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. Author manuscript; available in PMC: 2011 Dec 15.
Published in final edited form as: Dev Cell. 2010 Nov 18;19(6):807–818. doi: 10.1016/j.devcel.2010.10.015

Hdac1 and Hdac2 act redundantly to control p63 and p53 functions in epidermal progenitor cells

Matthew LeBoeuf 1,2, Anne Terrell 1, Sohum Trivedi 1, Satrajit Sinha 3, Jonathan A Epstein 4, Eric N Olson 5, Edward E Morrisey 6, Sarah E Millar 1,*
PMCID: PMC3003338  NIHMSID: NIHMS250145  PMID: 21093383

Summary

Epidermal and hair follicle development from surface ectodermal progenitor cells require coordinated changes in gene expression. Histone deacetylases alter gene expression programs through modification of chromatin and transcription factors. We find that deletion of ectodermal Hdac1 and Hdac2 results in dramatic failure of hair follicle specification and epidermal proliferation and stratification, phenocopying loss of the key ectodermal transcription factor p63. While expression of p63 and its positively regulated basal cell targets is maintained in Hdac1/2 deficient ectoderm, targets of p63-mediated repression, including p21, 14-3-3σ and p16/INK4a, are ectopically expressed, and HDACs bind and are active at their promoter regions in normal undifferentiated keratinocytes. Mutant embryos display increased levels of acetylated p53, which opposes p63 functions, and p53 is required for HDAC inhibitor-mediated p21 expression in keratinocytes. Our data identify critical requirements for HDAC1/2 in epidermal development, and indicate that HDAC1/2 directly mediate repressive functions of p63, and suppress p53 activity.

Keywords: Hdac1, Hdac2, epidermis, mouse, hair follicle, p63, p53, p16/INK4a

Introduction

The development of complex tissues from embryonic progenitor cells requires imposition of coordinated changes in gene expression programs. Conversely, development of malignancies involves broad suppression of differentiation programs and reversion to a more embryonic-like state. The accessibility and well-studied biology of the epidermis, and the prevalence of skin cancers, make it an ideal and important system in which to study these processes.

In mouse embryos, epidermal differentiation begins at approximately E8.5, when the single-layered ectoderm begins to express simple epithelial keratins KRT8 and 18, and the epidermal lineage-determining transcription factor p63 (Koster et al., 2007). Expression of basal cell keratins KRT5 and KRT14 is initiated at E9.5 (Byrne et al., 1994). Starting at E12.5, asymmetric cell divisions perpendicular to the plane of the epidermis are occasionally observed and are associated with the beginnings of epidermal stratification (Lechler and Fuchs, 2005). By birth, the epidermis is composed of basal, suprabasal, granular and outer cornified layers, and forms a functional barrier that is essential for postnatal survival. Terminally differentiated cornified cells are continuously shed and replenished through the mitotic activity of basal progenitor cells which both self-renew, and give rise to suprabasal progeny that enter the stratification program. While most embryonic surface ectodermal cells undergo epidermal stratification, a subset of these cells interacts with the underlying mesenchyme to form a variety of ectodermal appendage organs (Mikkola and Millar, 2006).

p63 is essential for ectodermal appendage specification, epidermal proliferation and asymmetric cell division, and epidermal development, acting upstream of other early regulators of these processes (Koster, 2010; Laurikkala et al., 2006; Lechler and Fuchs, 2005; Mills et al., 1999; Truong and Khavari, 2007; Yang et al., 1999a). Studies of p63-mutant thymus in vivo, and clonogenic assays in keratinocytes, support a key role for p63 in maintaining the proliferative potential of stem cells in stratified epithelia (Senoo et al., 2007).

Two classes of p63 isoforms are transcribed, TAp63 and ΔNp63, that respectively contain or lack an N-terminal transactivating domain. Each class includes multiple splice variants with differing activities, complicating mechanistic analysis (Blanpain and Fuchs, 2007). The predominant p63 isoform in embryonic epidermis, ΔNp63α, can function as either an activator or a repressor, depending on the target gene (McDade and McCance, 2010). ΔNp63α regulates the proliferation of epidermal progenitor cells by directly repressing expression of anti-proliferative target genes, such as 14-3-3σ and p21 (Westfall et al., 2003; Truong et al., 2006). ΔNp63α also directly represses expression of the cell cycle regulators p16/Ink4a and p19/Arf, and defects in proliferation, epidermal stratification and ectodermal appendage development are partially rescued in p63 mutants by concomitant loss of p16/Ink4a or, to a lesser extent, by loss of p19/Arf (Su et al., 2009). In addition to its repressive functions, ΔNp63α directly activates genes important for epidermal differentiation, including Krt14, Ikkα, Fasn, Claudin1, and Gata3 (Chikh et al., 2007; Koster et al., 2007; Lopardo et al., 2008; Marinari et al., 2009; Romano et al., 2009). In vitro experiments suggest that p63 acts in opposition to the related transcription factor p53 in regulating epidermal proliferation, but controls differentiation by a separate, p53-independent mechanism (Truong et al., 2006).

Lineage-determining transcription factors generally act in concert with chromatin regulators that permit access to sequence-specific binding sites and allow inheritance of gene expression programs (Kim et al., 1999). However, interactions of p63 with chromatin modifying factors have yet to be described. Genetic analyses in the mouse, and experiments in organotypic culture, have revealed requirements for the histone methylase EZH2, and the DNA methyltransferase DNMT1, respectively, in maintaining epidermal progenitor cell proliferation (Ezhkova et al., 2009; Sen et al., 2010), but loss of these factors is associated with premature differentiation of basal cells, a phenotype distinct from that observed in p63-null embryos.

Histone deacetylases (HDACs) remove histone acetylation marks, resulting in compaction of chromatin structure and transcriptional repression (Ruthenburg et al., 2007). HDACs operate by direct association with DNA binding factors, and by incorporation into large multifunctional repressor complexes such as Sin3, NuRD, coREST and PRC2 (Brunmeir et al., 2009). In addition to functions in chromatin remodeling, HDACs deacetylate certain transcription factors, such as p53, resulting in their decreased activity (Higashitsuji et al., 2007; Tang et al., 2008). HDACs form a large family, of which Class I HDACs, including the closely related proteins HDAC1 and HDAC2, show the strongest histone deacetylase activity (Haberland et al., 2009). While HDAC1 and HDAC2 perform non-redundant roles in early development, these proteins co-exist in repressive complexes, and tissue-specific deletion at later developmental stages reveals redundant functions for Hdac1 and Hdac2 in multiple different cell types (Brunmeir et al., 2009).

Several lines of evidence suggest important roles for histone deacetylation in epidermal development. Epidermal-specific deletion of Mi-2β, encoding an ATP-dependent chromatin-remodeling enzyme associated with HDAC1 and 2 in the NuRD complex, results in failure of hair follicle specification and gradual loss of basal epidermal progenitor cells (Kashiwagi et al., 2007). Class I and II HDACs also associate with the chromatin remodeling ATPase BRG1, which is required for keratinocyte terminal differentiation (Indra et al., 2005). Treatment of adult skin with the HDAC inhibitor trichostatin A (TSA) causes hair follicle stem cells to proliferate and exit the stem cell compartment (Frye et al., 2007). However, the precise functions of specific HDAC isoforms in the epidermis in vivo have not been elucidated and their requirements for embryonic epidermal development are unknown.

