1 Department of Cell Biology and Cancer Center, University of Massachusetts
Medical School, Worcester, Massachusetts 01655-0106, USA
2 Department of Physiology, University of Massachusetts Medical School,
Worcester, Massachusetts 01655-0106, USA
* Author for correspondence (e-mail: gary.stein{at}umassmed.edu)
Accepted 5 August 2002
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Summary |
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Key words: Intranuclear targeting, Runt homology factors, Green fluorescent protein, Fluorescence recovery after photobleaching, Nuclear matrix
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Introduction |
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The dynamics by which proteins traverse and localize within the nucleus may
be critical for their biological activity. One important question relates to
the relative mobility and compartmentalization of RUNX transcription factors
in subnuclear foci in living cells. Previous studies using fluorescence
recovery after photobleaching (FRAP) assays have shown that green fluorescent
protein (GFP) fused to the splicing factor ASF rapidly associates with
splicing compartments and is less mobile than GFP alone
(Phair and Misteli, 2000).
Other nuclear proteins, such as GFP-histone H2B in chromatin
(Kanda et al., 1998
;
Phair and Misteli, 2000
) and
GFP-lamin B receptor in the nuclear envelope
(Ellenberg et al., 1997
), are
even more immobile. Hence, an emerging concept is that the mobility of
proteins is directly coupled to their function and whether or not they are
architecturally linked to specific subnuclear compartments.
To understand the relative mobility of RUNX transcription factors in the nucleus and the dynamics of their association with subnuclear sites, we used time-lapse microscopy and FRAP analysis. Our key result is that RUNX1 and RUNX2 transcription factors are targeted to and dynamically associate with common subnuclear foci that remain stationary within the nuclear space. Furthermore, we show that a C-terminal truncation of RUNX2 that removes the subnuclear targeting signal increases the mobility of the protein to that of EGFP alone. These findings suggest that the dynamic association of RUNX proteins in stationary foci provides a mechanism for formation of regulatory complexes that are essential for RUNX-dependent cell differentiation and embryonic development.
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Materials and Methods |
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Plasmids
cDNA for enhanced green fluorescent protein (EGFP; Clontech, Palo Alto, CA)
was cloned into pcDNA3 (Invitrogen, Carlsbad, CA) and fused to full-length
RUNX2 (amino acids 1-513), RUNX2361 (amino acids 1-361) and RUNX1
(amino acids 27-480) cDNAs. EGFP-RUNX2 and EGFP-RUNX2
361 were generated
by inserting a PCR-amplified EGFP cDNA into pcDNA3 and then cloning either
RUNX2 or RUNX2
361 cDNAs. Briefly, the pEGFP-C3 vector (Clontech) was
used as a template for EGFP cDNA PCR amplification using the forward primer
5'-TCTAGAGGTACCATGGTGAGCAAGGGC-3', which contains a KpnI
restriction site and the reverse primer
5'-ATAGAATTCGGATCCCTTGTACAGCTCGTC-3' with engineered
BamHI and EcoRI sites. The KpnI and EcoRI
sites were used to insert the amplified EGFP cDNA fragment into pcDNA3. RUNX2
and RUNX2
361 cDNAs (Javed et al.,
2001
) were subsequently subcloned into this plasmid at the
3' end of EGFP using the BamHI site and either XbaI or
XhoI sites, respectively. Similarly, the EGFP-RUNX1 plasmid was
generated by inserting a PCR-amplified EGFP cDNA product into pcDNA3 and then
adding the RUNX1 cDNA. EGFP was amplified by PCR using the oligonucleotides
5'-GGATCCGGTACCATGGTGAGCAAGGGCGAGGAG-3' as the forward primer and
5'-GAATT-CTCTAGACTTGTACAGCTCGTCCATGCC-3' as the reverse primer.
The PCR product was digested with KpnI and XbaI and then
ligated to a similarly digested pcDNA3 vector to generate pcDNA3-EGFP. The
XbaI/XbaI fragment of RUNX1 (amino acids 27-480) was then
inserted into this plasmid. All clones were then manually sequenced using
Sequenase version 2.0 kit (Amersham Pharmacia, Piscataway, NJ).
Transcription assays
HeLa cells were plated in six-well plates at a density of
0.6x106 cells per plate and transiently transfected at 50%
confluency using, in each well, 5 µl of Superfect reagent (Qiagen,
Valencia, CA), 500 ng of each expression vector (as shown in
Fig. 1D), 50 ng of the minimal
osteocalcin (OC) promoter -83-OC-Luciferase
(Towler et al., 1994), which
was used as a control for transfection efficiency, and 2.5 µg of the rat
-1.1 kb OC promoter-CAT reporter gene
(Schepmoes et al., 1991
).
