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J. Biol. Chem., Vol. 277, Issue 52, 50860-50866, December 27, 2002
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From the
Received for publication, April 19, 2002, and in revised form, September 25, 2002
MRG15 is a novel chromodomain protein that is a
member of a family of genes related to MORF4. MORF4 (mortality
factor on chromosome 4) induces senescence in a subset of human tumor
cell lines. Our previous results indicated that MRG15 (MORF-related
gene on chromosome 15) could derepress the B-myb promoter by
association with Rb. In this study, sucrose gradient analysis
demonstrated that MRG15 was present in two distinct nuclear protein
complexes, MAF1 (MRG15-associated factor 1) and MAF2. Rb was associated
with MRG15 and PAM14 (a novel coil-coil protein) in MAF1, and a histone
acetyl transferase, hMOF, was an MRG15 partner in MAF2. Analysis
of deletion mutants of MRG15 indicated that the leucine zipper at the
C-terminal region of MRG15 was important for the protein associations
in MAF1 and that the N-terminal chromodomain was required for the
assembly of the MAF2 protein complex. Consistent with these data was
the fact that a histone acetyltransferase activity associated
with MRG15 was lost when the chromodomain was deleted and that both mutant MRG15 proteins failed to activate the B-myb promoter. The various mechanisms by which MRG15 could activate gene transcription are discussed.
Replicative senescence, the final non-proliferative state reached
by normal cells in culture, is considered a model for aging at the
cellular level. Senescence is dominant over the phenotype of
indefinite division exhibited by tumor cell lines (1). In an effort to
identify senescence-related genes,
MORF4,1 (MORtality Factor on
chromosome 4) was cloned as a gene capable of inducing a senescent
phenotype in a subset of immortal human cell lines (2). MORF4 is a
member of a family of genes, and two of these, MRG15 and MRGX
(MORF4-related genes on chromosomes 15 and X, respectively), which have
a high degree of homology to MORF4, are expressed in human cells.
Analysis of the deduced amino acid sequences of the three predicted
proteins has indicated the existence of helix-loop-helix and leucine
zipper domains, features typical of those found in transcriptional
regulators, as well as regions thought to be involved in
protein-protein interaction. MRG15 has a 96% similarity to MORF4 in
amino acid sequence but fails to induce senescence upon introduction
into immortal cells. The most striking structural difference between
the two proteins is the presence of an N-terminal extension in MRG15
that includes a chromodomain. This region and/or nine single amino acid
changes could therefore be responsible for the differential behavior of the two proteins. The fact that MORF4 is a truncated protein raises the
hypothesis that it could have a dominant negative effect when transfected into cells, resulting in loss of cell proliferation.
Proteins containing a chromodomain characterized to date have been
found to be chromatin remodeling factors involved in causing conformational changes in chromatin by ATP-dependent
movement of nucleosomes and modification of histones (3). Although the function of the chromodomain is not completely understood, some evidence suggests it is involved in protein-protein interaction (4-6),
and it has also been reported to have RNA binding properties (7). Human
MRG15 has overall similarity with the Drosophila msl-3
(male-specific lethal 3) protein, and several recent reports have shown
that they are evolutionarily related (8-10). In Drosophila, msl-3 is required for dosage compensation by the transcriptional enhancement of genes on the male X chromosome (11, 12). Although human
orthologues of most of the members of the proteins involved in sex
dosage compensation in flies exist (8), the processes they are involved
in have not yet been elucidated.
We have previously reported that MRG15 interacts with Rb and is
involved in the activation of the B-myb promoter under E2F control
(13). The action of Rb as a transcriptional repressor of E2F-responsive
genes involved in G1/S transition explains in part its function as a
tumor suppressor (14). The G1/S Rb-dependent arrest is
overcome during the normal cell cycle by phosphorylation of Rb by
cyclin-dependent kinases, which results in destabilization of the Rb/E2F complexes. In this report we demonstrate that MRG15 associates with other nuclear proteins in at least two distinct complexes, MAF1 and MAF2 (MRG15-associated factors 1 and 2, respectively). MRG15 interacts with Rb in the MAF1 complex. PAM14, a
novel protein isolated by us from a yeast two-hybrid screen using MRG15
and MRGX as bait, is another member of this complex. The MRG15 leucine zipper is necessary for the interaction with Rb as well as for activation of the B-myb promoter (13). These results suggest that MRG15
may disrupt E2F/Rb complexes by direct interaction with Rb. In addition
we have found that the hMOF protein, a MYST histone acetylase
(15), is a MRG15 partner in MAF2. hMOF is homologous to the
Drosophila MOF protein, which is also involved in the
enhancement of transcription of genes on the X male chromosome by
acetylation of histone H4 (16, 17). Cells expressing an MRG15 mutant
lacking the chromodomain failed to appropriately assemble the MAF2
complex. We detected a histone acetyltransferase (HAT) activity
associated with wild type MRG15; however, a mutant construct minus the
chromodomain did not have this activity. Additionally, the
chromodomain-minus mutant failed to activate the B-myb promoter, suggesting that this complex is also involved in transcriptional regulation, in this case most likely through chromatin remodeling and
histone acetylation. Thus both MAF1 1 and -2 can affect expression of
the B-myb promoter and, thereby, cell cycle progression.
