University of Georgia, Rhodes Center, 425 River Road, Athens, GA 30602-2771, USA
* Author for correspondence (e-mail: sdalton{at}uga.edu)
Accepted 22 December 2004
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SUMMARY |
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Key words: ES cells, Self-renewal, Myc, Pluripotency
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Introduction |
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Murine ES cells are typically cultured in the presence of fetal calf serum
(FCS) or a defined cocktail of supplements that in conjunction with LIF,
support proliferation and stem cell maintenance. Although LIF has a
well-characterized promaintenance function, other factors present in serum
have generally been overlooked in terms of performing a specific role in
promoting self-renewal. Ying and co-workers
(Ying et al., 2003) recently
reported, however, that factors in FCS such as bone morphogenic proteins
(BMPs) may work in collaboration with LIF to promote self-renewal. In the
presence of LIF, BMPs antagonize neural differentiation
(Ying et al., 2003
) and, in
parallel, promote self-renewal through suppression of ERK/MAPK signaling
(Qi et al., 2004
). Maintenance
of a stable stem cell state therefore appears to require multiple inputs that
impose lineage-specific differentiation blocks
(Chambers et al., 2003
;
Mitsui et al., 2003
;
Ying et al., 2003
).
Although LIF/STAT3 signaling is crucial for murine ES cell maintenance,
this pathway does not appear to have a role in human ES cell self-renewal
(Humphrey et al., 2004),
indicating the existence of alternate self-renewal mechanisms. Sato and
co-workers (Sato et al., 2004
)
recently defined a role for Wnt-dependent signaling in self-renewal of human
and murine ES cells that functions independently of LIF and STAT3. Moreover,
suppression of GSK3ß, an antagonist of Wnt signaling, is sufficient to
maintain self-renewal and pluripotency of human and murine ES cells in the
absence of LIF and Wnt (Sato et al.,
2004
). These observations signify a common mechanism of
self-renewal that may be further applicable to adult stem cell populations
that require Wnt-dependent signaling (Reya
et al., 2003
).
Although LIF and Wnt promote self-renewal by activation of separate
signaling pathways, we reasoned that they would converge on a common
target(s). We hypothesized that Myc could be a common effector on which these
signals converge because the Myc gene is a transcriptional target of STAT3 in
a number of biological contexts (Kiuchi et
al., 1999; Shirogane et al.,
1999
; Bowman et al.,
2001
), and signals transduced by Wnt can activate the Myc
transcription through a ß-catenin/TCF-dependent mechanism
(He et al., 1998
). Myc belongs
to a family of helix-loop-helix/leucine zipper transcription factors and
together with its obligatory binding partner, Max, performs roles in control
of cell proliferation, transformation, growth, differentiation and apoptosis.
A potential role for Myc in ES cell maintenance is suggested by two reports.
First, expression of an RLF/L-myc minigene that frequently arises from a
chromosomal translocation event in human small lung carcinomas, delays ES cell
differentiation and interferes with early embryonic development
(MacLean-Hunter et al., 1994
).
Second, elevated Myc activity is able to block the differentiation of multiple
cell lineages (Selvakumaran et al.,
1996
; Canelles et al.,
1997
; Pelengaris et al.,
1999
; Schreiner et al.,
2001
; Knoepfler et al.,
2002
). These lines of evidence prompted us to investigate whether
Myc plays a role in ES cell self-renewal downstream of LIF and/or Wnt. In this
report, we show that elevated Myc activity is required for ES cell maintenance
and that Myc is a key effector of the LIF/STAT3 self-renewal pathway. Our data
indicate that signals transduced by LIF and possibly Wnt, converge on Myc to
maintain ES cell identity.
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Materials and methods |
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ES cell maintenance, differentiation assays and colony forming assays
For long-term maintenance, mycER and mycT58AER cells were grown
in ES complete media supplemented with 4OHT (100 nM or 2-10 nM, respectively)
and re-fed every second day. For differentiation of ES cells as embryoid
bodies (EB), cells were trypsinized and replated on bacteriological dishes at
1.6x105 cells/cm2 and grown in ESC media (-LIF)
plus or minus 4OHT to allow for EB formation. Cells were re-fed every second
day and expanded after 4 days of culture. mycER cells were passaged by limited
trypsinization, avoiding generation of single cell suspensions. AP activity
was assayed with Alkaline Phosphatase Substrate Kit I (Vector Laboratories) by
scoring 100 individual ES cell colonies. AP-positive colonies were uniform and
dome-shaped in morphology, and consisted of densely packed AP-positive cells.
