© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
RESEARCH ARTICLE
Histone deacetylase 1 and 2 regulate Wnt and p53 pathways in the
ureteric bud epithelium
ABSTRACT
Histone deacetylases (HDACs) regulate a broad range of biological
processes through removal of acetyl groups from histones as well as
non-histone proteins. Our previous studies showed that Hdac1 and
Hdac2 are bound to promoters of key renal developmental regulators
and that HDAC activity is required for embryonic kidney gene
expression. However, the existence of many HDAC isoforms in
embryonic kidneys raises questions concerning the possible
specificity or redundancy of their functions. We report here that
targeted deletion of both the Hdac1 and Hdac2 genes from the
ureteric bud (UB) cell lineage of mice causes bilateral renal
hypodysplasia. One copy of either Hdac1 or Hdac2 is sufficient to
sustain normal renal development. In addition to defective cell
proliferation and survival, genome-wide transcriptional profiling
revealed that the canonical Wnt signaling pathway is specifically
impaired in UBHdac1,2−/− kidneys. Our results also demonstrate that loss
of Hdac1 and Hdac2 in the UB epithelium leads to marked
hyperacetylation of the tumor suppressor protein p53 on lysine 370,
379 and 383; these post-translational modifications are known to boost
p53 stability and transcriptional activity. Genetic deletion of p53 partially
rescues the development of UBHdac1,2−/− kidneys. Together, these data
indicate that Hdac1 and Hdac2 are crucial for kidney development.
They perform redundant, yet essential, cell lineage-autonomous
functions via p53-dependent and -independent pathways.
KEY WORDS: Branching morphogenesis, Histone, Kidney, Ureteric
bud, Wnt, p53 (Trp53), Mouse
INTRODUCTION
Histone deacetylases (HDACs) are a superfamily of enzymes important
for modulation of chromatin structure and function via removal of
acetyl moieties from lysine residues of histones as well as non-histone
proteins. In higher eukaryotes, 18 HDAC isoforms have been
identified. Based on sequence homology to yeast HDAC genes, they
are divided into four classes: the class I RPD3-like HDACs (1-3 and 8);
the class II HDA1-like HDACs (4-7, 9 and 10); Sirtuins 1-7; and the
class IV HDAC11 (de Ruijter et al., 2003; Denu and Gottesfeld, 2012).
HDACs were originally assumed to be global transcriptional
co-repressors, but it subsequently became clear that HDACs regulate
gene expression in a highly selective manner and exhibit both
repressive and activating effects. HDAC inhibition or deletion of
HDAC genes often results in alterations in a small subset of genes
(<10%), and approximately as many are downregulated as are
upregulated (Smith, 2008). Mechanistically, HDACs themselves
lack intrinsic DNA binding activity and are recruited to target genes
through association with various transcriptional complexes (e.g. the
Sin3, NuRD, Co-REST and SMRT/N-CoR complexes) (Marks
et al., 2003). As such, the specificity of HDACs in gene regulation
depends on the partner proteins that they associate with under
different pathophysiological conditions. In addition to histones,
HDACs can also deacetylate an increasing number of non-histone
proteins, including p53, STAT3, Yin Yang transcription factor
(YY1), GATA1, E2F1 and Hsp90 (Glozak et al., 2005; Kruse and
Gu, 2009; Spange et al., 2009). Deacetylation of these proteins has
been shown to affect multiple aspects of their function, such as
protein stability, DNA binding affinity and transcriptional activity,
adding an extra layer of complexity to the biological roles of HDACs.
In line with recent studies showing that HDACs play both unique
and redundant roles in the control of distinctive developmental
pathways during embryogenesis, our previous studies revealed that
the expression of key developmental renal regulators (e.g. Osr1,
Eya1, Pax2/8, Wt1 and Wnt9b) is dependent on intact HDAC
activity (Chen et al., 2011; Haberland et al., 2009). Treatment of
cultured mouse embryonic kidneys with Scriptaid, a general
inhibitor of class I and II HDACs, or MS-275, a selective inhibitor
of HDAC1-3, impairs ureteric bud (UB) branching morphogenesis
and nephrogenesis, two central processes of metanephric
development. Our results also demonstrated that Hdac1-3 are
enriched in the undifferentiated metanephric mesenchyme (MM)
and UB branches, but are reduced upon differentiation, implicating
their crucial role in mouse kidney development.
Among class I and class II HDACs, HDAC1 and HDAC2 are
evolutionarily close to each other, sharing a high degree of sequence
similarity (∼85% identity at the amino acid level) except for their
C-terminal domain. HDAC1 and HDAC2 can form homo- or
heterodimers and are found together in almost all nuclear protein
complexes, including three well-characterized co-repressor
complexes: Sin3, NuRD and Co-REST (Brunmeir et al., 2009).
Studies using a variety of genetic models of Hdac1 and Hdac2
indicate that they exert both overlapping and non-redundant
functions in different cell types at specific developmental stages
(Haberland et al., 2010; Lagger et al., 2002; LeBoeuf et al., 2010;
Ma et al., 2012; Montgomery et al., 2007; Ye et al., 2009). In this
study, by deleting different combinations of Hdac1 and Hdac2
alleles in the UB cell lineage, we revealed the redundant yet
essential cell-autonomous functions of Hdac1 and Hdac2 in the
ureteric epithelium during mouse kidney development. Moreover,
our findings illustrate the developmental importance of HDACmediated control of p53 acetylation.
1
Department of Pediatrics, Section of Pediatric Nephrology, Tulane University
2
Health Sciences Center, New Orleans, LA 70112, USA. Department of Molecular
Medicine, Laval University, Qué bec, QC, Canada G1R 2J6.