Here we show that HDAC1 and HDAC2 are expressed in a dynamic and overlapping fashion in developing skin and hair follicles. Deletion of both genes in the epidermis results in a phenotype that strikingly resembles that of p63-null skin, and de-repression of negatively regulated ΔNp63 target genes including the senescence factor p16/Ink4a. HDAC1/2 are present at the promoter regions of ΔNp63-repressed targets in keratinocytes, and histones in these regions are hyperacetylated following HDAC inhibition, indicating a direct requirement for HDAC1/2 in ΔNp63-mediated repression. In addition, p53 is hyperacetylated in Hdac1/2 mutant epidermis, and experiments in keratinocytes indicate that increased p53 function contributes to the anti-proliferative effects of Hdac1/2 deletion via induction of p21 expression. These data reveal redundant and essential roles for Hdac1/2 in controlling the activities of key regulators of epidermal development.

Results

HDAC1 and HDAC2 are expressed in dynamic and overlapping patterns in developing skin

Analysis of HDAC1 and HDAC2 expression in epidermal development revealed homogeneous expression of both proteins in epidermal nuclei at E13, prior to stratification of the epidermis (Figure 1A, B). At later developmental stages, HDAC1 and HDAC2 were expressed in all epidermal cells, but localized most strongly to nuclei in outer, differentiating cell layers (Figure 1C–J), and in the leading edges of developing hair follicles (Figure 1E, F).

Figure 1. HDAC1 and HDAC2 display overlapping, dynamic expression in developing epidermis and hair follicles.

Figure 1

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Immunohistochemistry (brown) for HDAC1 and HDAC2 at the stages indicated. (A, B) HDAC1 and HDAC2 are expressed in all cells of the E13 single layer epidermis. (C, D) HDAC1 and HDAC2 are ubiquitously expressed in E14.5 epidermis and hair follicle placodes (arrows). (E, F) HDAC1 and HDAC2 are expressed most strongly at the leading edge of E16.5 hair follicles (black arrows) and in differentiating epidermal cells (blue arrows). (G–J) At E18.5, HDAC1 and HDAC2 are expressed strongly in differentiating epidermal cells (blue arrows). Dashed black lines indicate dermal-epidermal junctions. Scale bar in (J) applies to all images.

Tissue-specific deletion of either Hdac1 or Hdac2 alone does not impact epidermal development or homeostasis

To delineate the functional requirements for Hdac1 and Hdac2 in epidermal development, we utilized KRT14-Cre transgenic mice in which Cre recombinase is efficiently expressed prior to hair follicle development or epidermal stratification (Liu et al., 2007), in combination with conditional loss of function alleles of either Hdac1 or Hdac2 (Montgomery et al., 2007). Consistent with the phenotypes of previously described tissue-specific Hdac1 or Hdac2 single mutants (Haberland et al., 2009), KRT14-Cre Hdac1fl/fl and KRT14-Hdac2fl/fl mice were viable and fertile, and displayed no gross or histological skin abnormalities. Similarly, compound heterozygous, KRT14-Cre Hdac1fl/fl Hdac2fl/+ and KRT14-Cre Hdac1fl/+ Hdac2fl/fl mice showed no gross or histological defects in epidermal or hair follicle development or homeostasis (Supplemental Figure S1 and data not shown).

Hdac1/2 epidermal mutants display multiple, severe ectodermal defects

To determine whether loss of both Hdac1 and Hdac2 resulted in epidermal abnormalities, we generated KRT14-Cre Hdac1fl/fl Hdac2fl/fl (DcKO) mice that lacked all four functional alleles in the epidermis. DcKO mice died perinatally with multiple and dramatic ectodermal defects (Figure 2). Immunostaining of DcKO mutant skin revealed absence of both HDAC1 and HDAC2 proteins in surface epithelia by E14.5 (Figure 2A–F). Consistent with key functions of HDAC1/2 in histone deacetylation, levels of histone H3 acetylated at lysine 9 (H3K9Ac) were markedly increased in E14.5 DcKO compared with control littermate epidermis (Figure 2G, H).

Figure 2. Embryos lacking epidermal HDAC1 and HDAC2 display striking defects in epidermal and ectodermal appendage development.

Figure 2

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(A–F) Immunofluorescence (green) reveals expression of HDAC1 and HDAC2 in the epidermis and developing hair follicles of control E14.5 and E16.5 Hdac1fl/fl Hdac2fl/fl littermate skin (A, C, E) and their absence in KRT14-Cre Hdac1fl/fl Hdac2fl/fl (DcKO) epidermis (B, D, F) (arrows). (G, H) Immunofluorescence (green) reveals increased levels of H3K9Ac in DcKO (H) compared with control (G) epidermis (arrows). (I, J) Open eyes, thin smooth skin, and failure of digit septation in a DcKO embryo at E18.5 (J) compared with a control KRT14-Cre Hdacfl/+ Hdac2fl/fl littermate (I) (arrows). (K–X) Hematoxylin and eosin staining reveals defects in stratification and ectodermal appendage development in DcKO compared with control littermate embryos. (K, L) absence of hair follicle placodes in E14.5 DcKO epidermis (L) compared with control skin (K, arrow). (M–P) DcKO epidermis remains as a single cell layer at E16.5 (M, N) and E18.5 (O, P) instead of stratifying, and lacks hair follicles. (Q–T) Abnormal DcKO dental lamina morphology at E16.5 (Q, R, arrows) and degeneration of incisor (black arrows) and molar (blue arrows) dental structures at E18.5 (S, T). (U, V) Failure of filiform papilla morphogenesis in E18.5 DcKO tongue surface ectoderm (arrows). (W, X) Sections through E18.5 hindlimbs reveal digit formation and failure of epidermal differentiation between the digits in the DcKO (arrows). Nuclei in (A–H) are DAPI-stained (blue). Dashed lines mark dermal-epidermal borders. Size bar in (F) applies to (A–F); size bar in (H) applies to (G, H); size bar in (P) applies to (K–P); size bars in (R, T, V, X) also apply to their respective littermate controls. See also Figure S1 and Figure S2.

DcKO embryos displayed thin, smooth skin; failure of eyelid fusion; and failure of limb digit separation (Figure 2I, J). Histological analysis showed that, instead of stratifying, the epidermis remained as a single layer throughout embryogenesis, and lacked any signs of hair follicle development (Figure 2K–P). Tooth development was initiated, consistent with the early timing of this process relative to KRT14-Cre activity (Liu et al., 2008); however, dental structures were abnormal in DcKO mutants at E16.5 (Figure 2Q, R), and degraded by E18.5 (Figure 2S, T). Formation of keratinized filiform papillae in tongue epithelium was absent in DcKO mutants, and like the epidermis, tongue surface ectoderm remained as a single layer throughout embryogenesis (Figure 2U, V). Histological analysis of DcKO embryonic limbs revealed failure of epidermal differentiation between the digits that is required for digit separation (e.g. Ingraham et al., 2006) (Figure 2W, X), and analysis of mutant eyes at E18.5 showed that eyelid development was arrested (Supplemental Figure S2).

Hdac1/2 are required for suprabasal epidermal differentiation and initiation of hair follicle development

To dissect the molecular basis for the abnormalities observed in DcKO embryos, we first examined the expression of molecular markers for epidermal stratification. The basal cell keratin, KRT14, was expressed similarly in control and DcKO epidermis at E14.5. At later embryonic stages, KRT14 was confined to basal cells in controls, and remained expressed in the ectoderm of DcKO embryos. By contrast, KRT10, which marks suprabasal cells in control embryos from approximately E15, was not expressed in DcKO epidermis at any stage. Loricrin, a marker for the granular layer, was readily detected in control embryos at E16.5, but was not expressed at any stage in DcKO embryos (Figure 3A). To determine whether epidermal cells revert to a less differentiated state in DcKO mutants, we asked whether the simple epithelial keratin, KRT18, is ectopically expressed. Immunostaining at E16.5 revealed absence of KRT18 expression in both control and DcKO epidermis (Supplemental Figure S3A, B). Thus, basal epidermal development is initiated and maintained in DcKO embryos, but differentiation and stratification of the epidermis completely fails.

