Department of Biochemistry, Tufts University
School of Medicine, Boston, Massachusetts 02111
Differentiation is a coordinated process of irreversible cell cycle
exit and tissue-specific gene expression. To probe the functions of the
retinoblastoma protein (RB) family in cell differentiation, we isolated
HBP1 as a specific target of RB and p130. Our previous work showed that
HBP1 was a transcriptional repressor and a cell cycle inhibitor. The
induction of HBP1, RB, and p130 upon differentiation in the muscle
C2C12 cells suggested a coordinated role. Here we report that the
expression of HBP1 unexpectedly blocked muscle cell differentiation
without interfering with cell cycle exit. Moreover, the expression of
MyoD and myogenin, but not Myf5, was inhibited in HBP1-expressing
cells. HBP1 inhibited transcriptional activation by the MyoD family
members. The inhibition of MyoD family function by HBP1 required
binding to RB and/or p130. Since Myf5 might function upstream of MyoD,
our data suggested that HBP1 probably blocked differentiation by
disrupting Myf5 function, thus preventing expression of MyoD and
myogenin. Consistent with this, the expression of each MyoD family
member could reverse the inhibition of differentiation by HBP1. Further
investigation implicated the relative ratio of RB to HBP1 as a
determinant of whether cell cycle exit or full differentiation
occurred. At a low RB/HBP1 ratio cell cycle exit occurred but there was
no tissue-specific gene expression. At elevated RB/HBP1 ratios full
differentiation occurred. Similar changes in the RB/HBP1 ratio have
been observed in normal C2 differentiation. Thus, we postulate that the
relative ratio of RB to HBP1 may be one signal for activation of the
MyoD family. We propose a model in which a checkpoint of positive and negative regulation may coordinate cell cycle exit with MyoD family activation to give fidelity and progression in differentiation.
 |
INTRODUCTION |
During terminal differentiation,
cells undergo an irreversible cell cycle withdrawal followed by the
expression of tissue-specific markers to specify the final phenotype.
The progression of differentiation is a precisely coordinated event in
which the irreversible cell cycle arrest is tightly coupled to the
expression of the tissue-specific genes. This communication of general
and tissue-specific pathways guarantees fidelity by ensuring that
appropriately arrested and viable cells proceed to the last steps in
tissue biogenesis. A lapse in this coordination can give uncontrolled
proliferation of otherwise-differentiated cells, a hallmark of
preneoplastic changes that may eventually result in cancer.
Alternatively, this lapse can yield defects in development, in which
the balance of proliferation and differentiation must be tightly
maintained. Despite the importance of these effects, the cellular and
molecular mechanisms underlying the coordination of general and
tissue-specific events in differentiation have not been extensively
addressed.
Muscle cells represent the best-characterized differentiation system,
with the landmark discovery of the MyoD transcription factor
family (MyoD, myogenin, Myf5, and Mrf4) as critical regulators of
muscle determination and differentiation (reviewed in reference 32). The major function of this family of basic
helix-loop-helix proteins is to form heterodimers with ubiquitous basic
helix-loop-helix E proteins which then activate muscle-specific genes.
The transcriptional activation is achieved through direct binding to
E-box elements (CANNTG), which are present in the promoters of
numerous muscle-specific genes and of each MyoD family member (54,
57). There is considerable complexity with extensive functional
redundancy and autoactivation among MyoD family members. Gene knockout
studies have demonstrated that Myf5, MyoD, and myogenin are critical
for normal muscle differentiation during development. Either Myf5 or
MyoD is required, and recent studies have suggested that Myf5 may be
upstream of MyoD (reviewed in reference 36). The
elegant characterization of the MyoD family function provides a
necessary backdrop for the elucidation of the important mechanisms that
activate the tissue-specific differentiation pathways. Additionally,
the C2C12 muscle cell line represents a feasible model system for
probing fundamental mechanisms of differentiation.
The retinoblastoma family of growth suppressor proteins (RB,
p130, and p107) are critical players in general cell cycle
regulation (see reference 11 and reviews within). A
major paradigm is that RB blocks G1 progression by
inhibiting the E2F family of transcription factors. E2Fs are required
for the activation of numerous genes that are necessary for
G1-to-S progression (see reference 11 and reviews within). However, the functions of RB, p130, and p107 are
not limited to G1 regulation and are important in
other cellular processes, such as differentiation and apoptosis
protection (reviewed in reference 53). These diverse
functions suggest a critical and broad role in cellular
regulation.
During differentiation in muscles and other tissues, RB and p130
expression is increased but p107 expression declines markedly. In
quiescent and differentiated cells, the major E2F complex contains p130
(7, 8, 25, 43). We have shown that this p130-E2F complex
coincides with transcriptional repression through E2F elements
(43). This E2F-p130 complex is distinctive for
G0 but not G1 (45). These
observations are also consistent with the view that terminal
differentiation may be an "irreversible G0" state. In
addition, complexes of RB and E2F4 have been observed in certain
differentiated cells (17). RB appears to be critical for
maintaining the irreversible cell cycle arrest that occurs with
differentiation, since RB
/ differentiated muscle
cells could reenter the cell cycle (30, 41, 56). Recent
studies with transgenic mice have demonstrated that a threshold level
of RB is necessary for the characteristic irreversible cell cycle
arrest during muscle development in animals (56).
Recent studies have indicated a surprising function for RB in
tissue-specific gene expression in adipocyte and muscle differentiation (4, 30). The expression of tissue-specific genes in
adipocyte and muscle differentiation is dictated by the functions of
C/EBP and MyoD family transcription factors, respectively (reviewed in references 32 and 55). RB is
necessary for adipocyte differentiation, as RB/
cells fail to undergo adipogenesis. One molecular mechanism is a direct
physical interaction of RB with C/EBP-
, resulting in the
transcriptional activation of adipocyte-specific genes (4). In muscle tissue, RB augments the transcriptional ability of MyoD in
the expression of muscle-specific genes (30). In muscle
tissue, the molecular mechanism probably does not occur through a
direct physical interaction since efforts to demonstrate a direct
interaction have had mixed results (14, 30). Recent work by
Kaelin and Lee has provided new evidence that the function of RB in
MyoD activation and E2F regulation can be uncoupled by selective
mutations. Both the N-terminal and pocket regions are required for the
activation of differentiation in the cellular and animal models
(37, 42).
Because of their dual roles in both cell cycle control and
differentiation, RB and its targets are excellent candidates for studying the mechanisms that coordinate general cell cycle and tissue-specific regulation during differentiation. While E2F is certainly one RB and p130 target in differentiation, other cellular targets may also be necessary for cell cycle arrest and
differentiation. A simple argument is that the concentration of RB
family members is vastly greater than that of the E2Fs. To address the
complex role of RB in differentiation, we and others have recently
isolated HBP1 as a novel target of p130 and RB from differentiated
muscle cells and from developing murine tissues (19, 49).
HBP1 is a new member of the sequence-specific HMG box proteins, which include LEF1, SRY, and TCF and which have all been linked to
differentiation and signaling (for reviews, see references 13,
15, and 31). HBP1 is a sequence-specific
transcriptional repressor that contains the consensus LXCXE, or RB
interaction, motifs. However, HBP1 interacts only with RB and p130 and
not with p107 (49). Three independent observations suggest
potential functions of HBP1 in muscle differentiation. First, HBP1, RB,
and p130 are all increased with differentiation (8, 23, 49).
Second, expression of the HBP1 protein represses the promoter of the
N-MYC gene, a protooncogene that becomes downregulated in
differentiating cells. Third, HBP1 expression can elicit cell cycle
arrest, which is a necessary feature of terminal differentiation. Thus,
our previous work suggests that HBP1 may promote the early stages of
differentiation by facilitating cell cycle arrest through
transcriptional repression of key cell cycle genes (49).
In the present study we have uncovered an additional and unexpected
function of HBP1 in differentiation. Because overexpression of HBP1
gave efficient cell cycle arrest and HBP1 was normally induced with
differentiation, we expected that HBP1 expression would enhance
differentiation. Surprisingly, the expression of HBP1 blocked full
terminal differentiation in muscle cells without blocking cell cycle
exit. The HBP1 phenotype was distinct from the findings of studies with
oncogenes that inhibited differentiation by preventing the required
initial step of cell cycle exit. The HBP1 block was selective,
as the expression of myogenin and MyoD was abolished. Yet,
Myf5 expression was normal, which suggested that HBP1
might inhibit Myf5 functions to prevent expression of MyoD.
Consistently, HBP1 was able to block transcriptional activation of the
MyoD family and the reexpression of MyoD family members did restore
differentiation in HBP1-expressing lines. Further investigation
revealed that the relative ratio of RB to HBP1 appeared to dictate the
progression of differentiation. Low RB/HBP1 ratios yielded cell
cycle exit but inhibited tissue-specific gene expression. In contrast,
high RB/HBP1 ratios yielded full differentiation. Similar changes
were manifested in endogenous C2 muscle cell differentiation. Our
experimental observations are consistent with a new model in which the
relative ratio of RB to HBP1 may constitute a signal for MyoD family
regulation. These and other new studies suggest that the coordination
of cell cycle arrest with tissue-specific gene expression in
differentiation may involve positive and negative regulation by HBP1
and RB.
| |
MATERIALS AND METHODS |
Plasmids. (i) Mammalian expression constructs.
pEF-BOS HBP1
wild-type and mutant constructs were as described previously
(49) (Fig. 1). pEMSV-MyoD and pEMSV-myogenin were kindly
provided by E. Olson. pCMV-HA-Myf5 was subcloned from EMSV-Myf5 (provided by E. Olson). pCMV-HA-RB was a gift from Q. Sheng and B. Schaffhausen.
(ii) CAT reporter constructs.
MCK 4800-CAT was a
generous gift from E. Olson. 4R-CAT was a generous gift from
the late Harold Weintraub (51).
Cell culture, establishment of stable cell lines, and
transfections.
C2C12 myogenic cells were cultured in
Dulbecco's modified Eagle medium (DMEM) supplemented with 15%
(vol/vol) fetal calf serum (FCS). Myogenic differentiation was
induced by growing cells in DMEM supplemented with 2% FCS.
To establish stable HBP1 cell lines, C2C12 cells were transfected by
the calcium phosphate precipitation method with 30 µg of pEF-BOS HBP1
and 3 µg of TK-hygro. Stably transfected cells were selected in
culture medium containing 250 µg of hygromycin B per ml (Calbiochem).
Colonies of hygromycin-resistant cells were isolated approximately 10 days after selection and were propagated. HBP1 stable cell lines were
screened by immunoprecipitation-Western analysis. Two HBP1 cell lines,
designated B1-C2 and B2-C2, were further analyzed.
To reverse the nondifferentiation phenotype, 10 µg of
pEMSV-MyoD, pEMSV-Myf5, or pEMSV-myogenin was transfected
into either B1-C2 or B2-C2 together with 5 µg of Rous sarcoma
virus-
-galactosidase (-Gal) as a cotransfection marker. The
cells were exposed to the DNA precipitates for 24 h in DMEM plus
15% FCS and were then grown in DMEM plus 2% FCS for an additional
40 h. Immunofluorescence staining was subsequently performed on
the transfected cells.
To establish stable RB and HBP1 cell lines, B1-C2 cells were
transfected by the calcium phosphate precipitation method with 30 µg
of pCMV-RB and 3 µg of pEF-1-puro. Stably transfected cells were
selected in culture medium containing 6 µg of puromycin per ml
(Calbiochem). Colonies of puromycin-resistant cells were isolated approximately 7 days after selection and were propagated. RB cell lines
were screened by Western analysis by using an anti-RB monoclonal antibody (Pharmingen). One RB cell line, designated B1-B9, and three
control puromycin-resistant lines were analyzed for differentiation.
Transient assay for differentiation.
The effect of
overexpression of HBP1 on C2C12 differentiation was evaluated in a
transient-transfection experiment. C2C12 cells grown on coverslips were
transfected with plasmids encoding wild-type or mutant HBP1, Myf5, and
-Gal. Within 24 h of transfection, the cells were grown in
medium supplemented with 2% FCS for another 40 h. Cells were
fixed and immunostained for -Gal and myosin heavy chain (MHC) to
visualize transfected and differentiated cells, respectively. The
percentage of differentiated cells was determined as a ratio of
double-positive (MHC+ [differentiated];
-Gal+ [transfected]) to total -Gal-positive
(transfected) cells. Each transfection was repeated three times, and
cells on two different coverslips from each experiment were counted.
Generally, 200 to 300 cells were counted for each experiment.
Immunoprecipitation and Western blotting.
Cells were lysed
on plates in TNN buffer (50 mM Tris-HCl, pH 7.4; 100 mM NaCl; 5 mM
EDTA; 0.5% Nonidet P-40; 1 µg pepstatin per ml; 0.5 mM EGTA; 200 µM phenylmethylsulfonyl fluoride [PMSF]; 0.5 mM dithiothreitol
[DTT]; 1 µg of leupeptin per ml). Cell lysates were precleared with
protein A-Sepharose beads before antibodies were added. The immune
complexes were formed for 1 h at 4°C with gentle agitation and
then collected onto protein A-Sepharose beads by gentle agitation at
4°C for another hour. After washes with TNN buffer, the beads were
boiled in sodium dodecyl sulfate (SDS) sample buffer for 10 min, and
the supernatant was analyzed by SDS-polyacrylamide gel electrophoresis
(PAGE) and Western blot analysis enhanced chemiluminescence (with ECL;
Renaissance).
For detection of HBP1 transgene product or transfected HBP1, anti-HBP1
rabbit antisera were used for immunoprecipitation, and monoclonal
antihemagglutinin (anti-HA) antibody 12CA5 was used to detect the
HA-tagged HBP1 transgene product by Western blot analysis. For
detection of HA-Myf5, anti-Myf5 rabbit antisera were used for
immunoprecipitation and 12CA5 was used in the Western analysis. For
detection of the RB transgene product, cell lysates were
immunoprecipitated with anti-RB antibodies (Santa Cruz), and RB was
detected with a monoclonal anti-RB antibody (Pharmingen) by Western
blot analysis.
For Western blotting of myogenin and MHC, antimyogenin monoclonal
antibody F5D and anti-MHC monoclonal antibody FS9 were used, respectively (generous gifts of Woody Wright and Frank Stockdale, respectively).
Immunofluorescence staining.
C2C12 cells, grown on
coverslips (Fisher Scientific), were fixed in 30% methanol-70%
acetone for 20 min at 
20°C. The coverslips were air dried and were
rehydrated in phosphate-buffered saline (PBS) for 3 min. Each coverslip
was covered with 50 µl of appropriately diluted primary antibodies
and incubated at 37°C for 1 h in a humidified chamber. After
several washes with PBS, each coverslip was covered with 50 µl of
appropriately diluted secondary antibodies and then incubated at 37°C
for 1 h in a humidified chamber. The coverslips were then washed
three times with PBS, and the cells were counterstained with 50 µl of
Hoechst dye for 5 min at room temperature. The coverslips were washed
once in PBS and once in distilled water and mounted on slides with 50%
glycerol-50% distilled water. Immunofluorescent cells were visualized
under a microscope and counted.
For detection of transfected cells, rabbit anti--Gal (final
concentration, 36 µg/ml; 5 Prime
3 Prime) was used as the
primary antibody combined with rhodamine-conjugated goat
anti-rabbit secondary antibodies (Jackson). For detection of
differentiated cells, anti-myosin heavy-chain monoclonal antibody FS9
was used as primary antibody combined with fluorescein
isothiocyanate-conjugated donkey anti-mouse secondary antibodies
(Jackson).
For detection of cells that incorporated bromodeoxyuridine (BrdU),
cells on coverslips were first treated with 2 N HCl for 1 h at
37°C before incubation with primary mouse anti-BrdU antibody (Boehringer Mannheim). BrdU-positive cells were stained with
fluorescein-conjugated donkey anti-mouse secondary antibodies.
T2 RNase protection assay.
Total cellular RNA was isolated
with Trizol reagent (Sigma) according to the manufacturer's directions
and treated with RNase-free DNase (Amersham) to remove residual DNA. T2
RNase protection assays were performed as previously described
(48). The probes were derived from murine MyoD (gift of the
late H. Weintraub [51]), murine Myf5 (gift of Yukang
Wang, murine HBP1 cDNA (49), or pTRI-GAPDH-mouse template
(Ambion).
CAT assays.
C2C12 cells were scraped off the plates 48 h after transfection and were lysed in lysis buffer (250 mM Tris-HCl,
pH 7.5; 1 mM EDTA; 1 µg of pepstatin per ml; 0.5 mM EGTA; 200 µM
PMSF; 1 µg of leupeptin per ml) by four cycles of freezing and
thawing. The amounts of CAT protein in cell extracts were determined
with a chloramphenicol acetyltransferase (CAT) enzyme-linked
immunosorbent assay kit (Boehringer Mannheim) according to the
manufacturer's specifications and with a linear standard curve. All
transcription data were normalized for transfection efficiency with
-Gal, whose activity was quantitated by an ONPG
(o-nitrophenyl--D-galactopyranoside) assay
and by using a linear standard curve. The normalized reporter activity
was expressed as a ratio of nanograms of CAT to units of -Gal.
Cell labeling and immunoprecipitations.
C2C12 cells
that were grown in 100-mm-diameter tissue culture plates were
differentiated for 4 days in DMEM complemented with 2% FCS. After
cells were washed twice with methionine-free DMEM (GIBCO), the cells
were preincubated in methionine-free DMEM for 20 min. The cells were
then labeled for 4 h with 1 mCi of 35S-methionine
label per 100-mm dish in methionine-free DMEM supplemented with 2%
FCS. The cells were washed with cold PBS and lysed in TNN buffer on ice
for 20 min. The cell lysates were collected and were briefly
centrifuged at 6,000 rpm at 4°C. The supernatants were pooled,
precleared by incubation with protein A-Sepharose beads, and divided
into three portions. One-twelfth of the extract was used as a control
for total RB expression by a double immunoprecipitation with anti-RB
antibodies (Santa Cruz). The remaining 11/12 of the extract were
divided into two portions and immunoprecipitated with anti-HBP1
antibodies for 1 h at 4°C. The immune complexes were collected
onto protein A-Sepharose beads over a 1-h period at 4°C, and the
beads were washed three times with TNN buffer. The beads were then
boiled in release buffer (50 mM Tris-HCl, pH 7.4; 1% SDS; 5 mM
DTT) for 15 min and then chilled on ice. The supernatants were
collected, and TNN buffer was added to each sample to a total volume of
1 ml. The second immunoprecipitations were carried out overnight at
4°C with anti-RB or control (anti--Gal) antibodies, and the immune
complexes were again collected onto protein A-Sepharose beads. After
the beads were washed four times with TNN buffer, they were boiled in
SDS sample buffer and the proteins were resolved by SDS-7% PAGE and
visualized with a phosphorimager after a 3-month exposure.
| |
RESULTS |
HBP1 inhibits differentiation by altering the MyoD family
expression pattern.
Differentiation can be divided into two
stages: initiation of an irreversible cell cycle arrest and expression
of tissue-specific genes. Our studies have implicated a role for HBP1
in cell cycle control during C2C12 muscle cell differentiation
(49). To directly investigate the potential role of HBP1 in
the full muscle differentiation program, we established two stable C2
cell lines that constitutively expressed HBP1. The HA-tagged rat
HBP1 transgene products in these two mouse lines, designated
B1-C2 and B2-C2, were detected by immunoprecipitation with anti-HBP1
antibodies followed by Western analysis with an anti-HA antibody (Fig.
2). Because rat and mouse HBP1 could be
distinguished in RNase protection assays, we determined that the
relative expression of exogenous rat HA-HBP1 was modestly increased
over the endogenous levels in the stable lines (3.7-fold in B1-C2 and
5.7-fold in B2-C2 [data not shown]). The overall levels of HBP1 in
the cell lines were similar to the expression of HBP1 in fully
differentiated C2 myotubes (49).
When these two cell lines were subjected to differentiation conditions,
a surprising result was that terminal differentiation was completely
inhibited. As shown in Fig. 3, the
expression of several molecular markers of differentiation
MyoD,
myogenin, and MHCwas undetectable in B1-C2 and B2-C2, suggesting that
the expression of HBP1 had blocked terminal differentiation. In
contrast, the differentiation markers were easily detected in a control
hygromycin-resistant line, verifying the expected efficient
differentiation under the same conditions. In addition to defective
expression of differentiation markers, B1-C2 and B2-C2 cells did
not undergo the differentiation-specific event of myotube formation,
even after prolonged incubation (8 days) in differentiation media.
Myotube formation began at 72 h in normal C2C12 cells and three
control hygromycin-resistant lines (data not shown).
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FIG. 2.
Detection of HBP1 transgene product in B1-C2 and B2-C2
cells. Cell lysates from each cell line were prepared, and the level of
HBP1 transgene product in each cell line was detected by
immunoprecipitation with anti-HBP1 antibodies followed by Western
analysis with anti-HA (12CA5) antibody as described in Materials and
Methods. The analyzed lysates are depicted in the figure as follows:
lane 1, normal C2 cells; lane 2, B1-C2 HBP1 expressing line; lane 3, B2-C2 HBP1 expressing line.
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Although differentiation was clearly blocked in the HBP1-expressing
cell lines, this did not reflect a global inhibition in the expression
of all MyoD family members. While MyoD and myogenin expression was
clearly absent, Myf5 expression was surprisingly unaffected in the HBP1
cell lines (Fig. 3). This apparently normal Myf5 expression also
indicated that the inhibition of muscle differentiation by HBP1
expression was not due to reversal of the myogenic phenotype, since
Myf5 served as a marker of the myogenic lineage. The expression of Mrf4
was not tested, since its expression is limited to mature muscle
fiber and is not generally manifested in tissue culture (reviewed
in reference 32). Thus, we conclude that HBP1
blocks differentiation by interfering with the expression of MyoD
and myogenin but not the expression of Myf5. Consistent with our
finding, genetic studies in mice have indicated that Myf5 is
functionally upstream of MyoD during muscle development (36,
47).
To confirm that the differentiation block in B1-C2 and B2-C2 was not
due to aberrant clonal selection of cell lines, we verified our results
in an experiment involving transient expression of HBP1. The design of
the transient differentiation is based on the observation that complete
differentiation in normal C2 cells was a relatively slow process (~96
h). The expression of Myf5 (or other MyoD family members) could
accelerate the terminal differentiation program to give complete
differentiation at ~48 h. Thus, the time line of the experiment was
transfection of undifferentiated cells followed by a 40-h incubation in
differentiation medium. The transfected cells were marked by the
coexpression of -Gal. By immunofluorescent staining, we then counted
the cells that were doubly positive for the expression of -Gal
(transfected cells) and for MHC (differentiated cells). The percentage
of differentiation was determined as the portion of MHC-positive cells
in the transfected cell population.
As shown in Fig. 4A, Myf5 did induce
nearly complete differentiation in the transfected population (lane 1).
Coexpression of HBP1 and Myf5 decreased the percentage of
differentiated cells (lane 2, 40 ± 10%), whereas coexpression of
the DNA binding domain of HBP1 (
HBP403-513) caused little inhibition
of differentiation (lane 3, 87.6%). Under the assay conditions,
20 ± 8% of control transfected cells exhibited differentiation
in the absence of exogenous Myf5 expression, and this level of
differentiation constituted the baseline of the assay (lane 4). As
shown in Fig. 4B, the inhibition of differentiation by HBP1 was not due
to loss of exogenous Myf5 expression, which was similar irrespective of
HBP1 coexpression (Fig. 4B, lanes 2 to 4). This experiment
demonstrated that the differentiation efficiency in the presence
of HBP1 approached that of the control in which there was no
exogenous Myf5 expression (compare columns 2 and 4 in Fig. 4A).
Therefore, we conclude that HBP1 can efficiently inhibit terminal
differentiation when expressed either stably or transiently and provide
independent support for a functional role of HBP1 as a dominant
inhibitor in C2C12 differentiation.
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FIG. 3.
Expression levels of terminal differentiation markers.
The levels of MHC, myogenin, MyoD, and Myf5 were scored in B1-C2,
B2-C2, and control cell lines. For myogenin and MHC, the protein levels
were detected by Western blot analysis with monoclonal antimyogenin
(F5D) and monoclonal anti-MHC (FS9) antibodies, respectively, in cell
lysates prepared from each line. For MyoD and Myf5, the RNA levels were
quantitated in a T2 RNase protection assay with total RNA that was
isolated from each cell line. GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) was employed as an RNA loading control. The tested cell
lines are indicated: lanes 1 and 2, B1-C2; and lanes 3 and 4, B2-C2;
and lanes 5 and 6, hygromycin-resistant control line without HA-HBP1.
The odd numbers represent undifferentiated conditions (15% serum) and
are denoted "U.". The even numbers represent differentiated
conditions (2% serum) and are denoted "D." A representative
experiment is shown here, and identical results were obtained in two
independent analyses.
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To understand the molecular basis for the block in differentiation, we
first sought to determine whether HBP1-expressing cells could exit the
cell cycle. Many oncogenes could inhibit differentiation by preventing
cell cycle exit (for a review, see reference 22). Our existing evidence already strongly suggested that HBP1 enhanced, rather than prevented, cell cycle exit. First, in contrast to oncogenes
that could stimulate S-phase entry, the expression of HBP1 directly
inhibited cell cycle progression in C2 cells (49). Second,
in a growth suppression assay, HBP1 led to a threefold inhibition of
colony formation, suggesting that HBP1 had a moderate growth
suppression effect rather than enhancing proliferation (unpublished
data). In contrast, expression of oncogenes that could inhibit
differentiation by preventing cell cycle arrest (e.g., RAS [18,
33]) generally gave increased colony formation in these same
assays. Third, the B1-C2 and B2-C2 HBP1-expressing lines
exhibited somewhat reduced growth rates (data not shown), a
finding again inconsistent with oncogenic transformation. Lastly, we
used BrdU incorporation as an assay for S phase. As shown in Fig.
5, the efficiencies of cell cycle exit in
response to serum deprivation were equivalent in normal C2C12, control
hygromycin-resistant, and HBP1-expressing B1-C2 and B2-C2 lines. Thus,
we conclude that the block of differentiation by HBP1 is not due to
defects in cell cycle exit. These data further suggest that HBP1
affects events downstream of cell cycle exit in the full
differentiation pathway.
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FIG. 4.
Overexpression of HBP1 inhibited C2C12 differentiation
in a transient-differentiation assay. A transient-differentiation assay
was devised to score the effects of HBP1 on C2C12 differentiation. The
basis of the assay was that expression of MyoD family members could
accelerate C2 cell differentiation. (A) C2C12 cells grown on coverslips
were transfected with plasmids encoding wild-type or mutant HBP1, Myf5,
and -Gal. After 24 h, the transfected cells were cultured in
medium supplemented with 2% FCS for another 40 h. Cells were
fixed and immunostained for -Gal and MHC. The percentage of
differentiated cells (MHC positive) among the transfected cells
(-Gal positive) was determined. Each transfection was repeated three
times, and cells on two different coverslips from each experiment were
counted. The total number of cells counted for each experiment was 200 to 300. All lanes contain -Gal and the following additions: lane 1, Myf5 (filled column); lane 2, Myf5 and wild-type HBP1 (vertical
stripes); lane 3, Myf5 and HA-HBP403-513 (open column); and lane 4, no addition (horizontal stripes). (B) To measure protein expression
levels, the lysates from a parallel experiment were analyzed for the
expression of HA-Myf5, HA-HBP403-513, and HA-HBP1 by using
immunoprecipitation with anti-Myf5 or anti-HBP1 antibodies,
respectively, followed by Western blot analyses of immune complexes
with the anti-HA antibody. The constructs and antibodies are described
in Materials and Methods. A representative experiment is shown. (Ba)
Western blot showing Myf5 expression. The assay was performed as
described for panel B. Cells were transfected with -Gal and Myf5
(lane 4), Myf5 and HBP1 (lane 3), Myf5 and HBP403-513 (lane 2), or
no other expression vector (lane 1). (Bb) Western blot showing HBP1
expression. Cells were transfected with -Gal, Myf5 and HBP1 (lane 1)
or Myf5 (lane 2). (BC) Cells were transfected with -Gal and
Myf5 and HBP403-513 (lane 1) or Myf5 (lane 2).
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Expression of MyoD family members restores full
differentiation.
The previous experiments suggested that HBP1
uncoupled the tightly coordinated processes of cell cycle exit and of
tissue-specific gene expression. We next asked if the inhibition of
differentiation by HBP1 could be reversed by expression of MyoD family
members. On a practical level, a positive outcome would predict the
location of the differentiation step that was affected by HBP1 and
would further argue that HBP1 did not block differentiation by
nonspecific and pleiotropic means. Instead, ectopic expression of HBP1
probably interfered with "appropriate" signals to activate
tissue-specific genes. Mechanistically, any restoration of
differentiation by MyoD family members would be informative. A positive
result might suggest that HBP1 blocked the transcriptional function of
Myf5 and the subsequent activation of the MyoD and myogenin promoter via E-box elements (5, 50, 54, 57). Thus, we hypothesized that reexpression of Myf5, MyoD, or myogenin should complement the
differentiation defect imposed by HBP1. A positive result would further
suggest that these MyoD family members could be downstream of HBP1.
We transiently expressed Myf5, MyoD, and myogenin into control
hygromycin-resistant or HBP1-expressing cells and scored MHC expression
as a marker for differentiation. As described above, a -Gal
expression vector was used as a cotransfection marker, and a
double immunostaining assay was used to quantitate the percentage of
transfected cells (-Gal positive) that were also differentiated (MHC
positive). As controls, the expression of Myf5, MyoD, or myogenin led
to efficient differentiation (Fig. 6A and
B), whereas -Gal alone gave little
differentiation (5 to 10%; indicated by "No" in Fig. 6). Together,
these data verified that differentiation was controlled by the
exogenous MyoD family member.
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FIG. 5.
Suppression of S phase in HBP1-expressing cells upon
serum deprivation. The HBP1-expressing cell lines (B1-C2 and B2-C2) and
the control lines were assayed for the ability to exit the cell cycle
upon serum deprivation. BrdU incorporation was used as a measure of the
S phase, and quiescent cells should exhibit a reduction in
BrdU-positive cells. All cell lines were grown on coverslips in
differentiation medium for the indicated time period followed by a 1-h
pulse of BrdU labeling. Cells were subsequently fixed and immunostained
for BrdU. The percentages of BrdU-positive cells were determined by
counting the cells from several random fields; approximately 200 to 300 cells were counted for each column. As controls, normal C2 and control
hygromycin-resistant C2 cell lines were utilized. The open, filled, and
diagonal striped bars represent the proliferating, 6-day-deprived, and
9-day-deprived cell populations for each cell line, respectively.
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As shown in Fig. 6C and D, the reexpression of Myf5, MyoD, or myogenin
could partially restore differentiation in both of the HBP1-expressing
lines, as determined by an increase in the number of MHC-positive
cells. In the absence of any MyoD family member expression, there was
no detectable differentiation in the HBP1-expressing lines, consistent
with the initial analysis in Fig. 2. Myogenin was the least effective
for unknown reasons. From Fig. 6 we conclude that the HBP1
differentiation block is not a result of a nonspecific impairment in
the intrinsic differentiation program generated during the selection of
the cell lines. Furthermore, these results suggest that MyoD family may
act downstream of HBP1 in a muscle differentiation pathway.
HBP1 blocks transcriptional activation by the MyoD family.
If
HBP1 blocked differentiation upstream of the MyoD family, a logical
mechanism would be interference with the transcriptional activation
functions of the MyoD family. This potential mechanism would abolish
not only expression of downstream muscle-specific genes but also
autoactivation among MyoD family members (5, 50, 54, 57). To
assess potential regulation by HBP1 of transcriptional activation by
MyoD family members, both natural differentiation-specific (muscle
creatine kinase [MCK]) and synthetic E-box promoters (4R-CAT) were utilized in standard transcriptional assays. The natural promoter provided information on transcriptional regulation in the
context of a complex promoter regulated by differentiation. The
synthetic promoter measured effects directly through the isolated E-box
element that was specific for MyoD family members. While the roles of
individual MyoD family members have been unraveled in genetic and
developmental studies, all members were functionally equivalent in
tissue culture with respect to transcriptional activation (reviewed in
reference 32). Thus, our experiments could only address whether HBP1 could block the general transcription function of
the MyoD family and could not evaluate the functions of any specific
member. Therefore, we used Myf5, MyoD, and myogenin interchangeably in
our transcription assays and obtained similar results.
As expected, the expression of MyoD, Myf5, and myogenin led to
significant activation of the synthetic E-box (4R-CAT) and of the
natural MCK promoters. Noticeably, the coexpression of HBP1 resulted in
a large inhibition of activation in each case (Fig.
7A). By Western analysis, the transfected
MyoD family members were expressed at the same levels regardless of
HBP1 expression (data not shown). Thus, the inhibition of HBP1 was not
a result of defective MyoD family expression. The N-terminal repression domain of HBP1 was required for inhibition of the MyoD family transactivation (as represented by myogenin). Despite efficient expression (Fig. 4B), the HBP1 mutant HBP403-513 was not functional (Fig. 7B). Thus, we conclude that expression of HBP1 can elicit efficient inhibition of transcriptional activation by MyoD family members and that this function depends upon the N-terminal domain of
HBP1.
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FIG. 6.
Expression of MyoD family members can overcome
inhibition of differentiation in the B1-C2 and B2-C2 lines. The purpose
of this experiment was to determine whether MyoD family members could
rescue the differentiation defect imposed by HBP1. A modification of
the transient-differentiation assay in Fig. 4 was used. Expression
vectors encoding MyoD, Myf5, or myogenin were transiently transfected
into either control or HBP1-expressing cell lines (B1-C2 or B2-C2). A
-Gal expression vector was cotransfected to identify the transfected
cells in each experiment. At 24 h posttransfection, cells were
grown in differentiation medium for an additional 40 h before they
were stained with antibodies. Double immunostaining for differentiated
and transfected cells was performed with anti-MHC monoclonal antibody
and anti--Gal antisera, followed by staining with
fluorescein-conjugated anti-mouse immunoglobulin G and
rhodamine-conjugated anti-rabbit immunoglobulin G, respectively. The
percentages of differentiated and transfected cells were quantitated
from approximately 200 to 300 cells for each experimental point. The
indicated cell lines are as described in the legend to Fig. 5. The
percentage of MHC-positive cells was determined for MyoD (horizontal
stripes), Myf5 (open column), myogenin (diagonal stripes), or -Gal
(filled; denoted "No").
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While HBP1 functioned as a sequence-specific transcriptional
repressor (49), the apparent inhibition of MyoD
transcriptional activation was probably not a result of DNA
binding. First, HBP1 did not bind to the E-box element in an in vitro
gel shift assay (data not shown). Second, it was important to note that
the inhibition by HBP1 was specific for transcriptional activation by
MyoD family members and did not reflect an inhibition of general
transcriptional function. Other control promoters such as B-MYB were
not repressed upon HBP1 expression (49). Similarly, the
basal activity of the E-box reporter was also not directly affected by
HBP1 expression (see Fig. 8C, lane 2). Third, our preliminary evidence
on direct physical interactions of HBP1 and MyoD family members was
mixed. We could demonstrate a specific physical interaction of HBP1
with all MyoD family members in glutathione S-transferase
(GST) capture assays, but specific in vivo interactions were not
detected (data not shown). While the block in transcriptional
activation was specific, HBP1 might target other cofactors
necessary for MyoD transcriptional activation (see Discussion), but the
precise physical interactions were not yet clear. While functionally
important, the physical interactions might also be transient and
impossible to detect by immunoprecipitations that required stable
contacts. However, these and previous studies suggest that
HBP1 may elicit repression in different contexts but that not all
promoters are equally affected.
Expression of RB reverses the HBP1 inhibition of MyoD
family transcriptional activation and of cell differentiation.
The observations in Fig. 2 to 7 suggested that HBP1, a target for RB
and p130, had an apparent negative role in differentiation. Since
previous work suggested a cooperative role of RB and MyoD in promoting
muscle differentiation (30), we sought to determine whether
RB could overcome the block in differentiation and in MyoD family
activation by HBP1. A positive outcome would suggest that the
activation of terminal differentiation genes might result from both
positive activation by MyoD family members and RB-mediated relief of
negative repression by HBP1.
To address whether RB could activate the tissue-specific aspects of
differentiation, we generated RB cell lines in the background of
one HBP1 cell line, B1-C2. We reasoned that the relatively low level of
HBP1 expressed in this line could be more easily counteracted by RB
expression. Using a second antibiotic (puromycin) selection marker, we
isolated 10 lines from B1-C2 cells transfected with CMV-HA-RB. Only one
(B1-B9) contained a detectable amount of RB transgene as determined by
anti-HA immunoblot analysis (data not shown), a finding consistent with
the known potent growth suppression ability of RB. This line contained
a higher overall level of RB compared to control and C2C12 lines (Fig.
8A). The ability of this line to
differentiate was examined by detection of MHC-expressing cells by
using the immunostaining assay. We found that when subjected to
differentiation conditions this cell line could now partially
differentiate, containing ~15% of MHC-positive cells (Table
1). Three control sister cell lines were
isolated by double antibiotic selection, but they had no detectable RB transgene expression. None of these control lines was able to differentiate appreciably (Table 1; Fig. 8A, lane 3; and data not
shown). The expression of the HA-HBP1 transgene was intact in all of
these cell lines as shown by immunoprecipitation-Western analysis (Fig.
8B, lanes 1 and 2, and data not shown). This indicated that the partial
reversal of differentiation in the HA-HBP1/RB cell line was not due
to the loss of HBP1. Thus, the elevated RB expression level shown in
Fig. 8A correlated well with the reversal of the HBP1-imposed
differentiation defect in the B1-B9 line.
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FIG. 7.
Inhibition by HBP1 of MyoD family transcriptional
activation. (A) HBP1 can inhibit MyoD family activation of a natural
differentiation-specific MCK-CAT or a simplified muscle-specific 4R-CAT
reporter constructs. MCK-CAT denotes a CAT reporter construct driven by
the ~3 kb of the differentiation-specific muscle creatine kinase
promoter (46). 4R-CAT denotes a simplified and
muscle-specific reporter in which CAT expression is driven by four
reiterated MyoD family binding sites (E-box elements) upstream of a
minimal thymidine kinase promoter (51). The effect of HBP1
on transcriptional activation by either MyoD (lanes 1 and 2), Myf5
(lanes 3 and 4), or myogenin (lanes 5 and 6) was quantitated in C2
cells as described in Materials and Methods. In each set,
transcriptional activities in the presence or absence of HBP1 were
denoted by open or filled columns, respectively. (B) Inhibition of MyoD
family transcriptional activation requires the N-terminal region of
HBP1. Myogenin was used as a representative member of the MyoD family,
and the relative inhibition by wild-type HBP1 (open column) and by
HBP403-513 (horizontal stripes) (see Fig. 1 for description) was
compared by using assays similar to those described for panel A.
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Consistent with these findings, while HBP1 blocked MyoD-dependent
transcription (Fig. 8C, lane 6), the reexpression of RB could also
restore MyoD-dependent activation of transcription (Fig. 8C, lanes 7 and 8). Neither RB nor HBP1 alone affected the basal activity of 4R-CAT
(Fig. 8C, lanes 2 and 3). It should be noted that the RB-positive C2
cells differed from previous studies in which RB/
fibroblasts were used to show the dependence of RB on MyoD
transcription. No exogenous effects of RB on MyoD activity were
observed in the RB-positive C2C12 line (30) (Fig. 8C,
compare lanes 4 and 5). We also verified that MyoD transcriptional
activation did not occur in the RB-negative C33A cervical
carcinoma line (data not shown); however, this would not provide
a feasible test system for muscle differentiation. In Fig. 8,
the use of the C2C12 system allowed a direct and concurrent test of
HBP1 regulatory functions on MyoD transcriptional activation and on
overall cell differentiation.
To further explore the mechanisms by which RB opposed HBP1 inhibition
of differentiation, we examined functions of HBP1 mutants that were
deficient in RB and p130 binding. For reference, HBP1 contained both
LXCXE and IXCXE RB interaction motifs; mutation of either one retained
interaction with RB family members. Mutation in both motifs abolished
binding to RB and p130. In the context of sequence-specific repression
of the N-MYC promoter, both motifs were necessary for full
repression (49; see Fig.
1 for a summary).
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FIG. 8.
Expression of RB reverses the HBP1-mediated inhibition
of differentiation and of MyoD transcriptional activation. Whereas
Table 1 represents a direct quantitation of differentiation in the test
cell lines, this figure depicts the relative expression levels of
exogenous and endogenous RB protein. The presence of HA-RB transgene
product was detected in lysates from each cell line by
immunoprecipitation with anti-RB antibodies followed by Western
analysis of immune complexes with a monoclonal anti-RB antibody (as
described in Materials and Methods). This protocol allowed a direct
comparison of "overexpressed" RB levels relative to endogenous RB.
C2 cell line B1-B9 represents a line coexpressing HBP1 and RB; the C2
cell line B1-B2 represents a line expressing HBP1 only, but it was
isolated with selection conditions identical to those for B1-B9. (A)
Expression of RB and HA-RB in cell lines. Lanes: 1, C2C12 transiently
transfected with HA-RB expression vector; 2, B1-B9 (HBP1+RB); 3, B1-B2
(control hygromycin- and puromycin-resistant line; HBP1 only); 4, B1-C2
(HBP1 only); 5, C2C12; 6, C2C12 cell extracts immunoprecipitated with
anti--Gal antibodies as a negative control. The position of the RB
protein is indicated and was determined in the positive control (lane
1). (B) Expression of HBP1 in cell lines. The levels were quantitated
by Western blotting with an anti-HA antibody of an anti-HBP1
immunoprecipitation. This control experiment was performed to ensure
that the reversal of the differentiation phenotype by RB was not due to
the loss of HBP1 expression. Lanes: 1, B1-B9 (HBP1+RB); 2, B1-B2
(HBP1 only). (C) Effect of RB on HBP1-mediated inhibition of MyoD
activation of 4R-CAT. The transcriptional activities were determined by
transient-transfection assays in C2 cells by using specific promoter
constructs together with wild-type or mutant HBP1 and RB expression
vectors. Rous sarcoma virus--Gal was used as an internal
transfection control to normalize transfection efficiency. The
transfection output is expressed as a normalized ratio of CAT protein
to -Gal activity, and the combinations of expressed proteins are
indicated. One representative experiment is shown in each graph, and
each quantitation represents duplicate transfections that varied by
<10%. Each experiment was repeated three to five times. Protein
expression levels were equivalent in all transfections by Western
blotting or immunoprecipitation followed by Western analyses (data not
shown).
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Intriguingly, the inhibition of MyoD family transcriptional activation
by HBP1 depended on the ability to bind RB and p130 (Fig.
9). This function was retained in an HBP1
mutant with a single LXCXE mutation (pmLXCXE) (Fig. 9A, lane 3) but was
abolished by mutation of both RB interaction motifs (pmL/IXCXE)
(Fig. 9A, lane 4). The expression levels of wild-type and mutant HBP1
were similar as shown by anti-HBP1 immunoprecipitation following
anti-HA Western analysis (data not shown).
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FIG. 9.
Regulation of MyoD family transcriptional activation by
HBP1 and RB. (A) Effect of HBP1 mutants on MyoD activation of a MCK-CAT
reporter construct. The role of RB binding in the inhibition of MyoD
family activation was tested by using the indicated mutants of HBP1. As
described in Fig. 1 and reference 49, the wild-type
HBP1 and pm-LXCXE are functional in RB binding and in repression of the
N-MYC promoter, but the pm-L/IXCXE is defective in both
functions. The indicated proteins were expressed in conjunction with
MyoD: lane 1, no HBP expression vector (filled bar); lane 2, wild-type
HBP1 (open bar); lane 3, pm-LXCXE (diagonal stripes); and lane 4, pm-L/I XCXE (horizontal stripes). (B) In vivo association of RB and
HBP1 in differentiated C2C12 myotubes. HBP1 was shown to interact with
RB in differentiated C2C12 myotubes. C2C12 were completely
differentiated for 4 days in DMEM supplemented with 2% FCS. Cells were
metabolically labeled with 35S-methionine, and cell lysates
were collected for double immunoprecipitations as described in
Materials and Methods. The first immunoprecipitations were carried out
with anti-RB antibodies (lane 1) or anti-HBP1 antibodies (lanes 2 and
3), and the second immunoprecipitations were carried out with anti-RB
antibodies (lanes 1 and 2) and anti--Gal antibodies (lane 3). Note
that the amount of extracts used in lane 1 was approximately one-sixth
of that used in lane 2 or 3.
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We have already shown that HBP1 could specifically interact with RB and
p130 in transfected cells (49). However, the demonstration of a physical interaction between endogenous HBP1 and RB or p130 in
muscle cells would support the proposed functional connection between
HBP1 and RB (and/or p130) in terminal differentiation. While simple
in design, the low abundance of HBP1 posed a significant technical
hurdle. HBP1 was present at an ~10-fold-lower level than E2F, which
was already a rare protein (47a, 52). Because the expression
of endogenous RB and HBP1 was maximal in differentiated myotubes, we
reasoned that this would be an optimal cell type for detecting HBP1-RB
complexes.
Fully differentiated C2 cells were labeled with
35S-methionine, and coimmunoprecipitation was used to
assess the endogenous interaction of RB and HBP1. As shown in
Fig. 9B, the position of authentic RB was determined by
immunoprecipitation with anti-RB antibodies in C2 extracts (lane 1). A
double immunoprecipitation with anti-HBP1 followed by anti-RB was
performed. As shown in Fig. 9B (lane 2), full-length RB was present in
the test immunoprecipitation, suggesting a specific association of HBP1
with RB. No RB-specific band was evident in the control
immunoprecipitation in which an irrelevant antibody (anti--Gal
antibody) was used (Fig. 9B, lane 3). A similar strategy was attempted
with p130, but the poorer quality of the anti-p130 antibodies precluded
definitive analysis and the detection of endogenous complexes with
HBP1. From Fig. 9, we conclude that endogenous HBP1 and RB complexes
do exist, although at a rare abundance. This experiment extends
and confirms previous results and suggests that our experimental
system may mimic endogenous conditions.
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DISCUSSION |
This study focuses on the roles of RB and HBP1 in the
coordination of cell cycle exit and tissue-specific gene expression in
full muscle differentiation. We have shown that the expression of
HBP1 inhibited muscle cell differentiation without blocking cell
cycle exit. This study differs significantly from previous studies that
have utilized oncogenes to block cell differentiation (e.g.,
E1A, RAS, mdm-2, and cyclin D1 [12, 35, 44]). In these cases, oncogenes prevented cell cycle exit, a necessary first
step in differentiation. Thus, HBP1 and RB must be involved in steps
that coordinate cell cycle exit and tissue-specific gene expression.
Because this study revealed unexpected results, we tried to eliminate
several concerns. The first involved the use of cell lines. While
ectopic expression of HBP1 in cell lines was necessary for our studies,
we emphasize that exogenous HBP1 levels were modest. The level in the
HBP1-expressing line was approximately the endogenous level obtained
upon differentiation induction. Additionally, our experiments excluded
the possibility that the observations in the HBP1-expressing lines were
an artifact of stable cell line selection. We showed that the
inhibition of differentiation was corroborated under both stable and
transient expression, which argued against unique aspects of either
experimental system. A second concern was that the HBP1-expressing cell
lines were inherently defective in differentiation due to multiple,
uninteresting, pleiotropic mutation. However, reexpression of MyoD and
RB resulted in the expression of muscle differentiation markers and
indicated that the HBP1-expressing lines retained the inherent ability
to differentiate. A third concern was that the HBP1 protein was simply
sequestering RB in an E1A-like manner. E1A and other viral oncogenes
bind RB and enhance cell cycle entry (reviewed in reference
9). In contrast, the increased expression of HBP1
gave cell cycle exit, but not reentry. However, HBP1 did block
tissue-specific gene expression in differentiation. Since RB is
involved in both cell cycle and tissue-specific regulation during
differentiation (reviewed in reference 53), the
selective effect of HBP1 is incompatible with a general E1A-like
sequestration of RB.
Regulation of specific MyoD family members.
Both cellular
differentiation and transcriptional activity assays indicated that
ectopic HBP1 expression blocked differentiation by interfering with
tissue-specific gene expression. Our transcriptional experiments
indicated that HBP1 abrogated the transcriptional activation by Myf5
and other MyoD family members. Consistent with this, ectopic expression
of HBP1 inhibited muscle differentiation with a loss of MyoD and
myogenin expression. Strikingly, Myf5 expression remained intact. A
feature of the MyoD family is autoactivation. For example, MyoD can
activate the myogenin promoter (54). Thus, HBP1 expression
probably blocked differentiation by inhibiting Myf5 function and
preventing the subsequent activation of the MyoD and myogenin promoters
(Fig. 10B). Consistently, reexpression of MyoD or myogenin did rescue the differentiation-defective phenotype of HBP1 expression.
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FIG. 10.
Summary model of HBP1 and RB functions in
differentiation. We postulate that muscle differentiation can be
divided into general cell cycle exit (G0) and
tissue-specific gene expression (terminal differentiation) coordinated
by the RB family (RB and p130) and their targets, such as HBP1. A high
p130/HBP1 or low RB/HBP1 ratio may favor the cell cycle exit
but act as a negative signal for terminal differentiation. This
transient suspension of differentiation may eventually be relieved by
activation of RB, resulting in a high RB/HBP1 ratio. This complex
regulatory mechanism may be an effective means for ensuring fidelity in
differentiation by ensuring that only viable and arrested cells proceed
to the irreversible expression of genes that specify individual tissue
phenotypes.
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Is there evidence supporting differential functions for the highly
homologous Myf5 and MyoD transcription factors? Recent studies have
suggested a distinction in myogenesis, and Myf5 may lie functionally
upstream of MyoD. First, Myf5 expression precedes MyoD in murine
development, and MyoD expression is delayed in the absence of Myf5
(3, 39). However, either Myf5 or MyoD is required for full
muscle development, since deletion of both abolishes muscle formation
(40). Second, recent data now suggest that two parallel
pathways governed by Myf5 and Pax3 may function upstream of MyoD to
activate its expression. A Pax3/
Myf5/ mouse not only lacks muscle but, importantly,
lacks MyoD expression, while MyoD expression is retained when either
gene alone is deleted (24, 47; reviewed in reference
36). A direct investigation on the location of the
HBP1 block will require the use of animal systems, since tissue culture
systems do not recapitulate the subtle differences between MyoD and
Myf5.
The unusual pattern of MyoD family members has also been observed in
studies with CDK5, a differentiation-specific cyclin-dependent kinase
(CDK) family member. The expression of a dominant negative CDK5 in
Xenopus sp. inhibited muscle differentiation by blocking MyoD expression, but Myf5 expression was normal. Myogenin was not
tested (34). Direct CDK5 expression enhanced muscle
differentiation in C2 cells, which nicely complemented the
Xenopus studies (20). Remarkably, CDK5 and HBP1
are both induced within 24 h of C2 cell differentiation
(34, 49). The induction kinetics and unusual phenotype
in differentiation inhibition suggest that CDK5 and HBP1 may
conceivably target a step that resides between Myf5 and MyoD. However,
the outcomes of CDK5 and HBP1 activity have opposite predictions, but
this intriguing scenario remains to be tested.
Although our study has demonstrated a clear inhibition of Myf5 function
by HBP1, an open question is whether a direct physical interaction
occurs between HBP1 and Myf5. We have detected a physical complex of
HBP1 and Myf5 in vitro, but in vivo interaction experiments have been
uninformative (data not shown). Alternatively, HBP1 may inhibit Myf5
function by targeting Mef2c or E proteins, which are cofactors for Myf5
transcriptional activation (26, 27, 29).
Coordination of differentiation by HBP1 and RB.
We have shown
that RB can reverse the HBP1-mediated inhibition of MyoD family
transcriptional activation and differentiation. The simplest
explanation is that the relative ratio of RB to HBP1 dictates whether
only cell cycle arrest or full differentiation occurs. At a lowered
RB/HBP1 ratio cell cycle exit, but not tissue-specific gene
expression, persists. The interaction with RB and p130 is central to
HBP1-mediated inhibition and suggests that the active inhibitor may be
a complex of HBP1 with p130 or RB. Additionally, experimentally
increasing the ratio of RB to HBP1 could partially rescue the HBP1
inhibition of differentiation and of MyoD transcriptional activity. In
normal C2 cell differentiation, the accumulation of the
underphosphorylated form of RB occurs just prior to the onset of
tissue-specific gene expression. This suggests that the accumulation of
the underphosphorylated RB may be one signal for activation of
MyoD and of tissue-specific gene expression. Thus, our
experimental system mimics the normal C2 cell differentiation. Because
the RB regulation of MyoD family members is probably indirect, the
elevated ratio of RB to HBP1 may provide the signal for activation of
the MyoD family and of tissue-specific gene expression (see model Fig.
10). The importance of RB has also been underscored by recent work in
which the functions of RB in MyoD activation and in E2F regulation
could be uncoupled (37, 42). Activation of E2F does not
require the N-terminal region, yet it is necessary for full muscle
differentiation. Additionally, specific pocket mutants that were
defective in E2F regulation still supported differentiation. Thus, both
the N-terminal and pocket regions are required for the activation of
differentiation in both cellular and animal models.
Our data do not exclude the possible involvement of another RB family
member, p130, in the negative signaling pathway during differentiation.
Indeed, we speculate that HBP1 and p130 may act as active inhibitors of
MyoD-like master regulators and block expression of the
differentiation-specific genes. In the adipocyte system, Classon et al.
have recently demonstrated that loss of p130 and p107 results in an
unexpected increase in differentiation. Due to functional compensation,
both p130 and p107 must be deleted to yield this phenotype. Their
results suggest opposite functions for RB and p130 and are consistent
with an inhibitory function for p130 in differentiation (6).
Preliminary data obtained in our laboratory suggest that the expression
of p130 alone cannot support muscle differentiation in C2 cells,
despite efficient cell cycle arrest (42a).
In the present study, HBP1 inhibited MyoD family transcriptional
functions; this negative function also required binding to either p130
or RB. The collective data do raise the novel possibility that HBP1 and
p130 are active inhibitors of tissue-specific gene expression during
differentiation. Yet, both HBP1 and p130 are active inducers of cell
cycle exit. These dual functions of HBP1 and p130 may ensure that cell
cycle exit is complete prior to expression of tissue-specific genes.
Perhaps p130 is involved in negative regulation but RB is necessary for
positive activation of tissue-specific gene expression. Thus, RB and
p130 could conceivably have opposite roles in the progression of
differentiation.
While the least complex model for RB family member function has been
provided, further investigation is clearly needed to firmly establish
these opposite roles for RB and for p130 or p107. A considerable
complication is that there is extensive functional compensation by
other RB family members when one is mutated (16, 28). HBP1
binds both p130 and RB, and we cannot distinguish the functions of
these related proteins in the C2 cell line, in which all three proteins
are upregulated during differentiation. The ideal reagents for testing
the functions in differentiation will be RB/
p107/ p130/ and/or
HBP1/ cells or mice, but neither currently
exists.
A differentiation checkpoint?
The paradoxical induction of
proteins that induce cell cycle arrest but block differentiation may be
a general feature, since three distinct examples have now been
described. In adipocytes, the apparent negative inhibitor induced upon
differentiation is GADD153/CHOP, and this protein shows striking
functional similarity to HBP1. GADD153/CHOP was originally
isolated as a gene that was induced with growth arrest and DNA damage
and was later shown to be an inhibitor of the C/EBP transcription
factor family (38). Like MyoD in muscle, members of the
C/EBP family are critical factors in adipocyte
differentiation and are positively regulated by RB (4).
First, like HBP1, CHOP expression is normally induced in adipocyte
differentiation, but ectopic expression paradoxically blocks adipocyte
differentiation. Similar to our studies, the reexpression of the master
regulator C/EBP- can also restore differentiation in
CHOP-expressing cells, suggesting that CHOP also blocks
differentiation by blocking the master regulator (2). Second, like HBP1, direct expression of CHOP leads to cell cycle arrest
(1, 49). Like HBP1, CHOP also has ubiquitous tissue distribution, which suggests that negative regulation of
differentiation may not be limited to adipocyte or muscle, respectively
(1, 21, 47a). While HBP1 is regulated by RB and p130, it is
not known whether this is true for CHOP. Recently, the p202 protein has
been described as another inducible yet negative inhibitor of
differentiation. Like HBP1 and CHOP, expression of p202 elicits cell
cycle arrest but blocks differentiation. Intriguingly, p202 also binds
RB (10).
To explain the observations in the current work and in the literature,
we propose the existence of a differentiation checkpoint that regulates
coordination of cell cycle exit and tissue-specific gene expression.
This hypothetical checkpoint would consist of both positive and
negative regulation involving RB, p130, and their target proteins. In
the early phase of differentiation (about 24 h), cell cycle exit
predominates but there is no tissue-specific differentiation. Both p130
and HBP1 levels are high in this early phase. We propose that p130 and
HBP1 simultaneously elicit cell cycle arrest but actively block the
activation of MyoD and tissue-specific gene expression. The lower ratio
of RB to HBP1 may signal that the environment for tissue-specific gene
expression is inappropriate until cell cycle exit is complete. The
functional similarities of three distinct proteins (HBP1, CHOP, and
p202) predict the existence of a general inhibitory pathway that
provides cell cycle arrest but may transiently suspend tissue-specific
gene expression during differentiation. Additionally, the repressor
complex E2F4-p130 may also contribute to cell cycle exit in this early
phase (see, for example, references 8 and
43).
When cell cycle exit is completed, there would be activation of MyoD
and of tissue-specific genes at about h 48 of C2 differentiation. We
hypothesize that a positive signal is generated by the higher ratio of
RB to HBP1. The accumulation of the underphosphorylated form of RB may
contribute to the activation of MyoD, C/EBP, and other regulators
of tissue-specific gene expression. In this way, both negative and
positive regulation of tissue-specific gene expression would ensure
that cell cycle exit is complete prior to activation of MyoD and other
global regulators.
We emphasize that this differentiation checkpoint is meant as a
framework for generating future studies. The results presented in the
current study and in the literature do support the notion of negative
and positive regulation in the coordination of cell cycle exit and
tissue-specific gene expression in a full differentiation pathway.
While the mechanics of MyoD family members are well understood, the
mechanisms underlying their activation are still unclear. A component
of the regulatory signal must be the completion of cell cycle exit,
since this necessarily precedes tissue-specific gene expression. Thus,
the dual functions in regulating cell cycle exit and tissue-specific
gene expression suggest that proteins such as HBP1 and RB might be
excellent candidates in a checkpoint that coordinates the progression
and fidelity during differentiation. How other MyoD cofactors such as
Mef2C and p300 fit into an RB-p130-mediated pathway is unclear. In any
case, the current studies do provide a new view on the role of RB
family members in cell differentiation. An important future goal is the
elucidation of the precise mechanisms by which cell cycle exit signals
to activate MyoD family transcription.
We thank the following colleagues for their generosity: Eric
Olson (myogenin, MyoD, Myf5, and MCK CAT), Woody Wright (antimyogenin F5D), Frank Stockdale (continuous supply of anti-MHC), and Yukang Wang
(murine Myf5). We thank Eric Paulson, Andrew Leiter, Brian Schaffhausen, and Larry Feig for many helpful discussions. We especially thank Marie Classon, Ed Harlow, Bill Kaelin, and Wen-Hwa Lee
for providing information prior to publication.
The work was supported by grants to A.S.Y. from the AHA, the NIH
(GM44634), and the Digestive Disease Center at New England Medical
Center (NIDDK, P30 DK-34928). A.S.Y. is an Established Investigator of
the American Heart Association.
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Abstract
Introduction
Present address: Howard Hughes Medical Institute and Children's
Hospital, Division of Hematology and Oncology, Boston, MA 02115.
