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Vol. 14, Issue 7, 2706-2715, July 2003
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*Molecular Oncogenesis Laboratory, Experimental
Oncology Department, Regina Elena Cancer Institute, Rome, Italy;
Laboratory of Clinical Pathology, Regina Elena
Cancer Institute, Rome, Italy; and
Dipartimento di Biologia Animale,
Università di Modena e Reggio, Modena, Italy
Submitted September 18, 2002;
Revised February 26, 2003;
Accepted March 7, 2003
Monitoring Editor: Marvin P. Wickens
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The CCAAT
motif is present in 25% of eukaryotic promoters and NF-Y has been shown to
bind >120 of these (CCAAT-containing) promoters
(Mantovani, 1998). In
accordance with the widespread presence of CCAAT boxes, NF-Y subunits are
extremely conserved and have been identified in several eukaryotic kingdoms
(Mantovani, 1999).
The NF-Y complex supports the basal transcription of a class of regulatory
genes responsible for cell cycle progression, among which are cyclin A, B1,
B2, cdc25B, cdc25C, and cdk1 (Zwicker et al.,
1995a,b;
Bolognese et al.,
1999; Farina et al.,
1999; Korner et al.,
2001). Consistent with this, the overexpression of a dominant
negative mutant of the NF-YA subunit that inhibits DNA binding of the
endogenous NF-Y results in retardation of fibroblast growth
(Hu and Maity, 2000). We
previously demonstrated that the cyclin B1 promoter activity is switched off
during myogenic differentiation due to the loss of a functional NF-Y complex
(Farina et al.,
1999). This evidence supports the hypothesis that the
transcription of several cell cycle regulatory genes is down-regulated in
differentiated muscle cells trough the loss of NF-Y activity.
The differential expression of NF-YA, resulting in an alteration of NF-Y
CCAAT binding activity, has been observed in several cell lines both during
cell cycle progression and under specific conditions. NF-YA expression is
modulated during the cell cycle, being high in G1, further increasing in S and
then decreasing in the G2/M phase
(Bolognese et al.,
1999). Reduction of NF-YA expression has been reported in IMR-90
fibroblasts after serum deprivation and in human monocytes
(Chang and Liu, 1994;
Marziali et al.,
1997). Complete down-regulation of NF-YA protein expression occurs
in terminally differentiated C2C12 muscle cells. The loss of NF-YA expression
results in the loss of a functional NF-Y complex, suggesting that, although
NF-Y is a ubiquitous transcription factor, differential expression of NF-YA
subunit can occur during growth and differentiation of different cell lines
(Farina et al.,
1999).
Permanent down-regulation of the NF-YA subunit could play a particularly important role in reducing the expression of several cell cycle control genes in differentiated muscle cells. In the present study, we show that NF-YA is not expressed in the nuclei of adult skeletal and cardiac muscle tissues, whereas NF-YB and NF-YC are still expressed. Consistent with this, by chromatin immunoprecipitation and genomic footprinting experiments, we demonstrate that in vivo cell cycle regulatory genes are targets for the NF-Y binding activity only in proliferating muscle cells. Interestingly, forced expression of NF-YA in cells committed to differentiate leads to a reduction in the down-regulation of cyclin B1, cyclin A, and cdk1 expression. Moreover, exogenous NF-YA protein interferes with the induction of p21, cyclin D3, and myogenin expression occurring early during muscle differentiation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), a clone derived from the C2C12 cell line
(Yaffe and Saxel, 1977), was
cultured in DMEM containing 10% fetal bovine serum (proliferating; P).
Differentiation was induced by plating the cells into collagen-coated dishes
and switching them for 72 h (terminally differentiated; TD) to serum-free (SF)
medium: DMEM supplemented with ReduSer (Upstate Biotechnology, Lake Placid,
NY) to a final concentration of 5 µg/ml human insulin, 5 µg/ml human
(holo) transferrin, and 5 ng/ml sodium selenite. Cytosine arabinoside (50
µM; Ara-C) was added to the SF medium to eliminate undifferentiated cells.
As previously described (Tiainen et
al., 1996), under these conditions more than 90% of the cells
become terminally differentiated (TD). For the analysis of cell cycle control
and muscle-specific gene expression after overexpression of NF-YA protein
(Figure 6), the cells were
induced to differentiate in the absence of Ara-C for 6, 12, 24, and 48 h.
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Total Cell and Tissue Lysates and Western Blot Analysis
Total cell extracts were prepared by incubating C2C12 cells in 0.15 M NaCl
buffer containing 50 mM Tris-HCl, pH 8, 5 mM EDTA, 1% NP-40, 0.5% deoxycholic
acid, 10 µg/ml leupeptin, 4 µg/ml pepstatin, 5 µg/ml aprotinin, 50 mM
NaF, 1 mM Na-orthovanadate, and 1 mM phenylmethylsulfonyl fluoride (PMSF).
BDF1 wild-type adult mice were sacrificed by vertebral dislocation. Organs
were dissected, immediately frozen in liquid nitrogen, and stored at
–70°C until used. Purified muscle and fibroblast were obtained by
treatment of the tissue with trypsin, collagenase, and mechanical disruption.