*Author for correspondence ([email protected])
RESULTS
Concurrent deletion of Hdac1 and Hdac2 in the UB cell
lineage causes renal hypodysplasia
Received 28 May 2014; Accepted 28 January 2015
In order to delete the Hdac1 and Hdac2 genes specifically from
the UB lineage, we crossed Hdac1flox/flox, Hdac2flox/flox and
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DEVELOPMENT
Shaowei Chen1, Xiao Yao1, Yuwen Li1, Zubaida Saifudeen1, Dimcho Bachvarov2 and Samir S. El-Dahr1, *
RESEARCH ARTICLE
Hoxb7-CreEGFP transgenic mice (Montgomery et al., 2007; Zhao
et al., 2004). Previous studies have shown that Hoxb7-directed GFP
expression is observed in the Wolffian duct at embryonic day (E)
10.0 and its derivatives, the UB and its branches, but not in the MM
lineage (Zhao et al., 2004). To test the efficacy of Hoxb7-driven
Cre-mediated excision, we examined the expression of Hdac1 and
Hdac2 proteins by immunohistochemistry in wild-type and mutant
kidney tissues at E13.5. Consistent with our previous report, Hdac1
and Hdac2 are expressed in both the UB and MM cells in wild-type
mice (Fig. 1A,E). By contrast, in UBHdac1,2−/− mice (Hoxb7-Cretg/+;
Hdac1flox/flox; Hdac2flox/flox), Hdac1 and Hdac2 are not detected in
Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
the UB cells but are maintained in the surrounding mesenchymal
cells (Fig. 1B,F). In accordance with the key functions of Hdac1 and
Hdac2 in histone deacetylation, the acetylation levels of histone H3
(lysine 9 and 14, or lysine 9 specifically) and H4 (lysine 5, 8, 12 and
16) are substantially increased in the ureteric cells of UBHdac1,2−/−
kidneys (Fig. 1I-N). Collectively, these results demonstrate efficient
deletion of Hdac1 and Hdac2 from the UB branches. It is worth
noting that knockout of Hdac1 and Hdac2 in oocytes leads to no
apparent change in histone H3K9 acetylation (Ma et al., 2012).
Thus, our results suggest that Hdac1 and Hdac2 might have different
histone residue specificity in different cell types at specific
developmental stages.
Our results revealed that mice with no more than three deleted
alleles of Hdac1 and Hdac2 exhibit no significant abnormalities in
kidney development (supplementary material Fig. S1); moreover,
these mice survive to adulthood without any overt abnormalities in
growth or development. By contrast, concurrent deletion of all four
alleles of Hdac1 and Hdac2 results in early postnatal lethality by
2-4 weeks of age (supplementary material Fig. S2). Histological
analysis of kidney tissue from UBHdac1,2−/− mice at postnatal day
(P) 0 showed absence of the nephrogenic zone, lack of corticomedullary patterning, and the formation of multiple epithelial cysts
(Fig. 2A-F). In line with the histological observations,
immunofluorescence staining demonstrated that the UBHdac1,2−/−
neonates completely lack Six2-positive and Pax2-positive cells
(Fig. 2G-J).
UBHdac1,2−/− kidneys exhibit stunted UB growth and branching
Genome-wide transcriptome analysis of UBHdac1,2−/− kidneys
Fig. 1. UB-specific deletion of Hdac1 and Hdac2 causes hyperacetylation
of histones H3 and H4 at E13.5. Wild-type (WT) and UBHdac1,2−/− kidney
sections were immunostained with antibodies against Hdac1 (A,B), Hdac2 (E,F),
acetylated histone H3 (I,J), histone H3 acetylated at K9 (K,L), acetylated
histone H4 (M,N), cytokeratin (CK) (A,B,E,F,I-N) and with DAPI (C,D,G,H).
UB, ureteric bud.
To further elucidate the developmental pathways regulated by Hdac1
and Hdac2, we carried out a genome-wide microarray analysis on
RNA samples extracted from wild-type and UBHdac1,2−/− kidneys at
E13.5. The raw and analyzed data have been deposited in the NCBI
Gene Expression Omnibus (GEO) under accession number
GSE35432. The results revealed that 496/41,000 probes (∼1.2%)
are significantly altered in UBHdac1,2−/− kidneys (by ≥1.4-fold,
P<0.05, n=4), of which 226 transcripts (0.55%) were upregulated
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DEVELOPMENT
To begin to define the embryological mechanisms leading to this
phenotype, we monitored UB morphogenesis in a real-time manner
in vivo and in vitro, taking advantage of the GFP fluorescence in the
UB tissue driven by the Hoxb7 promoter. UBHdac1,2−/− mice
exhibited attenuated UB branching as early as E13.5 (Fig. 3A), and
started to show degeneration of the UB tissue after 1-2 days in
culture (Fig. 3B). Previous studies have shown that Hdac1 and
Hdac2 regulate apoptosis and proliferation in a wide range of cells.
To examine whether increased apoptosis and decreased proliferation
contribute to the observed defects in UB branching morphogenesis,
we examined the status of cell apoptosis and proliferation at E13.5,
when the abnormal UB branching is first noted. Quantification of
active caspase 3 (aCasp3)-positive cells revealed a significant
increase in the number of apoptotic UB cells in UBHdac1,2−/− versus
wild-type kidneys at E13.5 (Fig. 4A-E). Consistently, staining of
phospho-histone γH2AX, a marker of DNA double-strand breaks,
showed that UBHdac1,2−/− kidneys exhibited increased DNA
damage in UB cells (Fig. 4F-I). Analysis of phospho-histone H3
( pH3), a marker of mitosis, revealed that the rate of cell proliferation
was decreased by 37.5% in UBHdac1,2−/− relative to wild-type
kidneys (Fig. 4J-N). This is consistent with the known proproliferative functions of Hdac1 and Hdac2. Taken together, these
results indicated that Hdac1 and Hdac2 are crucial for UB cell
growth, survival and branching morphogenesis.
RESEARCH ARTICLE
Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
Fig. 2. Renal cystic dysplasia in newborn mice with combined Hdac1 and
Hdac2 deficiency in the ureteric cell lineage. (A,B) Gross anatomy of WT
and UBHdac1,2−/− kidneys at P0. (C-F) PAS staining of kidney sections at P0
shows loss of the nephrogenic zone and the formation of cysts in UBHdac1,2−/−
kidneys. (E,F) High-magnification images of C,D. The dashed green line
delineates the nephrogenic zone (NZ). (G-J) In UBHdac1,2−/− kidneys, there
is complete loss of Six2-positive and Pax2-positive cells. Note the cysts
originating from the UB in UBHdac1,2−/− kidneys (H,J).
(range 1.4- to 7.5-fold) and 270 (0.66%) downregulated (range 1.4to 3.8-fold) (Fig. 5A; see GSE35432).
To analyze whether certain pathways or biological processes are
especially sensitive to the loss of Hdac1 and Hdac2 in the UB cells,
Ingenuity Pathway Analysis (IPA) was performed on the
differentially expressed transcripts. This analysis indicated that the
most significantly enriched genes participate in: (1) cell
morphology; (2) cellular growth and proliferation; (3) cellular
development; (4) cell death and survival and (5) cellular movement
(Fig. 5B). The most affected canonical pathways include: (1) basal
cell carcinoma signaling; (2) Wnt/β-catenin signaling; (3) sonic
hedgehog signaling; (4) human embryonic stem cell pluripotency
and (5) tight junction signaling (Fig. 5C). The complete list of genes
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for each category and pathway is shown in supplementary material
Tables S1 and S2.