ES colonies were classified as being negative in the absence of AP staining or
when a mixture of stained and unstained cells were observed with a
corresponding flattened, non-uniform colony morphology. Colony forming assays
were performed by plating 500 cells at clonal density in ESC medium, followed
by scoring colonies for AP activity after 5 days.
Antibodies, flow cytometry and immunoblot analysis
The following antibodies were used: phospho T58 Myc (Cell Signaling
Technology), anti-Myc (N-262; Santa Cruz), anti-Oct4 (N-19; Santa Cruz),
anti-tubulin (Serotech), anti-HDAC1 (Zymed), anti-Cdk2 (M2; Santa Cruz) and
anti-GSK3ß (Transduction Laboratories). The anti-HDAC1 antibody detects
both HDAC1 and HDAC2 and accordingly, a doublet is seen by immunblot analysis.
For flow cytometry, ES cells (2x106) were washed with
1xPBS and fixed in 2% paraformaldehyde in 1xPBS for 10 minutes at
room temperature. Cells were then washed (1xPBS), incubated with
anti-SSEA1 mouse monoclonal antibody (Chemicon, 1:100) at 4°C for 30
minutes, washed twice and resuspended in anti-mouse Alexa-488 secondary
antibody (1:1,000; Molecular Probes) in 1% BSA/PBS at 4°C for 30 minutes.
Finally, cells were washed twice, resuspended in 1% BSA/1xPBS and
analysis performed using a Beckman Coulter FC500 flow cytometer.
Cells were lysed by incubating for 1 hour on ice in lysis buffer consisting of 50 mM HEPES pH 7.5, 1 mM EDTA, 1 mM EGTA, 250 mM NaCl, 10% glycerol, 0.1% Tween-20, 10 µg/ml TPCK, 50 µg/ml TLCK, 170 µg/ml PMSF, 0.5 mg/ml leupeptin, 0.7 mg/ml pepstatin and phosphatase inhibitor cocktail II (Calbiochem) with occasional vortexing/pipetting. Whole-cell extracts (20-40 µg total protein) were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes (BioRad), blocked with 3% BSA in TBSE (Tris HCl pH 7.5, 150 mM NaCl, 2 mM EDTA and 0.1% NP40) and probed with the indicated antibodies at 4°C.
GSK3ß kinase assays
Cell lysate (300 µg protein at 1 mg/ml) in cell lysis buffer was tumbled
with 3 µg of primary antibody for 3 hours at 4°C. Protein A Sepharose
beads (30 µl) were added and tumbled for a further 1 hour at 4°C. Beads
were washed three times with 1 ml of cold lysis buffer then once with 1 ml of
kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 2.5 mM
EGTA and protease inhibitor cocktail II, Calbiochem) and resuspended in 30
µl of kinase buffer with or without the addition of GSK3 inhibitor II (3
µM in ethanol, Calbiochem) or ethanol. Reactions were incubated at 30°C
for 20 minutes with 10 µCi of [-32P] ATP and 5 µg of
myelin basic protein (MBP, Upstate Biotech) at 30°C for 20 minutes.
Reactions were terminated by the addition of 2xSDS load buffer then
resolved by SDS PAGE. Kinase activities were quantitated by phosphorimager
analysis.
Plasmids, northern blot, RT-PCR analysis and ChIP assays
Details of all plasmid constructs are available on request. Total RNA from
ES cells was prepared with TRIzol Reagent (InVitrogen), Northern blot analysis
and probe synthesis were as described previously
(Stead et al., 2002). Primer
sequences for RT-PCR analysis and PCR conditions were as described previously
(Oka et al., 2002
). Chromatin
immunoprecipitation assays were performed essentially as described by
Fernandez et al. (Fernandez et al.,
2003
). Sequences of primers used for RT-PCR and ChIP analysis are
available on request.
Generation and analysis of chimeric mice
4OHT-maintained R1-EGFP/mycT58AER ES cells (129 background) were
injected into blastocyst stage C57BL/6 embryos and reimplanted into
pseudopregnant females as described by Hogan et al.
(Hogan et al., 1994). Embryos
were analyzed for GFP fluorescence of whole embryos (12.5 dpc) and by coat
color of adult mice.