Figure 3. Loss of epidermal Hdac1/2 causes failure of epidermal stratification, absence of hair follicle specification, and progressive defects in proliferation and cell survival.

Figure 3

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(A) Immunofluorescence (red) with antibodies to the basal layer marker KRT14, the suprabasal marker KRT10 and the granular layer marker loricrin, reveals expression of KRT14, but lack of KRT10 and loricrin in DcKO epidermis at E14.5 E18.5. The weak signal in E16.5 DcKO skin incubated with anti-KRT10 is due to background fluorescence in the periderm. (B) Whole mount in-situ hybridization (blue-purple) for Ctnnb1 reveals formation of hair follicle pre-placodes in E14.0 control littermate skin (arrows), and their absence in DcKO skin. (C) Immunohistochemistry for β-catenin (brown) and immunofluorescence for LEF1 (green) reveal upregulated expression in developing hair follicle placodes (black or white arrows), and lower or absent expression in inter-placodal epidermis (blue or yellow arrows) in controls, and uniform low level expression in DcKO epidermis (black or white arrows). (D) Immunofluorescence for phospho-histone H3 (PH3) (green) reveals decreased proliferation in DcKO epidermis at E14.5 and almost absent proliferation at E16.5 (arrows). E16.5 samples were co-stained with anti-KRT14 (red) to mark basal cells. (E) TUNEL staining (green) reveals increased cell death in DcKO embryos at E16.5 (arrows) but not at E14.5 compared to littermate controls. (F) Quantification of the percentage of PH3 positive basal layer cells reveals a statistically significant decrease in proliferation in DcKO compared with control embryos at E14.5, and a further decrease in the relative rate of proliferation at E16.5. 20 fields of view were counted for each genotype. (G) Quantification of the number of TUNEL positive cells per field of view at 40× magnification reveals a statistically significant increase in the number of apoptotic basal cells in DcKO embryos at E16.5, but not at E14.5. 20 fields of view were counted for each genotype. Statistical data are represented as mean +/− SEM. Dashed lines indicate dermal-epidermal junctions. NS, not statistically significant. See also Figure S3.

Like epidermal stratification, initiation of hair follicle development requires a global switch in the program of ectodermal differentiation. To determine the stage at which hair follicle development is blocked in DcKO mutants, we carried out whole mount in situ hybridization for Ctnnb1 (encoding β-catenin), a very early marker for hair follicle placode initiation (Zhang et al., 2009). Patterned expression of Ctnnb1, corresponding to hair follicle pre-placodes, was observed in control epidermis at E14.0, but was absent in DcKO mutants, indicating that placode development was not initiated (Figure 3B). Similarly, nuclear β-catenin protein and its transcriptional partner LEF1 were elevated in control hair follicle placodes, and downregulated in intervening epidermis at E14.5 (Jamora et al., 2003; Zhang et al., 2009), while DcKO epidermis displayed uniform, low level expression of nuclear β-catenin and LEF1 (Figure 3C). Vibrissa and mammary placode formation is initiated prior to efficient expression of KRT14-Cre (Liu et al., 2007). Elevated Ctnnb1 expression marked developing vibrissae and mammary buds in DcKO as well as control embryos (Supplemental Figure S3C, D). These observations indicate that Hdac1/2 are not required to maintain elevated Ctnnb1 expression, but rather are necessary to allow hair follicle fate acquisition. Thus Hdac1/2 are required for at least two different types of developmental transition in embryonic surface ectoderm: stratification and hair follicle initiation.

DcKO embryos display progressive defects in proliferation and apoptosis

To determine whether proliferation was affected by Hdac1/2 deletion, we analyzed the percentage of phospho-histone H3 (PH3) positive basal cells in mutant and control littermate epidermis. At E14.5, proliferation was decreased in DcKO mutants to approximately 56% of the levels seen in controls (20 fields of view counted at 40x magnification for each genotype; p<1×10−4). By E16.5 this percentage had decreased to only 17% of control levels (p<1×10−5) (Figure 3D, F; see also Supplemental Figure S3E-H). The progressive nature of this proliferation defect is consistent with failure of self-renewal of progenitor cells (Sen et al., 2010; Senoo et al., 2007).

To determine whether programmed cell death contributed to the Hdac1/2 mutant phenotype we utilized a TUNEL assay. Interestingly, there was no statistically significant difference in the rate of programmed cell death between DcKO and control epidermis at E14.5 (20 fields of view counted at 40× magnification for each genotype; p=0.32), indicating that the initial defects observed in DcKO skin were not due to increased apoptosis. However, by E16.5, an approximately 6-fold increase in the rate of basal cell apoptosis was observed in DcKO compared with control epidermis (p<0.4×10−5) (Figure 3E, G).

Hdac1/2 are not required for maintaining expression of ΔNp63 and its positively regulated basal cell targets

The ectodermal defects observed in DcKO embryos were strikingly reminiscent of many of the phenotypes described in embryos lacking p63, including lack of epidermal stratification, decreased proliferation, failure of hair follicle specification, defective eyelid development, absence of tongue filiform papillae, and defective dental development (Laurikkala et al., 2006; Lechler and Fuchs, 2005; Mills et al., 1999; Yang et al., 1999b). Of particular note, similar to p63-null skin, Hdac1/2 deficient embryonic epidermis exhibited failure of proliferation in the absence of premature differentiation.

We therefore asked whether expression of p63 is maintained following deletion of Hdac1/2. Immunostaining using an antibody that recognizes all p63 isoforms revealed expression of p63 in basal cells and in some immediately suprabasal cells in control embryos at E14.5 – E18.5. In DcKO embryos, strong expression of p63 was observed in the epidermis at E14.5, and was maintained at E16.5 and E18.5 (Figure 4A). To determine whether similar p63 isoforms were expressed in control and DcKO skin, we used antibodies specific for ΔNp63 and TAp63 isoform classes (Romano et al., 2009). ΔNp63 isoforms predominated in both control and DcKO epidermis at E14.5 (Figure 4B, C). As previously reported, TAp63 isoforms were not detected in control skin at this stage (Laurikkala et al., 2006; Romano et al., 2009), and were also not detectable in the mutant (Figure 4D, E).

Figure 4. Hdac1 and Hdac2 are required for repression of negatively regulated ΔNP63 target genes.

