G1 cyclins link proliferation, pluripotency and differentiation of
embryonic stem cells
Lijun Liu
1,2
, Wojciech Michowski
1,2
, Hiroyuki Inuzuka
3
, Kouhei Shimizu
3
, Naoe Taira
Nihira
3
, Joel M. Chick
4
, Na Li
1,2
, Yan Geng
1,2
, Alice Y. Meng
5
, Alban Ordureau
4
, Aleksandra
Kolodziejczyk
1,2
, Keith L. Ligon
5,6
, Roderick T. Bronson
7
, Kornelia Polyak
8
, J. Wade
Harper
4
, Steven P. Gygi
4
, Wenyi Wei
3
, and Piotr Sicinski
1,2
1
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215,
USA
2
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
3
Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts 02115, USA
4
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
5
Department of Pathology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA
6
Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02215, USA
7
Department of Biomedical Sciences, Tufts University Veterinary School, North Grafton,
Massachusetts 01536, USA
8
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215,
USA
Abstract
Progression of mammalian cells through the G1 and S phases of the cell cycle is driven by D-type
and E-type cyclins. According to the current models, at least one of these cyclin families must be
present to allow cell proliferation. Here, we show that several cell types can proliferate in the
absence of all G1 cyclins. However, upon ablation of G1 cyclins, embryonic stem (ES) cells
attenuated their pluripotent characteristics, with majority of cells acquiring the trophectodermal
cell fate. We established that G1 cyclins, together with their associated cyclin-dependent kinases
(CDKs) phosphorylate and stabilize core pluripotency factors Nanog, Sox2 and Oct4. Treatment of
Correspondence and requests for materials should be addressed to P.S. (peter_sicinski@dfci.harvard.edu).
Author Contributions L.L. and P.S. conceived the study. L.L. designed and performed the experiments with help from collaborators.
W.M. contributed
in vitro
and
in vivo
kinase assays, viral transduction and conceptually helped with design of experiments. H.I., K.S.,
N.T.N., and W.W. contributed analyses of endogenous Nanog/Oct4/Sox2 phosphorylation, interaction with Pin1, and analyses of
mutant proteins. J.M.C. and S.P.G. contributed mass spectrometric analyses. N.L. initiated the study and bred compound mutant
animals, Y.G. performed cultures of human cancer cells, A.Y.M. and K.L.L. provided immunostaining analyses of teratomas, K.L.L.
provided human patient-derived primary glioblastoma cells, A.O. and J.W.H. contributed analyses of polyubiquitination of
endogenous proteins, A.K. helped with in-cell kinase assays, R.T.B. analyzed and interpreted all histological specimens, K.P. provided
human breast cancer cell lines and helped with the design of breast cancer analyses. L.L. and P.S. wrote the paper. P.S. directed the
study.
P.S. is a consultant and a recipient of a research grant from Novartis.
HHS Public Access
Author manuscript
Nat Cell Biol
. Author manuscript; available in PMC 2017 September 01.
Published in final edited form as:
Nat Cell Biol
. 2017 March ; 19(3): 177–188. doi:10.1038/ncb3474.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
murine ES cells, patient-derived glioblastoma tumor-initiating cells, or triple-negative breast
cancer cells with a CDK-inhibitor strongly decreased Sox2 and Oct4 levels. Our findings suggest
that CDK-inhibition might represent an attractive therapeutic strategy by targeting glioblastoma
tumor-initiating cells, which depend on Sox2 to maintain their tumorigenic potential.
Progression of cells through the G1 phase as well as entry into and passage through the
DNA-synthesis (S phase) of the cell cycle is driven by a class of proteins called G1 cyclins.
Two families of G1 cyclins operate in mammalian cells, D-type (D1, D2 and D3), which
activate the cyclin-dependent kinases CDK4 and CDK6 and E-type (E1 and E2), which
activate CDK2 (ref. 1).
Analyses of mice lacking all three D-type cyclins (D1
−/−
D2
−/−
D3
−/−
) revealed that these
proteins are essential for proliferation only in a few selected compartments, such as
hematopoietic cells. Importantly, the overwhelming majority of cells in cyclin D-null
embryos proliferated normally, revealing that normal cell cycle progression can take place in
the absence of D-cyclins
2
.
Furthermore, studies of embryos lacking cyclins E1 and E2 (E1
−/−
E2
−/−
) revealed a
requirement for these proteins only in specific compartments, such as placenta and heart.