Total tissue extracts were prepared by homogenization in lysis buffer (20 mM
Tris-HCl, pH 8, 1% NP-40, 10% glycerol, 137 mM NaCl, 5 mM EDTA pH 8, 5 mM EGTA
pH 7, 1 mM Naorthovanadate, 50 mM NaF, 5 µg/ml Aprotinin, 10 µg/ml
Leupeptin, 10 mM PMSF) on dry ice. Total C2C12 lysate (50 µg) and 150 µg
of total tissue lysate were separated by SDS-PAGE (12.5%) and electroblotted
onto nitrocellulose. After blocking with 5% nonfat dried milk, the filters
containing tissue lysates were immunoreacted with each of the following
antibodies: anti-NF-YA rabbit polyclonal antibody (0.3 µg/ml) (Rockland,
Gilbertsville, PA), anti-NF-YB rabbit polyclonal antibody (0.3 µg/ml)
(Mantovani et al.,
1994), anti-NF-YC rabbit polyclonal antibody (0.3 µg/ml)
(Mantovani et
al.,1994), anti-Hsp70 mouse monoclonal antibody (mAb) (0.25
µg/ml) (StressGen Biotechnologies, Victoria, Canada). The filters
containing C2C12 lysates were immunoreacted with 0.3 µg/ml anti-NF-YA
rabbit polyclonal antibody (Rockland), 0.1 µg/ml anti-cyclin B1 rabbit
polyclonal antibody, 0.1 µg/ml anti-cyclin A rabbit polyclonal antibody,
0.1 µg/ml anti-cdk1 p34 rabbit polyclonal antibody, 1 µg/ml
anti-myogenin rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, CA), 1:500 anti-mouse p21 (kindly provided by C. Schneider, Laboratorio
Nazionale CIB, Trieste, Italy), rabbit polyclonal antibody 0.25 µg/ml
anti-Hsp70 mouse mAb (StressGen, Biotechnologies), following the
manufacturer's directions. Peroxidase activity of the appropriate secondary
antibodies was visualized by enhanced chemiluminescence detection system
(Amersham Biosciences UK, Little Chalfont, Buckinghamshire, United
Kingdom).
Immunohistochemistry
BDF1 wild-type adult mice were sacrificed by vertebral dislocation, and
tissues were fixed in 100% methanol at 4°C, dehydrated, and embedded in
paraffin. Five-micrometer-thick serial sections were mounted on slides,
deparaffinized in xylene, and rehydrated. Sections were washed in
phosphate-buffered saline (PBS), incubated 30 min. in 1 M glycine, pH 7.8, at
4°C, 10 min in 3% H2O2, and incubated with 5
µg/100 µl anti-NF-YA rabbit polyclonal antibody (Rockland) and 2
µg/100 µl anti-NF-YB rabbit polyclonal antibody
(Mantovani et al.,
1994) for 1 h at room temperature. Sections were washed four times
in PBS, 1% bovine serum albumin (BSA), 0.1% Triton X-100 and incubated with a
peroxidase-conjugated goat anti-rabbit antibody as secondary antibody.
Sections were then washed in PBS, 0.2% BSA, 0.1% Triton X-100 and
counterstained with hematoxylin. Peroxidase activity of the secondary
antibodies was visualized by reaction with 3,3'-diaminobenzidine and
H2O2 in sodium phosphate buffer.
Electromobility Shift Assays (EMSAs)
Electromobility shift assays were performed in a 25 µl of DNA binding
reaction that contained 150 µg of murine tissues extracts, 6 fmol of
labeled duplex oligonucleotide, binding buffer (20 mM Tris-HCl, pH 7.8, 60 mM
KCl, 0.5 mM EDTA pH 8, 0.1 mM dithiothreitol, 3 mM MgCl2), 1.5
µg of poly(dI-dC), and 10 mM spermidine. The reaction was carried out on
ice for 30 min and the protein–DNA complexes were subjected to native
electrophoresis on 5% polyacrylamide, 0.5 x TBE gels. The following
oligonucleotides used as probes or as autocompetitor (300 fmol) consensus
sites are underlined: B1up CCAAT, 5'-CCG CAG CCG CCA ATG GGA
AGG GAG TGA; B1down CCAAT, 5'-GAA CAG GCC AAT AAG GAG GGA G;
and unrelated competitor (1500 fmol): cyclin B1 (SP1) up, 5'-GGG CTG TGG
CCC CGC CCC TCT C, cyclin B1 (SP1) down, 5'-GGG GAG AGG GGC GGG GCC ACA
G. Production and purification of recombinant NF-YA protein were described
previously (Mantovani et al.,
1994).
Formaldehyde Cross-linking and Chromatin Immunoprecipitations
Formaldehyde cross-linking and chromatin immunoprecipitations were
performed as described previously
(Fontemaggi et al.,
2001). Formaldehyde was added directly to proliferating and
terminally differentiated C2C12 cells at a final concentration of 1% at
22°C for 10 min. The reaction was stopped by addition of glycine to a
final concentration of 0.125 M. The cells were then rinsed with cold 1x
PBS, incubated with 0.2x trypsin-EDTA in 1x PBS, and scraped.
Cells were collected by centrifugation, washed in cold 1x PBS plus 0.5
mM PMSF, and resuspended in lysis buffer (5 mM piperazine N,N bis
zethone sulfonic acid, pH 8, 85 mM KCl, 0.5% NP-40). Nuclei were solicited in
the sonication buffer (0.1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8, 0.5%
deoxycholic acid) 1 time for 10 min by using microultrasonic cell disruptor.
The chromatin was sheared to an average size of 500 base pairs, and
immunoprecipitation was performed with protein G-agarose (KPL, Gaithersburg,
MD). The chromatin solution was precleared by adding protein G for 1 h at
4°C, aliquoted (each aliquot corresponding to 2 x 106
cells), and incubated with 2 µg of affinity-purified rabbit polyclonal
antibodies anti-NF-YB 2 µg of affinity-purified rabbit polyclonal
antibodies anti-NF-YA (Santa Cruz Biotechnology), and 2 µg of
affinity-purified rabbit polyclonal antibodies anti-E2F1 (Santa Cruz
Biotechnology) overnight at 4°C with mild shaking. Before use, protein G
was blocked with 1 µg/µl sheared herring sperm DNA and 1 µg/µl BSA
for 3 h at 4°C and then incubated with chromatin and antibody for 2 h at
4°C. Immunoprecipitates were washed 10 times by adding 1 ml of
immunoprecipitation buffer. The control sample supernatant (not
immunoprecipitated with antibody) was collected as input sample, before
washing. Immunoprecipitates were eluted from the beads with elution buffer (1%
SDS, 50 mM NaHCO3, 1.5 µg/ml sonicated salmon sperm) at 37°C
for 30 min with vigorous shaking. The samples were heated at 65°C
overnight to reverse formaldehyde cross-links and ethanol precipitated.
Recovered material was treated with 25 µl of proteinase K buffer (1.25%
SDS, 50 mM Tris, pH 7.5, 25 mM EDTA, pH 8), 1.5 µl of proteinase K (Roche
Diagnostics, Mannheim, Germany), 100 µl of 1x TE (10 mM Tris, pH 7.5,
1 mM EDTA, pH 8), extracted with phenol/chloroform/isoamyl alcohol (25:24:1),
and ethanol precipitated. The pellets were resuspended in 30 µl of
H2O and analyzed by using polymerase chain reaction (PCR). Total
input sample was resuspended in 100 µl of H2O and diluted 1:100
before PCR. For PCR analysis on cyclin B2 and cyclin A, the following
oligonucleotides were used: cyclin B2 up3, 5'-TGT AGA CAA GGA AAC AAC
AAA GCC TGG TGG CC; cyclin B2 down2, 5'-CAG CCA CTC CGG TCT GCG ACA;
cyclin A up2, 5'-CTG TAA GAT TCC CGT CGG GCC TTC G; and cyclin A down2,
5'-GTA GAG CCC AGG AGC CGC GAG.
In Vivo DNA Footprinting
The in vivo DNA footprinting was performed by using ligation-mediated PCR
as described previously (Dey et
al., 1992). P and TD cells were treated with 0.1% dimethyl
sulfate (DMS) for 2 min. After DMS treatment, cells were washed three times
with cold PBS. DNA was isolated and cleaved with piperidine. Labeled PCR
products were resolved on a 6% polyacrylamide-8 M urea sequencing gel. In
vitro control DNA samples consisted of purified naked DNA from HeLa cells
treated with 0.125% DMS for 2 min and cleaved with piperidine as described
above. The following oligonucleotides were used. For cyclin B2, first primer,
TM = 45°C 5'-ATATCAGGGACTAGAATTTG; second primer, TM = 61.4°C
5'-GACTGTAGACAAGGAAACAACAAAGCCTG; and third primer, TM = 65°C
5'-TGTAGACAAGGAAACAACAAAGCCTGGTGGCC. For cyclin A, first primer, TM =
54°C 5'-GCGGGAGGAGCGTAGAG; second primer, TM = 65.8°C
5'-GTAGAGCCCAGGAGCCGCGAG; and third primer, TM = 70°C
5'-AAGATTCCCGTCGGGCCTTCGCTCG.
DNA Transfections, Chloramphenicol Acetyltransferase (CAT), and
Luciferase Assays
Stable transfections were performed as described previously
(Farina et al.,
1999). CAT or Luc-reporter constructs carrying the cyclin B1
(Piaggio et al.,
1995), cdk1 (Dalton,
1992), cyclin A (Schulze
et al., 1995), cyclin B2
(Bolognese et al.,
1999), cdc25A (Vigo et
al., 1999), cdc25C (Manni
et al., 2001), and cyclin E
(Botz et al., 1996)
were stably transfected by calcium phosphate in C2C12 cells. The obtained
polyclonal cell lines were analyzed for CAT or luciferase activity. Transient
transfections of p21 (kindly provided by G. Blandino, Regina Elena Cancer
Institute, Rome, Italy), and myogenin (kindly provided by P.L. Puri,
University of Rome, La Sapienza, Italy), promoters were performed by
calcium-phosphate precipitation in proliferating C2C12 cells. Transient NF-YA
overexpression were performed using the FuGENE 6 transfection reagent (catalog
no. 1814443; Roche Diagnostics). Cells (1 x 106) were
transfected with 10 µg of the eukaryotic expression vector containing the
human wild-type cDNA for the NF-YA subunit under the control of the simian
virus 40 promoter (NF-YA-polyII), or the vector alone (polyII), by using 40
µl of FuGENE 6 transfection reagent. Cells were incubated with transfection
medium for 16 h, and differentiation was induced after transfection by adding
SF medium in the absence of Ara-C for 6, 12, 24, and 48 h. The transfection
efficiency (
40%) was evaluated by scoring green fluorescent
protein-positive cells in a parallel sample transfected with green fluorescent
protein vector under the same experimental conditions.
Creatine Kinase Assay
Creatine kinase activity was assayed spectrophotometrically using a
diagnostic kit (Sigma-Aldrich, St. Louis, MO). Briefly, 0-, 6-, 12-, 24-, and
48-h cultures of NF-YA or vector alone (polyII)-transfected cells grown in
differentiation medium were washed twice with ice-cold PBS and scraped. Cells
were spun at 1000 x g and resuspended in PBS containing 0.1%
Tween 20 and incubated on ice for 15 min. The lysates were centrifuged at
10,000 x g for 15 min at 4°C, and the protein concentration
in the supernatant was determined using the Bio-Rad protein assay reagent.
Protein content in the samples was then adjusted to 0.4 µg/µl with PBS
containing 0.1% Tween 20, and, typically, 100 µl of sample was used for
creatine kinase assay according to the manufacturer's instructions. The
activity of the enzyme was determined by measuring the absorbance at 510 nm
and expressed as units per milligram of protein. A unit of creatine kinase
activity was defined as (A510 units/min·1000)/6.22
(extinction coefficient).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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CCAAT Binding Activity of NF-Y Is Lost in Adult Muscle Tissues
To assess whether loss of the NF-YA subunit leads to loss of the CCAAT
binding activity of NF-Y, we performed electromobility shift assays with
extracts from adult mouse tissues, by using an oligo containing the cyclin B1
CCAAT box (Farina et al.,
1999). The results demonstrate that the DNA binding activity of
NF-Y is lost in skeletal and cardiac muscle extracts, whereas, as expected, it
is present in the other tested tissues
(Figure 2A). The lack of NF-Y
binding to the CCAAT box in muscle and heart is due to the loss of the NF-YA
protein as demonstrated by EMSA experiments performed in the presence of
recombinant purified NF-YA protein. As shown in
Figure 2B, the addition of 150
ng of purified NF-YA protein to skeletal muscle (lane 5) and heart (lane 8)
extracts restores the binding activity of the NF-Y complex. The addition of
purified NF-YA protein to brain extract (lane 2) does not substantially modify
the complex mobility. Together, the results reported in Figures
1 and
2 demonstrate that of NF-YA
protein cannot be detected in terminally differentiated muscle tissues and
clearly indicate that NF-YA is the limiting factor for the DNA binding
activity of NF-Y complex in these tissues.
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NF-Y Does Not Bind Cell Cycle-dependent Promoters in Terminally
Differentiated Cells In Vivo
NF-Y supports the basal transcription of a class of regulatory genes
responsible for cell cycle progression, among which are cyclin A, B1, B2,
cdc25B, cdc25C, and cdk1 (Zwicker et al.,
1995a,b;
Bolognese et al.,
1999; Farina et al.,
1999; Korner et al.,
2001; Manni et al.,
2001). To investigate the DNA-binding activity of NF-Y on cell
cycle target promoters in terminally differentiated cells, we performed
chromatin immunoprecipitation experiments on P and TD C2C12 skeletal muscle
cells. Proliferating cells were induced to differentiate by growth factors
withdrawal for 72 h. P and TD cells were treated with formaldehyde to
cross-link proteins to DNA. After sonication, the cross-linked chromatin from
equivalent numbers of P and TD cells was immunoprecipitated by using
anti-NF-YB or anti-NF-YA antibodies. As negative controls, we included a
reaction lacking primary antibody and one containing an anti-E2F1 antibody.
E2F1 is a nuclear transcription factor that does not have a binding site on
the cyclin B2 promoter. After immunoprecipitation, enrichment for the
endogenous cyclin B2 and A promoter fragments in each sample was assessed by
PCR amplification by using primers amplifying the cyclin B2 promoter region
from –145 to +90 base pairs and the cyclin A promoter region from
–143 to +89 base pairs, respectively. As shown in
Figure 3, the anti-NF-YB and
the anti-NF-YA antibodies did not immunoprecipitate the cyclin B2 (lanes 7 and
8) and cyclin A (lanes 19 and 20) promoters from differentiated chromatin. As
expected, both promoters were present in the NF-YB and NF-YA
immunoprecipitates in chromatin from proliferating cells (lanes 1, 2, 13, and
14). The anti-E2F1 antibody was unable to immunoprecipitate cyclin B2 promoter
from both cell types (lanes 3 and 9), whereas it immunoprecipitated the cyclin
A promoter in proliferating cells (lane 15). This result is in agreement with
recent data demonstrating that E2F1 binds to the cyclin A promoter in
proliferating glioblastoma cells
(Takahashi et al.,
2000).
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It has been described that the CCAAT displacement protein is able to
displace NF-Y from several promoters containing CCAAT boxes
(Nepveu, 2001). Thus, we asked
whether in differentiated cells the CCAAT boxes of the cyclin B2 and cyclin A
promoters are bound by proteins, other than NF-Y. To answer this question, we
performed in vivo genomic footprinting analysis of both promoters. P and TD
C2C12 cells were treated with the DNA-alkylating reagent DMS, and methylated G
residues were identified using the ligation-mediated PCR technique
(Dey et al., 1992).
As shown in Figure 4, in
proliferating cells the CCAAT boxes of both the cyclin B2 and cyclin A
promoters were protected from DMS methylation (lanes 3 and 6). In contrast,
the CCAAT boxes and the flanking regions of these promoters were devoid of
interactions with proteins in differentiated cells (lanes 2 and 4).
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Together, these results provide further in vivo evidence for a lack of NF-Y binding activity to the CCAAT boxes of the cell cycle target promoters in skeletal muscle differentiated cells.
Promoter Activities of NF-Y-Target Cell Cycle Genes Are
Down-Regulated in Differentiated Muscle Cells
We queried whether the lack of NF-Y binding activity to the CCAAT boxes
leads to down-regulation of the activities of CCAAT-containing promoters in
terminally differentiated cells. To this end, C2C12 cells were stably
transfected with CAT reporter constructs carrying human cyclin B1
(Piaggio et al.,
1995), and human cdk1 (Dalton,
1992) promoter fragments, and luciferase reporter constructs
carrying human cyclin A (Schulze et
al., 1995), murine cyclin B2
(Bolognese et al.,
1999), human cdc25A (Vigo
et al., 1999), human cdc25C
(Manni et al., 2001),
murine cyclin E promoter fragments. Except for cyclin E promoter, it has been
demonstrated that NF-Y contributes to support the basal activity of these
promoters in proliferating cells (Zwicker et al.,
1995a,b;
Botz et al., 1996;
Huet et al., 1996;
Kramer et al. 1997;
Plet et al., 1997;
Liu et al., 1998;
Farina et al., 1999;
Yun et al., 1999
Jung et al., 2001;
Manni et al., 2001;
Sciortino et al.,
2001). Figure 5
shows that all of promoter constructs are capable of driving transcription
only in proliferating cells, whereas their activities are down-regulated in TD
cells. Interestingly, the cyclin E promoter fragment that does not contain
CCAAT boxes is still active in TD cells.
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Forced Expression of NF-YA During the Muscle Differentiation Program
Inhibits the Down-Regulation of Several Cell Cycle Gene Expression and the
Induction of p21, Myogenin, and Creatine Kinase Activity
Normal differentiation of myoblasts is accompanied by a change in the
expression of both cell cycle and muscle-specific genes. Initiation of
muscle-specific gene expression during differentiation of muscle cells is
preceded by withdrawal of the cells from the cell cycle
(Crescenzi et al.,
1990; Sorrentino et
al., 1990). Cell cycle arrest is a requirement for muscle
differentiation, and it is accomplished by down-regulation of cyclins, with
the exception of cyclin D3, which is up-regulated during differentiation and
contributes to cell cycle withdrawal
(Cenciarelli et al.,
1999). To investigate whether the presence of NF-YA could
influence the gene expression profile of differentiated cells, we transiently
transfected proliferating C2C12 cells with either an empty vector or a vector
encoding NF-YA and then induced the cells to differentiate by withdrawing
growth factors for 6, 12, 24, and 48 h. We did not tested the effect of NF-YA
in terminally differentiated cells (72 h of growth factor withdrawal) because
we have reported previously that in C2C12 cells transfected with NF-YA, this
protein cannot be detected after terminal differentiation
(Farina et al.,
1999). Moreover, because the rationale of this experiment was that
the presence of NF-YA could retain the cells in the cell cycle, the cells were
induced to differentiate in the absence of Ara-C. Indeed, in the presence of
Ara-C the proliferating cells would be killed. In this experimental system, we
examined the endogenous expression of several cell cycle regulatory genes by
immunoblot. As shown in Figure
6A, the cells transfected with the NF-YA subunit (+) displayed an
impairment in the down-regulation of cyclin B1 and cyclin A protein expression
compared with cells transfected with the empty vector (–), as manifested
by the presence of these proteins after 48 h of growth factor withdrawal. In
cells transfected with the empty vector cdk1 expression started to be
down-regulated after 24 h of growth factor withdrawal, when endogenous NF-YA
is still present, thus indicating that cdk1 expression is controlled by
factors different than NF-Y. However, the down-regulation of cdk1 expression
is, at least in part, impaired by the presence of NF-YA after 48 h of growth
factor withdrawal. Moreover, the presence of the NF-YA protein merely delayed,
at least at early times, the up-regulation of the cell cycle inhibitor p21 and
slightly reduced the up-regulation of cyclin D3. Consistent with this, NF-YA
overexpression leads to a delay in the induction of myogenin, an early muscle
differentiation marker, whereas there was no effect on MyoD expression
(Sassoon, 1993;
Buckingham, 1994;
Tiainen et al.,
1996). We next examined the activity of creatine kinase, a
muscle-specific enzyme marker, in cell lysates. As shown in
Figure 6B, cells transfected
with the empty vector exhibited an approximately eightfold increase in
creatine kinase activity up to 48 h of culture in differentiation medium.
NF-YA-transfected cells showed only an approximate twofold greater creatine
kinase activity after 48 h of growth factor withdrawal. The results presented
herein indicate that the down-regulation of NF-YA expression is strictly
required for the correct modulation of the cell cycle and muscle-specific gene
expression during differentiation of muscle cells. These results also suggest
that NF-YA interferes, at least in part, with the myogenic differentiation
program of C2C12 cells. To further investigate whether NF-YA directly
regulates transcription of genes involved in specifying myogenic fate, we used
a dominant-negative NF-YA protein, YA13m29
(Mantovani et al.,
1994). On transfection in mammalian cells, this vector expresses a
mutant protein containing a triple amino acids substitution in the NF-YA DNA
binding subdomain enabling the subunit to interact with the NF-YB·NF-YC
dimer. The resulting trimer is inactive in terms of CCAAT recognition. The
dominant-negative or the wild-type NF-YA proteins were cotransfected with
constructs carrying p21 or myogenin promoters driving the expression of the
luciferase reporter gene in proliferating C2C12 cells, and the promoter
activities were determined (Figure 7, A and
B). As expected for promoters that do not contain CCAAT boxes, the
dominant-negative as well as the wild-type NF-YA proteins do not modulate the
activities of both promoters. In contrast, the activities of p21 and myogenin
promoters are induced by p53 and serum deprivation, respectively, as described
previously (Edmondson et al.,
1992; D'Orazi et al.,
2002).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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,b;
Bolognese et al.,
1999; Korner and Muller, 2001;
Manni et al., 2001).
We have already demonstrated that the loss of a functional NF-Y complex in
differentiated muscle cells leads to down-regulation of the cyclin B1 promoter
(Farina et al.,
1999). We now extend this observation to the promoters of the
other cell cycle control genes that are known to be regulated by NF-Y during
the cell cycle. Moreover, the results of the in vivo chromatin
immunoprecipitation experiments on the cyclin B2 and cyclin A promoters
indicate that down-regulation of these genes in differentiated cells could be
due to the absence of NF-Y on their promoters. Interestingly, the lack of NF-Y
binding to cyclin B2 and cyclin A promoters in differentiated cells does not
lead to the binding of other proteins to the CCAAT boxes as demonstrated by in
vivo genomic footprinting experiments. These results provide evidence for a
model in which NF-Y serves as a common transcription factor in proliferating
muscle cells (myoblasts) for several key cell cycle control genes. Permanent
down-regulation of NF-YA expression in differentiated cells (myotubes) leads
to the inhibition of the expression of these genes. A recent analysis of a
large database of 1031 human promoters indicated that the CCAAT box is present
in 63% of them (Suzuki et al.,
2001). It will be of interest to investigate whether the
transcription from other CCAAT-containing promoters, in postmitotic skeletal
muscle cells, is down-regulated by the same common mechanism presently
described.
In adult muscle tissues, the expression of NF-YA protein is down-regulated,
whereas NF-YB and -C are still expressed. It has been demonstrated that the
NF-Y histone-like dimer NF-YB/C can form complexes with H3-H4 histones for
whose formation the CCAAT box is not required. Moreover, the NF-Y histone-like
dimer associates with DNA during nucleosome formation in the absence of NF-YA
(Caretti et al.,
1999). Proteins involved in histone acetylation are found
associated with NF-YB (Currie,
1998, Li et al.,
1998). This evidence supports the hypothesis that the NF-YB/C
heterodimer could serve the dual function of binding NF-YA to activate
transcription of CCAAT-containing promoters and, thanks to the histone-like
domains, associating DNA during nucleosome formation. Thus, skeletal muscle
cells represent an excellent system to study whether the NF-YB/C dimer exerts
a biological role in the absence of NF-YA.
Initiation of muscle-specific gene expression, i.e., myogenin, during
differentiation of muscle cells, is preceded by withdrawal of the cells from
the cell cycle. Exit from the cell cycle is accomplished by down-regulation of
several cyclins and cdks and by up-regulation of cell cycle inhibitors, such
as p21, and cyclin D3 (Perry and Rudnick,
2000). We demonstrate herein that ectopic expression of NF-YA
protein could interfere with the onset of both cell cycle withdrawal and early
muscle-specific program. Although NF-YA directly binds the endogenous
promoters of the analyzed cell cycle regulatory genes, cyclin A and cyclin B2,
as well as cyclin B1 (Sciortino et
al., 2001), and cdk1 (A. Gurtner, personal observation),
NF-YA does not directly interfere with the muscle differentiation program.
Indeed, our results indicate that NF-YA does not modulate the transcriptional
activity of p21 and myogenin promoters. However, unrestricted expression of
NF-YA interferes, although in an indirect way, with the differentiation
process as indicated by the inhibition of creatine kinase activity occurring
during differentiation in the presence of NF-YA.
Together, the above-mentioned results are consistent with a scenario in
which NF-YA expression inhibits the differentiation program and keeps the
myoblasts in the cell cycle. Because the induction of p21, cyclin D3, and
myogenin (Molkentin and Olson,
1996; Cenciarelli et
al., 1999), as well as the down-regulation of the of cell
cycle control genes (Perry and Rudnick,
2000), are important events in the induction of muscle
differentiation, our data suggest that NF-Y exerts a biological role in the
switch from proliferation to differentiation of muscle cells.
Although the presence of NF-YA in cells committed to differentiate could lead to conflicting signals, no apparent apoptosis was seen in our experimental conditions. It remains to be determined whether expression of NF-YA in the presence of differentiation stimuli for periods of time longer than those tested herein leads to apoptosis. Indeed, the level of NF-YA drops between 24 and 48 h in the transfected cells and after 72 h of growth factor withdrawal it becomes undetectable (P. Fuschi, unpublished data). This could explain why overexpression of NF-YA can only delay rather than inhibit differentiation.
Our previous results indicate that the regulation of NF-YA expression
depends on a posttranscriptional level of regulation
(Farina et al.,
1999). Based on these considerations, we tested the effect of the
specific calpain I and II and catepsin inhibitor LN-acetyl-leu-leu-norleucinal
on NF-Y stability (A. Gurtner, I. Manni, and P. Fuschi, unpublished data).
These preliminary experiments show that there is a cytoplasmatic increase of
NF-YA protein, suggesting that the degradation of NF-YA involves a
ubiquitine-proteasome pathway. The study of the regulation of NF-Y expression
in adult muscle tissues, in particular of the NF-YA subunit, will be an
important step toward a full understanding of cell cycle control in these
cells.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Corresponding author. E-mail address:
piaggio{at}ifo.it.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Bolognese, F., et al. (1999). The cyclin B2 promoter depends on NF-Y, a trimer whose CCAAT-binding activity is cell cycle regulated. Oncogene 18, 1845–1853.[CrossRef][Medline]
Botz, J., Zerfass-Thome, K., Spitkovsky, D., Delius, H., Vogt, B., Eilers, M., Hatzigeorgiou, A., and Yansen-Durr, P. (1996). Cell cycle regulation of the murine cyclin E gene depends on an E2F binding site in the promoter. Mol. Cell. Biol. 16, 3401–3409.[Abstract]
Buckingham, M.E. (1994). Muscle: the regulation of myogenesis (review). Curr. Opin. Genet. Dev. 4, 745–751.[CrossRef][Medline]
Caretti, G., Motta, M.C., and Mantovani, R. (1999).
NF-Y associates with H3–H4 tetramers and octamers by multiple
mechanisms. Mol. Cell. Biol.
19,
8591–8560.
Cenciarelli, C., De Santa, F., Puri, P.L., Mattei, E., Ricci, L.,
Bucci, F., Felsani, A., and Caruso, A.M. (1999). Critical role
played by cyclin D3 in the MyoD-mediated arrest of cell cycle during myoblast
differentiation. Mol. Cell. Biol.
19,
5203–5217.
Chang, Z.F., and Liu, C.J. (1994). Human thymidine
kinase CCAAT-binding protein is NF-Y, whose A subunit expression is
serum-dependent in human IMR-90 diploid fibroblasts. J. Biol.
Chem. 269,
17893–17898.
Crescenzi, M., Fleming, T.P., Lasser, A.B., Weintraub, H., and
Aaronson, S.A. (1990). MyoD induces growth arrest independent of
differentiation in normal and transformed cells. Proc. Natl. Acad. Sci.
USA 87,
8442–8446.
Currie, R.A. (1998). NF-Y is associated with the
histone acetyltransferases GCN5 and P/CAF. J. Biol. Chem.
273,
1430–1434.
Dalton, S. (1992). Cell cycle regulation of the human cdc2 gene. EMBO J. 11, 1797–1804.[Medline]
Dey, A., Thornton, A.M., Lonergan, M., Weissman, S.M., Chamberlain,
J.W., and Ozato, K. (1992). Occupancy of upstream regulatory
sites in vivo coincides with major histocompatibility complex class I gene
expression in mouse tissues. Mol. Cell. Biol.
12,
3590–3599.
D'Orazi, G., et al. (2002). Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis. Nat. Cell Biol. 4, 11–19.[CrossRef][Medline]
Edmondson, D.G., Cheng, T.C., Cserjesi, P., Chakraborty, T., and
Olson, E.N. (1992) Analysis of the myogenin promoter reveals an
indirect pathway for positive autoregulation mediated by the muscle-specific
enhancer factor MEF-2. Mol. Cell. Biol.
12,
3665–3677.
Farina, A., Manni, I., Fontemaggi, G., Tiainen, M., Cenciarelli, C., Bellorini, M., Mantovani, R., Sacchi, A., and Piaggio, G. (1999). Down-regulation of cyclin B1 gene transcription in terminally differentiated skeletal muscle cells is associated with loss of functional CCAAT-binding NF-Y complex. Oncogene 18, 2818–2827.[CrossRef][Medline]
Fontemaggi, G., Gurtner, A., Strano, S., Higashi, Y., Sacchi, A.,
Piaggio, G., and Blandino, G. (2001). The transcriptional
repressor ZEB regulates p73 expression at the crossroad between proliferation
and differentiation. Mol. Cell. Biol.
21,
8461–8470.
Hu, Q., and Maity, S.N. (2000). Stable expression of a
dominant negative mutant of CCAAT binding factor/NF-Y in mouse fibroblast
cells resulting in retardation of cell growth and inhibition of transcription
of various cellular genes. J. Biol. Chem.
275,
4435–4444.
Huet, X., Rech, J., Plet, A., Vie, A., and Blanchard, J.M. (1996). Cyclin A expression is under negative transcriptional control during the cell cycle. Mol. Cell. Biol. 16, 3789–3798.[Abstract]
Jung, M.S., Yun, J., Chae, H.D., Kim, J.M., Kim, S.C., Choi, T.S., and Shin, D.Y. (2001). p53 and its homologues, p63 and p73, induce a replicative senescence through inactivation of NF-Y transcription factor. Oncogene 20, 5818–5825.[CrossRef][Medline]
Korner, K., Jerome, V., Schmidt, T., and Muller, R.
(2001). Cell cycle regulation of the murine cdc25B promoter:
essential role for NF-Y and a proximal repressor element. J. Biol.
Chem. 276,
9662–9669.
Kramer, A., Carstens, C.P., Wasserman, W.W., and Fahl, W.E.
(1997). CBP/cycA, a CCAAT-binding protein necessary for
adhesion-dependent cyclin A transcription, consists of NF-Y and a novel Mr
115, 000 subunit. Cancer Res.
57,
5117–5121.
Li, Q., Herrler, M., Landsberger, N., Kaludov, N., Ogryzko, V.V., Nakatani, Y., and Wolffe, A.P. (1998). Xenopus NF-Y pre-sets chromatin to potentiate p300 and acetylation-responsive transcription from the Xenopus hsp70 promoter in vivo. EMBO (Eur. Mol. Biol. Organ.) J. 17, 6300–6315.[CrossRef][Medline]
Liu, Q., Yan, H., Dawes, N.J., Lu, Y., and Zhu, H.
(1998). Transcriptional activation of the p34cdc2 gene by cdc2
promoter binding factor/nuclear factor-Y in fetal rat ventricular myocytes.
Circ. Res. 82,
251–260.
Manni, I., Mazzaro, G., Gurtner, A., Mantovani, R., Haugwitz, U.,
Krause, K., Engeland, K., Sacchi, A., Soddu, S., and Piaggio, G.
(2001). NF-Y mediates the transcriptional inhibition of the
cyclin B1, cyclin B2, and cdc25C promoters upon induced G2 arrest. J.
Biol. Chem. 276,
5570–5576.
Mantovani, R. (1998). A survey of 178 NF-Y binding
CCAAT boxes. Nucleic Acids Res.
26,
1135–1143.
Mantovani, R. (1999). The molecular biology of the CCAAT-binding factor NF-Y. Gene 239, 15–27.[CrossRef][Medline]
Mantovani, R., Li, X.Y., Pessara, U., Hoof van Huijsduijnen, R.,
Benoist, C., and Mathis, D. (1994). Dominant negative analogs of
NF-YA. J. Biol. Chem. 269,
20340–20346.
Marziali, G., Perrotti, E., Ilari, R., Testa, U., Coccia, E.M., and Battistini, A. (1997). Transcriptional regulation of the ferritin heavy-chain gene: the activity of the CCAAT binding factor NF-Y is modulated in heme-treated Friend leukemia cells and during monocyte-to-macrophage differentiation. Mol. Cell. Biol. 17, 1387–1395.[Abstract]
Molkentin, J.D., and Olson, E.N. (1996). Combinatorial
control of muscle development by basic helix-loop-helix and MADS-box
transcription factors. Proc. Natl. Acad. Sci. USA
93,
9366–9373.
Nepveu, A. (2001). Role of the multifunctional CDP/Cut/Cux homeodomain transcription factor in regulating differentiation, cell growth and development. Gene 270, 1–15.[CrossRef][Medline]
Perry, R.L., and Rudnick, M.A. (2000). Molecular mechanisms regulating myogenic determination and differentiation. Front. Biosci. 5, 750–767.
Piaggio, G., Farina, A., Perrotti, D., Manni, I., Fuschi, P., Sacchi, A., and Gaetano, C. (1995). Structure and growth-dependent regulation of the human cyclin B1 promoter. Exp. Cell. Res. 216, 396–402.[CrossRef][Medline]
Plet, A., Huet, X., Algarte, M., Rech, J., Imbert, J., Philips, A., and Blanchard, J.M. (1997). Relief of cyclin A gene transcriptional inhibition during activation of human primary T lymphocytes via CD2 and CD28 adhesion molecules. Oncogene 14, 2575–2583.[CrossRef][Medline]
Sassoon, D.A. (1993). Myogenic regulatory factors: dissecting their role and regulation during vertebrate embryogenesis (review). Dev. Biol. 156, 11–23.[CrossRef][Medline]
Schulze, A., Zerfass, K., Spitkovsky, D., Middendorp, S., Berges,
J., Helin, K., Yansen Durr, P., and Henglein, B. (1995). Cell
cycle regulation of the cyclin A gene promoter is mediated by a variant E2F
site. Proc. Natl. Acad. Sci. USA
92,
11264–11268.
Sciortino, S., Gurtner, A., Dey, A., Sacchi, A., Ozato, K., and Piaggio, G. (2001). Cyclin B1 gene is actively transcribed during mitosis in HeLa cells. EMBO Rep. 2, 1018–1023.[CrossRef][Medline]
Sorrentino, V., Pepperkok, R., Davis, R.L., Ansorge, W., and Philipson, L. (1990). Cell proliferation inhibited by MyoD1 independently of myogenic differentiation. Nature 345, 813–815.[CrossRef][Medline]
Suzuki, Y., et al. (2001). Identification and
characterization of the potential promoter regions of 1031 kinds of human
genes. Genome Res. 11,
677–684.
Takahashi, Y., Rayman, J.B., and Dynlacht, B.D.
(2000). Analysis of promoter binding by the E2F and pRB families
in vivo: distinct E2F proteins mediate activation and repression. Genes
Dev. 14,
804–816.
Tiainen, M., Pajalunga, D., Ferrantelli, F., Soddu, S., Salvatori, G., Sacchi, A., and Crescenzi, M. (1996). Terminally differentiated skeletal myotubes are not confined to G0 but can enter G1 upon growth factor stimulation. Cell Growth Diff. 7, 1039–1050.[Abstract]
Vigo, E., Müller, H., Prosperini, E., Hateboer, G.,
Cartwright, P., Moroni, M.C., and Helin, K. (1999). CDC25A
phosphatase is a target of E2F and is required for efficient E2F-induced S
phase. Mol. Cell. Biol. 19,
6379–6395.
Yaffe, D., and Saxel, O. (1977). Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270, 725–727.[CrossRef][Medline]
Yun, J., Chae, H.D., Choy, H.E., Chung, J., Yoo, H.S., Han, M.H.,
and Shin, D.Y. (1999). p53 negatively regulates cdc2
transcription via the CCAAT-binding NF-Y transcription factor. J. Biol.
Chem. 274,
29677–29682.
Zwicker, J., Gross, C., Lucibello, F.C., Truss, M., Ehlert, F.,
Engeland, K., and Muller, R. (1995a). Cell cycle regulation of
cdc25C. transcription is mediated by the periodic repression of the
glutamine-rich activators NF-Y and Sp1. Nucleic Acids Res.
23,
3822–3830.
Zwicher, J., Lucibello, F.C., Wolfraim, L.A., Gross, C., Truss, M., Engeland, K., and Muller, R. (1995b). Cell cycle regulation of cdc25C transcription is mediated by the periodic repression of the glutamine-rich activators NF-Y and Sp1. EMBO J. 14, 4514–4522.[Medline]
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gal reporter
construct. Values are the means ± standard deviations of four
independent experiments.