Figure 4

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(A) Immunofluorescence with pan-p63 antibody (green) reveals expression in the basal layer of littermate controls and in the single layered DcKO epidermis at E14.5, E16.5 and E18.5 (arrows). (B-E) Expression of ΔNP63 (B, C, arrows) and undetectable expression of TAP63 (D, E) in both control (B, D) and DcKO (C, E) epidermis at E14.5. (F–K) Positively regulated p63 targets FASN (F, G) (green), CDC25c (H, I) (brown), and claudin1 (J, K) (green) and are similarly expressed in single-layered DcKO (G, I, K) and basal control (F, H, J) epidermis (arrows). (L–O) Immunofluorescence for 14-3-3σ at E14.5 (L, M) (green signal) and E16.5 (N, O) (red signal) reveals its expression in control suprabasal cells (N, yellow arrow) and absence in control basal cells (N, white arrow). In DcKO skin, 14-3-3σ is expressed in the single-layered epithelium at both E14.5 (M) and E16.5 (O) (yellow arrow). (P–S) p21 immunostaining (brown) is slightly more intense in DcKO (Q, S) compared with control basal epidermis (P, R) at both E14.5 (P, Q) and E16.5 (R, S) (arrows). (T–W) p16/INK4a (brown immunostaining) is dramatically upregulated in DcKO (U, W) compared with control (T, V) epidermis at both E14.5 (T, U) and E16.5 (V, W) (arrows). Dashed lines indicate dermal-epidermal boundaries. (X) CHiP assays with extracts of untreated HEKs and HEKs treated with TSA for 6 hours or 12 hours, and antibodies to HDAC1, HDAC2, H3K9Ac, or rabbit IgG negative control, as indicated. Primers amplifying sequences that encompass GAPDH negative control promoter regions or p63 binding sites in the promoter regions of the 14-3-3σ, p21, and p16/INK4a genes were used for PCR as indicated. All panels in (A) were photographed at the same magnification; scale bar in (E) applies to (B–E); scale bars in (G, I, K, M, O) also apply to their respective littermate controls; scale bar in (W) applies to (P–W). See also Figure S4 and Supplemental Table.

As p63 isoform expression appeared unchanged by deletion of Hdac1/2, we asked whether Hdac1/2 might mediate p63 function. Positively regulated targets of ΔNp63 in epidermal basal cells include Krt14 (Romano et al., 2009), fatty acid synthase (Fasn) (Lefkimmiatis et al., 2009), the cell cycle progression regulator Cdc25c (Lefkimmiatis et al., 2009), claudin1 (Lopardo et al., 2008) and Ikkα (Candi et al., 2006; Marinari et al., 2009). As shown in Figure 3A, maintenance of KRT14 expression was unaffected by Hdac1/2 deletion. Similarly, FASN, CDC25, Claudin1 and IKKα were expressed in DcKO epidermis (Figure 4F–K and Supplemental Figure S4A–D). Consistent with absence of stratification, GATA3, a positively regulated target of TAp63 in suprabasal cells (Chikh et al., 2007), was not expressed in DcKO epidermis (Figure S4E, F).

ΔNp63-repressed targets are upregulated in Hdac1/2-deficient epidermis

As expression of positively regulated basal cell ΔNp63 targets was unaffected by Hdac1/2 deletion, we next examined expression of 14-3-3σ and p21, which are direct targets of ΔNp63-mediated repression (Westfall et al., 2003). In control epidermis at E14.5, 14-3-3σ was expressed in emerging suprabasal layers but was largely absent from the basal layer. However, E14.5 DcKO embryos displayed weak ectopic expression of 14-3-3σ in the basal layer (Figure 4L, M). This difference was more pronounced at E16.5, when 14-3-3σ was strongly expressed in suprabasal cells, but absent from the basal layer in controls, and DcKO mutants displayed strong ectopic 14-3-3σ expression in basal cells (Figure 4N, O). Similarly, P21 was weakly expressed in control basal cells at E14.5 and E16.5 and displayed increased immunoreactivity in mutant epidermis (Figure 4P–S).

The Ink4a/Arf locus produces two independent proteins: p16/INK4a, which specifically binds and inhibits the cyclin-dependent kinases 4 and 6, preventing phosphorylation of the retinoblastoma tumor suppressor protein; and p19/ARF which stabilizes p53. P63 has been reported to directly repress both p16/Ink4a and p19/Arf (Su et al., 2009). However, ectodermal defects in p63-null embryos, including proliferation, stratification and hair follicle specification, are rescued to a greater extent by concomitant deletion of p16/Ink4a than by loss of p19/Arf (Su et al., 2009). Interestingly, expression of p19/ARF appeared unaffected by Hdac1/2 mutation (Figure S4G–J). However, p16/INK4a was dramatically over-expressed in DcKO embryos at both E14.5 and E16.5 (Figure 4T–W). These data indicate that key repressive functions of ΔNp63, including suppression of p16/Ink4a, require HDAC1/2.

To determine whether the requirement for HDAC1/2 in ΔNp63-mediated repression is direct, we first asked whether HDAC1/2 are present at the promoters of the 14-3-3σ, p21 and p16/INK4a genes in undifferentiated primary human epidermal keratinocytes (HEKs). Chromatin immunoprecipitation (ChIP) assays with antibodies to HDAC1 and HDAC2, and PCR primers that amplify promoter regions containing p63-binding sites for each of these genes, revealed specific binding of both HDAC1 and HDAC2 in each case (Figure 4X, left panels). We next wanted to determine whether HDACs are required for histone acetylation at these promoter regions. To address this question, we carried out ChIP assays using the same primers to amplify 14-3-3σ, p21 and p16/INK4a promoter sequences, antibodies to H3K9Ac, and extracts of keratinocytes that had been cultured with or without the HDAC inhibitor TSA. These experiments revealed increased histone acetylation at the 14-3-3σ, p21 and p16/INK4a promoters in TSA treated compared with control treated cells (Figure 4X, right panels). Quantification of bound H3K9Ac levels by real-time PCR coupled with ChIP revealed that the increases in H3K9Ac at the p21, 14- 3-3σ, and p16INK4a promoter regions were statistically significant (p<0.05 for all TSA treated samples compared with control treated, except for 14-3-3σ in samples treated with TSA for 6 hours, for which p=0.05) (Figure S4K). By contrast, levels of acetylated histone H3 at the negative control GAPDH promoter were not significantly increased in TSA treated compared with control treated cells (Figure S4K). Thus HDACs are specifically active at ΔNp63-repressed promoters. These data are consistent with a direct requirement for HDAC1/2 in ΔNp63-mediated repression of cell cycle inhibitory genes, and suggest that proliferative, stratification and ectodermal appendage defects in Hdac1/2-null epidermis are due at least in part to failure of ΔNp63-repressive functions.

HDAC1/2 suppress P53 hyperacetylation in embryonic epidermis

P53 positively regulates several targets of p63 repression, including 14-3-3σ and p21 (el-Deiry et al., 1993; Hermeking et al., 1997), which are ectopically or over-expressed in DcKO epidermis. In addition, levels of PCNA, which is indirectly suppressed by P53 (Saifudeen et al., 2002), and a P53-activated direct target GADD45A (Rosemary Siafakas and Richardson, 2009), were respectively decreased and increased, in DcKO compared with control epidermis at E16.5 (Figure 5A–H). In vitro studies suggest that P53 activity is repressed in an HDAC1 dependent manner through removal of acetylation marks from its C-terminal lysine residues (Higashitsuji et al., 2007; Ito et al., 2002; Tang et al., 2008). We therefore asked whether acetylated P53 levels were increased in DcKO epidermis in vivo. Immunofluorescence revealed marked increases in the levels of p53 acetylated at either lysine 379 or lysine 386 in DcKO compared with control littermate epidermis at both E14.5 and E16.5 (Figure 5I–P). Thus Hdac1/2 are necessary to suppress hyper-acetylation of P53 in embryonic epidermis, and lack of this activity may contribute to upregulation of cell cycle inhibitory genes and the observed proliferative defects and apoptosis in DcKO embryos.

Figure 5. HDAC1/2 suppress hyper-acetylation of p53 in embryonic epidermis.

Figure 5

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(A–D) PCNA (green) is expressed in control basal cell and some suprabasal cell nuclei at E14.5 (A) and E16.5 (C, arrows). PCNA is expressed in DcKO epidermis at E14.5 (B), but is downregulated by E16.5 (D). (E–H) GADD45A (green) is expressed in control suprabasal but not basal cells at E14.5 (E) and E16.5 (G). In single-layered DcKO epidermis, GADD45A is weakly expressed at E14.5 (F) and gives a strong signal at E16.5 (H, arrows). (I–P) p53-AcK379 (I–L) and p53-AcK386 (M–P) (green) are undetectable in control basal nuclei at E14.5 (I, M) or E16.5 (K, O), and are weakly detected in a subset of differentiating nuclei at E16.5 (K, O). DcKO epidermis displays strong staining for acetylated p53-AcK379 (J, L) and p53-AcK386 (N, P) at both E14.5 (J, N) and E16.5 (L, P) (arrows). Dashed lines indicate dermal-epidermal junctions. Size bars represent 25μm.

p53 is required for upregulation of p21 in keratinocytes in response to HDAC inhibition

To test more directly whether increased levels of acetylated p53 might be required for the observed upregulation of epidermal p21, we inhibited HDAC function in HEKs by treatment with TSA. 24 hours of TSA treatment resulted in a statistically significant decrease in proliferation (Figure 6A, D, G and Supplemental Figure S5C), as well as dramatic increases in the intensity of immunoflourescence for acetylated p53 and for P21 (Figure 6B, C, E, F). shRNA-mediated knockdown of p53 or p21 expression resulted in reduced immunostaining for the corresponding protein (Figure 6H, I, K, L), decreased transcript levels to approximately 25% of those observed in control knockdown cells (Figure S5A, B), and inhibition of the anti-proliferative effects of TSA (Figure 6G and Supplemental Figure S5C). Knockdown of p21 in TSA-treated cells did not affect total p53 levels (Figure 6H, J). By contrast, knockdown of p53 prevented accumulation of p21 protein and mRNA in response to TSA treatment (Figure 6K, M, N). Thus p53 is required for elevated p21 levels in response to HDAC inhibition in keratinocytes.

Figure 6. p53 is required for elevated p21 expression in response to HDAC inhibition in keratinocytes.

Figure 6

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(A–F) TSA treatment of HEKs causes decreased proliferation, and increased expression of acetylated p53 and p21. HEKs treated with 500nM TSA in DMSO (D–F) or DMSO alone (A–C) were subjected to immunofluorescence (green) using antibodies to PH3 (A, D), p53-AcK379 (B, E) or p21 (C, F). (G) The TSA-induced decrease in proliferation requires p53 and p21. HEKs were transduced with retroviruses carrying control knockdown (KD), p53 KD or p21 KD shRNAmir constructs as indicated, and treated with 50nm TSA in DMSO (light grey bars) or DMSO only (dark grey bars). The percentage of PH3-positive nuclei is indicated for each condition. 10 fields of view were counted per sample at 40x magnification and the experiment was performed in duplicate. (H–N) p53 is required for the TSA-induced increase in p21 expression. HEKs were transduced with retroviruses carrying control (H, K), p53 (I, M) or p21 (J, L) knockdown shRNAmirs and treated with 50nM TSA. Immunofluorescence with antibodies to total p53 (H–J) (red) or p21 (K–M) (green), reveals efficient knockdown of p53 or p21 with the corresponding shRNAmir (H, I, K, L), and decreased p21 expression in p53 knockdown compared with control knockdown cells (K, M). Knockdown of p21 does not affect expression of p53 (H, J). (N) Quantification of p21 mRNA levels by real-time PCR reveals a statistically significant increase in p21 mRNA expression in TSA-treated control knockdown cells compared with DMSO-treated control knockdown cells (dark grey bars) (p<0.05), and no TSA-induced increase in p21 expression in either p21 (light grey bars) or p53 (medium grey bars) knockdown cells. (O) Model for interactions of HDAC1/2, ΔNp63 and p53 in embryonic epidermis. ΔNp63 and HDAC1/2 cooperate to directly repress expression of p16/INK4a and may also directly contribute to repression of p21. HDAC1/2 independently deacetylate p53, suppressing its ability to induce apoptosis and activate target genes including p21. Data in (G, N) are represented as mean +/− SEM; asterisks indicate statistical significance; NS, not statistically significant. See also Figure S5.

These data are consistent with a model in which increased levels of p53 activity contribute to proliferative defects in Hdac1/2-deficient epidermis. However, the relatively mild effects of loss of Hdac1/2 on expression of p21 in DcKO mutants, compared with dramatic upregulation of the p53-independent gene p16/Ink4a by E14.5 (Figure 4P–W), indicate that p53-independent loss of ΔNP63 repressor functions plays a major role. While deletion of HDAC1/2 should immediately relieve ΔNp63-mediated repression of p16/Ink4a, accumulation of critical levels of acetylated p53 is likely required for increased p53 activity and p53-mediated target gene activation. This less direct mechanism (Figure 6O) may account in part for our observation that activation of some p53 targets is delayed relative to the increase in p16/INK4a expression. As p53 is well established as a pro-apoptotic protein, this indirect mechanism may also explain the relatively late increase in apoptosis observed in Hdac1/2 epidermal mutants (Figure 3E, G).

Discussion

Here we show that Hdac1/2 perform essential functions in ectodermal appendage and epidermal development, and are required for basal epidermal cells to acquire suprabasal or hair follicle placode fates. Consistent with specific defects resulting from Hdac1/2 deletion in other tissues, KRT14-Cre-mediated loss of Hdac1/2 produced an epidermal phenotype strikingly similar to that of embryos lacking the epidermal master regulator, p63. Krt14 is a direct, positively regulated p63 target (Romano et al., 2009), and KRT14-Cre-mediated deletion of Hdac1/2 occurs after initiation of p63 expression, but prior to development of a suprabasal epidermal layer or specification of hair follicles (Liu et al., 2007). In line with this, both p63 and KRT14 were expressed in Hdac1/2 epidermal mutants, and defects in morphogenesis of the limbs and ectodermal appendages such as mammary glands, vibrissae and teeth that develop before onset of high levels of KRT14-Cre expression, were less severe than the epidermal stratification and hair follicle phenotypes.

Consistent with HDAC1/2’s functions in gene repression, we found that, while p63-activated basal layer genes were unaffected by Hdac1/2 deletion, p63-suppressed genes, in particular p16/Ink4a, were markedly dysregulated. We further showed that HDAC1/2 bind the promoter regions of p63-repressed genes in undifferentiated keratinocytes, and that histone acetylation is specifically increased in these regions following treatment with the HDAC inhibitor TSA. Together, these results indicate that HDAC1/2 directly mediate p63’s repressive functions. Loss of the repressive functions of this key epidermal factor in Hdac1/2 mutants may mask other less central roles of Hdac1/2 at early stages of epidermal development, accounting for the striking similarity of p63-null and Hdac1/2 mutant phenotypes. HDAC1/2 are likely to play additional roles at subsequent embryonic stages and in adult skin and hair follicles. These may be revealed by inducible epidermal-specific deletion of Hdac1/2 later in embryonic or postnatal life.

Interestingly, concomitant loss of p16/Ink4a partially rescues multiple defects in p63-null epidermis, including progenitor cell proliferation, stratification and hair follicle development (Su et al., 2009). Suppression of p16/INK4a expression is essential for self-renewal of multiple types of progenitor cells (Akala et al., 2008; Molofsky et al., 2006; Nishino et al., 2008). The partial rescue of stratification and hair follicle defects in p63−/− p16/Ink4a−/− double mutants suggests that this suppression is also essential for asymmetric cell division and cell fate decisions in embryonic epidermis. These data further suggest that the dramatic increase in p16/INK4a levels in Hdac1/2 mutant epidermis contributes in a major way to the observed defects in epidermal development and hair follicle specification.