Again, the majority of cell types proliferated normally, revealing that E-cyclins are
dispensable for proliferation of most cell types
3,4
. Collectively, these findings led to the
current model that G1 cyclins can perform overlapping functions, and that at least one class
of G1 cyclins (D-type or E-type) must be present to allow mammalian cell proliferation.
In this study we decided to test this notion by generating embryonic stem (ES) cells, tissues
and chimeric embryos lacking all five G1 cyclins. These studies revealed that, contrary to
the prevailing view, G1 cyclins are not uniformly required for cell proliferation, but they
play essential, direct roles in maintaining cell stemness and in regulating cell fate
specification.
RESULTS
Generation of G1 cyclin-deficient ES cells
We interbred cyclin D1
−/−
, D2
−/−
, D3
−/−
, E1
F/F
(conditional cyclin E1 knockout) and E2
−/−
mice and generated cyclin D1
+/−
D2
+/−
D3
+/−
E1
F/F
E2
+/−
animals. We then intercrossed these
mice, harvested blastocysts and cultured them
in vitro
to derive pluripotent ES cells (Fig.
1a). We succeeded in generating one cell line of the desired D1
−/−
D2
−/−
D3
−/−
E1
F/F
E2
−/−
genotype (expected ratio: 1:256), and one additional independent ES cell line heterozygous
at the cyclin D2 locus (D1
−/−
D2
+/−
D3
−/−
E1
F/F
E2
−/−
). The latter cell line was then converted
to the D1
−/−
D2
−/−
D3
−/−
E1
F/F
E2
−/−
genotype by re-targeting the remaining cyclin D2 allele
(Supplementary Fig. 1a, b, Table 1).
ES cells proliferate in the absence of G1 cyclins
We introduced Cre recombinase into cyclin D1
−/−
D2
−/−
D3
−/−
E1
F/F
E2
−/−
ES cells, thereby
acutely deleting cyclin E1 and rendering cells devoid of all G1 cyclins (D1
−/−
D2
−/−
D3
−/−
E1
Δ/Δ
E2
−/−
). Very unexpectedly
Liu et al. Page 2
Nat Cell Biol
. Author manuscript; available in PMC 2017 September 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Ablation of G1 cyclins in mouse embryonic fibroblasts
These observations were very surprising, given the prevailing view that at least one class of
G1 cyclins is required for cell proliferation. However, most of current cell cycle models are
based on analyses of cultured mouse embryonic fibroblasts (MEFs). To extend our
observations to this cell type, we utilized tetraploid blastocyst complementation method,
which allows one to generate embryos derived entirely from mutant ES cells
3
(Fig. 2a).
We injected D1
−/−
D2
−/−
D3
−/−
E1
F/F
E2
−/−
ES cells into tetraploid blastocysts, and collected
embryos at day 13.5 of gestation. Mutant embryos were viable and displayed normal
appearance (Fig. 2a), indicating that a single G1 cyclin (cyclin E1) is sufficient for normal
cell proliferation and development up to this stage. We then derived MEFs from the mutant
embryos and transduced cells with Cre recombinase, thereby acutely shutting off cyclin E1
expression and rendering MEFs devoid of all G1 cyclins. Strikingly, ablation of all G1
cyclins completely blocked proliferation of MEFs (Fig. 2b–e, Supplementary Fig. 1c). We
concluded that while ES cells can proliferate in the absence of G1 cyclins, in MEFs at least
one G1 cyclin protein must be expressed to allow cell cycle progression.
Attenuation of stem cell pluripotency upon shutdown of G1 cyclins
Staining of
in vitro
cultured ES cells with alkaline phosphatase (AP), a marker of
undifferentiated pluripotent stem cells
5
, revealed that ablation of G1 cyclins resulted in a
strong decrease in AP staining. Whereas in control ES cells ~90% of colonies were
uniformly AP-positive, this fraction was reduced to ~30% following ablation of all G1
cyclins (Fig. 3a, b).
To probe this phenomenon at the molecular level, we analyzed protein levels of core
pluripotency factors Nanog, Oct4 and Sox2 in Q-KO ES cells. These proteins are essential to
maintain cell stemness, and their depletion causes the loss of the pluripotent state
6–9
. We
found that ablation of G1 cyclins resulted in a downregulation of all three pluripotency
factors (Fig. 3c). Importantly, the levels of transcripts encoding Nanog, Oct4 and Sox2 were
unchanged in Q-KO ES cells (Fig. 4a, Supplementary Fig. 2a), indicating that G1 cyclins
maintain the levels of these proteins at the posttranscriptional level.