Further analysis using the Biological Networks Gene Ontology
(BiNGO) tool revealed that many genes involved in: (1) tube
development; (2) Wnt receptor signaling pathway; (3) ureteric bud
development; (4) cell-cell adhesion; (5) kidney development and (6)
positive regulation of cell proliferation are downregulated in
UBHdac1,2−/− kidneys (Table 1). Interestingly, we found decreased
expression of a group of cytokeratins, including Krt7, Krt8, Krt18,
Krt19 and Krt23 (Table 2), which is consistent with our
immunostaining results using a pan-cytokeratin antibody (Fig. 1I-N).
Cytokeratins, the largest intermediate filament protein group, are
recognized as peptide fingerprints for the classification of epithelial
cells and are implicated in cytoplasmic organization and cellular
communication. In embryonic kidneys, Krt8 and Krt18 are
specifically expressed in the ureteric epithelium, whereas Krt23 is
specifically expressed in the UB tips.
We validated the microarray results by quantitative real-time PCR
(qPCR) and in situ hybridization (ISH) of known developmental
regulators in the E13.5 kidney. No change was observed in the
mRNA expression of Bmyc, cRet, Wnt11, Etv4, Emx2 and Six2
between UBHdac1,2−/− and wild-type kidneys (note that Six2 was
downregulated later at P0; Fig. 1). By contrast, Axin2, Shh, Tcf7,
Wnt4 and Lef1 were downregulated, and the stromal gene Meis1 was
upregulated, confirming our microarray results (Fig. 5D and Fig. 6).
It should be noted that qPCR and especially ISH demonstrated a
significant decrease of Wnt7b and Wnt9b, whereas the microarray
failed to detect this decrease. This is likely to be a sensitivity issue:
microarray of whole kidney RNA is not a sensitive approach to
detect focal changes in gene expression in the ureteric epithelium.
β-catenin is downregulated in the UB cells of UBHdac1,2−/−
kidneys
β-catenin is a multifunctional protein that plays a crucial role in UB
branching morphogenesis as well as nephrogenesis. One role of
β-catenin is to coordinate cell-cell adhesion through the formation
DEVELOPMENT
Fig. 3. Defective UB branching morphogenesis in UBHdac1,2−/− kidneys.
(A) Heterozygous (HET; UBHdac1−/−,2+/− or UBHdac1+/−,2−/−) and UBHdac1,2−/−
kidneys from the same litters were dissected at E11.5, E12.5 and E13.5, and
visualized by Hoxb7-driven GFP fluorescence. Attenuated branching is seen
in UBHdac1,2−/− from E13.5. (B) The kidneys were isolated at E12.5 and cultured
in vitro for up to 72 h. The UBHdac1,2−/− tissue shows evidence of degeneration
after 24-48 h in culture.
RESEARCH ARTICLE
Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
of adherens junctions with E-cadherin and α-catenin. Another
function of β-catenin is to regulate gene transcription, primarily
through interactions with the Tcf/Lef family of transcription factors.
This function is under the control of the canonical Wnt signaling
pathway. Either deletion or overexpression of β-catenin in UB cells
leads to kidney hypoplasia (Bridgewater et al., 2008, 2011; Marose
et al., 2008). Hdac1 and Hdac2 have been reported to regulate
β-catenin in epidermal progenitor cells and oligodendrocytes. In
epidermal progenitor cells, Hdac1 and Hdac2 are required for the
elevation of β-catenin during hair follicle fate acquisition.
Epidermis that is deficient of Hdac1 and Hdac2 displays uniform,
low-level expression of β-catenin (LeBoeuf et al., 2010).
Conversely, deletion of Hdac1 and Hdac2 in oligodendrocytes
results in the stabilization and nuclear translocation of β-catenin,
leading to activation of the canonical Wnt/β-catenin pathway (Ye
et al., 2009). Our immunofluorescence results showed that β-catenin
is dramatically decreased in the UB cells of UBHdac1,2−/− kidneys at
E13.5 and thereafter (Fig. 7). These data are consistent with our
observation made above that Wnt signaling is repressed in
UBHdac1,2−/− kidneys.
To exclude the possibility that we missed an initial activation of
Wnt signaling upon loss of Hdac1 and Hdac2 in our animal model,
we examined the acute response of cultured embryonic kidneys
(E13.5) to HDAC inhibitor, using Axin2, a direct target of canonical
Wnt signaling, as a read-out. As indicated by ISH, Axin2 is
expressed at a high level in the UB cells and at a low level in the
surrounding mesenchymal cells (Fig. 8A). As expected, Axin2 is
rapidly induced by the GSK3β inhibitor LiCl, which stabilizes
β-catenin and thus activates the canonical Wnt signaling pathway
(Fig. 8A). We found that a 6-h treatment with HDAC inhibitor
substantially decreases the mRNA level of Axin2 with or without
LiCl (Fig. 8B,C). By contrast, expression of cRet is not altered
(Fig. 8D). These results suggest that the repression of canonical Wnt
signaling in the UB cells of UBHdac1,2−/− kidneys is likely to be a
direct rather than secondary effect. As discussed below and shown
in supplementary material Table S5, several genes in the Wnt
pathway are regulated by Hdac1 and Hdac2 and are also directly
bound by p53 in the developing kidney, linking HDAC-Wnt to p53.
Deletion of Hdac1 and Hdac2 in the ureteric cells leads to
p53 hyperacetylation
At least two studies have shown that Hdac1 and Hdac2 are
responsible for deacetylation of p53 (Trp53) in epidermal
progenitor cells (LeBoeuf et al., 2010) and oocytes (Ma et al.,
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DEVELOPMENT
Fig. 4. Conditional deletion of Hdac1 and Hdac2 increases
cell apoptosis associated with DNA damage and
decreases cell proliferation in the UB lineage. (A-D)
Apoptotic cells were identified using anti-active caspase 3
(aCasp3) antibody. (E) Quantitative analysis of UB cell
apoptosis as a percentage of the total number of UB cells.
There is a ∼7-fold elevation in the level of UB cell apoptosis in
UBHdac1,2−/− compared with wild-type kidneys: WT
0.54±0.21% versus UBHdac1,2−/− 3.93±0.77% apoptotic
ureteric cells; n=5, *P=0.0029 (t-test), error bars indicate
s.e.m. Note that the baseline of apoptosis in wild-type kidneys
is very low. (F-I) Representative images showing the induction
of DNA damage ( phospho-histone γH2AX immunostaining) in
UB cells of UBHdac1,2−/− kidneys. (J-M) Proliferating cells were
identified using an anti-phospho-histone H3 ( pH3) antibody.