As mentioned above, the defects in Hdac1/2 mutant epidermis were remarkably similar to those described for p63-null skin. One difference, however, was that p19/ARF is upregulated in p63 mutants, but its levels were not substantially altered by Hdac1/2 deletion. We discovered that HDAC1/2 are required to suppress hyper-acetylation of p53 in embryonic epidermis. As p53 indirectly suppresses levels of p19/ARF (Kamijo et al., 1998; Stott et al., 1998), the competing effects of loss of p63-mediated repression and increased p53 activity may account for the relatively unchanged levels of p19/ARF in Hdac1/2 compared with control epidermis. In addition to its possible contribution to regulation of p19/ARF, experiments in keratinocytes demonstrated that p53 is required for elevated p21 expression in response to HDAC inhibition. Thus it is likely that hyper-acetylated p53 contributed to the observed upregulation of p21 and proliferative defects in Hdac1/2 mutant epidermis.

Loss of Hdac1/2 function in embryonic epidermis lead to a gradual decrease in proliferation rates relative to controls, and initially was not associated with increased apoptopsis, suggesting that, as in p63-null ectoderm (Senoo et al., 2007), failure of basal progenitor cell self-renewal contributes to the phenotype. Loss of either the epidermal histone methylase EZH2 or the DNA methyltransferase DNMT1 also results in defects in progenitor cell proliferation (Ezhkova et al., 2009; Sen et al., 2010). Epidermis lacking Ezh2 displays increased p16/INK4a expression (Ezhkova et al., 2009), consistent with association of HDAC1/2 and EZH2 in the polycomb repressor complex PRC2 (van der Vlag and Otte, 1999). However, perhaps due to compensating activity of the related gene Ezh1, Ezh2-deficient epidermis displays a much less severe phenotype than that caused by loss of p63 or Hdac1/2, and is capable of stratifying and producing a functional barrier (Ezhkova et al., 2009).

Notably, unlike the effects of Hdac1/2 deletion or absence of p63, loss of Ezh2 or DNMT1 is associated with premature differentiation of basal cells (Ezhkova et al., 2009; Sen et al., 2010). Thus it is possible that Hdac1/2 control progenitor cell differentiation in part through mechanisms distinct from those utilized by Ezh2 and DNMT1. Alternatively, persistence of methylation marks in Hdac1/2 mutants might block precocious activation of differentiation genes.

Unlike loss of EZH2 and DNMT1, lack of epidermal Mi-2β, which associates with HDAC 2intheNuRDcomplex does not result in inappropriate activation of differentiation genes; instead differentiation markers are expressed appropriately and the epidermis stratifies normally. However, as in Hdac1/2-deficient epidermis, Mi-2β mutants display defects in hair follicle specification and a gradual decline in basal cell proliferation (Kashiwagi et al., 2007). These common phenotypes may reflect functions of the NuRD complex in hair follicle induction and progenitor cell self-renewal. Precisely how HDAC1/2 and p63 interact with Mi-2β EZH1/2, DNMT1, and other chromatin modification factors to coordinate gene expression in the developing epidermis will be a fascinating area for further study.

In summary, our data reveal essential roles for Hdac1/2 in proliferation, stratification and cell fate decisions in the embryonic epidermis, and indicate that this occurs in part through mediating the repressive functions of p63, and in suppressing p53 activity. As increased expression of HDACs in squamous cell carcinoma (SCC) predicts poor outcome, and HDACi can induce growth arrest in SCC lines as well as in normal keratinocytes, HDACs likely play key roles in SCC as well as in epidermal morphogenesis (Chang et al., 2009; Prystowsky et al., 2009; Saunders et al., 1999). Loss of Hdac1/2 function results in de-repression of cell cycle inhibitory and senescence factors in embryonic basal cells, suggesting specific targeting of these factors in undifferentiated epidermal tumors as a potentially powerful therapeutic tool. Determining the effects of Hdac1/2 deletion in adult epidermis and in skin tumor models will be critically important in further testing the utility of this approach.

Experimental Procedures

Generation of mice

Mice carrying Hdac1 and Hdac2 floxed alleles (Montgomery et al., 2007) were crossed with transgenic KRT14-Cre line 43 mice (Andl et al., 2004). Genotyping was as described previously (Andl et al., 2004; Montgomery et al., 2007). All experiments were performed with approved animal protocols according to institutional guidelines established by the University of Pennsylvania IUCAC committee.

Histology, immunostaining and in situ hybridization

Histology, immunostaining and whole mount in situ hybridization were carried out as described previously (Zhang et al., 2009). Details of the antibodies used are provided in Supplemental Methods.

Chromatin immunoprecipitation

ChIP was carried out according to the manufacturer’s instructions using the EZ ChIP kit (Millipore) or the Magnify CHiP System (Invitrogen) and extracts of neonatal foreskin HEKs cultured in low calcium medium (Li et al., 2005). For analyses of bound H3K9Ac, HEKs were cultured in 0nM or 500nM TSA in DMSO for 6 or 12 hours. Cells were fixed in 1% formaldehyde, lysed, and chromatin was sheared using a Bioruptor UCD-200 (Diagenode) to ~200 base pair fragments. Chromatin was immunoprecipitated with antibodies to HDAC1 (Abcam), HDAC2 (Abcam), H3K9Ac (Abcam) or Rabbit IgG (Millipore). Real-time PCR experiments coupled with ChIP utilized the StepOnePlus system and software (Applied Biosystems) and SYBR Green reagents (DNA Master). Changes were determined based on the 2−Δ ΔCT method. See Supplemental Table for PCR primer sequences.

Assays for proliferation and P53-AcK379 expression in HEKs

Neonatal foreskin HEKs in low calcium medium (Li et al., 2005) were plated at 20% confluency, treated with either 500nM TSA in DMSO or DMSO alone for 24 hours, fixed with 4% paraformaldehyde and immunostained with antibodies to PH3 (1:200) (Millipore), or P53-AcK379 (1:200) (Abcam).

shRNAmir knockdown experiments

Neonatal foreskin HEKs were transduced with retroviruses carrying shRNAmir non-silencing (Cat# RHS4971), p53 (Cat# RHS4917-99781715), or p21 (Cat# RHS4917-99780100) vectors (OpenBiosystems). Following puromycin selection, cells were treated with control (DMSO) or 50nM TSA in DMSO for 24 hours, fixed, permeabilized, and immunostained with anti-PH3 (Millipore), p21 (Abcam), p53 (Abcam), or P53-AcK379 (Abcam). Each experimental condition was performed in duplicate and the entire experiment was carried out twice. Detailed methods are provided in Supplemental Experimental Procedures.

Quantification of transcript levels by real-time PCR

Total RNA was purified using an RNeasy kit (Qiagen), reverse-transcribed using First-Strand cDNA Synthesis Kit (GE, Amersham), and subjected to real-time PCR using the StepOnePlus system and software (Applied Biosystems) and SYBR Green reagents (DNA Master). See Supplemental Experimental Procedures for detailed methods and primer sequences.

Highlights.

  • Deletion of ectodermal Hdac1/2 blocks epidermal development, phenocopying loss of p63

  • Targets of ΔNp63-mediated repression are upregulated in Hdac1/2 epidermal mutants

  • HDACs specifically bind and are active at ΔNp63-repressed promoters

  • HDAC1/2 are also required to suppress p53 hyperacetylation in embryonic epidermis

Supplementary Material

01

Acknowledgments

We thank Leroy Ash and Tzvete Dentchev for histology and members of the Millar, Morrisey and Epstein labs for helpful discussions. Research in S.E.M.’s lab is supported by RO1AR47709; RO1HD053829; RO1AR055241 and RC1DE020337. M.L. was supported by T32AR007465 and T32HD007516. J.A.M. and E.E.M. received support from UO1HL100405 (J.A.M. and E.E.M.) and RO1HL071546 (J.A.M.).