To gauge the contribution of the D-type and E-type cyclins to maintenance of the pluripotent
state, we compared wild-type, cyclin D-null (D1
−/−
D2
−/−
D3
−/−
), E-null (E1
Δ/Δ
E2
−/−
) and
Q-KO ES cells. We observed that wild-type ES cells expressed low levels of D-type cyclins,
whereas cyclin E was strongly expressed, as reported before
10
(Fig. 3d, e). Ablation of D-
type cyclins did not affect the levels of cyclin E (Fig. 3d), and resulted in only a slight
decrease in the percentage of undifferentiated AP-positive ES cell colonies (see D-KO in
Fig. 3a, f, g), and modest reduction of Nanog, Oct4 and Sox2 levels (Supplementary Fig.
2b). Cyclin E-null (E1
Δ/Δ
E2
−/−
) ES cells displayed very strong compensatory upregulation
of all three D-type cyclins (Fig. 3e), a modestly reduced fraction of AP-positive colonies
(see E-KO in Fig. 3a, f, g), and modestly reduced levels of Nanog, Oct4 and Sox2 proteins
(Supplementary Fig. 2b). We concluded that G1 cyclins cooperate to maintain the levels of
Nanog, Oct4 and Sox2 proteins in ES cells via a posttranscriptional mechanism, with E-
cyclins playing the primary role and D-cyclins representing a likely ‘backup’ mechanism.
Liu et al. Page 3
Nat Cell Biol
. Author manuscript; available in PMC 2017 September 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
To better characterize the population of Q-KO ES cells, we stained control and Q-KO cells
with antibodies against SSEA-1 (a marker of undifferentiated mouse stem cells), as well as
Oct4 and Nanog, and analyzed cells by flow cytometry. As expected, nearly all control ES
cells were positive for SSEA-1. In contrast only 45–50% of Q-KO ES cells were SSEA-1-
positive, with majority of Q-KO cells being SSEA-1-negative (Fig. 4b, c). As expected, 95%
of control ES cells expressed high Nanog and Oct4 levels. In contrast, approximately 40% of
Q-KO ES cells were Oct4
low
and Nanog
low
, and the rest was Oct4/Nanog-negative (Fig. 4b,
c). Hence, ablation of G1 cyclins led to an attenuation of markers that characterize
undifferentiated pluripotent stem cell state.
Ablation of G1 cyclins promotes trophectodermal differentiation
In addition to maintaining pluripotency, Oct4 and Sox2 proteins actively repress the
trophectodermal cell fate. Oct4 was shown to block transcriptional upregulation of a caudal-
related homeobox protein Cdx2, a key regulator of the trophectoderm lineage that confers
the trophectodermal specification
11–13
. Consequently, reduced Oct4 (or Sox2) expression in
ES cells triggers differentiation of ES cells into trophectoderm, by transcriptionally
upregulating Cdx2 and another regulator of trophectodermal differentiation, Eomesodermin
(Eomes)
6,9,11,14,15
. Given strongly reduced levels of Oct4 and Sox2 proteins upon ablation
of G1 cyclins, we tested expression of trophectodermal markers in Q-KO ES cells.
We stained ES cells with antibodies against Cdx2, Eomes and another trophectodermal cell
marker, Hand1, and analyzed by flow cytometry. As expected, control ES cells were
negative for each of these markers. In contrast, 50–60% of Q-KO ES cells were Cdx2- and
Eomes-positive, and expressed low levels of Hand1 (Fig. 4d, e). Immunostaining of intact
ES cell colonies for Cdx2 confirmed increased Cdx2 protein levels in Q-KO cells (Fig. 4f,
g). As expected, upregulation of the trophectodermal markers (Cdx2, Eomes, Ehox) in Q-
KO cells occurred at the transcriptional level (Fig. 4a, Supplementary Fig. 2a). Importantly,
markers of mesodermal, endodermal and ectodermal lineages remained unchanged upon
ablation of G1 cyclins, except for an upregulation of a neuroectodermal marker, Pax6 (Fig.
4a, Supplementary Fig. 2a, and Table 2).
To further extend these findings, we used RNA-sequencing to compare transcript abundance,
in an unbiased fashion, between Q-KO and control ES cells. We found that 66 transcripts
were strongly (>4 fold) upregulated and 16 downregulated upon ablation of G1 cyclins
(Supplementary Fig. 2c, d, Table 3). Several of these upregulated transcripts corresponded to
trophectodermal/trophoblast genes, in addition to strongly increased levels of Cdx2 and
Eomes (Supplementary Table 3). Collectively, these results indicate that ablation of G1
cyclins leads to the attenuation of pluripotency, and promotes trophectodermal cell fate.