(N) Quantitative analysis showing that the UB cell proliferation
rate, quantitated as the percentage of pH3-positive among
total UB cells, was decreased by ∼37.5% in UBHdac1,2−/−
kidneys: WT 6.78±1.00% versus UBHdac1,2−/− 4.24±0.31%
proliferating ureteric cells; n=4, *P=0.0047 (t-test), error bars
indicate s.e.m.
Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
Fig. 5. Aberrant gene expression in UBHdac1,2−/− kidneys. (A) Microarray analysis of gene expression in UBHdac1,2−/− kidneys at E13.5. A small subset of
genes (1.2% of 41,000 probes) are significantly altered (>1.4-fold; P<0.05, n=4 experiments) in UBHdac1,2−/− kidneys. The heat maps display gene expression by
log2-transformed fold change (black=0); green indicates lower expression, red indicates higher expression. (B) Molecular functions analysis was performed
with IPA. Significantly (P<0.05) enriched biological processes are shown. (C) IPA depicts the most affected canonical pathways in UBHdac1,2−/− kidneys. Numbers
above bars indicate number of altered genes. (D) Validation of microarray results by real-time PCR. Data are presented as mean±s.e., n≥3, *P<0.05 (t-test).
2012). Acetylation is a key determinant of p53 function by increasing
protein stability, DNA binding affinity and transcriptional activity
(Brooks and Gu, 2011; Meek and Anderson, 2009). Previous studies
in our laboratory demonstrated that p53 is a key renal development
regulator that controls cell proliferation, differentiation and
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apoptosis pathways (Hilliard et al., 2011, 2014; Saifudeen et al.,
2002, 2009). Gain-of-function experiments showed that excessive
p53 levels mediated by deletion of Mdm2 in the UB or MM led to
bilateral renal dysplasia, which could be rescued by concurrent
removal of p53 (Hilliard et al., 2011, 2014). We therefore asked
DEVELOPMENT
RESEARCH ARTICLE
Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
Table 1. Selected functional categories of genes downregulated in
UBHdac1,2–/– kidneys
GO ID
P-value
Description
Genes in test set
35295
3.91E–09
Tube
development
Gsta3, Bmp2,
Foxa1, Gja1, Hp,
Calb1, Shh,
Epcam, Pgr,
Wnt4, Arg2,
Tgm2, Ptch1,
Hhip, Adamts2
Wnt2, Wnt10a,
Fzd10, Wnt4,
Tcf7, Lef1, Frzb,
Axin2, Apcdd1,
Shh
Epcam, Gsta3,
Wnt4, Bmp2,
Arg2, Calb1, Shh
Mia, Amigo2, Pvrl4,
Cdh16, Cldn3,
Pkhd1, Cldn1,
Lef1, Thy1
Col4a4, Wnt4,
Pkhd1, Shh
Wnt2, Cyp7b1,
Fosl2, Btc, Tgm2,
Avpr1a, Shh
16055
1.82E–08
Wnt receptor
signaling
pathway
1657
2.65E–07
Ureteric bud
development
16337
1.34E–06
Cell-cell adhesion
1822
1.23E–02
8284
1.05E–02
Kidney
development
Positive
regulation of
cell proliferation
whether Hdac1 and Hdac2 modulate p53 acetylation as well as its
function in UB cells. Immunofluorescence revealed marked
increases in the levels of p53 acetylated at lysine 370, 379 and
383 (corresponding to lysine 373, 382 and 386 in human,
Table 2. Selected mRNA transcripts decreased in UBHdac1,2–/– kidneys
Probe set ID
Gene
symbol
A_52_P49014
A_51_P287100
A_51_P417643
A_51_P335801
A_51_P171616
Shh
Cdh16
Foxa1
Calb1
Wnt10a
A_52_P377941
Pkhd1
A_52_P61864
Wnt2a
A_52_P244702
Tcf7
A_51_P501145
Lef1
A_52_P54176
Axin2
A_51_P130475
Wnt4
A_51_P312348
A_51_P242399
A_51_P324814
A_51_P356642
A_51_P287198
Krt7
Krt8
Krt18
Krt19
Krt23
Gene name
Sonic hedgehog
Cadherin 16
Forkhead box A1
Calbindin 1
Wingless-type
MMTV
integration
site 10a
Polycystic kidney
and hepatic
disease 1
Wingless-type
MMTV
integration
site 2a
Transcription
factor 7
Lymphoid
enhancer
binding factor 1
Axis inhibition
protein 2
Wingless-type
MMTV
integration site 4
Keratin 7
Keratin 8
Keratin 18
Keratin 19
Keratin 23
Fold
change
P-value
–2.90
–2.82
–1.84
–1.73
–1.66
1.24E–05
2.18E–07
9.52E–04
6.72E–06
3.68E–05
–1.59
2.82E–03
–1.44
2.56E–03
–1.43
6.24E–03
–1.43
3.77E–03
–1.42
1.79E–04
–1.41
2.70E–03
–2.19
–2.02
–2.20
–1.84
–1.55
5.26E–08
4.68E–07
3.33E–06
1.80E–03
1.79E–03
respectively) in the ureteric epithelium of UBHdac1,2−/− kidneys at
E13.5 (Fig. 9A-D). As anticipated, we also observed an
accumulation of total p53 protein in the ureteric epithelium
(Fig. 9E,F).
Germline deletion of p53 partially rescues kidney
development in UBHdac1,2−/− mice
Considering the observed upregulation of p53 in Hdac1/Hdac2deficient UB cells, we hypothesized that p53 mediates the renal
defects observed in UBHdac1,2−/− mice, and employed a genetic
rescue approach that involved crossing the Hoxb7-Cre tg/+;
Hdac1flox/flox; Hdac2flox/flox mice to p53−/− mice. Gross and
histological analyses of the kidney at P0 revealed that removal of
p53 on the UBHdac1,2−/− background partially rescued kidney
development in a gene dosage-dependent manner (Fig. 10).
Remarkably, Mendelian proportions of UBHdac1,2−/−; p53 +/− and
UBHdac1,2−/−; p53 −/− mice were retrieved at P30, indicating that
elimination of one allele of p53 is sufficient to rescue the early
postnatal lethality of UBHdac1,2−/− mice (Table 3). Thus, although a
proportion of UBHdac1,2−/−; p53 +/– and UBHdac1,2−/−; p53 –/– mice
exhibited no overt histological rescue, a measurable improvement in
renal survival must occur in these mice that allows their survival for
at least 30 days. Remarkably, two of the 11 UBHdac1,2−/−; p53 +/−
mice were still alive and fertile at 13 months of age and their kidneys
exhibited relatively normal histology (supplementary material
Fig. S3A-D). These results suggest that hyperacetylation of p53
upon loss of Hdac1 and Hdac2 is one of the important mediators of
the congenital renal dysgenesis observed in UBHdac1,2−/− mice.
To begin to identify the potential targets of p53 leading to the
renal dysgenesis, we cross-referenced our UBHdac1,2−/− microarray
data with p53 ChIP-seq in E15.5 mouse kidneys (Li et al., 2013).
Among the 496 altered genes in E13.5 UBHdac1,2−/− kidneys, 121
are bound by p53 in their promoter regions (supplementary material
Table S3). The Hdac1/2-regulated and p53-bound genes include
Lef1, Axin2, Tcf7 (Wnt signaling), Ptch1 (Shh signaling), several
pro-apoptosis and cell cycle regulatory genes, kinesin family
members (important for ciliary function), calbindin and keratin
genes. IPA revealed that the top five molecular and cellular
functions regulated by these genes are: (1) cell death and survival;
(2) molecular transport; (3) cellular growth and proliferation; (4)
cellular development and (5) cell cycle (supplementary material
Table S4); and the top five affected canonical pathways are: (1) Vdr/
Rxr activation; (2) neuroprotective role of Thop1 in Alzheimer’s
disease; (3) Wnt/β-catenin signaling; (4) protein kinase A signaling
and (5) sonic hedgehog signaling (supplementary material Table S5
and Fig. S4). Hypergeometric distribution analysis demonstrated
that cell death and survival genes are highly over-represented (51 of
121 genes, P=0.00037).
Based on the above analyses, we further examined how deletion
of p53 affected UB cell survival in UBHdac1,2−/− mice.
Immunostaining results demonstrated that UBHdac1,2−/−; p53 –/–
mice had a similar increase in γH2AX-positive UB cells, whereas
three of four UBHdac1,2−/−; p53 –/– kidneys showed fewer aCasp3positive UB cells, when compared with UBHdac1,2−/− kidneys at
E14.5 (4.73±0.47% UBHdac1,2−/− versus 2.73±0.35% UBHdac1,2−/−;
p53 –/– apoptotic ureteric cells, P=0.0249) (Fig. 10C,D).
Direct effects of HDAC inhibition on cell growth and survival
To test more directly the cell-autonomous role of p53
hyperacetylation upon loss of HDAC activities, we treated
HCT116 p53 (TP53)+/+ and p53−/− isogenic human colon cancer
cells with the class I-specific HDAC inhibitor MS-275, which
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deficient for Hdac1 and Hdac2 (Wilting et al., 2010). By contrast,
p53 –/– cells underwent significantly less apoptosis than p53+/+ cells
(3.52% versus 6.18%, respectively) after a 48-h treatment with
10 µM MS-275 (supplementary material Fig. S5C,E), indicating
that MS-275 induces apoptosis partially through p53 in HCT116
cells. Western blot analyses confirmed that MS-275 effectively
increased the level of acetylated histone H3 and p53 in p53+/+ cells
and the level of acetylated histone H3 in p53 –/– cells (supplementary
material Fig. S5F). Moreover, the total level of p53 is also increased
in p53+/+ cells (supplementary material Fig. S5F). Cleavage of poly
(ADP-ribose) polymerase 1 (PARP1) by caspases is a hallmark of
apoptosis; it promotes apoptosis by preventing DNA repair under
normal conditions or stress. When we compared the HCT116 cell
lines, we identified a strong induction of cleaved PARP1 in p53+/+
cells but a much weaker induction in p53 –/– cells (supplementary
material Fig. S5F). These findings are consistent with a recent report
from Sonnemann et al. (2014), showing that HDAC inhibition in
HCT116 cells is partially dependent on p53.
DISCUSSION
This study investigated the functions of Hdac1 and Hdac2 in the
ureteric epithelium during kidney development. Our results
demonstrate that: (1) Hdac1 and Hdac2 perform redundant yet
essential functions in UB branching morphogenesis; (2) Hdac1 and
Hdac2 are required for the canonical Wnt signaling pathway in
kidney development; and (3) Hdac1 and Hdac2 are required to
suppress hyperacetylation of p53 in the ureteric epithelium, which
partially accounts for the UBHdac1,2−/− phenotype.
Fig. 6. In situ hybridization of renal developmental regulators in UB cells
at E13.5. (A-D) The expression of cRet, which encodes a tyrosine kinase
receptor for Gdnf, and its downstream target Wnt11 is unaltered in UBHdac1,2−/−
kidneys. (E-J) Axin2, Wnt9b and Wnt7b are dramatically reduced in UBHdac1,2−/−
kidneys. (K,L) Expression of Emx2 is maintained in the UB epithelium in
UBHdac1,2−/− kidneys.
preferentially inhibits HDAC1, 2 and 3. Light microscopy analysis
revealed that MS-275 induces notable growth arrest and cell death in
both p53+/+ and p53−/− cells, although p53−/− cells are clearly more
resistant to MS-275 than p53+/+ cells (supplementary material
Fig. S5A).
Next, we quantified the status of the cell cycle and of apoptosis
using flow cytometry. We found that the anti-proliferative effects of
MS-275 are not dependent on p53 function in HCT116 cells. MS275 induced G1 and G2 phase arrest in both cell lines, with a
decrease in S-phase cells to similar degrees (supplementary material
Fig. S5B,D and Table S6), consistent with a previous report that p53
is dispensable for cell cycle arrest in mouse embryonic fibroblasts
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Kidney development is dependent on reciprocal epithelialmesenchymal interactions between the UB and the MM, which
drive iterative branching of the UB and subsequent induction of
nephrons. Our previous studies showed that pharmacological
inhibition of class I and II HDACs by Scriptaid, or of Hdac1, 2
and 3 by MS-275, causes disruptions of the reciprocal signaling
pathways, leading to growth arrest and apoptosis. As many HDAC
isoforms (e.g. Hdac1, 2 and 3) are expressed in the UB and MM, it
was important to determine the distinct functions of individual
enzymes in specific cell lineages. In this study, we set out to
elucidate the developmental roles of Hdac1 and Hdac2 in the
ureteric epithelium using a conditional knockout strategy. Although
global deletion of either Hdac1 or Hdac2 leads to a lethal phenotype
(Lagger et al., 2002; Montgomery et al., 2007), demonstrating the
unique roles of these two enzymes in early embryogenesis,
redundancy of Hdac1 and Hdac2 function has been found in
many somatic cell types (e.g. oligodendrocytes, Schwann cells,
cardiomyocytes, basal cells and adipocytes) (Haberland et al., 2010;
Jacob et al., 2011; LeBoeuf et al., 2010; Montgomery et al., 2007;
Ye et al., 2009). In line with the previous studies, our results reveal
that one allele of either Hdac1 or Hdac2 is sufficient to support
normal kidney development, whereas loss of all four alleles leads to
renal hypodysplasia and early postnatal lethality.
Hdac1 and Hdac2 are required for the canonical Wnt
signaling pathway in kidney development
Genome-wide profiling revealed that changes in gene expression are
highly specific in UBHdac1,2−/− mice at E13.5. Only 226 transcripts
(0.55%) were upregulated and 270 transcripts (0.66%) were
downregulated (using a cut-off of 1.4-fold, P<0.05). This is
consistent with the increasing evidence showing that Hdac1 and
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Hdac1 and Hdac2 perform redundant yet essential functions
in the UB lineage
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Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
Hdac2 selectively control specific gene expression programs in
different tissues. For example, deletion of Hdac1 and Hdac2 in the
heart resulted in dysregulation of only ∼1.6% of the transcriptome
(Montgomery et al., 2007). Given the apparent cell apoptosis and
growth arrest observed, it is not surprising that genes regulating cell
death and survival and cellular growth and proliferation are among
the most notably dysregulated functional categories. More
interestingly, we found that the canonical Wnt signaling pathway
is among the most affected pathways in UBHdac1,2−/− kidneys.
The canonical Wnt signaling pathway controls both UB
branching morphogenesis and nephrogenesis. Specifically, Wnt7b
is expressed in the UB trunk and is essential for the establishment of
a cortico-medullary axis during later stages of kidney development.
In Wnt7b mutant mice, cortical epithelial development is normal but
the medullary zone fails to form (Yu et al., 2009). Wnt9b, another
member of the Wnt family secreted by the UB trunk epithelium,
plays an indispensable role in the activation of Wnt4, which is both
necessary and sufficient for the induction of renal vesicles from
renal progenitor cells. Wnt9b is also important for the proliferation/
renewal of the renal progenitor cells. Genetic deletion of Wnt9b in
mice results in failure of nephron induction and premature
exhaustion of the progenitor pool (Carroll et al., 2005; Karner
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Fig. 7. β-catenin is downregulated in UBHdac1,2−/− kidneys. Immunofluorescence shows that β-catenin is initially expressed at normal levels in UBHdac1,2−/−
kidneys at E12.5 (A-F), but is decreased at E13.5 (G-L) and E14.5 (M-T). At E14.5, calbindin staining is too weak to label the UB cells in UBHdac1,2−/− kidneys
and Hdac2 staining is used instead.
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Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
et al., 2011). Our results show significant decreases in Wnt7b,
Wnt9b and Wnt4 expression in UBHdac1,2−/− kidneys at E13.5,
associated with the downregulation of a number of Wnt target
genes, such as Axin2, Tcf7 and Lef1. Consistent with these findings,
immunofluorescence results demonstrate that membrane-associated
β-catenin is greatly decreased in the UB cells of UBHdac1,2−/−
kidneys. β-catenin exists in the cell in two pools: membrane
associated and cytosolic. On the membrane, as a component of
adherens junctions (AJs), β-catenin links cadherins to the
cytoskeleton, whereas in the cytoplasm β-catenin is an essential
mediator of the Wnt signaling pathway (Perez-Moreno and Fuchs,
2006). Although the interplay between the two pools remains to be
fully elucidated, time-lapse microscopy using photoactivatable
GFP-tagged β-catenin has revealed that the membrane-associated
pool of β-catenin can be internalized together with E-cadherin,
Fig. 9. Deletion of Hdac1 and Hdac2 in UB cells causes p53
hyperacetylation and accumulation. (A-D) Immunofluorescence detection
of p53 acetylation at lysine 370, 379 and 383 in wild-type and UBHdac1,2−/−
kidneys at E13.5. UBHdac1,2−/− kidneys display strong staining for lysine 370,
379 (B) and 383 (D) p53 acetylation specifically in UB cells. (E,F) Detection of
p53 by DAB staining reveals elevated p53 expression in UB cells in
UBHdac1,2−/− kidneys.
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accumulates at the perinuclear endocytic recycling compartment
(ERC) upon AJ dissociation, and can be translocated into the
nucleus upon Wnt pathway activation (Kam and Quaranta, 2009).
Therefore, it is conceivable that decreased levels of membraneassociated β-catenin reduce the availability of cytosolic β-catenin
and thus lead to the decrease in Wnt signaling in UBHdac1,2−/−
kidneys. Moreover, we observed that a 6-h treatment of cultured
embryonic kidney with HDAC inhibitor leads to a prompt reduction
of Axin2, a direct target of Wnt, supporting the idea that Hdac1 and
Hdac2 function in the activation of Wnt signaling in the embryonic
kidney.
It is also of note that Shh, a hedgehog homolog, is one of the most
suppressed genes in UBHdac1,2−/− kidneys. During kidney
development, Shh is initially expressed in the distal part of the UB,
and then in the urothelium. Shh acts as a paracrine signal to promote
mesenchymal cell proliferation, and regulates the pattern of
mesenchymal differentiation. Conditional deletion of Shh in the UB
results in renal hypoplasia, hydronephrosis and hydroureter in
newborn pups (Yu et al., 2002). However, such a phenotype is not
observed in UBHdac1,2−/− mice. It is likely to be masked by the more
severe dysplastic phenotype of UBHdac1,2−/− kidneys, or the residual
level of Shh is still sufficient to support mesenchymal cell proliferation
and differentiation. Taken together, our results demonstrate that Hdac1
and Hdac2 play a crucial role in maintaining essential ureteric genes
for renal growth and differentiation.
It should be noted that, although it might be difficult to
completely dissociate the direct effects of HDAC inactivation
from the secondary effects of cell loss due to apoptosis, there are
several clues that support the notion that many of the gene
expression changes are specific. First, ISH and microarrays were
performed at E13.5, prior to the observed degeneration of the UB
tree. Several genes, such as Tcf7, Wnt7b, Wnt9b, Axin2 and Shh,
were specifically altered. Second, if the effects of Hdac1/2
inactivation on gene expression were generalized or non-specific
due to cell death, we would have expected a much more substantial
change in the number of genes altered, as opposed to the few percent
of differentially expressed genes observed in the study. Third, our
results cannot be fully explained by excessive cell death of UB tip
cells since the expression of cRet, Wnt11, Bmyc and Etv4, four
specific markers of UB tips, is unaltered in UBHdac1,2−/− mice.
Hdac1 and Hdac2 are required to suppress hyperacetylation
of p53 in the ureteric epithelium
Although lysine acetylation was originally discovered in histones,
these are not the only proteins that can be acetylated. p53 was the
first non-histone protein found to be regulated by acetylation (Gu
and Roeder, 1997; Luo et al., 2000). The acetylation of multiple
lysine residues in the C-terminus (K305, K370, K372, K373,
DEVELOPMENT
Fig. 8. Inhibition of HDAC activity in cultured metanephroi suppresses Axin2. Paired E13.5 metanephroi were cultured in the presence of 15 mM LiCl or
vehicle control (PBS) (A), 2 µg/ml Scriptaid (HDACi) or Nullscript control (B,D), or in the presence of 15 mM LiCl with or without 2 µg/ml Scriptaid (HDACi) (C) for
6 h. Expression of Axin2 (A-C) and cRet (D) was evaluated by whole-mount ISH. The HDAC inhibitor substantially decreases the Axin2 mRNA level, with or
without LiCl, whereas cRet is unaffected.
Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
Fig. 10. Elimination of p53 partially rescues kidney development in UBHdac1,2−/− mice. (A) Representative images of kidneys from newborn mice of the
indicated genotypes. Whole-mount (top), transverse sections (middle) and higher magnification (40x, bottom) are shown. (B) Quantification of the renal
phenotype of mice of the indicated genotypes, as illustrated in A. The degree of morphological rescue was judged by microscopy and classified as:
significant rescue, where kidneys are notably larger and form fewer cysts than UBHdac1,2−/−; p53 +/+; moderate rescue, where kidneys (either unilateral or
bilateral) are not notably larger but form fewer cysts; no overt rescue, where there is no obvious difference to UBHdac1,2−/−; p53 +/+ kidneys. (C) Quantitative
analysis of UB cell apoptosis (aCasp3 immunostaining) as a percentage of total UB cells indicates that there are fewer apoptotic cells upon p53 deletion
from UBHdac1,2−/−. (D) By contrast, UBHdac1,2−/−; p53 –/– mice show a similar elevation in γH2AX-positive UB cells to UBHdac1,2−/− mice. (C,D) Error bars denote
mean±s.e.m.
K381, K382 and K386) and DNA-binding domain (K164) of
human p53 is significantly enhanced in response to stress
and required for its stabilization, transcriptional activation and
transcription-independent function in apoptosis (Brooks and Gu,
2011). p53 in which all eight lysine residues are substituted (8KR)
loses the ability to mediate cell cycle arrest and apoptosis (Tang
et al., 2008). There is also evidence that HDAC1 is recruited to p53
by an MDM2-containing protein complex, and HDAC1-mediated
deacetylation of p53 is required for degradation of p53 (Ito et al.,
2002).
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Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
Genotype
Observed/total
number of mice
Predicted/total
number of mice
UBHdac1,2−/−; p53 +/+
UBHdac1,2−/−; p53 +/–
UBHdac1,2−/−; p53 –/–
1/81*
11/81
5/81
5/81
10/81
5/81
The genotypes shown were generated by initial crossing of p53 –/–; Hdac1flox/flox;
Hdac2flox/flox and Hoxb7-CreEGFP mice followed by cross-breeding of offspring.
UBHdac1,2−/− refers to Hoxb7-Cre tg/+; Hdac1flox/flox; Hdac2flox/flox. The numbers of
living mice of each genotype were counted at P30. Chi-square one-tailed
analysis was used to compare the observed values with those predicted by
Mendelian inheritance patterns. UBHdac1,2−/−; p53 +/+ mice show postnatal
lethality (P=0.048 observed versus expected), whereas UBHdac1,2−/−; p53 +/–
(P=0.408 observed versus expected) and UBHdac1,2−/−; p53–/– (P=0.500
observed versus expected) mice were rescued from postnatal lethality.
*Note that the one UBHdac1,2−/−; p53 +/+ mouse observed at P30 was smaller and
weak compared with its littermates, and later died at the age of 3 months.
Consistent with two recent studies showing that double knockout
of Hdac1 and Hdac2 enhances p53 acetylation in mice epidermal
progenitor cells and oocytes, our results demonstrated that loss of
Hdac1 and Hdac2 in the ureteric epithelium leads to marked
hyperacetylation and accumulation of p53. However, to our
knowledge, the present study is the first to show rescue of
organogenesis, albeit partial, upon concomitant germline deletion
of p53 in vivo. This finding is in line with our previous report that
excessive p53 accumulation in the UB or renal progenitor cells
(mediated by deletion of Mdm2) causes bilateral renal dysplasia,
while concomitant elimination of p53 rescues the renal phenotype
and animal survival. With regards to the underlying cellular
mechanisms, our immunostaining results demonstrate that p53 is
partially responsible for UB cell apoptosis in UBHdac1,2−/− kidneys
at E14.5. In support of these results, bioinformatics analysis
revealed that 121/496 dysregulated genes in UBHdac1,2−/− kidneys
are bound by p53 in their promoter regions and are thereby
potentially regulated by p53. Moreover, over 40% of these genes are
highly enriched in the category of cell death and survival. As to
the apparent paradox of longer survival in the triple UBHdac1,2−/−;
p53−/− null mutants yet no significant morphological rescue, we
suggest that ‘histological’ and ‘functional’ rescues might not be
identical in this setting. Loss of p53 might have improved functional
but not structural variables, e.g. glomerular filtration or tubular
transport functions. This might account for the overall longer
survival of UBHdac1,2−/−; p53+/− or UBHdac1,2−/−; p53−/− mice,
which did not show structural rescue.
Together, these results suggest that Hdac1 and Hdac2 are required
to suppress the hyperacetylation and activation of p53 in the UB
lineage, thus protecting cells from apoptosis in renal development.
Further studies using p53 lysine-specific mutant mice are needed to
address the question of whether the pro-apoptotic function of p53
is dependent on its acetylation during kidney development. In
summary, our data reveal that Hdac1 and Hdac2 play an essential
role in cell proliferation, survival and transcriptional regulation in
the ureteric epithelium during kidney development, and indicate
that this occurs, in part, through p53-mediated pathways.
MATERIALS AND METHODS
and Hdac2 genes, singly or in combination, specifically in the UB
epithelium. For the rescue experiment, p53 –/– mice (Jackson Laboratory)
were first crossed to the Hdac1flox/flox; Hdac2flox/flox mice, and then further
crossed to Hoxb7-CreEGFP transgenic mice to remove p53 on the
UBHdac1,2−/− background.
Histology and immunohistochemistry
Kidneys were fixed in 10% buffered formalin, embedded in paraffin, and
sectioned at 4 μm. Histology analyses were performed by standard periodic
acid-Schiff (PAS) staining and Hematoxylin and Eosin (H&E) staining.
Immunofluorescence was performed as previously described (Chen et al.,
2011). Primary antibodies and working concentrations are listed in
supplementary material Table S7. The peroxidase-based Vectastain ABC
Elite Kit (Vector Laboratories) was used for DAB detection.
Organ culture
Embryonic kidneys were aseptically micro-dissected from timed-pregnant
mice and cultured on polycarbonate Transwell filters (0.4 μm pore size,
Corning Costar) over medium [DMEM/F-12 containing 10% fetal bovine
serum (FBS)] at 37°C and 5% CO2.
RNA extraction
Kidneys were harvested at E13.5 and stored in RNAlater RNA stabilization
reagent (Qiagen). Wild-type and UBHdac1,2−/− kidneys were each divided
into four random pools (n=4). Total RNA was then isolated using the
RNeasy Mini Kit (Qiagen).
Genome-wide microarray analysis
Microarray analysis was performed according to established protocols
(Schanstra et al., 2007). Briefly, fluorescently labeled cRNA was generated
from 0.5 μg total RNA in each reaction using the Agilent Fluorescent Direct
Label Kit and 1.0 mM Cyanine 3′- or 5′-labeled dCTP (PerkinElmer).
Hybridization was performed using the Oligonucleotide Microarray
Hybridization and In Situ Hybridization Plus Kit (Agilent). The labeled
cRNA was hybridized to Agilent 44K whole mouse genome
oligonucleotide microarray (containing ∼41,000 probes) as previously
described (Schanstra et al., 2007). The arrays were scanned using a duallaser DNA microarray scanner (Agilent). The data were then extracted from
images using Feature Extraction software 6.1 (Agilent). Microarray data are
available at GEO under accession number GSE35432.
Data analysis
MultiExperiment Viewer v4.9 software was used to generate lists of
genes differentially expressed between wild-type and UBHdac1,2−/−
kidneys, using P≤0.05 and a minimum 1.4-fold change in gene
expression. Genes were classified according to their function using IPA
software and BiNGO classification systems as previously described
(Chen et al., 2011).
qPCR
Quantitative real-time PCR (qPCR) was performed using the One-Step
Brilliant Quantitative RT-PCR Master Mix Kit (Applied Biosystems). Realtime PCR reaction mix contained 200 nm forward primer, 200 nm reverse
primer, and 60 ng total RNA. Relative levels of mRNA were normalized to
Gapdh. The primers used for qPCR are listed in supplementary material
Table S8.
Whole-mount in situ hybridization (ISH)
ISH was performed using digoxigenin-labeled antisense probes on kidney
tissue fixed with 4% paraformaldehyde as previously described (Chen et al.,
2011).
Mice
Mice bearing conditional null alleles of Hdac1 and Hdac2 (Hdac1flox/flox
and Hdac2flox/flox) were obtained from the laboratory of Eric Olson
(Montgomery et al., 2007) and were crossed to Hoxb7-CreEGFP
transgenic mice (Hoxb7-Cretg/+) (Zhao et al., 2004) to delete the Hdac1
1190
Cell culture and treatments
HCT116 p53+/+ and p53–/– cells were obtained from Dr Hua Lu (Tulane
University, New Orleans, LA, USA, and Johns Hopkins University Cell
Center, Baltimore, MD, USA). Cells were grown in high-glucose DMEM with
DEVELOPMENT
Table 3. One-month (P30) survival rate of Hdac1,2 mutant offspring
carrying no, one or two p53 mutant alleles
Development (2015) 142, 1180-1192 doi:10.1242/dev.113506
stable glutamine supplemented with 10% FBS and 10 mg/ml antibiotics
(penicillin and streptomycin), under 5% CO2 and saturated moisture. Cells
were treated with vehicle control DMSO or 10 µM MS-275 for 24, 48 or 72 h.
Analysis of cell cycle and apoptosis using flow cytometry
Cells were treated with vehicle control DMSO or 10 µM MS-275 for 48 h.
At the time of harvesting, cells were digested with 0.05% trypsin and
resuspended in phosphate-buffered saline (PBS). For cell cycle analysis,
2×105 cells were stained with 50 µg/ml propidium iodide (PI) using the
Coulter DNA Prep Reagents Kit (Beckman Coulter). For evaluation of the
early stages of apoptosis, 2×105 cells were stained with Annexin V-FITC
and PI using the Annexin V-FITC Apoptosis Detection Kit (Sigma). Cells
were then analyzed using a Beckman Coulter FC500 flow cytometer and
CXP software (Beckman Coulter) for acquisition and analysis. The cell
cycle distribution was further analyzed by ModFit LT 3.0 software (Verity
Software House).
Western blot
Whole-cell proteins were extracted using RIPA buffer (Invitrogen) and
nuclear proteins were extracted using the Nuclear Extract Kit (Active
Motif ). Primary antibodies and working concentrations are listed in
supplementary material Table S7.
Acknowledgements
flox/flox
flox/flox
We thank Dr Eric Olson for the Hdac1
and Hdac2
mice; Dr Carlton Bates
for the Hoxb7-CreEGFP transgenic mice; and Dr Hua Lu for providing HCT116 cell
lines. We acknowledge the support of the Tulane Renal and Hypertension Center of
Excellence, Center for Stem Cell Research & Regenerative Medicine and the Center
for Gene Therapy.
Competing interests
The authors declare no competing or financial interests.
Author contributions
S.S.E. conceived the project, supervised the studies, analyzed the data, read, edited
and approved the manuscript; S.C. and X.Y. performed the majority of the
experiments; D.B. performed the microarray experiments; S.C. and Y.L. performed
microarray data analysis; Z.S. provided p53 knockout mice and analyzed and
discussed the data; S.C. performed experiments, analyzed data and wrote the
manuscript.
Funding
This work was supported by National Institutes of Health grants [R01DK079886,
P50DK096373 and P30GM103337]. Deposited in PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.113506/-/DC1
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