Footnotes

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References

  1. Akala OO, Park IK, Qian D, Pihalja M, Becker MW, Clarke MF. Long-term haematopoietic reconstitution by Trp53−/−p16Ink4a−/−p19Arf−/− multipotent progenitors. Nature. 2008;453:228–232. doi: 10.1038/nature06869. [DOI] [PubMed] [Google Scholar]
  2. Andl T, Ahn K, Kairo A, Chu EY, Wine-Lee L, Reddy ST, Croft NJ, Cebra-Thomas JA, Metzger D, Chambon P, et al. Epithelial Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development. Development. 2004;131:2257–2268. doi: 10.1242/dev.01125. [DOI] [PubMed] [Google Scholar]
  3. Blanpain C, Fuchs E. p63: revving up epithelial stem-cell potential. Nat Cell Biol. 2007;9:731–733. doi: 10.1038/ncb0707-731. [DOI] [PubMed] [Google Scholar]
  4. Brunmeir R, Lagger S, Seiser C. Histone deacetylase HDAC1/HDAC2-controlled embryonic development and cell differentiation. Int J Dev Biol. 2009;53:275–289. doi: 10.1387/ijdb.082649rb. [DOI] [PubMed] [Google Scholar]
  5. Byrne C, Tainsky M, Fuchs E. Programming gene expression in developing epidermis. Development. 1994;120:2369–2383. doi: 10.1242/dev.120.9.2369. [DOI] [PubMed] [Google Scholar]
  6. Candi E, Terrinoni A, Rufini A, Chikh A, Lena AM, Suzuki Y, Sayan BS, Knight RA, Melino G. p63 is upstream of IKK alpha in epidermal development. J Cell Sci. 2006;119:4617–4622. doi: 10.1242/jcs.03265. [DOI] [PubMed] [Google Scholar]
  7. Chang HH, Chiang CP, Hung HC, Lin CY, Deng YT, Kuo MY. Histone deacetylase 2 expression predicts poorer prognosis in oral cancer patients. Oral Oncol. 2009;45:610–614. doi: 10.1016/j.oraloncology.2008.08.011. [DOI] [PubMed] [Google Scholar]
  8. Chikh A, Sayan E, Thibaut S, Lena AM, DiGiorgi S, Bernard BA, Melino G, Candi E. Expression of GATA-3 in epidermis and hair follicle: relationship to p63. Biochem Biophys Res Commun. 2007;361:1–6. doi: 10.1016/j.bbrc.2007.06.069. [DOI] [PubMed] [Google Scholar]
  9. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993;75:817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]
  10. Ezhkova E, Pasolli HA, Parker JS, Stokes N, Su IH, Hannon G, Tarakhovsky A, Fuchs E. Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell. 2009;136:1122–1135. doi: 10.1016/j.cell.2008.12.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frye M, Fisher AG, Watt FM. Epidermal stem cells are defined by global histone modifications that are altered by Myc-induced differentiation. PLoS ONE. 2007;2:e763. doi: 10.1371/journal.pone.0000763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haberland M, Montgomery RL, Olson EN. The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet. 2009;10:32–42. doi: 10.1038/nrg2485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hermeking H, Lengauer C, Polyak K, He TC, Zhang L, Thiagalingam S, Kinzler KW, Vogelstein B. 14-3-3 sigma is a p53-regulated inhibitor of G2/M progression. Mol Cell. 1997;1:3–11. doi: 10.1016/s1097-2765(00)80002-7. [DOI] [PubMed] [Google Scholar]
  14. Higashitsuji H, Masuda T, Liu Y, Itoh K, Fujita J. Enhanced deacetylation of p53 by the anti-apoptotic protein HSCO in association with histone deacetylase 1. J Biol Chem. 2007;282:13716–13725. doi: 10.1074/jbc.M609751200. [DOI] [PubMed] [Google Scholar]
  15. Indra AK, Dupe V, Bornert JM, Messaddeq N, Yaniv M, Mark M, Chambon P, Metzger D. Temporally controlled targeted somatic mutagenesis in embryonic surface ectoderm and fetal epidermal keratinocytes unveils two distinct developmental functions of BRG1 in limb morphogenesis and skin barrier formation. Development. 2005;132:4533–4544. doi: 10.1242/dev.02019. [DOI] [PubMed] [Google Scholar]
  16. Ingraham CR, Kinoshita A, Kondo S, Yang B, Sajan S, Trout KJ, Malik MI, Dunnwald M, Goudy SL, Lovett M, et al. Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6) Nat Genet. 2006;38:1335–1340. doi: 10.1083/ng1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ito A, Kawaguchi Y, Lai CH, Kovacs JJ, Higashimoto Y, Appella E, Yao TP. MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation. EMBO J. 2002;21:6236–6245. doi: 10.1093/emboj/cdf616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jamora C, DasGupta R, Kocieniewski P, Fuchs E. Links between signal transduction, transcription and adhesion in epithelial bud development. Nature. 2003;422:317–322. doi: 10.1038/nature01458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kamijo T, Weber JD, Zambetti G, Zindy F, Roussel MF, Sherr CJ. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc Natl Acad Sci U S A. 1998;95:8292–8297. doi: 10.1073/pnas.95.14.8292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kashiwagi M, Morgan BA, Georgopoulos K. The chromatin remodeler Mi-2beta is required for establishment of the basal epidermis and normal differentiation of its progeny. Development. 2007;134:1571–1582. doi: 10.1242/dev.001750. [DOI] [PubMed] [Google Scholar]
  21. Kim J, Sif S, Jones B, Jackson A, Koipally J, Heller E, Winandy S, Viel A, Sawyer A, Ikeda T, et al. Ikaros DNA-binding proteins direct formation of chromatin remodeling complexes in lymphocytes. Immunity. 1999;10:345–355. doi: 10.1016/s1074-7613(00)80034-5. [DOI] [PubMed] [Google Scholar]
  22. Koster MI. p63 in Skin Development and Ectodermal Dysplasias. J Invest Dermatol. 2010 doi: 10.1038/jid.2010.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koster MI, Dai D, Marinari B, Sano Y, Costanzo A, Karin M, Roop DR. p63 induces key target genes required for epidermal morphogenesis. Proc Natl Acad Sci U S A. 2007;104:3255–3260. doi: 10.1073/pnas.0611376104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Laurikkala J, Mikkola ML, James M, Tummers M, Mills AA, Thesleff I. p63 regulates multiple signalling pathways required for ectodermal organogenesis and differentiation. Development. 2006;133:1553–1563. doi: 10.1242/dev.02325. [DOI] [PubMed] [Google Scholar]
  25. Lechler T, Fuchs E. Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature. 2005;437:275–80. doi: 10.1038/nature03922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lefkimmiatis K, Caratozzolo MF, Merlo P, D’Erchia AM, Navarro B, Levrero M, Sbisa E, Tullo A. p73 and p63 sustain cellular growth by transcriptional activation of cell cycle progression genes. Cancer Res. 2009;69:8563–8571. doi: 10.1158/0008-5472.CAN-09-0259. [DOI] [PubMed] [Google Scholar]
  27. Li W, Marshall C, Mei L, Dzubow L, Schmults C, Dans M, Seykora J. Srcasm modulates EGF and Src-kinase signaling in keratinocytes. J Biol Chem. 2005;280:6036–6046. doi: 10.1074/jbc.M406546200. [DOI] [PubMed] [Google Scholar]
  28. Liu F, Chu EY, Watt B, Zhang Y, Gallant NM, Andl T, Yang SH, Lu MM, Piccolo S, Schmidt-Ullrich R, et al. Wnt/beta-catenin signaling directs multiple stages of tooth morphogenesis. Dev Biol. 2008;313:210–224. doi: 10.1016/j.ydbio.2007.10.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu F, Thirumangalathu S, Gallant NM, Yang SH, Stoick-Cooper CL, Reddy ST, Andl T, Taketo MM, Dlugosz AA, Moon RT, et al. Wnt-beta-catenin signaling initiates taste papilla development. Nat Genet. 2007;39:106–112. doi: 10.1038/ng1932. [DOI] [PubMed] [Google Scholar]
  30. Lopardo T, Lo Iacono N, Marinari B, Giustizieri ML, Cyr DG, Merlo G, Crosti F, Costanzo A, Guerrini L. Claudin-1 is a p63 target gene with a crucial role in epithelial development. PLoS ONE. 2008;3:e2715. doi: 10.1371/journal.pone.0002715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Marinari B, Ballaro C, Koster MI, Giustizieri ML, Moretti F, Crosti F, Papoutsaki M, Karin M, Alema S, Chimenti S, et al. IKKalpha is a p63 transcriptional target involved in the pathogenesis of ectodermal dysplasias. J Invest Dermatol. 2009;129:60–69. doi: 10.1038/jid.2008.202. [DOI] [PubMed] [Google Scholar]
  32. McDade SS, McCance DJ. The role of p63 in epidermal morphogenesis and neoplasia. Biochem Soc Trans. 2010;38:223–228. doi: 10.1042/BST0380223. [DOI] [PubMed] [Google Scholar]
  33. Mikkola ML, Millar SE. The mammary bud as a skin appendage: unique and shared aspects of development. J Mammary Gland Biol Neoplasia. 2006;11:187–203. doi: 10.1007/s10911-006-9029-x. [DOI] [PubMed] [Google Scholar]
  34. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature. 1999;398:708–713. doi: 10.1038/19531. [DOI] [PubMed] [Google Scholar]
  35. Molofsky AV, Slutsky SG, Joseph NM, He S, Pardal R, Krishnamurthy J, Sharpless NE, Morrison SJ. Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing. Nature. 2006;443:448–452. doi: 10.1038/nature05091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Montgomery RL, Davis CA, Potthoff MJ, Haberland M, Fielitz J, Qi X, Hill JA, Richardson JA, Olson EN. Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis, growth, and contractility. Genes Dev. 2007;21:1790–1802. doi: 10.1101/gad.1563807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nishino J, Kim I, Chada K, Morrison SJ. Hmga2 promotes neural stem cell self-renewal in young but not old mice by reducing p16Ink4a and p19Arf Expression. Cell. 2008;135:227–239. doi: 10.1016/j.cell.2008.09.01. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Prystowsky MB, Adomako A, Smith RV, Kawachi N, McKimpson W, Atadja P, Chen Q, Schlecht NF, Parish JL, Childs G, et al. The histone deacetylase inhibitor LBH589 inhibits expression of mitotic genes causing G2/M arrest and cell death in head and neck squamous cell carcinoma cell lines. J Pathol. 2009;218:467–477. doi: 10.1002/path.2554. [DOI] [PubMed] [Google Scholar]
  39. Romano RA, Ortt K, Birkaya B, Smalley K, Sinha S. An active role of the DeltaN isoform of p63 in regulating basal keratin genes K5 and K14 and directing epidermal cell fate. PLoS One. 2009;4:e5623. doi: 10.1371/journal.pone.0005623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rosemary Siafakas A, Richardson DR. Growth arrest and DNA damage-45 alpha (GADD45alpha) Int J Biochem Cell Biol. 2009;41:986–989. doi: 10.1016/j.biocel.2008.06.018. [DOI] [PubMed] [Google Scholar]
  41. Ruthenburg AJ, Li H, Patel DJ, Allis CD. Multivalent engagement of chromatin modifications by linked binding modules. Nat Rev Mol Cell Biol. 2007;8:983–994. doi: 10.1038/nrm2298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Saifudeen Z, Marks J, Du H, El-Dahr SS. Spatial repression of PCNA by p53 during kidney development. Am J Physiol Renal Physiol. 2002;283:F727–733. doi: 10.1152/ajprenal.00114.2002. [DOI] [PubMed] [Google Scholar]
  43. Saunders N, Dicker A, Popa C, Jones S, Dahler A. Histone deacetylase inhibitors as potential anti-skin cancer agents. Cancer Res. 1999;59:399–404. [PubMed] [Google Scholar]
  44. Sen GL, Reuter JA, Webster DE, Zhu L, Khavari PA. DNMT1 maintains progenitor function in self-renewing somatic tissue. Nature. 2010;463:563–567. doi: 10.1038/nature08683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Senoo M, Pinto F, Crum CP, McKeon F. p63 Is essential for the proliferative potential of stem cells in stratified epithelia. Cell. 2007;129:523–536. doi: 10.1016/j.cell.2007.02.045. [DOI] [PubMed] [Google Scholar]
  46. Stott FJ, Bates S, James MC, McConnell BB, Starborg M, Brookes S, Palmero I, Ryan K, Hara E, Vousden KH, et al. The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2. EMBO J. 1998;17:5001–5014. doi: 10.1093/emboj/17.17.5001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Su X, Cho MS, Gi YJ, Ayanga BA, Sherr CJ, Flores ER. Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf. EMBO J. 2009;28:1904–1915. doi: 10.1038/emboj.2009.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tang Y, Zhao W, Chen Y, Zhao Y, Gu W. Acetylation is indispensable for p53 activation. Cell. 2008;133:612–626. doi: 10.1016/j.cell.2008.03.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Truong AB, Khavari PA. Control of keratinocyte proliferation and differentiation by p63. Cell Cycle. 2007;6:295–299. doi: 10.4161/cc.6.3.3753. [DOI] [PubMed] [Google Scholar]
  50. Truong AB, Kretz M, Ridky TW, Kimmel R, Khavari PA. p63 regulates proliferation and differentiation of developmentally mature keratinocytes. Genes Dev. 2006;20:3185–3197. doi: 10.1101/gad.1463206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. van der Vlag J, Otte AP. Transcriptional repression mediated by the human polycomb-group protein EED involves histone deacetylation. Nat Genet. 1999;23:474–478. doi: 10.1038/70602. [DOI] [PubMed] [Google Scholar]
  52. Westfall MD, Mays DJ, Sniezek JC, Pietenpol JA. The Delta Np63 alpha phosphoprotein binds the p21 and 14-3-3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay-Wells syndrome-derived mutations. Mol Cell Biol. 2003;23:2264–2276. doi: 10.1128/MCB.23.7.2264-2276.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature. 1999a;398:714–718. doi: 10.1038/19539. [DOI] [PubMed] [Google Scholar]
  54. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature. 1999b;398:714–718. doi: 10.1038/19539. [DOI] [PubMed] [Google Scholar]
  55. Zhang Y, Tomann P, Andl T, Gallant NM, Huelsken J, Jerchow B, Birchmeier W, Paus R, Piccolo S, Mikkola ML, et al. Reciprocal requirements for EDA/EDAR/NF-kappaB and Wnt/beta-catenin signaling pathways in hair follicle induction. Dev Cell. 2009;17:49–61. doi: 10.1016/j.devcel.2009.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]

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