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Results |
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Responsiveness of the Myc gene to LIF signaling and the direct recruitment
of STAT3 to the Myc promoter suggested that STAT3 trans-activates the Myc gene
in vivo. To address this, we evaluated the responsiveness of the endogenous
Myc gene to activation by STAT3-ER which in the presence of 4-hydroxytamoxifen
(4OHT), can maintain ES cells in the absence of LIF
(Matsuda et al., 1999). By
adding 4OHT to cultures, the labile form of ER fusions can be switched to a
biologically active state by releasing them from chaperone proteins (see
Eilers et al., 1989
). In the
case of nuclear factors, this involves their steroid-induced translocation to
the nucleus. D3 ES cells were grown with LIF and then in the absence of LIF
for 36 hours. 4OHT or LIF was then re-added to cultures for a further 24
hours, and Myc mRNA levels determined to evaluate the responsiveness of the
endogenous Myc gene to activated STAT3 and restored LIF signaling. Following
withdrawal of LIF, endogenous Myc mRNA levels decreased but this was reversed
following readdition of LIF (Fig.
1C). Restoration of STAT3 signaling by addition of 4OHT (-LIF) had
a similar effect within 24 hours (Fig.
1C), indicating that STAT3 trans-activates the Myc gene in ES
cells in vivo. These results demonstrate that the Myc gene is a direct
transcriptional target of LIF/STAT3 signaling in ES cells.
Maintenance of Myc levels in ES cells requires suppression of T58 phosphorylation
For Myc to be a regulator of self-renewal we predicted that its activity
would rapidly decline following LIF withdrawal. To investigate this, we
evaluated Myc protein levels in ES cells and during EB differentiation. Levels
of Myc protein were elevated in LIF-maintained ES cells but declined markedly
by day 1 of LIF withdrawal and even further over days 1-3
(Fig. 2A). The downregulation
of Myc protein occurs well before Oct4 mRNA levels are extinguished and prior
to the appearance of mRNAs for Fgf5 and brachyury
(Fig. 1A). Downregulation of
Myc protein is therefore associated with loss of LIF signaling and the early
stages of differentiation.
|
As phosphorylation at T58 could not be detected in ES cell lysates, this
suggested that signals involved in Myc degradation, such as that generated by
GSK3ß, were suppressed in ES cells. If so, we predicted that Myc would
have enhanced stability compared with other cell types where its half-life
(t1/2) is 20-30 minutes or less. Cycloheximide chase
experiments in ES cells showed that in the presence of LIF, Myc has a half
life of 105 minutes (Fig. 2B).
Enhanced Myc stability is therefore an important determinant of Myc levels in
ES cells. By comparison, the t1/2 values of Cdk2 and
cyclin D3 were over 180 minutes and 25 minutes, respectively. Parallel
experiments showed that Myc t1/2 in NIH 3T3 fibroblasts is
less than 30 minutes (P.C. and S.D., unpublished).
To further demonstrate that increased turnover of Myc correlates with the
activation of GSK3ß and T58 phosphorylation, we differentiated ES cells
as EBs and evaluated the stability of Myc in the presence or absence of
proteosome inhibitor MG132. The cell line used also stably expressed Myc fused
to the steroid-binding domain of the estrogen receptor, making it possible to
evaluate the role of post-transcriptional control in Myc regulation, because
it is expressed from the constitutive CAG promoter, which is active throughout
differentiation in vitro and in all tissues in vivo
(Okabe et al., 1997;
Pratt et al., 2000
). At day 2
after LIF withdrawal, levels of Myc and mycER collapsed but this was
completely blocked by addition of MG132
(Fig. 2C). Besides indicating
that the decline of Myc levels is proteosome dependent, these results show
that post-transcriptional controls are crucial in determining Myc levels
during differentiation. This point is underscored by the parallel collapse of
mycER levels, even though transcription of the fusion protein was driven by
the constitutively active CAG promoter
(Okabe et al., 1997
;
Pratt et al., 2000
). Recovery
of detectable Myc in day 2, day 3 and day 4 EBs treated with MG132 indicates
that residual Myc transcription generates a labile pool of Myc protein that is
rapidly degraded and not readily detected by immunoblot analysis.
To confirm that downregulation of Myc protein levels following LIF withdrawal involved a T58-dependent mechanism, we evaluated levels of a Myc estrogen receptor (ER) fusion protein and the corresponding mycT58AER fusion driven by the CAG promoter in differentiating EBs. A comparison of cell lines expressing both forms of mycER fusion, in the presence of 4OHT, revealed that the T58A mutant persisted for at least 5 days after LIF withdrawal compared with levels of the T58 form, which had collapsed from day 1 (Fig. 2D). These observations indicate that downregulation of Myc during differentiation occurs through a T58-dependent mechanism.
As Wnt3a activity can contribute to self-renewal of ES cells
(Sato et al., 2004), we asked
if Myc was elevated under conditions of Wnt3a-dependent maintenance and if it
was phosphorylated on T58 after withdrawal of the Wnt3a signal. To test this
idea, we used L-fibroblast conditioned media (CM) containing secreted Wnt3a as
a source of Wnt (see Sato et al.,
2004
). Wnt3a CM supports self-renewal indefinitely in the absence
of LIF, in contrast to CM from a non-secreting L-fibroblast cell line that has
no maintenance activity (Sato et al.,
2004
). Under conditions where ES cells were maintained by Wnt3a
CM, Myc protein levels were elevated and unphosphorylated on T58; furthermore,
GSK3ß kinase activity was low (Fig.
2E). By day 1 after Wnt3a CM withdrawal, we observed marked
elevation of Myc T58 phosphorylation and GSK3ß kinase activity
(Fig. 2E). Activation of
GSK3ß and phosphorylation of T58 both occurred well before downregulation
of Oct4, consistent with earlier observations in LIF maintained ES cells. The
increase in GSK3ß kinase activity is therefore consistent with it
participating in T58 phosphorylation, resulting in Myc degradation following
Wnt3a CM withdrawal (see Discussion). These events are indistinguishable from
those that occur following LIF withdrawal (see
Fig. 2A).
Sustained Myc activity maintains ES cell self-renewal in the absence of LIF
Myc levels rapidly collapse following withdrawal of LIF, indicating that
this may be a requirement for ES cell differentiation. We therefore asked if
sustained Myc levels could promote self-renewal in the absence of
pro-maintenance factors such as LIF. We reasoned that elevated Myc transcript
levels and the absence of T58 phosphorylation could be important in
establishing conditions for ES cell self-renewal. To reproduce conditions in
ES cells where Myc levels are elevated, we constitutively expressed a form of
Myc that evaded T58 dependent proteolysis (mycT58AER) and compared
this with the effects of mycER in self-renewal assays.
Clonally selected puroR D3 ES cell lines expressing either Myc
or MycT58A ER fusion proteins were tested for their ability to
maintain ES cells over 12-15 days. Colony morphology, SSEA1 reactivity (flow
cytometry), alkaline phosphatase (AP) activity and marker transcript profiling
were used as initial readouts for ES cell maintenance. In the presence of
4OHT, mycER/mycT58AER ES cells retained high SSEA1 levels
(Fig. 3A), a uniform
dome-shaped colony morphology (Fig.
3B) and elevated alkaline phosphatase activity
(Fig. 3C) for the first 6 days
(two passages). mycER colonies became more flattened and heterogeneous with
time and eventually displayed low AP and SSEA1 levels although
mycT58AER cells retained these characteristics for over 12-15 days
(4-5 passages; Fig. 3A-C).
Including low levels of LIF (7.5 units/ml), which by themselves have only a
small effect on maintaining AP levels over 15 days, appeared to further delay
the loss of AP activity in mycER cells grown with 4OHT, indicating that LIF
can cooperate with Myc (Fig.
3C). Expression levels of mycT58AER were comparable
with that of endogenous Myc in the cell lines tested
(Fig. 3D). Transcript profiling
showed that mycT58AER cells retained elevated levels of ES cell
mRNA transcripts Oct4 and Rex1 in a 4OHT-dependent manner throughout the
experiment (Fig. 3E). Addition
of 4OHT to control cells had no role in promoting self-renewal, as determined
by colony morphology, transcript profiling and AP activity
(Fig. 3B,E). In parallel
experiments, cell lines constitutively expressing MycT58A from the
CAG promoter were shown to promote self-renewal for more than 15 days (P.C.
and S.D., unpublished), indicating that the ER per se is not contributing to
the ability of Myc to promote self-renewal. When these experiments were
repeated in R1 ES cells, we obtained similar results, indicating that
Myc-dependent self-renewal is not cell line-dependent (see data in Figs
4,
5). We also confirmed that
Myc-dependent self-renewal was completely independent of any STAT3 signaling
by showing that mycT58AER could bypass the dominant-negative
effects of STAT3Y705F (see Niwa
et al., 1998) (S.D., unpublished). These results indicate that the
stable form of Myc normally seen in ES cells is required for maintenance of
self-renewal and stem cell identity, and is likely to work downstream of
LIF/STAT3.
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To determine whether Myc-dependent self-renewal is reversible, we asked if ES cells maintained by mycT58AER for 14 days (-LIF) could differentiate following withdrawal of 4OHT. This was tested by plating cells in suspension under conditions where EBs could form for 7 days in the presence or absence of 4OHT. Under conditions where cells were grown in the presence of 4OHT, Oct4 mRNA and Oct4 protein remained elevated and differentiation markers such as Fgf5 and brachyury mRNAs remained low (Fig. 4D). After withdrawal of 4OHT, however, Oct4 mRNA and Oct4 protein levels decreased significantly, whereas Fgf5 and brachyury transcripts increased (Fig. 4D). These results indicate that myc-maintained ES cells differentiate when Myc is inactivated by removal of 4OHT.
As another test that Myc-dependent self-renewal was reversible, we performed colony forming assays on D3 and R1 ES cells grown for 30 days in the presence or absence of 4OHT (±LIF) to determine the proportion of stem cells present after inactivation of Myc. Cells were plated at clonal density under various conditions and evaluated by colony-forming assay. No major differences were observed between the number of colony-forming cells in LIF versus 4OHT maintained R1 or D3 ES cells (Fig. 4E), indicating that Myc can maintain the ES cell state at a comparable level with LIF. Removal of 4OHT (and LIF) led to a decrease in the number of colony-forming cells in bulk cultures, indicating that when Myc is inactivated, the stem cell pool declines as a result of Myc-dependent self-renewal being reversible.
The remaining scenario that could most probably account for Myc-dependent
self-renewal is that it exerts its effects by imposing a blockade on
differentiation. If this were the case, we would predict that ES cells would
become unstable under conditions when Myc activity is reduced, leading to
differentiation. To investigate this possibility we generated stable cell
lines expressing a mycER fusion where the Myc open reading frame lacked most
of its transactivation domain (myc40-178ER). This and
similar dominant-negative versions of Myc have previously been shown to
promote differentiation in other cell types without necessarily imposing a
cell cycle arrest (Canelles et al.,
1997
; Schreiner et al.,
2001
). Although myc
40-178ER ES cell colonies
could be maintained with LIF indefinitely in the absence of 4OHT,
morphologically they showed obvious signs of differentiation and loss of AP
staining following the addition of 4OHT to bulk cultures
(Fig. 5A,B). This was
associated with a significant decrease in the ability to form AP-positive
colonies when cells were plated at clonal density
(Fig. 5C). These data suggest
that the proportion of self-renewing stem cells had significantly declined as
a consequence of decreased Myc activity, indicating that differentiation had
occurred. These findings were confirmed by showing that expression of specific
transcripts associated with pluripotency, such as Nanog and Oct4, declined
following addition of 4OHT and that mesoderm (brachyury), GATA4
(extra-embryonic endoderm) transcripts were expressed at higher levels
(Fig. 5D). A predominance of
extra-embryonic endoderm and mesoderm is typically produced following LIF
withdrawal from adherent ES cell cultures
(Niwa et al., 2000
) and
although mRNA markers for these lineages were detected, we did not perform
analysis at the single cell level to determine the percentage of different
cell types being generated. We interpret our data to indicate that abrogation
of Myc activity is incompatible with maintenance of ES cell self-renewal.
Together with our previous findings, we conclude that Myc is necessary and
sufficient for ES cell self-renewal.