Contribution of Q-KO ES cells to different lineages in vivo
To gauge the ability of Q-KO ES cells to contribute to different lineages
in vivo
, we
knocked-in a cDNA encoding green fluorescent protein (GFP) into the ubiquitously
expressed
Rosa26
locus
16
in Q-KO ES cells. We then injected GFP
+
Q-KO ES cells into
wild-type blastocysts and implanted the chimeric embryos for further development (Fig. 5a).
Consistent with our observation that ablation of G1 cyclins triggered an upregulation of
Liu et al. Page 4
Nat Cell Biol
. Author manuscript; available in PMC 2017 September 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
trophectodermal markers (Fig. 4a, d–g, Supplementary Fig. 2a and Table 3), which specify
the lineage that forms the placenta, we found that Q-KO ES cells preferentially contributed
to embryonic placentas
in vivo
(Fig. 5b). On the other hand, Q-KO ES cells contributed
poorly to the embryo proper, however we detected a clear contribution of GFP
+
cells to
neural cells (Fig. 5c).
We extended these analyses by subcutaneously injecting Q-KO ES cells into
immunocompromised nu/nu mice. Under these conditions ES cells proliferate, differentiate
and form teratomas composed of tissues derived from ectodermal, endodermal and
mesodermal lineages
17
. As expected, teratomas derived from control ES cells displayed
roughly equal proportion of these three lineages. In contrast, tumors derived from Q-KO ES
cells showed a predominance of the neural tissue (Fig. 6, upper panel). Immunostaining of
teratoma sections revealed the Q-KO ES cells give rise to neural stem/progenitor cells
(Nestin
+
), immature postmitotic neuronal precursors (beta III tubulin
+
), mature neurons
(NeuN
+
), astrocytes (GFAP
+
), oligodendrocytes (Olig2
+
), as well as endodermal (GATA6
+
)
and mesodermal lineages (skeletal muscle, cartilage and bone) (Fig. 6 and Supplementary
Fig. 3). Collectively, these analyses reveal that Q-KO ES cells retain the ability to give rise
to several different lineages
in vivo
.
Decreased protein stability and increased polyubiquitination of pluripotency factors in Q-
KO ES cells
To elucidate the exact role of G1 cyclins in maintaining Nanog, Oct4 and Sox2 protein levels
in ES cells, we blocked new protein synthesis by treating wild-type and Q-KO ES cells with
cycloheximide, and followed the decay of these proteins over time. We found that ablation
of all G1 cyclins decreased the half-life and strongly accelerated degradation of Nanog, Oct4
and Sox2 proteins (Supplementary Fig. 4a–d). Importantly, treatment of Q-KO cells with a
proteasomal inhibitor MG132 partially restored normal Nanog, Oct4 and Sox2 protein
levels, indicating that these proteins undergo accelerated proteasomal degradation in the
absence of G1 cyclins (Supplementary Fig. 4e).
Since degradation of Nanog, Oct4 and Sox2 proteins is triggered by polyubiquitination
18–20
,
we analyzed polyubiquitination status of the endogenous Nanog and Oct4 proteins in Q-KO
cells. We observed that ablation of G1 cyclins strongly increased polyubiquitination of both
proteins (Fig. 7a, b). Collectively these analyses indicate that G1 cyclins promote the
stability of core pluripotency proteins by inhibiting their polyubiquitination, and by
protecting them from proteasomal degradation.
G1 cyclins-CDKs directly phosphorylate and stabilize Nanog, Oct4 and Sox2 proteins
We next asked how at the molecular level G1 cyclins regulate the stability of Nanog, Oct4
and Sox2. The best documented function of G1 cyclins and their associated cyclin-
dependent kinases is to phosphorylate target proteins
1
. Therefore, we used
in vitro
kinase
reactions with purified recombinant proteins to test the ability of cyclin E-CDK2 and D3-
CDK6 kinases to phosphorylate Nanog, Oct4 and Sox2. We found that all three pluripotency
factors were readily phosphorylated by these kinases (Fig. 7c). We also verified the ability of
cyclin E-CDK2 to phosphorylate Nanog, Oct4 and Sox2 proteins
in vivo
using cells
Liu et al. Page 5
Nat Cell Biol
. Author manuscript; available in PMC 2017 September 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript