1882 RESEARCH ARTICLE
DEVELOPMENT AND STEM CELLS
Development 140, 1882-1891 (2013) doi:10.1242/dev.089649
© 2013. Published by The Company of Biologists Ltd
Rapid and widespread suppression of self-renewal by
microRNA-203 during epidermal differentiation
Sarah J. Jackson1,*, Zhaojie Zhang1,*, Dejiang Feng1, Meaghan Flagg1, Evan O’Loughlin1, Dongmei Wang1,
Nicole Stokes2, Elaine Fuchs2 and Rui Yi1,‡
SUMMARY
MicroRNAs (miRNAs) play important roles in differentiation of stem cells. However, the precise dynamics of miRNA induction during
stem cell differentiation have not been visualized and molecular mechanisms through which miRNAs execute their function remain
unclear. Using high-resolution in situ hybridization together with cell lineage and proliferation markers in mouse skin, we show that
miR-203 is transcriptionally activated in the differentiating daughter cells upon the asymmetric cell division of interfollicular progenitor
cells. Once induced, miR-203 rapidly promotes the cell cycle exit within 6 hours and abolishes self-renewal of the progenitor cells. With
an inducible mouse model, we identify numerous miR-203 in vivo targets that are highly enriched in regulation of cell cycle and cell
division, as well as in response to DNA damage. Importantly, co-suppression of individual targets, including p63, Skp2 and Msi2 by
miR-203 is required for its function of promoting the cell cycle exit and inhibiting the long-term proliferation. Together, our findings
reveal the rapid and widespread impact of miR-203 on the self-renewal program and provide mechanistic insights into the potent
role of miR-203 during the epidermal differentiation. These results should also contribute to understanding the role of miR-203 in
the development of skin cancer.
INTRODUCTION
Differentiation of somatic stem cells is a complex process that is
orchestrated by multiple mechanisms leading to changes in gene
expression. In turn, these changes at the molecular level drive stem
cells to abandon their state of self-renewal and produce terminally
differentiated cells with specific physiological functions (Blanpain
and Fuchs, 2009; He et al., 2009). Although extensive effort has
been devoted to identify master regulators that directly drive
differentiation programs, little is known about the kinetics of such
transition, e.g. when is a differentiation-related gene induced and
how does its expression compromise self-renewal and initiate
differentiation? Among many regulators, miRNAs are implicated
important roles in stem cell differentiation (Ivey and Srivastava,
2010; Yi and Fuchs, 2011). miRNAs are a family of small,
noncoding RNAs that are widely expressed in most animal species
ranging from sponge to human (Grimson et al., 2008). These tiny
riboregulators function by recruiting the RNA-induced silencing
complex (RISC) to their target mRNAs usually through base-pairing
between 5⬘ end sequences of miRNAs and mRNA sequences
located in the 3⬘ untranslated region (3⬘UTR) (Bartel, 2009). In turn,
miRNAs repress their target expression by inhibiting translation
initiation and destabilizing mRNAs (Bazzini et al., 2012). Despite
the evidence for functional significance of miRNAs in stem cell
differentiation (Melton et al., 2010; Murchison et al., 2005; Yi et
al., 2008), the dynamics of the molecular events that are controlled
by miRNAs remain poorly defined. In particular, the precise timing
1
Department of Molecular, Cellular, and Developmental Biology, University of
Colorado, Boulder, CO 80309, USA. 2Laboratory of Mammalian Cell Biology and
Development, Howard Hughes Medical Institute, The Rockefeller University, New
York, NY 10065, USA.
*These authors contributed equally to this work
Author for correspondence ([email protected])
‡
Accepted 25 February 2013
of miRNA induction during differentiation is not known. Although
many distinct miRNAs have been detected in stem cell lineages,
these results are usually obtained by quantifying miRNAs in
isolated cell populations that lack spatiotemporal resolution to
distinguish the exact expression pattern of miRNAs at the single-cell
level. Furthermore, the underlying mechanism for the role of
miRNAs in differentiation, namely how many targets are regulated
by an miRNA and how the regulation of many targets by an miRNA
contributes to its function, has yet to be elucidated.
In the epidermis of mouse skin, stem/progenitor cells reside in
the basal layer in touch with the basement membrane (Blanpain and
Fuchs, 2009). These progenitor cells divide either horizontally to
give rise to two basal cells (symmetric cell division, SCD) or
perpendicularly to one basal cell and one suprabasal cell
(asymmetric cell division, ACD) (Lechler and Fuchs, 2005). The
basal cell retains the stem cell/progenitor property, whereas the
suprabasal cell embarks upon epidermal differentiation (Lechler and
Fuchs, 2005). Thus, the epidermis is an excellent model for
examining kinetics and mechanisms that underlie differentiation
processes.
We and others have identified miR-203 as a skin-specific miRNA
that plays an important role in the epidermal differentiation both in
vivo and in vitro (Lena et al., 2008; Yi et al., 2008). To distinguish
whether the induction of miR-203 is an early event that drives the
epidermal differentiation, we have developed a high-resolution in
situ hybridization technique and precisely define spatiotemporal
expression patterns of miR-203 together with protein markers for
skin lineages and proliferation during the epidermal differentiation.
We have also established an inducible mouse model that enables us
to control the expression level and timing of miR-203. We show
that miR-203 has an immediate impact on the cell cycle exit and
also abolishes long-term cell proliferation. We further identify a
large number of in vivo mRNA targets of miR-203 that are highly
enriched in the regulation of self-renewal. By enhancing the
expression of the targets of miR-203 (including Skp2, a cell cycle
DEVELOPMENT
KEY WORDS: Epidermal differentiation, miRNA, Self-renewal, Mouse
miR-203 inhibits self-renewal
MATERIALS AND METHODS
Animals
miR-203-inducible mouse was generated by standard pronuclear injection
of pTRE2-miR-203 expression plasmid in a FVB background. This strain
was subsequently bred to K14-rtTA to create the inducible mouse model.
Two independent pTRE2-miR-203/K14-rtTA founder lines were generated
and validated for the experiments. Mice were bred and housed according to
the guidelines of IACUC at a pathogen-free facility at the University of
Colorado (Boulder, CO, USA).
In situ hybridization, Immunofluorescence and antibodies
In situ hybridization of miRNAs was performed as previously described
(Yi et al., 2008) with modifications to signal development. Briefly, double
DIG-labeled miR-203 probe (Exiqon, Denmark) was used for hybridization
at 46°C for 2 hours and the signal was developed with the TSA amplification
systems with FITC-conjugated reagent (PerkinElmer, USA). For co-staining
with other protein markers, the developed in situ slides were treated with
DNase I (25 units/ml; Sigma, USA) for 1 hour at 37°C, then incubated with
primary antibodies against BrdU (1:500; Abcam, USA), Krt5 (1:500;
Covance, USA), PH3 (1:1000; Cell Signaling, USA) or β4 integrin (1:200;
BD Biosciences, USA). Subsequent antibody co-staining was performed as
described previously (Yi et al., 2008).
5⬘ RACE and luciferase assay
5⬘ RACE for miR-203 primary transcripts was carried out with the SMART
mRNA Amplification kit (Clontech, USA) following the manufacturer’s
instruction. Four independent clones were sequenced for the identification
of the TSS of miR-203. The promoter region was amplified with mouse
genomic DNA and cloned into a pGL3-basic vector (Promega, USA). The
luciferase assay was carried out by transfecting 20 ng of the luciferase
reporters as indicated together with 380 ng of an empty Krt14 plasmid as
well as 2 ng of a Renilla luciferase reporter into a 24-well plate. The
luciferase activity was measured 48 hours post-transfection.
For the 3⬘UTR luciferase assay, 3⬘UTR fragments of individual targets
were obtained by PCR amplification (supplementary material Table S2)
from total skin cDNA and cloned into the 3⬘ end of a pGL3-control vector
(Promega, USA). The luciferase assay for wild-type and mutant 3⬘UTRs
was carried out as described previously (Yi et al., 2008).
Microarray and target analyses
Total RNA (500 ng) isolated from the basal epidermis of two pairs of miR203 induced and control mice were used for microarray analysis with Mouse
Genome 430 2.0 array (Affymetrix, USA) following the manufacturer’s
instruction. Microarray data were analyzed with the Bioconductor software
(Gentleman et al., 2004). Probesets that were called ‘present’ and had known
annotations were collected for downstream analysis.
The 5⬘UTR, CDS and 3⬘UTR of all genes annotated in the Knowngene
table of the mouse genome database (mm9) were downloaded from the
UCSC Genome Browser (Kent et al., 2002). The sequences obtained were
used for miR-203 target analysis by searching for perfect matches for 7mer motifs that are derived from mature miR-203 sequences.
Microarray data have been deposited in Gene Expression Omnibus with
accession number GSE45121.
Cell culture and cell cycle analysis
Primary keratinocytes were cultured as previously described (Yi et al.,
2008). Inducible keratinocytes were isolated from pTRE2-miR-203/K14rtTA transgenic mice. miR-203 expression was induced by adding
doxycycline to the media at a concentration of 3 μg/ml. For induction for
longer than 24 hours, fresh doxycycline was added daily. The cell cycle
profile was determined by following the instruction of the BD Pharmingen
BrdU kit.
Characterization of the functions of miR-203 targets
The rescue experiments were conducted using retroviral MIGR constructs
containing cDNA of miR-203 targets [Msi2, p63 (Trp63 – Mouse Genome
Informatics), Skp2 and Vav3], as well as an empty MIGR vector control.
The knockdown of p63, Msi2 and Skp2 was carried out with two shRNA
constructs (Sigma-Aldrich, USA) for each gene with cultured keratinocytes.
Puromycin-resistant cells (3000 cells) were plated for the colony formation
assay.
RNA-Seq and quantification
Total RNA (~0.5-2 μg) isolated from the basal and suprabasal cells was used
for RNA-Seq. The RNA-Seq experiment was performed as previously
described (Wang et al., 2013). The relative expression was calculated by
normalizing to Hprt.
Statistical analysis
P values were calculated using an unpaired t-test using the online calculator
from GraphPad software. Error bars indicate s.d.
RESULTS
miR-203 is inversely correlated with epidermal
proliferation
miR-203 is thought to have an important role during epidermal
differentiation (Yi et al., 2008). However, the precise dynamics of
the upregulation of miR-203 remain unclear. We set out to
determine the correlation between the expression and epidermal
differentiation of miR-203 with high-resolution in situ
hybridization. During mouse embryonic skin development, a highly
proliferative stem/progenitor cell population resides at the basal
layer and gives rise to differentiating daughter cells, collectively
called suprabasal cells (Blanpain and Fuchs, 2009). We first
examined the expression of miR-203 in the developing epidermis by
correlating its in situ signal with the proliferative cells, as marked
by phosphorylated (Ser-10) Histone H3 (PH3) at embryonic day 15
(E15), a period when the basal progenitor cells begin to stratify and
produce the differentiated suprabasal cells (Blanpain and Fuchs,
2009; Lechler and Fuchs, 2005). The signals of miR-203 were
largely restricted to the cytoplasm of the differentiated suprabasal
cells (Fig. 1). The punctate localization of miR-203 signals
throughout the cytoplasm was reminiscent of the established
patterns for the RISC, as determined by immunofluorescence
staining of argonaute proteins (Liu et al., 2005). Furthermore, the
specificity of in situ signals was confirmed by the absence of signals
in the Dicer knockout skin (supplementary material Fig. S1), where
all miRNAs, including miR-203, are absent (Yi et al., 2008). These
observations confirm that our in situ hybridization specifically
detects miR-203 with subcellular resolution. At E15, although most
proliferative cells were already restricted in the basal layer, limited
cellular proliferation did occur at the suprabasal layer immediately
adjacent to the basal layer (Fig. 1A-C). By doing so, it allows rapid
expansion of developing epidermis at this stage (Lechler and Fuchs,
2005). When examined closely, these proliferative cells at the
suprabasal layer expressed a lower level of miR-203 than did the
cells located further away from the basal layer (Fig. 1A-C).
Moreover, the expression of miR-203 appeared to vary among
individual suprabasal cells at this stage, suggesting an ongoing
transition from proliferation to terminal differentiation (Fig. 1A-C).
When examined at later stages, e.g. E17 and postnatal day 6 (P6),
the expression of miR-203 was uniformly high in all suprabasal
layers when all proliferative cells are restricted in the basal layer
(Fig. 1D-E). Together, these results demonstrate that the temporal
DEVELOPMENT
regulator; Msi2, a RNA-binding protein; and p63, a transcription
factor required for skin stem cells) in the presence of miR-203 both
individually and combinatorially, we demonstrate that co-repression
of these targets is required to mediate the widespread inhibition of
self-renewal by this miRNA. Together, our studies provide
mechanistic insights into the activation and function of miR-203
during the epidermal differentiation.
RESEARCH ARTICLE 1883
1884 RESEARCH ARTICLE
Development 140 (9)
induction of miR-203 is inversely correlated with cell proliferation
in the epidermis.
Unlike the interfollicular progenitor cells, which constantly
progress through the cell cycle, hair follicle stem cells are quiescent
(Blanpain et al., 2004; Nowak et al., 2008; Tumbar et al., 2004). To
distinguish whether miR-203 is associated simply with the lack of
proliferation or specifically with the differentiation, we
characterized its expression in hair follicle stem cells. As shown in
Fig. 1F, miR-203 was absent from the hair follicle stem cells that are
attached to the basement membrane marked by β4 integrin but was
highly expressed in the terminally differentiated cells located in the
adjacent companion layer (Hsu et al., 2011). Together, these results
demonstrate that miR-203 is intimately correlated with the
epidermal differentiation in a spatiotemporally specific manner.
miR-203 is transcriptionally activated in
differentiating cells upon asymmetric cell division
When the basal cells divide, they divide either symmetrically or
asymmetrically to balance maintenance and differentiation of the
epidermal lineages (Poulson and Lechler, 2010). To define precisely
the spatial induction pattern of miR-203, we next examined the
expression of miR-203 more closely in postnatal skin. Because
epidermal cell division becomes increasingly rare postnatally, a
relatively long BrdU pulse can label both daughter cells derived
from a single cell division. The two daughter cells should be easily
recognized as dual-labeled cells adjacent to each other, whereas the
neighboring cells are mostly unlabeled because of the overall
scarcity of dividing cells. Indeed, when we pulse P6 animals with
BrdU for 12 hours, we observed many dual-labeled cells in the
epidermis either horizontally (Fig. 2A-C) or perpendicularly
localized (Fig. 2D-F). When we co-stained miR-203 with BrdU as
well as keratin 5 (K5), we found that miR-203 is not expressed in
the daughter cells of the SCD but is robustly induced in the
suprabasal cell of the ACD (Fig. 2A-F). These results provide novel
insights to the precise correlation between the induction of miR203 and the onset of epidermal differentiation. In particular, the
simultaneous detection of miR-203 and dividing cells provides a
high-resolution picture for the rapid induction of miR-203 following
the ACD in the epidermis.
Because the expression of miRNAs can be controlled by
transcriptional regulation (Krol et al., 2010), we characterized the
transcription start site (TSS) and the promoter region of miR-203.
The miR-203 gene is located at an intergenic region on chromosome
12. With 5⬘ RACE, we determined that the TSS of miR-203 is
located ~100 bp upstream of the miR-203 hairpin, which is
immediately downstream of three highly conserved DNA sequences
in vertebrates (Fig. 2G). The conserved DNA fragments are located
within a CpG island characteristic of mammalian promoters. To test
whether the upstream sequences from the TSS are indeed the
promoter of miR-203, we cloned a fragment of ~3 kb DNA
sequences upstream of miR-203 into a promoterless luciferase
reporter (pGL-basic) and tested the luciferase activity in
undifferentiated primary keratinocytes (LowCa), where miR-203 is
expressed at low levels, and in differentiated primary keratinocytes
(HiCa), where miR-203 is expressed at high levels (Yi et al., 2008).
As expected, the promoter activity was significantly upregulated in
the differentiated primary keratinocytes (HiCa), while
demonstrating a basal level of expression in the undifferentiated
primary keratinocytes (LowCa) (Fig. 2H). Importantly, the
approximately ninefold upregulation of the promoter activity is
consistent with the expression pattern of mature miR-203 reported
in these cells (approximately eight- to 10-fold activation).
The promoter assay suggests that the ~3 kb DNA sequences
upstream of miR-203 contain binding sites for transcription factors
to activate miR-203 during the epidermal differentiation. We then
performed bioinformatic analysis to identify transcription factors
that have conserved binding sites in this region by using ECR
Browser (Ovcharenko et al., 2004). Intriguingly, we have identified
numerous conserved binding sites for transcription factors that are
directly involved in the epidermal differentiation (supplementary
material Fig. S2). Among them, C/EBP (Lopez et al., 2009), Hes1
(Moriyama et al., 2008), SRF (Luxenburg et al., 2011), AP2 (Wang
et al., 2008), IRF (Ingraham et al., 2006; Richardson et al., 2006)
and Blimp1 (Magnúsdóttir et al., 2007) have been reported to have
crucial roles in promoting epidermal differentiation, whereas E2F
(Dicker et al., 2000) and Lef1/Tcf3/Tcf4 (Nguyen et al., 2009) are
important for maintaining the progenitor fate of the basal epidermal
cells. Taken together, these observations suggest that miR-203 is
repressed in the basal progenitor cells and activated in the
suprabasal cells through the control of many transcription factors.
miR-203 has immediate and long-term impact on
cell proliferation
Having determined the precise dynamics of miR-203 induction in the
skin, we sought to determine the functional significance of the rapid
induction of miR-203. To achieve this, we established an inducible
DEVELOPMENT
Fig. 1. Spatiotemporal expression of miR-203 is determined
by in situ hybridization during embryonic skin
development. (A-C) miR-203 is restricted from actively dividing
cells. At E15, suprabasal cells in the epidermis are still actively
dividing, as marked by PH3 (B,C). miR-203 is highly expressed in
the non-dividing cells and expressed at low levels in the
dividing cell (outlined) in the suprabasal layers. (D) By E17,
actively dividing cells, marked by PH3, are restricted in the basal
layer, marked by β4 integrin, and miR-203 is highly expressed in
all suprabasal cells. (E,F) At P6, miR-203 is highly expressed in
differentiated cells but restricted from the basal progenitor cells
and the bulge hair follicle stem cells. Scale bars: 10 μm.
miR-203 inhibits self-renewal
RESEARCH ARTICLE 1885
Fig. 2. miR-203 is transcriptionally activated in
differentiating cells following asymmetric cell division.
(A-C) miR-203 remains repressed in both basal cells (marked by
BrdU) following symmetric cell division in the basal layer
(marked by K5). (D-F) miR-203 is rapidly induced in the
differentiating daughter cell upon asymmetric progenitor cell
division in the epidermis. Two daughter cells of the asymmetric
cell division are marked by BrdU in D and F. The daughter cell
remaining in the basal layer is marked by K5 in E and F. Scale
bars: 10 μm. (G) The TSS of miR-203, located ~100 bp upstream
of the miR-203 hairpin, is determined by 5⬘ RACE. Conserved
DNA patterns and a CpG island surrounding the conserved
DNA sequences are identified in the upstream region,
indicating the existence of a putative promoter. (H) The
promoter activity of the DNA fragment spanning ~3 kb region
upstream of the TSS is tested in undifferentiated (LowCa) and
differentiated (HiCa) keratinocytes. A basal-level promoter
activity is observed in the undifferentiated condition
(compared with the pGL-basic and the positive control pGLcontrol). A significant activation (approximately ninefold) is
detected in the differentiated condition. n=3, *P≤0.01. Data are
mean±s.e.m.
potential. We induced miR-203 transiently and terminated the
induction by washing away doxycycline after 24, 48 or 72 hours of
induction. Although the inhibitory effect on the colony formation by
a 24-hour exposure to miR-203 could be reversed, longer exposure
resulted in permanent loss of proliferation (Fig. 3E). In particular,
after a 72-hour induction, colony formation was completely
abolished, similar to cells under continuous miR-203 induction
(Fig. 3E). These data provide compelling evidence for a potent role
of miR-203 in restricting the proliferative potential during the
epidermal differentiation. Because an extended induction of miR203 can lead to the permanent loss of the proliferative capacity, it
suggests that miR-203 causes widespread and irreversible changes
to the gene expression in the epidermis.
miR-203 has a widespread impact on the selfrenewal program
To provide molecular insights on the role of miR-203 in the epidermal
differentiation, we identified in vivo miR-203 targets by microarray
profiling with the inducible mice. Recent studies directly link
miRNAs to the destabilization of mRNAs, and microarray analysis
has been successfully employed to identify miRNA targets (Giraldez
et al., 2006; Guo et al., 2010; Lim et al., 2005). Because miR-203 is
rapidly induced when the basal cells divide to give rise to the
suprabasal cells (Figs 1, 2) and plays a potent role in the
differentiation (Fig. 3), we reasoned that we could identify the targets
of miR-203 by examining downregulated genes shortly after the
induction of miR-203. We first validated the Dox-mediated induction
of miR-203 in vivo. As shown in Fig. 4A, 6 hours after the Dox
injection, miR-203 was induced in the basal cells only in the K14rtTA/TRE-miR-203 double-positive (DP) skin but not in the TRE-miR203 single-positive (SP) skin. Importantly, the induced miR-203 was
comparable with the endogenous expression in the suprabasal cells,
as determined by in situ hybridization (Fig. 4A). To establish the
optimal timing for detecting the changes to the targets of miR-203
and minimizing secondary effects, we monitored the mRNA level of
DEVELOPMENT
system to control miR-203 in the skin (Fig. 3A). Previously, skinspecific inducible transgenic mouse models driven by Keratin14-rtTA
(K14-rtTA) have been established to study transcription factors
(Nguyen et al., 2006). We adapted this approach by using the K14rtTA line crossed with pTRE2-miR-203 lines (Fig. 3A).
To characterize the immediate impact of miR-203 on the
epidermal differentiation, we isolated and established keratinocytes
from the K14-rtTA/pTRE2-miR-203 double positive (DP) skin. We
further validated an eightfold induction of miR-203 24-48 hours
after the addition of doxycycline to the DP cells (Fig. 3B). The
induced level of miR-203 is consistent with the levels of
endogenously expressed miR-203 during Ca2+-induced keratinocyte
differentiation (Lena et al., 2008; Yi et al., 2008).
Next, we examined the impact of miR-203 on the epidermal
proliferation. When we analyzed cell cycle progression with the
BrdU analysis, we observed a rapid cell cycle withdrawal (Fig. 3C).
Within 6 hours of the doxycycline addition, the percentage of cells
in S phase was reduced from 48% to 37%. By 24 hours, S-phase
cells made up only 15% of cells, whereas G1/G0 populations
increased to 78% from 43% (Fig. 3C). These results suggest that
miR-203 has an immediate impact in promoting the cell cycle exit.
We next asked how miR-203 modulates long-term proliferation. We
determined the colony formation capacity and tracked individually
colonies. As expected, non-induced control colonies grew
exponentially (Fig. 3D; supplementary material Fig. S3). By
contrast, colonies with induced miR-203 expression divided two or
three times within first 72 hours and then stopped proliferating
(Fig. 3D; supplementary material Fig. S3). Taken together, these
results demonstrate that the inhibition of keratinocyte proliferation
by miR-203 is reflected by both immediate inhibition of cell cycle
progression and long-term inhibition of self-renewal.
Upon differentiation, the epidermal stem/progenitor cells lose
proliferative potential. To further define the role of miR-203 during
the process, we asked whether a short exposure to miR-203 was
sufficient to drive the cell cycle exit and dampen the proliferative
1886 RESEARCH ARTICLE
Development 140 (9)
p63, a well-characterized miR-203 target (Lena et al., 2008; Yi et al.,
2008), at 12, 24 and 48 hours after Dox treatment. Overall, we
determined that the 24-hour time-point was ideal, which is the shortest
time-point that allows us to reliably detect the downregulation. Under
this condition, miR-203 was induced ~2.5 fold and p63 mRNA was
downregulated ~30% in the epidermis (Fig. 4B,C).
We used two pairs of biological duplicates to perform the
microarray analysis from the epidermal samples harvested from DP
and SP littermates at P4, 24 hours after the Dox injection. The
expression of H2BGFP is used for sorting epidermal cells
specifically. Collectively, we have identified 424 genes that are
consistently downregulated more than 19% in both experiments by
using p63 as the cut-off. We first determined whether the genes that
contain the matches to miR-203 seed sequences in their 3⬘UTRs
were enriched in the downregulated group. We also carried out three
analyses as control. First, we used all genes as the control for the
downregulated genes. Second, we used the 5⬘UTRs and the coding
regions as the control for the 3⬘UTRs. Finally, we used all possible
7-mer motifs from mature miR-203 sequences, e.g. 1-7, 2-8, 3-9,
etc., as the control for the seed region (2-8). As expected, we
observed very strong (approximately threefold) enrichment of
perfect matches to the seed region (2-8) in the 3⬘UTRs but not in the
5⬘UTR or coding regions of the downregulated genes (Fig. 4D). We
also observed strong enrichment (>2-fold) of matches to the 7-mer
motifs of number 1-7 and number 3-9 nucleotides of miR-203,
whereas no other 7-mer motifs of miR-203 are significantly
enriched (Fig. 4E; supplementary material Fig. S4). These
observations are consistent with the notion that miRNAs
preferentially destabilize their target mRNAs through the
recognition of the seed sequences (1-7, 2-8 and 3-9) at the 3⬘UTRs
(Bartel, 2009). Therefore, our analysis has successfully enriched
miR-203 targets in the downregulated genes.
We next examined the correlation between the match to seed
sequences [e.g. 7-mers, including 1-7, 2-8 and 3-9, and an 8-mer
(2-9)] and the level of mRNA downregulation as a measurement for
the strength of seed matches. Consistent with previous bioinformatic
analyses (Grimson et al., 2007; Lewis et al., 2005), 8-mer (2-9)
matches showed the strongest effect on the downregulation of target
mRNAs, whereas 7-mer matches (1-7, 2-8 and 3-9) also showed
strong effects (Fig. 4F). Overall, the 2-8 and 3-9 matches showed
stronger effects on the mRNA downregulation than did the 1-7
matches. We also examined the correlation between the number of
seed matches and the degree of mRNA downregulation. As
expected, mRNAs containing more than two seed matches showed
the greatest reduction followed by mRNAs with two and one seed
match (Fig. 4G). All together, these genome-wide analyses further
support the robustness of our target identification.
Among these miR-203 target candidates, we next focused on the
transcripts that contain perfect matches to 1-7, 2-8 and 3-9 seed
sequences of miR-203 in their 3⬘UTRs as the candidates for the
DEVELOPMENT
Fig. 3. miR-203 has an immediate and a long-term impact on cell cycle and proliferation. (A) Schematic illustration for the inducible miR-203
mouse model. (B) Induction of miR-203 in cultured inducible keratinocytes is consistent with the pattern of endogenous induction during in vitro
differentiation without affecting the expression of miR-205, an miRNA expressed in the keratinocytes. (C) Induction of miR-203 rapidly promotes the cell
cycle exit of the keratinocytes. There is a rapid increase of the G0/1 cell population and decrease of the S cell population 6, 12, 24 and 48 hours after the
Dox treatment. (D) Induction of miR-203 potently inhibits long-term cell proliferation. The number of non-induced cells doubles every 24 hours and
they form numerous productive colonies, whereas the miR-203-induced cells cease to divide and fail to generate any colonies. (E) Extended induction
of miR-203 irreversibly inhibits keratinocyte proliferation. Compared with the control cells (0h exposure), keratinocytes with a 24-hour exposure to miR203 still retain the proliferative capacity, whereas keratinocytes with a 48-hour exposure to miR-203 significantly lose the proliferative capacity and the
cells with a 72-hour exposure to miR-203 no longer have the capacity, similar to the cells with continuous exposure to miR-203. For all assays, n=3,
*P≤0.01, **P≤0.001. Data are mean±s.e.m.
miR-203 inhibits self-renewal
RESEARCH ARTICLE 1887
2009) and Cav1 (Ørom et al., 2012). Overall, we identified 261 out
of 424 downregulated genes (61.6%) that contain at least a single
match to the seed sequences of miR-203 in their 3⬘UTRs
(supplementary material Table S1). To provide molecular insights
into the functions of the targets of miR-203, we performed a pathway
analysis to classify these genes (Huang et al., 2009). Strikingly, the
processes of cell cycling, cell division and response to DNA damage
are the three events that produce the most increases in gene function
(Table 1). In our functional studies, miR-203 had strong inhibitory
effects on both cell cycle progression and long-term proliferation
(Fig. 3). Therefore, these results strongly argue that miR-203 directly
targets many factors for the control of cell proliferation.
targets of miR-203. Among these genes, we have identified numerous
miR-203 targets that are experimentally validated in the skin,
including p63 (Lena et al., 2008; Yi et al., 2008), Bmi1 (Wellner et al.,
Co-regulation of multiple targets is required for
the function of miR-203
We and others have previously identified p63 as a target of miR203 (Lena et al., 2008; Yi et al., 2008). However, it is not clear
whether the function of miR-203 is simply mediated through the
downregulation of p63 or whether the regulation of other targets
also contributes to the function of miR-203. In this study, we greatly
expanded the pool of miR-203 targets using a genome-wide
approach. It raises the issue of whether miR-203 functions by
targeting a single crucial gene, e.g. p63, or multiple targets
DEVELOPMENT
Fig. 4. Systematic identification of miR-203 targets by microarray and
bioinformatic analyses with the inducible mouse model. (A) Six hours
after Dox injection, miR-203 is readily induced in the basal progenitor cells
only in the DP but not in the SP control skin. All epidermal cells are marked
by H2BGFP; the basement membrane is marked by β4 integrin; miR-203 is
marked by red cytoplasmic signals. Scale bars: 10 μm. (B) miR-203 is
induced at the physiological level in the basal progenitor cells. The
expression of miR-205 is not affected by the induction of miR-203 by Dox
treatment. (C) Twenty-four hours after Dox treatment, p63 is
downregulated ~30% at the mRNA level, as measured by qPCR. (D) High
enrichment of miR-203 seed sequences is detected only in the 3⬘UTR of the
downregulated genes upon miR-203 induction. Neither 5⬘UTR nor the CDS
is enriched for the miR-203 seed sequences. (E) Perfect matches to 5⬘
sequences of miR-203, including numbers 1-7, 2-8 and 3-9 nucleotides but
not other regions of miR-203, e.g. numbers 16-22, are highly enriched in the
3⬘UTRs of the downregulated genes. (F) Effectiveness of seed matches on
the downregulation of target mRNAs. Changes in the mRNA level after miR203 induction are plotted with the cumulative distributions. Repression
from UTRs containing an 8-mer (2-9) site is most significant, whereas 7-mer
sites (2-8, 3-9, 1-7) also show strong repression, compared with all
expressed UTRs. (G) Effectiveness of the number of seed matches on the
downregulation of target mRNAs. Changes in the mRNA level after miR-203
induction are plotted with the cumulative distributions. Repression from
UTRs containing more than two seed matches is strongest, whereas
repression from UTRs containing 2 or 1 seed matches is also significant,
compared with UTRs without any seed match.
miR-203 directly regulates a large number of
targets
Having demonstrated that miR-203 may regulate many genes that
are crucial for cell cycle and cell division by our bioinformatic
analysis, we set out to determine the accuracy of our target
identification experimentally. We selected a group of 15 genes that
are downregulated by miR-203 at the mRNA level (from 19% to
65% downregulation). We cloned the 3⬘UTR from each of these 17
genes and performed the heterologous luciferase assay by testing
their response to the expression of miR-203. As a positive control,
we engineered a miR-203 reporter construct with 2× perfectly
matched sites at the 3⬘UTR of the luciferase gene. Perfect matched
sites usually lead to mRNA cleavage and, in turn, to significant
downregulation of the luciferase activity (Zeng et al., 2003). Indeed,
we observed a 95% repression in the luciferase activity, indicating
the efficacy of our assay (Fig. 5A). Among the 15 3⬘UTRs that we
tested, 12 of them (80%) showed statistically significant
downregulation of the luciferase activity. Notably, the lack of
repression by three of the 3⬘UTRs did not correlate with the level of
mRNA downregulation detected by microarray analysis. This
observation suggests that: (1) target sites may locate in other regions
of the mRNA, e.g. the coding region, which may be responsible for
the regulation; and (2) the extent of mRNA downregulation may
not be used as a sole determinant to validate individual targets.
To further validate the direct regulation of these candidates by miR203, we tested whether the predicted miR-203 target sites are
responsible for the observed repression. Among the 12 3⬘UTRs that
confer downregulation on the luciferase activity, we targeted eight
genes for point mutations at the predicated miR-203 binding sites
(supplementary material Table S1). Seven out of eight tested 3⬘UTRs
showed robust derepression of the luciferase activity when the miR203 sites were mutated (Fig. 5B). Together, these results validate that
the downregulated genes bearing miR-203 target sites in the 3⬘UTR
are highly enriched for bona fide miR-203 targets. They also confirm
that our combinatorial approach with the inducible mouse model and
bioinformatic analysis is highly effective for identifying the targets
of miR-203. Furthermore, these results support the observations that
miR-203 targets many genes in the epidermis.
Development 140 (9)
1888 RESEARCH ARTICLE
Table 1. Targets of miR-203 are highly enriched in the regulation of self-renewal
A GOterm analysis of the targets of miR-203 reveals high enrichment in the regulation of self-renewal pathway
GOTERM_BP_FAT
Cell cycle
Cell division
Response to DNA damage
P value
Gene numbers
26
14
13
–26
4.80×10
1.40×10–13
5.00×10–12
Fold enrichment
FDR
15.6
18.3
16.6
7.00×10–23
2.10×10–10
7.20×10–9
B Targets of miR-203 that are involved in the regulation of self-renewal
Bmi1, Ccnd3, Cdt1, Cep55, Chek2, Rbl1, Sept2, Sept6, Sgol1, Skp2, Smc2, Tfdp2, Tial1, p63, p73
Fen1, Hus1, Morf4L1, Pola1, Prmt6, Prpf19, Rad51Ap1, Shprh, Wrnip1, Xrn2
concurrently. We reasoned that if a single target is crucial for the
function of miR-203 function, then derepression of the target should
be sufficient to suppress the proliferation defects caused by the
expression of miR-203. In addition to p63, we selected Skp2 as a
representative gene for cell cycle regulation. We also chose Msi2, a
RNA-binding protein that has recently been implicated in the selfrenewal of hematopoietic stem cells (Hope et al., 2010; Kharas et
al., 2010), and Vav3, an oncogene with possible roles in cytoskeleton
organization and cell signaling (Hornstein et al., 2004; Tybulewicz,
2005), for the functional test.
We first examined whether a single miR-203 target could recover
the cell cycle and proliferation defects. We infected the miR-203
inducible keratinocytes with MSCV constructs for p63, Skp2, Msi2,
Vav3 or empty vector control. Twenty-four hours after infection, we
induced miR-203 expression by adding Dox (Fig. 6A). Forty-eight
hours after miR-203 induction, cell cycle was determined by BrdU
Fig. 5. Validation of miR-203 targets by 3⬘UTR luciferase assay.
(A) Twelve out of 15 targets are repressed by miR-203 when their 3⬘UTR is
tested in the luciferase assay. The positive control (2×miR-203) bears two
perfectly matched sites for full-length miR-203 and shows ~95%
repression. (B) When the predicted miR-203 target sites are specifically
mutated, seven out of eight targets are derepressed in the presence of
miR-203 in the 3⬘UTR luciferase assay. For all assays, n=3, *P≤0.05,
**P≤0.01, ***P≤0.001, N.S., not significant. Data are mean±s.e.m.
analysis. As shown in Fig. 6B, Msi2, p63 and Vav3 were unable to
rescue the impaired cell cycle progression, whereas Skp2 increased
the S-phase population in the presence of miR-203. We next
examined the ability of individual targets to compromise the
inhibition of long-term cell growth caused by miR-203 with colony
formation assay (Fig. 6C). Compared with the empty vectorinfected cells, where no colony formation was observed, cells
infected with Msi2, p63 and Skp2 formed small colonies, whereas
Vav3 showed a weaker effect (Fig. 6C). These results indicate that
the targets of miR-203 may have distinct functions in the regulation
of proliferation and co-suppression of multiple targets is required
for the function of miR-203.
To further test this hypothesis, we performed combinatorial
experiments in which we infected the cells with a cocktail of
Msi2+p63, Msi2+Skp2, p63+Skp2 or Msi2+p63+Skp2. Interestingly,
the slight recovery of the cell cycle progression was only observed
when Skp2 was included and no synergistic effect was observed
when Skp2 is combined with Msi2 and/or p63 (Fig. 6D).
Intriguingly, when Msi2, Skp2 and p63 were simultaneously
delivered to the cells in the presence of miR-203 induction, we
observed the best phenotypes in long-term cell proliferation, e.g.
larger colonies were formed (Fig. 6E). Taken together, these results
provide further evidence that co-regulation of multiple targets is
crucial for the function of miR-203 in potently driving the cell cycle
exit and restricting the proliferative potential.
Targets of miR-203 are required for the selfrenewal in vitro
To provide more insights about p63, Skp2 and Msi2, we
characterized their expression patterns in the basal and suprabasal
cells. Using E14 epidermis from K14-H2BGFP animals where all
epidermal cells are marked by the expression of H2BGFP, we
purified the basal and suprabasal cells as the H2BGFPhiα6hi and
H2BGFPloα6lo populations, respectively. Using RNA-Seq analysis,
we quantified their expression levels in the basal and suprabasal
cells (Fig. 7A). Both p63 and Skp2 were highly enriched in the basal
cells with over twofold basal/suprabasal ratio, whereas Msi2 was
mildly enriched in the basal cells with 1.23-fold basal/suprabasal
ratio (Fig. 7A). As control, Krt14 was over 20-fold enriched in the
basal cells, whereas Krt10 was ~50-fold enriched in the suprabasal
cells (Fig. 7A). These results suggest that these targets are highly
enriched in the basal progenitor cells and inversely correlated with
the expression of miR-203 in the epidermis. Furthermore, when we
induced miR-203 expression in vivo with a single Dox injection, we
observed 19%, 32%, 43% and 38% reduction of mRNAs for p63,
Msi2, Skp2 and Vav3, respectively (supplementary material Fig. S5).
These results further validate that miR-203 suppresses these targets
in vivo.
Because of the basal enrichment of p63, Skp2 and Msi2 and the
fact that their repression is required for the inhibition of self-renewal
DEVELOPMENT
Cell cycle and cell division
Response to DNA damage
miR-203 inhibits self-renewal
RESEARCH ARTICLE 1889
Fig. 7. miR-203 targets are required for self-renewal of cultured
keratinocytes. (A) p63, Skp2 and Msi2 are enriched in the basal cells, as
determined by RNA-Seq. The basal enrichment of Krt14 and the
suprabasal enrichment of Krt10 are used as controls. The numbers
indicate the basal/suprabasal ratio. (B) Knocking down individual targets
of miR-203 causes severe defects in self-renewal of cultured
keratinocytes.
Fig. 6. Different targets mediate distinct functions of miR-203.
(A) The experimental design for the functional test of the targets of miR203. (B) Skp2 but not other targets shows a recovery to the cell cycle exit
caused by miR-203 (n=3). (C) Msi2, p63 and Skp2 all partially recover the
loss of colony formation capacity caused by miR-203 (n=3). (D) Among
individual targets, only Skp2 shows a recovery to the cell cycle exit in the
combinatorial test (n=3). (E) Enhanced colony formation is observed
when multiple miR-203 targets are delivered to the cells. Msi2+p63+Skp2
shows the best result for the colony formation. *P≤0.01, **P≤0.001,
***P≤0.0001. Data are mean±s.e.m.
by miR-203, we investigated their function in the proliferation of
keratinocytes. We used two independent shRNAs to knockdown
each gene and test with colony formation assay. Consistent with
previous studies (Senoo et al., 2007), knockdown of p63 abolished
colony formation (Fig. 7B). Interestingly, knockdown of Skp2,
which is crucial for the cell cycle progression, also abolished colony
formation (Fig. 7B), whereas knockdown of Msi2 showed relatively
DISCUSSION
miRNAs are thought to have important roles in stem cell
differentiation (Ivey and Srivastava, 2010; Yi and Fuchs, 2011). In
somatic stem cells, the roles of miRNAs in promoting
differentiation have been reported in numerous tissues, including
skin (Botchkareva, 2012; Lena et al., 2008; Ning and Andl, 2012;
Yi et al., 2008), brain (Cheng et al., 2009; Zhao et al., 2009) and
skeletal muscle (Chen et al., 2010). However, two important
questions about the role of miRNAs in the differentiation of stem
cells remain unanswered. First, it is not clear when miRNAs are
activated during differentiation. A pertinent question is what is the
role of miRNA-mediated regulation during the transition of stem
cells towards their differentiating daughter cells? Our current study
has provided an important insight by revealing the rapid activation
of miR-203 during the epidermal differentiation. To visualize the
expression patterns of miR-203 at single-cell resolution, we have
developed a high resolution in situ hybridization technique. The
ability to co-stain miR-203 together with skin lineage markers e.g.
K5 and β4 integrin, and proliferation markers, e.g. BrdU and PH3,
allows us to demonstrate the immediate activation of miR-203 in
the differentiating daughter cell following the ACD of the epidermal
progenitor cells. We also characterize the promoter of miR-203 and
DEVELOPMENT
modest yet significant reduction in the long-term proliferation
(Fig. 7B). When all three genes were knocked down concurrently,
the cells failed to give rise to any colonies, as expected (data not
shown). These results demonstrate the crucial role of each target in
the epidermal proliferation and further highlight the potent impact
of miR-203 on the self-renewal by simultaneously repressing these
targets.
Development 140 (9)
1890 RESEARCH ARTICLE
Acknowledgements
We thank G. Voeltz for the confocal microscopy and the members of the Yi lab
for discussion; L. Greiner for managing the mouse colony; Y. Han for cell
sorting; J. Huntley for Illumina Sequencing; R. Henry and Functional Genomics
Facility for shRNA.
Funding
This publication was made possible by funding from the National Institutes of
Health [R00AR054704 and R01AR059697 to R.Y.] and the Howard Hughes
Medical Institute [E.F.], and it was partly supported by the American Cancer
Society [ACS IRG #57-001-50 to R.Y.]. Deposited in PMC for release after 12
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.089649/-/DC1
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DEVELOPMENT
miR-203 inhibits self-renewal