteristic cilia, which are clearly TAp63α visible in the sections from WT embryos. The abnormal number of 10 Doxy - + − − hair cells in p63 / mice suggests that p63 transcriptional target HA genes are essential for the normal development of the cochlea. 5 Tub 0 Selective Involvement of TAp63 Isoform in Cochlea Development. Relative Expression p21 Dll3 Gli2 p27 Hes1Hes7Hey1Jag1Jag2 HeyLHes5Sox2Hey2Prox1Atoh1 Cx26 Whereas ΔΝp63 is normally expressed in epithelia, TAp63 was Notch1Notch3 reported to be restricted to oocytes and cardiomyocytes (14–16). Fig. 2. Mice lacking Puma, Noxa, or both BH3-only proteins have normal To investigate the importance of each of these p63 isoforms in the hair cells in the cochlea; TAp63 induces Notch-related genes. Immunoﬂuo- − − − − cochlea, we performed RT-PCR on surgical extract from whole rescence staining of cochlear sections from E18.5 noxa / (A), puma / (B), − − − − cochlea from E18.5 WT mice. Interestingly, this analysis revealed and puma / ;noxa / (C) mouse embryos stained with MyoVIIa (green), Cx26 a strong expression of TAp63 in the cochlea (Fig. 1C), as well as in (red), and DAPI nuclei (blue). (Scale bars, 20 μm.) (D) Prox1, Hairy and en- oocytes (15), whereas, as reported, ΔΝp63 was the predominant hancer of split 5 (Hes5), and Atonal homolog 1 (Atoh1) mRNA levels are isoform expressed in the skin. regulated by TAp63α. Transcriptional activity (RT-qPCR) of TAp63α in doxy- According to this result, we analyzed the cochlear morphology cycline-inducible (24 h) TAp63α cell lines. Tet-on cells lacking p63-inducible − − − − β of selective TAp63 / mice. As shown in Fig. 1F, the TAp63 / constructs were used as a control, and -actin was used as an internal − − ^ΔΔ E mouse shows the same abnormalities of the full p63 / . Also in standard for 2 Ct calculations. ( ) Western blot of Tet-on induced SaOs2 cells, expressing HA-tagged TAp63α. p21 expression was analyzed as a posi- this case, in fact, supernumerary OHC and IHC are evident; the − − tive control, and expression of tubulin was used as a loading control. TAp63 / mouse cochlea show a higher number of HC (Fig. S2O). CELL BIOLOGY

TAp63−/− Mice Have Sensorineural Deafness. Auditory evoked po- −/− Hes7, Hes5, Atoh1, prospero homeobox 1 (Prox1), Jagged 1 tential analysis performed on a cohort of WT and TAp63 mice (Jag1), Jag2, Connexin 26 (Cx26), Notch1 and Notch3 have all showed an altered response to 8-KHz stimulation, with a variable been validated as differentially expressed genes by RT–quanti- fi delay of cerebral cortex potentials, especially on the fth wave (Fig. tative PCR (RT-qPCR) in this Tet-On inducible system as well as 1G). Furthermore, computerised axial tomography (CT-scan) in cells transiently transfected with expression constructs for analysis of mice cochleae demonstrated that this organ is lightly TAp63α (Fig. 2 D and E). Remarkably, Prox1 and Atoh1 were up- smaller in size in knockout compared with the WT mice (Fig. 1H; regulated ∼fivefold, and Hes5 by nearly 35-fold in response to sample images in Fig. S4). The analysis of IHC/OHC number, in α −/− TAp63 overexpression (Fig. 2 D). cochleae from TAp63 and WT mice, using at least three cochlea This suggests that TAp63 selectively transactivated genes may sections located near the apex, middle, and base of the organ, did be important in cochlear neuroepithelial development, and we not reveal defects in WT mice, whereas all of the TAp63 mice show focused specifically on Hes5, Atoh1 (Math1), and Prox1. We first 50% to approximately 70% sections with supernumerary IHC/ identified in silico p53-like responsive elements in their promoters OHC cells (Fig. 1I). This suggests that TAp63 ablation (or total p63 (Mathinspector software; Genomatix). The analysis of the Hes5 ablation) could have different penetrance, supporting the different −2,000 bp (from transcription start site, TSS) promoter region degrees of hearing loss. showed the presence of a putative p63 responsive element (RE) − p63 Regulation of the Cochlea Does Not Involve a Cell Death located at 1,098/1,066 bp upstream of the TSS (Fig. 3A). This Mechanism. TAp63 can trigger apoptosis, and this is dependent entire region was therefore cloned upstream of luciferase ORF and the resulting construct transfected into SaOs-2 cells. Cotrans- on the direct transcriptional induction of the proapoptotic BH3- fi only proteins Puma and Noxa (20). To test the possibility that the fection of WT TAp63 signi cantly enhanced luciferase reporter − − α additional IHC and OHC present in p63 / mice could result from activity, whereas DBD TAp63 mutants (R280C, R279H, S272N, the loss of p63-mediated apoptosis (21, 22), we examined co- R304W) were inactive, SAM domain mutants (G530V, I537T, − − − − − − − − fi α chleae from single Puma / , Noxa / , or double Puma / Noxa / Q536L) showed a signi cant loss of function, and a TAp63 knockout mice. Figure 2 A–C shows that, in all these mice, the N-terminal domain mutant (Q634X) showed a gain of function fi numbers of hair cells are normal. Therefore, the increased IHC activity (Fig. 3B) (26, 27). Con rming the in silico detection of a − − and OHC cells in the p63 / mice are unlikely to result from p63 binding site within the Hes5 promoter, ChIP assays on Saos-2 α α a failure of p63-mediated apoptosis, but alternative mechanisms Tet-On expressing TAp63 -HA showed binding of TAp63 to must be involved. this site (Fig. 3D, Lower); the MDM2 promoter was used as a positive control (Fig. 3D, Upper; Fig. 3C for protein control). TAp63 Regulates the Notch Pathway via Hairy and enhancer of split 5 Atoh1 regulatory sequences (28) have characteristic canonical (Hes5) and Atonal homolog 1 (Atoh1) in Cochlea Development. To promoter sequences that are located at the 5′ end of the gene and identify the molecular mechanisms leading to the morphological two downstream sequences, called Enhancer-A and Enhancer-B, − − defects in the cochlea of p63 / embryos, we performed a gene which are located ∼3 kb from the stop codon. These two sequences expression microarray analysis using SaOs-2 cells transfected with are separated by ∼400 bp and span ∼500 bp each. Their strong TAp63α or ΔNp63α. In particular, we searched for genes known conservation between human and mouse highlights their impor- to be involved in cochlear neuroepithelial development. A com- tance in gene expression regulation. Indeed, these enhancer se- parative gene function map provided evidence for the in- quences are responsible for timing and tissue specificity of gene volvement of the Notch pathway, which is known to be critical for expression during embryogenesis (28). The in silico analysis iden- cochlea development (23–25) (Fig. S5 A and B). Indeed, Hes1, tified p63RE within the promoter and Enhancer-A (Fig. 3E); the

Terrinoni et al. PNAS Early Edition | 3of6 A Promoter RE Hes5 gene

200 1000 2000 2200 2400 2600 2800 3000 3200 3400 45 B Luciferase activity TID 40

35 30 SaOs-2 TAp63α C SaOs-2 HA-TAp63α D 25 Input HA IgG SAM DBD Doxy - + 20 HA

Fold over control 15 MDM2 p21 10 Actin 5 Hes5 0

I537T G530V Q536LR280CR304WS272NR279H Q634X E pcDNA-HATAp63a Wt Q566fsX94 RE I 5’ Promoter RE II 3’

Atoh1 mRNA transcript Enhancer A Enhancer B 3000 4200 7200 H 7 Luciferase activity TID F SaOs-2 TAp63α G SaOs-2 HA-TAp63α 6 SAM DBD IgG Doxy - + Input HA 5 HA Atoh1 4 Enhancer 3’ 3 Atoh1 Atoh1 2 Promoter 5’ Fold over control p21 1

0 MDM2 Tub I537T G530V R279HR280CR304WS272N Q634X pcDNA-HATAp63a Wt Q566fsX94

Fig. 3. p63 drives Hes5 and Atoh1 promoters. (A) The Hes5 gene structure shows the presence of a putative p53/p63 RE localized at −988, −966 from the TSS, in the promoter sequence. All promoters analyses were performed by MathInspector professional release 8.0.5, March 2011; Matrix Family Library Version 8.3, October 2010. (B) SaOs2 cells were transiently cotransfected with expression constructs for WT TAp63α-HA or TAp63α-HA mutant (G530V, I537T, Q536L, R280C, R304W, S272N, R279H, Q566fsX94, and Q634X) plus the hHes5-luc reporter vector. There was an increase in luciferase activity in cells transfected with TAp63α-HA WT, more pronounced with the TAp63α-Q634X-HA mutant, but not in cells transduced with the vectors encoding mutants for the DBD (mean ± SD, n = 3). (C) Western blot analysis was performed to verify TAp63a protein expression. (D) ChIP analysis shows the binding of TAp63α to the putative p53/ p63-RE. ChIP on the MDM2 promoter was used as positive control. (E) The Atoh1 gene structure shows the presence of two p53 RE: RE-I5′ localized at −1,682, −1,660 from the TSS; and RE-II3′ in the Enhancer-A. (F) ChIP analysis of the Atoh1 p53-Res. TAp63α binds only to the p53-RE Atoh1 enhancer sequence; the MDM2 promoter was used as a positive control. (G) Western blot showing TAp63α protein expression. (H) Luciferase activity was increased by TAp63α-HA and TAp63α Q634X plasmids. SaOs2 cells were transiently cotransfected with an hAtoh1-luc expression vector, TAp63α-HA, and TAp63α-HA mutants (G530V, I537T, R280C, R304W, S272N, R279H, Q566fsX94, and Q634X) (mean ± SD, n = 3). p63RE in the Enhancer-A bound TAp63 by ChIP (Fig. 3F; Fig. 3G 4D). Studies in transfected TAp63 cells showed a significant Hes5 for protein control) and responded to TAp63 in a Luciferase assay, induction (Fig. S6D), in which a mechanism of nuclear relocali- though not to p63 mutants (Fig. 3H). These results support the zation is also evident (Fig. S6 A–C). hypothesis that this region is important for the expression of this Interestingly, the supernumerary hair cell phenotype observed − − − − p63 target gene in vivo (28). In contrast, analysis of the Prox1 in our p63 / mice (Fig. 1) is also seen in ASPP2 / mice (Fig. S6E) putative promoter did not reveal the presence of any responsive a regulator of p63 function (30), and is highly reminiscent of that − − element. found in Hes5 / mice (31). This is consistent with the notion that To test the possibility that TAp63α drives Hes5 transcription, the lack of TAp63-mediated regulation of the Hes5 promoter with we treated H1299 cells with LBH589, a histone deacetylase in- consequent reduction of Notch expression is responsible for this hibitor, to induce TAp63 (29) (Fig. 4A). LBH589 induced TAp63 developmental defect. expression by ∼15-fold, compared with ΔNp63, and resulted in the induction of both Hes5 (∼15-fold) and p21 (∼40-fold), the latter Discussion known to be a p63 target gene. To confirm the relationship be- Notch signaling is critical for the development of many tissues and tween TAp63 and Hes5, Atoh1, and Prox1, we extracted RNA organs. This pathway is activated by ligand–receptor interactions, − − from cochleae of p63 / E18.5 embryos and found that the levels resulting in transcriptional activation of target genes, including of Hes5 mRNA were considerably (∼10-fold) lower than in WT Hes1 and Hes5 (23, 24, 32). Specifically, the Notch pathway is mice (Fig. 4B); a reduction is also shown for Atoh1 and Prox1, involved in neuroepithelial differentiation of the inner ear, in the thereby providing a direct link between p63 and these genes in organ of Corti (25, 33, 34). Here, the auditory sensory epithelium is vivo. Furthermore, the protein analysis, using cochlear extracts, the specialized region of the cochlea that transduces sound. The − − revealed Hes5 and Atoh1 depletion in p63 / E18.5 samples (Fig. sensory neuroepithelia are characterized by a mosaic of supporting 4C), as well as Atoh1 induction in TAp63 transfected cells (Fig. cells and hair cells, both IHC and OHC, which are arranged into

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1214498110 Terrinoni et al. formation of the organ of Corti. Mice lacking Puma and/or Noxa, A 50 Ctr C Cochlea -/- the BH3-only proteins that are transcriptionally induced by LBH589 WT TAp63 40 TAp63 and essential for its ability to trigger apoptosis, had normal Hes5 cochlea and organs of Corti. This demonstrated that loss of 30 a nonapoptotic mechanism activated by TAp63 must be the cause − − of the defects in ear development in the p63 / embryos. Indeed, 20 Atoh1 H&E staining of E18.5 embryos showed disorganization of the cochlea, especially the scala medium and timpani, and a minor 10

Reletive Expression thickening of the stria vascularis, with supernumerary IHC and 0 Cx26 OHC, as well as an abnormal distribution of Cx26 (Fig. 1 D–F). Hes5 p21 TA/ΔNp63α This inhibition of hair progenitor cell by Notch signaling is therefore essential for correct morphological and functional de- B D H1299 600 Cx26 pcDNA TAp63α velopment of the inner ear. Accordingly, we propose a model Hes5 whereby TAp63 regulates the formation of the organ of Corti by 10 Prox1 transcriptional induction of Hes5/Atoh1 (Fig. S7A), thereby reg- Atoh1 p63 −/− 8 TAp63α ulating this process of lateral inhibition. Indeed, both Hes5 and − − p63 / mice develop supernumerary hair cells (Fig. S7B) (31) as 6 Atoh1 a result of abnormal differentiation of supporting cells into hair 4 cells, because the block by Hes5 has been removed by the ablation 2 of p63. Reletive Expression Parp To date, a developmental role for TAp63 has only been clearly 0 -/- documented in oocytes, where it promotes apoptosis upon DNA p63 WT damage (15, 20), or in the heart (14). Here we show that p63 is also Fig. 4. p63 drives Hes5 expression in the cochlea. (A) Treatment of H1299 important for the maturation of the cochlea; this activity of p63 cells with LBH589 for 24 h induced the expression of TAp63, resulting in the requires transcriptional regulation of cell differentiation and not transcriptional induction of Hes5, as determined by RT-qPCR analysis (mean ± the induction of its predominant apoptosis inducing target genes. SD, n = 3). (B) Transcriptional analysis on cochlea tissue biopsies from WT Auditory analysis in two new patients demonstrated a sensorineu- −/− and p63 mouse embryos (E18.5). Expression levels of Hes5 proved to be ral component in hearing loss previously not reported. Further CELL BIOLOGY dependent on TAp63, and Hes5 mRNA levels were ∼10 times lower in co- −/− − − studies performed on TAp63 mice, demonstrated a variable but chlea of p63 / embryos compared with those from WT embryos; addition- − − consistent sensorineural hearing loss, and histological examination ally, Prox1 and Atoh1 expression were lower in p63 / embryos. (C) Protein fi −/− con rmed the presence of supernumerary IHC/OHC, in all expression of Hes5 and Atoh1 in WT and TAp63 embryos. C shows a lower −/− −/− D TAp63 mice cochleae. These data imply that TAp63 mutations expression of Hes5 in TAp63 sample; Cx26 protein was used as control. ( ) fi Up-regulation of Atoh1 in TAp63 transfected H1299 cells. Parp protein was lead to modi cation of sensorineural epithelium by affecting the analyzed as loading control for nuclear protein extract. integrity of the Hes5/Atoh1 pathway and contribute with different degrees to the hearing loss seen in a subset of patients with ED. highly ordered rows. Several components of the Notch signaling Materials and Methods pathway are known to be expressed in the developing organ of Mice. p63 knockout mice (11) and mice lacking Puma, Noxa (40), or both Puma Corti. Importantly, interference with their expression or mutation and Noxa (41) and TAp63 (42), have been described. Mice lacking p63 were of the corresponding genes leads to an abnormal increase in hair generated electroporating JI Es cells, which were microinjected into blasto- cysts from C57BL/6 mice. Heterozygous mice have been crossed with C57BL/6 cells (25, 35), highlighting a role for Notch activation in the for more than 10 generations. Mice lacking Puma or Noxa were generated on regulation of progenitors that develop into hair cells. Previous a C57BL/6 background, using C57BL/6-derived ES cells. TAp63 knockout mice studies have established the importance of the basic helix–loop– were in a C57BL/6 background. Animal experiment have been performed in helix (bHLH) Notch target genes Hes1, Hes5, and Math1 in the the Department of Experimental Medicine and Surgery, and approved by the developing ear. Math1-deficient mice die shortly after birth (36) Department Board, according Italian law 116/92, September 29, 2011. with complete disruption of hair cell development (37). Hes1 and Hes5 were shown to negatively regulate the differentiation of Cell Culture, Transfection, and Plasmid. SaOs-2 cells with doxycycline-inducible inner ear hair cells and, accordingly, Hes5-deficient mice have expression of HA-TAp63a and HA-DNp63a were generated and treated as previously described (43). The luciferase reporter plasmid (hHes5-luc) was supernumerary OHC (31). modified from the pGL3-Basic vector (Promega) by inserting the putative We have previously demonstrated that p63 can regulate com- hHes5 promoter sequence containing the p53RE between Sac1 and Xho1 ponents of the Notch pathway by direct transcriptional induction of sites. Similarly, the amplified hAtoh1 enhancer sequence was inserted in Jag1 and Jag2 during epidermal differentiation (21), further im- pGL3 using NheI sites. plicating a link between p63 and Notch in epidermal development (38). Here we identify Hes5 and Atoh1 as two Notch-related Western Blot, Immunostaining, and Confocal Microscopy. See refs. 12 and 44 transcriptional targets of TAp63 that are involved in the differen- for embryo preparation and confocal analysis. Subsequently, sections were tiation of the organ of Corti. Hes5 is involved in the formation of incubated for 2 h with the following primary antibodies: anti-p63 (H129 sc- 8344 and H137 sc-8243; Santa Cruz), anti-MyoVIIa (25-6790, Proteus), anti- the hair cells in the organ of Corti (31), and here we demonstrate Sox2 (AB5603; Millipore), and anti-Cx26 (13-8100; Invitrogen). See ref. 26 for that it is primarily regulated by TAp63. TAp63 also regulates an- Western blot preparation. All membranes were incubated for 2 h with the other bHLH transcription factor, Atoh1, which also plays an im- following antibodies: anti-Hes5 (ab25374; Abcam), anti-Math1 or Atoh1 portant role in the differentiation of hair cells. Atoh1 is expressed in (ab27667; Abcam;), anti-Cx26 (13-8100; Invitrogen), anti-p63 (Y4A3 p3362; a transient population of actively proliferating progenitor cells (39), Sigma-Aldrich), and anti-Parp (sa250; Biomol). and Atoh1-deficient mice are characterized by the absence of au- Real-Time qPCR, Semiquantitative RT-PCR, and ChIP. ditory sensory hair cells (37). We show that TAp63 regulates Atoh1 See ref. 44 for PCR analysis and Table S1 for specific PCR primer sequences. SaOs TAp63α-inducible cells via binding to the enhancer A, a region known to be critical for × 6 fi (5 10 ) were cross-linked for 10 min in a solution containing 1% formalde- time- and tissue-speci c expression of this gene. The morphological hyde, and ChIP assays were performed using a MAGnify ChIP system (Invi- −/− comparison of the inner ear of p63 and WT mouse embryos trogen), according to the manufacturer’s instructions. Luciferase reporter also confirmed the involvement of p63 in the late stages of the assays and immunoblot analysis were performed as described previously (44).

Terrinoni et al. PNAS Early Edition | 5of6 Auditory Evoked Potential. Mice wereanesthetizedby i.m.injection ofketamine FIRB (RBIP06LCA9_0023, RBIP06LCA9_0C), AIRC (2008-2010_33-08) (#5471) (100 mg/kg) and xylazine (10 mg/kg) and placed in a dark, electrically shielded (2011-IG11955), Associazione Italiana Ricerca sul Cancro (AIRC) 5xmille ’ ﬁ room. Stainless steel electrodes were inserted s.c. into the vertex (positive pole), (#9979), Ministero dell Istruzione e Ricerca Scienti ca (MIUR)/Progetti di oneretroauricularregion(negativepole),andtheopposite retroauricularregion Ricerca di Interesse Nazionale (PRIN) 2008MRLSNZ_004, and Telethon Grant GGPO9133 (to G.M.); National Health and Medical Research Council of Aus- (ground) ofeachmouse. Acoustic stimuli, consisting oftone bursts atfrequencies tralia Program Grant 461221, Australia Fellowship; and Leukemia and Lym- of 8 kHz, were delivered to each mouse. Amplaid MK12 (Amplifon) instruments phoma Society Specialized Center of Research (SCOR) Grant 7413. Research was used to perform the analysis and elaborate data. described in this article was also supported in part by Min. Salute (Ricerca oncologica 26/07) Istituto Dermopatico dell’Immacolata (RF06 c.73, RF07 ACKNOWLEDGMENTS. We thank Dr. Eleonora Candi for constructive com- c.57, RF08 c.15, RF07 c.57) (to G.M.) and Independent Research Institutes ments, Mr. M. Cook for help with harvesting embryos, and Drs. A. Villunger, Infrastructure Support Scheme (IRISS) grants through the Australian Gov- C. L. Scott, and J. M. Adams for gifts of mice. This work was supported by the ernment and the Victorian State Government Operational Infrastructure Medical Research Council, United Kingdom; MIUR/PRIN (20078P7T3K_001)/ Support (OIS).

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