Plasmid Constructs--
The MRG15 cDNA was introduced into
the BamH1/XbaI sites of pcDNA3.1 to generate
pcDNA3.1 MRG15. The MRG15 open reading frame was then amplified
from this construct using primers that resulted in the incorporation of
HindIII and BamH1 sites at the 5'- and 3'-ends,
respectively. Primers were MRG15Hind,
5'-CCGAAGCTTCCACCATGGCGCCGAAGCAGGAC-3', and MRG15Bam,
5'-GAGTGAGAGGTGCCGGATCCACAGTTTCCGATG-3'.
After amplification, the amplicons were digested with
HindIII and BamH1 and cloned directionally into
the EGFP-N1 vector (Clontech), cut with the same
enzymes. The MRG15 EGFP-N1 chromodomain-minus construct was obtained by
amplifying the MRG15 EGFP-N1 construct with primers that amplified all
but the chromodomain region (amino acids 26-62) and introduced a
PstI site upon religation. Primers were MRG15chr-F,
5'-GCCCTGCAGCTCAAATACGTGGACACC-3', and MRG15chr-R, 5'-GCCCTGCAGAAGAAGAGGCCCATGAAGC-3'.
The MRG15 EGFP-N1 leucine zipper-minus was cloned by a similar strategy
to the chromodomain-minus mutant. Primers used to generate the MRG15
luminous-EGFP-N1 construct were MRG15leu-F, 5'-GCCCTGCAGGCCAGCGATTATGAAGTG-3', and MRG15leu-R,
5'-GCC- CTGCAGCTCATCCAGAGGTGTATA-3'.
The MRG15-HA construct was obtained using the primers MRGHA-F,
5'-TATGGATCCTCCACCATGGCGCCGAAGCAGGACCCG-3', and MRGHA-R,
5'-CTCATGGTAGCCTTTCGACACACAATGGGTATGCTGCAGGGYSYGCAGGGTCTGATGCGATCGGACATCAGA- TCTACT-3'.
The PCR product was cloned into the BamH1 and
XbaI sites of pcDNA3.1 Constructs were verified
by sequencing and restriction enzyme analysis.
Cell Lines and Transfections--
The immortal cell lines EJ
(bladder carcinoma-derived cells) and CMV-MJ-Hel1 (CMV-transformed
human fibroblasts) were maintained in minimum essential medium with
Hanks' salts supplemented with 10% fetal bovine serum. Cells were
transfected using LipofectAMINE Plus (Invitrogen) according to the
manufacturer's instructions. Stable clones expressing PAM14-HA were
obtained from EJ and CMV-MJ-Hel1 parental cell lines as previously
described (13). Cells stably expressing MRG15-HA were obtained by
transfecting EJ cells with pcDNA3.1 MRG15-HA. 24 h
post-transfection, 3,000-10,000 cells were plated into 60-mm dishes in
minimum essential medium with Earl's salts supplemented with 10%
fetal bovine serum and G418 (1 mg/ml). After a two-week incubation at
37 °C in 5% CO2, several clones were isolated and
maintained in medium containing G418. MRG15-HA expression was verified
by immunoblot analysis.
Antibodies--
Rabbit anti-MRG15 polyclonal antibody was raised
against a peptide (amino acids 54-64) in the chromodomain region
(Cocalico Biologicals Inc.). Polyclonal anti-hMOF was obtained as
previously described (15). Polyclonal rabbit anti-HA and rabbit anti-Rb were purchased from Santa Cruz. Mouse monoclonal anti-Rb and anti-E2F1 were from Neomarkers. Mouse monoclonal anti-HA CA125 and mouse anti-GFP
were from Roche Molecular Biochemicals.