Myc maintained ES cells retain wide-range differentiation potential
We next evaluated the ability of mycT58AER maintained (+4OHT)
EGFP R1 cells (Hadjantonakis et al.,
1998) to differentiate in an in vivo setting by asking if they
could contribute to the three embryonic germ layers following injection into
blastocyst stage embryos. Cells were first maintained in 4OHT for 30 days and
grown as EBs in the absence of LIF and 4OHT to confirm they differentiate in
vitro by monitoring the downregulation of Oct4 protein
(Fig. 6A). In parallel to this,
cells were directly injected into C57BL/6 blastocyst stage embryos.
mycT58AER cells maintained for 30 days in 4OHT were confirmed to be
AP positive and, when plated onto gelatin coated plastic in the absence of
4OHT, did not form domed-shaped colonies but formed non-uniform, flattened
monolayers indicative of differentiation
(Fig. 6B). Following injection
of EGFP-marked R1 ES cells into C57BL/6 blastocysts, embryos were analyzed at
12.5 dpc for their integration into the three germ layers. The extensive
integration of mycT58AER EGFP-marked cells throughout the embryo
indicates that they contributed to all three embryonic germ layers and hence,
retained pluripotency (Fig.
6C-F). A mock-injected embryo is shown to demonstrate negligible
autofluorescence (Fig. 6C,D).
Injected embryos that developed to term generated live chimeric animals as
judged by chimeric coat color (Fig.
6F). The relative distribution and degree of chimerism of
mycT58AER cells was indistinguishable from non-transfected EGFP ES
cell lines (Fig. 6F). From a
total of 18 injected embryos, more than 90% were judged to be chimeric by EGFP
incorporation. At least ten chimeric animals were generated for each cell
line, each displaying chimeric coat color. Similar results were obtained with
mycT58AER maintained D3 ES cell lines (P.C. and S.D., unpublished).
We cannot rule out the possibility that Myc-maintained ES cells may be more
predisposed to differentiate into certain lineages over others. We have shown
that they can differentiate into cell types representative of the three germ
layers and retain wide-range differentiation potential, even though germline
transmission was not demonstrated. These analyses confirm, however, that
Myc-dependent self-renewal is reversible and identify Myc as a central
maintenance factor of ES cell pluripotency.
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Discussion |
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A problem in deciphering the true function of Myc has been its promiscuous
role in a wide variety of transcriptionally regulated pathways that generate a
diversity of biological outcomes (see
Patel et al., 2004). The
identification of Myc target genes has been particularly problematic, with
over 20 transcription profiling papers failing to reach a clear consensus with
regards to bone fide genomic targets
(Oster et al., 2002
). The
multiplicity of such data underscores the pleiotropic functions of this
transcription factor, indicating that its biological roles are highly
cell-type specific and subject to complex regulation by extracellular signals
and by its binding to cellular co-factors. Analysis of binding sites in human
and Drosophila cells in vivo shows that Myc is associated with an
extraordinarily large number of genomic sites
(Fernandez et al., 2003
;
Orian et al., 2003
),
indicating that it can participate in the regulation of complex genetic
pathways. Although Myc may bind overlapping sets of genes under various
conditions in different cell types
(Fernandez et al., 2003
),
transcriptional responses to Myc binding differs markedly in a context and
cell type-dependent manner (Ellwood-Yen et
al., 2003
). In the case of ES cells, Myc family members may bind
and regulate a cluster of genes required for stem cell maintenance that would
be only partially overlapping with those regulated by Myc in other biological
contexts. Genes identified previously as being specifically associated with
the pluripotent state (Calaveri and
Scholer, 2003
) may in fact be part of the Myc regulatory hierarchy
in ES cells.
Although stem cell markers such as Oct4 and Nanog persist for several days
following LIF withdrawal, ES cells are committed to differentiate within 24 to
36 hours (Boeuf et al., 2001).
This implies that the commitment `decision' occurs long before Oct4 or Nanog
levels are extinguished. The ability of Myc to maintain self-renewal combined
with its rapid downregulation within the first 36 hours following LIF
withdrawal, suggests it to be a key determinant of commitment and could
provide the molecular basis underpinning the commitment phase. Hence, the
inactivation of Myc transcriptional regulators and the activation of pathways
required for Myc degradation could define a `point of no return' where cells
become committed to differentiate. We predict that if mycER was reactivated
within this `window' following LIF or 4OHT withdrawal, ES cells would retain
their stem cell identity but this would not be possible if restoration of Myc
activity was further delayed.
A direct role for Myc in development of pluripotent cells in the
preimplantation embryo has not been previously described nor has it been
implicated through gene knockout studies in the mouse
(Davis et al., 1993;
Stanton et al., 1992
), even
though Myc family members are expressed at elevated levels during this phase
of development (Downs et al.,
1989
). A likely explanation for this is that functional redundancy
among the Myc family masks the function of individual members during early
development in loss-of-function experiments
(Malynn et al., 2000
). This is
consistent with our observations that Nmyc1 can substitute for Myc in
promoting self-renewal of ES cells and that Nmyc1 is regulated in parallel to
Myc at the transcript and protein level (S.D., unpublished). It is not known,
however, if Myc and Nmyc1 are subject to the same regulatory pathways in
pluripotent cells.
Two lines of evidence support a case for involvement of Myc activity in the
development of pluripotent cells in the embryo. First, enforced expression of
an oncogenic form of L-myc (Rlf) blocks normal development of the embryonic
epiblast (MacLean-Hunter et al.,
1994). Second, the obligatory binding partner of Myc, Max, is
essential for early embryonic growth and development
(Shen-Li et al., 2000
). The
essential role of Max in the early embryo therefore implicates an important
role for Myc family members in development of the embryonic epiblast that
would be compatible with it having a role in maintenance and regulation of the
pluripotent state. Pools of maternally derived Myc family members persisting
during early embryonic development could also potentially provide additional
levels of redundancy to ensure the correct progression of pluripotent cells,
reinforcing the robust nature of embryonic development.
Multiple levels of control regulate the Myc dependent ES cell self-renewal pathway
Under conditions where ES cells are maintained by Wnt3a CM, Myc levels are
elevated, T58 is unphosphorylated and GSK3ß activity is suppressed. These
trends are rapidly reversed following Wnt3a CM withdrawal, similar to events
following LIF withdrawal. This raises the possibility that LIF and Wnt
signaling pathways converge on Myc as a common target to promote self-renewal.
Self-renewal of ES cells is dependent on the regulation of Myc at two levels.
First, through transcriptional activation of the Myc gene; and, second, by
establishment of conditions where Myc stability is enhanced through inhibition
of T58 phosphorylation (Fig.
7). Although the LIF-STAT3 pathway is clearly critical for
maintenance of Myc transcription in ES cells, it probably functions in
collaboration with additional signaling pathways, such as those generated by
serum-derived factors. The dual mode of Myc regulation is similar to that
recently reported for regulation of Nmyc1 in cerebellar precursor cells, where
it plays a role in promoting cell division
(Kenney et al., 2004). In this
scenario, transcription is activated by sonic hedgehog through an undefined
mechanism and by increased protein stability achieved through inhibition of
GSK3 by PI3 kinase.
|
Degradation of Myc requires its phosphorylation by GSK3ß on T58,,
leading to its ubiquitin-dependent degradation
(Sears et al., 2000;
Gregory et al., 2003
). The
requirement for a stable form of Myc in ES cell self-renewal is made more
intriguing as GSK3ß inhibition was recently shown to promote self-renewal
of mouse and human ES cells in the absence of LIF or Wnt
(Sato et al., 2004
). These
data suggested to us that suppression of GSK3ß and enhanced Myc stability
could be linked. A role for GSK3ß in promoting differentiation through
T58-dependent degradation is consistent with the kinetics of GSK3ß
activation and Myc T58 phosphorylation following LIF/Wnt3a CM withdrawal.
These observations point towards a scenario where maintenance of ES cells
requires elevated Myc levels, achieved in part through the suppression of
GSK3ß activity. Activation of GSK3ß following LIF/Wnt3a CM
withdrawal can then account for decreased Myc stability and loss of a stable
self-renewal pathway. The stability of Myc in ES cells in unprecedented and is
comparable with the stability of oncogenic forms of the protein associated
with Burkitt's lymphoma. Our findings also provide a potential mechanism for
how the GSK3 inhibitor 6-bromoindirubin-3'-oxime (BIO) promotes
self-renewal of ES cells (Sato et al.,
2004
). We predict that suppression of GSK3 activity by BIO, in the
absence of LIF/Wnt signaling, would establish conditions where Myc was
unphosphorylated on T58, leading to elevated Myc levels and enhanced stem cell
stability. This mechanism may also be applicable to human ES cells where BIO
has a pro-maintenance function (Sato et
al., 2004
).
Our findings establish a mechanism for how ES cells retain the ability to proliferate indefinitely while retaining their pluripotentiality. The identification of Myc as a key regulator of ES cell self-renewal raises two important questions. First, does Myc have a role in maintenance of human ES cells? Second, does Myc have a role in self-renewal in other stem cells that use Wnt-dependent mechanisms? These questions are currently being addressed.
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