Preparation of Cellular Fractions--
Nuclear and cytoplasmic
extracts were prepared essentially as described by Timchenko et
al. (18). Cells were harvested and resuspended in buffer A (25 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol), passed through a 29 gauge needle 5 times on ice, and centrifuged at 1800 × g for 10 min.
Supernatants (cytoplasmic fraction) were frozen at Analysis by Ultracentrifugation in Sucrose
Gradients--
300-700 µg of protein from nuclear extracts was
loaded on 5-20% linear sucrose gradients made in buffer Tris-HCl (25 mM). Samples were diluted with 1.5 volumes of 25 mM Tris-HCl, pH 7.5, before being loaded. Centrifugation
was performed in a Beckmann SW50 at 36,000 rpm for 14 h. Fractions
of 150-µl-200-µl were collected from bottom to top, and aliquots
were analyzed by SDS-PAGE and Western blot. Cytochrome c
(1.7 S), peroxidase (3.5 S), alkaline phosphatase (6.3 S), and catalase
(11.2 S) were used as sedimentation coefficient standards. Cytochrome
c was detected by colorimetry, and the enzymatic activity of
peroxidase, alkaline phosphatase,and catalase was determined in the fractions.
Immunoprecipitations--
Protein A/G BioMag beads
(PolySciences) were preincubated with the corresponding antibodies for
30 min on ice. Nuclear extracts were diluted 1:3 in 25 mM
Tris-HCl, pH 7.5. 200 µg of protein from nuclear extracts was
incubated with 40 µl of beads for 1 h at 4 °C with constant
rotation, These control beads were separated from the lysate, which was
subsequently incubated with the antibody-bound beads for 3 h at
4 °C with constant rotation. Antibody-bound beads were separated
from the remaining soluble proteins with a magnetic holder and washed
twice in buffer B diluted 1: 3 with 25 mM Tris-HCl, pH 7.5.
For the immunoprecipitations of the sucrose gradient fractions, nuclear
extract obtained from EJ-MRG15-HA cells harvested from 32T-150 tissue
culture flasks was loaded onto five 5-20% sucrose gradients, which
were run simultaneously as described above. 175-µl fractions were
collected from top to bottom. On a sixth gradient, peroxidase and
catalase were loaded as sedimentation standards and fractionated. 24 fractions were obtained from each gradient. Fractions 5-10,
corresponding to the position of peroxidase, and fractions 17-21,
corresponding to the position of fractions just below catalase, from
the five gradients were pooled. Protein concentration was raised to 0.5 mg/ml by addition of bovine serum albumin. Immunoprecipitation of the
pooled fractions was performed with A/G beads preincubated with 50 µg
of mouse anti-HA antibody after a preincubation with the same amount of
beads in the absence of antibody. Immunoprecipitates were washed,
denatured, and loaded on a 7.5% gel. Western blots were performed with
rabbit polyclonal anti-HA, anti-hMOF, and anti-Rb antibodies.
Immunostaining and Confocal Microscopy--
0.2 × 106 cells were plated on glass coverslips in 35-mm tissue culture dishes in
minimum essential medium with Earl's salts plus fetal bovine serum in
a 5% CO2 incubator. Transfections with the appropriate
constructs were done as described above. Cells were fixed with
formaldehyde (2%) and permeabilized with Triton-X100 (0.2%) for 10 min. Coverslips were then rinsed with phosphate-buffered saline (PBS)
and blocked with 2% goat serum in PBS for 30 min. After incubation
with the appropriate primary antibodies, immunostaining was performed
using Texas Red-conjugated goat anti-rabbit (1: 500) from Molecular
Probes. Staining with 1 mg/ml DAPI (4, 6-diamino-2-phenylindole) was
done for 10 min. Coverslips were mounted on microscope slides with
Prolong Antifade from Molecular Probes. Microscopy was performed using
an Applied Precision Delta Vision microscope (Issaquah, WA) fitted with
an Olympus I×70 microscope. Images were acquired via wide field
sectioning using fluorescent light. The stacked images, usually 20-25
sections, were subjected to point spread function analysis for better
image quality on Silicon Graphics software (SGI, Mountain View, CA).
Magnification of all cells was maintained at ×600. PAM14-HA was seen
as red, MRG15-GFP as green, 4_,6-diamidino-2-phenylindole nuclear
staining as blue.
HAT Activity Assays--
EJ cells transfected with either
control pCDNA3.1 MRG-15-HA, or Tip60-HA constructs were assayed for
histone acetyltransferase activity using the HAT Check kit (Pierce).
Equal amounts of protein were immunoprecipitated using an anti-HA
antibody, and the immunoprecipitates were incubated with the first 23 amino acids of histone H4 conjugated to biotin and
[14C]acetyl-CoA for one h at 30 °C. The mixture was
added to streptavidin beads and rocked for 30 min at room temperature.
The beads were subsequently washed four times with RIPA buffer,
resuspended into 500 µl of HAT buffer, and 14C activity
was measured with a scintillation counter.
Luciferase Assay--
These utilized the B-myb promoter reporter
constructs and the same experimental approach detailed previously (13).
Briefly, expression plasmids were transfected into EJ cells, which were harvested 24 h later with reporter lysis buffer (Promega). The luciferase activity was determined using the luciferase assay kit
(Promega) and a Monolight 2010 luminometer. The data were normalized to
the amount of protein in the samples as determined by the Bradford
assay (Bio-Rad). Luciferase assays were performed in triplicate to
verify reproducibility, and protein assays were performed in duplicate.
MRG15 Is Present in at Least Two Distinct Nuclear Multiprotein
Complexes--
From our previous work we had evidence of the presence
of MRG15, PAM14-HA, and Rb in independent immunoprecipitates obtained using antibodies against either HA or Rb. This suggested they were
present together in the same multiprotein complex (13). Using a cell
line stably expressing PAM14 tagged with HA, we immunodepleted the
tagged protein in nuclear extracts with an anti-HA antibody and
determined that about 30% of MRG15 was still present in the supernatant (data not shown). Because this indicated that MRG15 could
be associated with other proteins in more than one complex, we
fractionated nuclear extracts from EJ cells stably expressing either
MRG15-HA or PAM14-HA and CMV-MJ-Hel 1 cells stably expressing PAM14-HA
by ultracentrifugation on 5-20% linear sucrose gradients. The
presence of MRG15, PAM14, and Rb proteins was determined by SDS-PAGE
and Western blot analysis. In the stable clone expressing MRG15-HA, the
anti-HA antibody demonstrated the existence of at least two different
complexes containing MRG15: a more abundant component with a
sedimentation coefficient of 3.5 S, MAF1, and a minor component with a
sedimentation coefficient of 14 S, MAF2 (Fig.
1a). In all the clones the same
distribution of MRG15 was also detected by the anti-HA antibody, and
PAM 14 and Rb were found only in the fractions that corresponded to
MAF1 (Fig. 1, b and c). The peaks of Rb and MRG15
were not found in the same fraction in EJ-PAM14-HA cells (Fig.
1b), which could be because of clonal variation.
Alternatively, there may be more than one Rb/MRG15 complex in this
clone.
To eliminate the possibility that these two complexes were the result
of the disassembly of a unique multiprotein complex during the
preparation of the extract, we investigated the in vivo
distribution of MAF1 and -2 by immunostaining and confocal microscopy.
CMV-MJ-Hel 1 cells stably expressing PAM14-HA were transfected with a
construct encoding MRG15-GFP. GFP was visualized directly and PAM14-HA
by immunostaining with an anti-HA primary antibody and a secondary
anti-rabbit antibody conjugated to Texas Red fluorescent dye. The
images obtained by confocal microscopy demonstrated that MRG15 was
present throughout the nucleus (Fig. 2a), whereas PAM14 was more
abundant in the perinuclear region (Fig. 2b) where it
co-localized with MRG15 (Fig. 2c). The fact that
there was no co-localization of MRG15 and PAM14 in the inner part of
the nucleus supports the results from the fractionation studies
indicating that MRG15 is present in at least two nuclear complexes.
MRG15 Interacts with Both the Phosphorylated and Unphosphorylated
Forms of Rb--
We have previously established that the MRG15
interaction with Rb in a nucleoprotein complex mediates the activation
of the E2F-responsive promoter, B-myb (13), most likely through
derepression by the disruption of E2F-Rb complexes. The most common
mechanism responsible for this effect is the phosphorylation of Rb by
cyclin-dependent kinases, which hyperphosphorylate Rb to
the inactive form that loses affinity for E2F (14). Both the
phosphorylated and unphosphorylated forms of Rb cosediment with MRG15
at the position of MAF1. To determine whether MRG15 associated
preferentially with one of these forms of Rb, nuclear extracts of EJ
MRG15-HA cells immunoprecipitated with an anti-HA antibody were
analyzed with an antibody that recognizes Rb in both states. Both forms
of Rb were present in the immunoprecipitates (Fig.
3, lane 3), and some of the
phosphorylated molecules were present in the non-precipitated proteins
(Fig. 3, lane 1).
hMOF is an MRG15 Partner in MAF2--
The human MRG15 protein has
an overall homology with the msl-3 protein from Drosophila
(8) that is involved in dosage compensation by transcriptional
enhancement of the genes present on the X male chromosome (12). This
mechanism requires the recruitment of the multiprotein MSL complex and
two associated small RNA molecules, rho1 and rho2, to the X chromosome
(9). The MSL complex has multiple activities involved in chromatin
remodeling, including histone acetylation via a histone
acetyltransferase, MOF. A homologue to the Drosophila protein was
recently found in humans, hMOF, a chromodomain protein belonging to the
MYST family of acetyl transferases (15). MRG15 and another human
protein, the product of the msl-3L1 gene (19), have been proposed as
proteins potentially able to interact with hMOF (15). We therefore used
an anti-hMOF antibody in Western analysis of EJ-MRG15-HA nuclear
extracts immunoprecipitated with either anti-HA or anti-Rb antibodies.
The fractions obtained from sucrose gradient sedimentation were also
analyzed. hMOF was detected only in the anti-HA immunoprecipitates and
the sucrose gradient fractions corresponding to MAF2 but not MAF1 (Fig.
4, a and b) and was
absent in the Rb immunoprecipitates (Fig. 4a).
Immunoprecipitation Analysis of the Proteins in the Sucrose
Gradient Fractions--
To demonstrate that the proteins identified by
Western analysis in the sucrose gradient fractions (Figs. 1 and 4) were
indeed together in the proposed complexes (MAF1 and MAF2), we initially attempted immunoprecipitations of the individual fractions involving MAF1 and MAF2. Western analysis produced no results, most likely because of the low amount of protein present. We then decided to pool
the fractions related to MAF1 and MAF2 from five gradients produced
simultaneously prior to immunoprecipitation. We were successful in
identifying the presence of MRG15 with Rb in MAF1 and hMOF in MAF2
(Fig. 5). We did not identify the presence of PAM14 because it was necessary to run a 7.5% gel to visualize MRG15,
Rb, and hMOF, a percentage gel in which PAM14 is lost during electrophoresis. However, the data demonstrate that MRG15 associates with Rb in MAF1 and hMOF in MAF2.
The MRG15 Leucine Zipper Domain and the Chromodomain Are Necessary
for Appropriate Assembly of MAF1 and MAF2, Respectively--
Leucine
zipper and chromodomain regions are generally thought to be involved in
protein-protein interactions. We have previously determined that the
MRG15 leucine zipper was required for appropriate interaction with
PAM14 and Rb (13). To evaluate the effect of both regions in the
constitution of the MAF multiprotein complexes, we analyzed the
sedimentation behavior of the protein members of the MAFs when EJ cells
stably expressing PAM14-HA were transfected with plasmids containing
either wild type MRG15-GFP or deletion mutants lacking either the
leucine zipper or chromodomain regions.
The absence of the leucine zipper caused disassembly of MAF1 by
disrupting the association with Rb (Fig. 6)
and the displacement of MRG15 to a lower position in the gradient. We
did not detect PAM14 in any of the fractions, possibly because of the
instability of this protein when it loses association with MRG15. The
deletion of the leucine zipper resulted in no significant change in the MAF2 complex, whereas the absence of the chromodomain resulted in
complete loss of the MAF2 complex. This indicates that the leucine
zipper is necessary for the assembly of proteins in MAF1 and that the
chromodomain is required for the assembly of MAF2.
The MRG15 Chromodomain Is Associated with a HAT Activity and Is
Also Involved in the Activation of the B-myb Promoter--
Because we
had detected hMOF in immunoprecipitates as well as sucrose gradient
fractions of MRG15, we performed a HAT activity analysis. Nuclear
extracts from cells transfected with either empty vector pcDNA3.1
(Invitrogen), pcDNA3.1 MRG15-HA tagged at the C terminus, or a
plasmid expressing a HA-tagged TIP60 protein (a known co-activator
protein that has HAT activity) (19) were immunoprecipitated with an
anti-HA antibody and analyzed for HAT activity. The results, expressed
as the percentage of the total HAT activity, indicate that in the case
of MRG15-HA there was 2.2-fold increased HAT activity compared with
empty vector, similar to the 2.7-fold activity observed with TIP60-HA
(Fig. 7a). Interestingly, immunoprecipitates of cells transfected with GFP N-terminal-tagged MRG15 protein did not associate with HAT activity (data not shown), suggesting that the N-terminal region of MRG15 is required for this
association.
We had previously demonstrated that MRG15 could activate the B-myb
promoter and that the leucine zipper region was required for this
derepression, most likely through interaction with Rb. We tested the
chromodomain minus mutant in this assay to determine whether HAT
activity was involved in affecting the activity of this promoter and
indeed observed that the activation was abolished (Fig. 7b).
Collectively, these observations suggest that the B-myb promoter
activity is dependent on both the interaction of MRG15 with Rb and the
resulting inhibition of Rb-mediated transcriptional repression,
as well as a HAT-associated activity acting most likely via
histone acetylation.
The MRG15 gene had been identified (2) as a member of a family of
genes related to MORF4, a gene that induces replicative senescence in a
subset of immortal cell lines. The MRG15 protein is very similar to the
MORF4 protein except for the presence of an N-terminal extension that
harbors a chromodomain and nine changes in amino acids, most of which
are conserved. This strong homology suggests that MORF4 could have a
dominant negative effect on MRG15 action when transfected into cells.
Proteins containing a chromodomain are usually involved in chromatin
remodeling that leads to transcriptional activation or repression of a
large number of genes. This implicates MRG15 as a good target for
involvement in the permanent growth arrest elicited by MORF4.
Our previous findings have demonstrated that MRG15 is able
to derepress transcription of the luciferase gene under the control of
the B-myb promoter and that it associates with Rb and PAM14, a novel
protein (13). In this report we have established that MRG15 is present
in two distinct nuclear protein complexes, MAF1 and MAF2, in
association with proteins known to be involved in the transcriptional
activation of genes. MRG15 has a leucine zipper and a chromodomain,
motifs known to facilitate interaction with other proteins. The
analysis by sucrose gradients of nuclear extracts from cells expressing
deleted versions of the MRG15 protein, lacking one or the other of
these domains, has shown that the leucine zipper is required for the
appropriate assembly of proteins at MAF1 without a significant change
in MAF2 constitution. In MAF1, MRG15 associates with Rb, whereas E2F1
is not part of the complex, suggesting a derepressive nature of this
association. Thus MRG15 stimulation of the B-myb promoter most likely
involves the disruption of Rb-E2F complexes. PAM14 is a novel coil-coil
protein discovered in our laboratory by its ability to interact with
MRG15 in the yeast two-hybrid system. Its interaction with MRG15 has
been previously demonstrated by GST pull-down and immunoprecipitation
(13). In this report we have established that it is part of MAF1. In MAF2 we have found the association of MRG15 with the human hMOF, the
Drosophila homologue of which has been shown to have histone acetyl
transferase activity (17). In addition, immunoprecipitates of MRG15
have an associated HAT activity. Expression of a chromodomain-minus mutant MRG15 protein resulted in loss of the MAF2 complex and HAT-associated activity. This mutant protein was also unable to positively affect the activity of the B-myb promoter. These results suggest that both MAF1 and MAF2 are needed for B-myb promoter activity,
the former involving interaction with Rb through the leucine zipper
domain of MRG15 and the latter possibly through relaxation of chromatin
around the promoter via histone acetylation. Proteins containing a
chromodomain, such as the Drosophila msl-3 protein, are components of
multimeric complexes that modify chromatin structure via histone
acetylation at target sites specified by the complex (3, 17).
Specifically, these msl proteins paint the Drosophila X
chromosome in association with the rox RNAs (transcripts specific to
the X chromosome) and the histone acetyltransferase MOF to cause
hypertranscription in dosage compensation (17). Therefore, the presence
of the chromodomain in MRG15 suggests that it possesses the distinct
potential to act in transcriptional regulation, perhaps through an
interaction with specific RNAs or histone acetyltransferases. The
Saccharomyces cerevisiae homologue of MRG15, Eaf3p, is a
component of the yeast NuA4 HAT complex that contains Esa1p, the
S. cerevisiae homologue of MOF and Tip60 (20). Esa1p and the
NuA4 complex are essential for cell division and viability in budding
yeast (20). Therefore, the MRG15 protein may be a component of a HAT
complex homologous to the yeast NuA4 complex. The role of PAM14
is not clear at this time, but by comparison with other nucleoprotein
complexes that have been described, it could be acting as an adaptor
protein, facilitating interaction between proteins in the MAF1 complex.
The ability of MRG15 to increase B-myb promoter activity has
implications for a potential role for MRG15 in cell cycle progression. The B-myb protein is an established transcriptional activator that
promotes cell cycle progression through stimulation of multiple effectors of cell growth, including cyclin D1 and cdc2 (22, 23).
Furthermore, overexpression of the B-myb gene has been demonstrated to
promote bypass of p53-induced G1 arrest (23), suggesting that B-myb
also induces cell growth through inhibition of growth inhibitory
molecules. B-myb expression correlates with cell cycle progression in
that it is repressed during G0 and early G1 phases but stimulated in
late G1 and S phases (21). Significantly, this mRNA expression
pattern is similar to that of MRG15, in that there is a ~2-fold
increase in MRG15 mRNA levels at 4-8 h postserum stimulation of
normal quiescent fibroblasts, and these elevated mRNA levels
persist up to 28 h poststimulation (9). Young fibroblast cells
4-8 h poststimulation are in the early to mid-G1 phase of the cell
cycle and enter S phase at 16 h postexposure to serum, with
maximal DNA synthesis at 24 h poststimulation. Thus, like B-myb,
MRG15 expression correlates with cell cycle progression and therefore
represents a putative growth stimulatory factor. It is tempting to
speculate that such potential proliferative functions of MRG15 are
because of its ability to antagonize one or more activities of Rb in
addition to chromatin remodeling.
Western analysis of MRG15 protein levels has indicated that MRG15
protein levels decrease about 2-3-fold with increasing cell population
doublings and cell senescence in normal human fibroblasts (data not
shown). This further suggests that the function of MRG15 is
correlated with cell cycle progression. The fact that the MRG15 transcripts and protein are expressed in a wide variety of tissues and
are conserved across a large number of species suggests an essential
function for this protein. Future studies aimed at examination of the
effect of MRG15 on additional Rb/E2F-repressed promoters and
identification of additional cellular factors within the MRG15 protein
complexes should provide critical insight into the role of this protein
in the cell cycle and other processes.
We thank Dr. Kaoru Tominaga for help with this work.
*
This work was supported by NIA, National Institutes of
Health Grants R37AG05333 and PO1AG13663 (to O. M. P. S.), T32AG00183 (to J. K. L.), and GM15691 (to J. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Both authors contributed equally to this work.
¶
Present Address: University of California, Berkeley, Dept. of
Molecular and Cell Biology, 229 Stanley Hall, No. 3206, Berkeley, CA
94720-3206.
**
To whom correspondence should be addressed: Dept. of Cellular and
Structural Biology, The Sam and Ann Barshop Center for Longevity and
Aging Studies, University of Texas Health Science Center at San
Antonio, South Texas Centers for Biology in Medicine, 15355 Lambda Dr.,
Rm. 2.200, San Antonio, TX 78245-3207. Tel.: 210-562-5069; Fax:
210-562-5028; E-mail: smitho@uthscsa.edu.
Published, JBC Papers in Press, October 22, 2002, DOI 10.1074/jbc.M203839200
The abbreviations used are:
MORF, mortality
factor;
MRG, MORF-related gene;
MAF, MRG15-associated factor;
HAT, histone acetyltransferase;
msl-3, male-specific lethal 3;
HA, hemagglutinin;
GFP, green fluorescence protein;
Mof, males absent on
the first.
MRG15, a Novel Chromodomain Protein, Is Present in Two Distinct
Multiprotein Complexes Involved in Transcriptional Activation*
§,¶§,
, and**
Roy M. and Phyllis Gough Huffington Center
on Aging, Baylor College of Medicine, Houston, Texas 77030 and the
Department of Biology, Emory University,
Atlanta, Georgia 30322
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C or used
immediately. Pellets were washed in buffer A and resuspended in buffer
B (25 mM Tris-HCl, pH 7.5, 0.42 M NaCl, 1.5 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, 25% sucrose), incubated for 30 min on
ice, and centrifuged at 1800 × g for 10 min. For
sucrose gradient analysis, buffer B was made with 10% sucrose. The
supernatants were used immediately or frozen at 70 °C. All buffers
were supplemented with a protease inhibitor mixture (Calbiochem),
phenylmethylsulfonyl fluoride (0.5 mM), and the proteasome
inhibitor, MG132 (2 µM).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sucrose gradient analyses demonstrate MRG15
is in at least two distinct nucleoprotein complexes.
Nuclear proteins from EJ cells stably expressing
either MRG15-HA (a) or PAM14-HA (b) and
CMV-MJ-Hel 1 cells (c) stably expressing PAM14-HA were
fractionated on a 5-20% w/v sucrose gradient, and the fractions were
analyzed by SDS-PAGE and Western blot. An anti-HA polyclonal antibody
was used for detection of the HA-tagged proteins and a commercial
monoclonal antibody for detection of Rb. In cells expressing PAM14-HA,
MRG15 was detected using a polyclonal antibody raised against a peptide
in the chromodomain region. Cytochrome c (1.7S),
peroxidase (3.5S), alkaline phosphatase (6.3S), and catalase (11.2S)
were used as sedimentation coefficient standards.
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Fig. 2.
Partial co-localization of MRG15 with PAM14
in the cell nucleus. CMV-MJ-Hel 1 cells stably expressing PAM-HA
were transfected with the pEGFP-N1-MRG15 plasmid, fixed, and
immunostained using a polyclonal anti-HA antibody and Texas
Red-conjugated goat anti-rabbit as secondary antibody. DNA was stained
with DAPI. Localization of MRG15-GFP (a), of PAM14-HA
(b), and of the merged colocalization of the two proteins
(c) is shown.
View larger version (24K):
[in a new window]
Fig. 3.
MRG15 binds both the phosphorylated and
unphosphorylated forms of Rb. Nuclear proteins (200 µg) from EJ
cells expressing MRG15-HA were immunoprecipitated with the monoclonal
anti-HA antibody. Lane 1, half the supernatant following
immunoprecipitation; lane 2, proteins that bound
nonspecifically to the beads; lane 3, the immunoprecipitated
proteins subjected to SDS-PAGE and immunoblotted with a polyclonal
anti-HA antibody and a monoclonal anti-Rb antibody.
View larger version (31K):
[in a new window]
Fig. 4.
Western analysis of nuclear proteins from EJ
cells stably expressing MRG15-HA following immunoprecipitation with
either an anti-HA or anti-Rb antibody. a, Western analyses
of lane 1, initial nuclear extract; lane 2, the
supernatant following immunoprecipitation with anti-HA antibody;
lanes 3 and 6, proteins bound nonspecifically to
the beads; lane 4, proteins bound to the control monoclonal
anti-FLAG antibody; lane 5, proteins immunoprecipitated with
anti-HA antibody; and lane 7, proteins immunoprecipitated
with anti-Rb antibody. b, fractions from the nuclear
extracts in panel a following ultracentrifugation on a
sucrose gradient and analyzed by SDS-PAGE and Western blots using the
same antibodies as in panel a.
View larger version (30K):
[in a new window]
Fig. 5.
Immunoprecipitation analysis of the proteins
present in the sucrose gradient fractions. Five gradients of
nuclear extract from 32T-150-cm2 tissue culture
flasks of EJ-MRG15-HA were run simultaneously. Fractions 5-10 (which
corresponded to the position of MAF1 and the peroxidase sedimentation
standard in our many previous experiments) and fractions 17-21
(corresponding to MAF2 and the catalase sedimentation standard) from
all gradients were pooled and immunoprecipitated with an anti-HA
antibody. Western analysis revealed the presence of MRG15, Rb, and hMOF
proteins. Lane 1, nuclear extract; lane 2,
immunoprecipitation of fractions 5-10; lane 3,
postimmunoprecipitates of fractions 5-10; lane 4,
immunoprecipitation of fractions 17-21; lane 5,
postimmunopreciptates of fractions 17-21.
View larger version (32K):
[in a new window]
Fig. 6.
Sucrose gradient analysis of wild type
versus deletion mutants of MRG15. Fractions
obtained by sucrose gradient analysis of EJ cells stably expressing
PAM14-HA transfected with pEGFP-N1 MRG15 (WT), pEGFP-NI
MRG15 chromodomain minus ( CD), or pEGP-N1 MRG15 leucine
zipper minus (LZ) were run on 4-15% gradient minigels.
Proteins were analyzed by Western analysis using antibodies against
anti-HA, anti-GFP, and anti-Rb antibodies.
View larger version (92K):
[in a new window]
Fig. 7.
A HAT activity is present in
immunoprecipitates of wild type MRG15 but not the chromodomain-minus
mutant protein. a, nuclear extracts of EJ cells
transfected with either empty vector pcDNA3.1 MRG15-HA, or Tip60-HA
were immunoprecipitated with an anti-HA antibody, and HAT assays were
performed. b, EJ cells were transfected with 0.5 µg of a
luciferase-expressing plasmid under the control of the B-myb promoter
either alone or with 1 µg wt or chromodomain-deleted MRG15. Cells
were lysed 24 hrs post-transfection and the amount of luciferase
normalized to the protein in sample. The mutant B-myb promoter served
as a positive control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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