Reporter activities were determined 36-40 hours following transfection. Cells
were lysed with 250 µl of 1x Reporter lysis buffer (Promega, Madison,
WI). CAT activities were determined in 50 µl of cell lysate and normalized
to luciferase values. The significance of these results was assessed using the
analysis of variance (ANOVA) test, and the error bars are shown as the
standard error of the mean (s.e.m.).
|
Western blot analysis
HeLa cells were plated at a density of 0.7x106 in 100 mm
plates and transfected with 10 µg of expression plasmid and 40 µl of
Superfect reagent (Qiagen) following the manufacturer's recommendations. Cell
pellets were collected 20 hours after transfection and lysed in 300 µl of
lysis buffer containing 8 M urea, 0.1 M NaH2PO4, 0.1 M
Tris-HCl, pH 8.0 and a cocktail of protease inhibitors including, 1.2 mM
phenylmethanesulfonyl fluoride (PMSF), 0.5 µg/ml leupeptin, 0.7 µg/ml
pepstatin, 10 µg/ml trypsin inhibitor, 2 µg/ml TPCK, 40 µg/ml
bestatin, 17 µg/ml calpain inhibitor I, and 1 µg/ml E64 (Roche,
Indianapolis, IN). For each sample 20 µg of total protein was separated on
a 10% SDS-PAGE gel. EGFP proteins were detected using a GFP monoclonal
antibody (Clontech; 1:10,000 dilution). RUNX proteins were detected with
polyclonal antibodies against either RUNX2 (1:10,000 dilution) or RUNX1
(1:3,000 dilution) (Meyers et al.,
1996). Appropriate HRP-conjugated secondary antibodies (1:10,000
dilution) were detected using the Renaissance chemiluminescence kit (NEN,
Boston, MA). Cdk2 protein was detected using an
-cdk2 antibody (1:5,000
dilution) as a control for protein loading. The HRP-conjugated secondary
antibodies and the polyclonal cdk2 antibody were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA).
In situ immunofluorescence
SaOS-2 cells were grown on 0.5% (w/v) gelatin-coated coverslips and
cultured to 70% confluency. Cells were transiently transfected with 0.5 µg
of expression plasmid, 3 µl Plus reagent and 2.5 µl Lipofectamine
reagent (Life Technologies, Grand Island, NY) following the manufacturer's
protocol. Cells were harvested 18-20 hours post-transfection. Whole cell (WC)
and nuclear matrix-intermediate filament (NMIF) preparations were performed as
previously described (Javed et al.,
2000). Briefly, cells were fixed using formaldehyde (3.7%), then
permeablized with 0.5% Triton X-100 for whole cell preparations.
Nuclear-matrix-intermediate filament preparations were extracted twice for 15
minutes each with CSK buffer [100 mM NaCl, 0.3 M sucrose, 10 mM pipes, pH 6.8,
3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 2 mM Vanadyl
Ribonucleoside Complex (VRC), 0.8 mM 4-(2-Aminoethyl)benzenesulfonyl floride
(AEBSF)] and digested twice for 30 minutes each with 400-600 Units/ml of
RNase-free DNase I (Roche, Indianapolis, IN) in digestion buffer (CSK buffer
with 50 mM NaCl). Cells were extracted with 0.25 M ammonium sulfate in
digestion buffer for 10 minutes. Xpress-tagged RUNX2 proteins were detected
using a monoclonal
-mouse Xpress antibody (Invitrogen; 1:800 dilution)
and a Texas-red-conjugated donkey
-mouse secondary antibody (Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA; 1:200 dilution). Cells were
mounted in Vectashield antifade mounting media (Vector Laboratories,
Burlingame, CA). Fluorescence and transmitted light images were captured using
a Zeiss Axioplan 2 microscope with a 63x Zeiss Plan-Apochromat objective
(1.4 N.A.), a 100 W Hg lamp and a Hamamatsu digital charged-couple device
(CCD) camera interfaced with the MetaMorph Imaging System (Universal Imaging
Corp., Downingtown, PA).
BrUTP labeling
SAOS-2 cells were transfected with 0.25 µg EGFP-RUNX2 as described above
and labeled for BrUTP incorporation 18-20 hours following transfection. Cells
were incubated for 3 minutes with glycerol buffer (20 mM Tris-HCl pH 7.4, 5 mM
MgCl2, 0.5 mM EGTA, 25% glycerol) and then for 3 minutes with
glycerol buffer supplemented with 0.05% Triton X-100 and 4 mM AEBSF. Nascent
transcripts were labeled with BrUTP for 30 minutes at room temperature in
transcription buffer [2x Synthesis buffer (100 mM Tris-HCl pH 7.4, 20 mM
MgCl2, 1 mM EGTA, 200 mM KCl, 50% glycerol), 25 µM SAM, 500
µM each of ATP, CTP and GTP (Roche), 750 µM BrUTP (Sigma, St. Louis,
MO), 4 mM AEBSF and 40 Units/ml RNase Inhibitor (Roche)]. NMIF extractions
were performed on cells as described above. A rat monoclonal -BrdU
antibody (Accurate Chemical and Scientific Corp., Westbury, NY; 1:20 dilution)
and an Alexa 568 nm
-rat secondary antibody (Molecular Probes, Eugene,
OR; 1:500 dilution) were utilized to detect BrUTP labeling. A Leica SP1 laser
scanning confocal microscope interfaced with Scanware software and a Leica
100x Plan Apo 1.4 N.A. objective were used to capture confocal images.
Images were taken using an average of four to six sections per cell and 0.365
µm per section. The line scan function in MetaMorph was used to show points
of colocalization in a particular area of the nucleus.
Time-lapse imaging
SaOS-2 cells were plated at a cell density of 2x106 in 100
mm plates containing gelatin-coated 40 mm coverslips (Bioptechs, Butler, PA).
Cells were then transiently transfected using 4 µg of either EGFP,
EGFP-RUNX1, EGFP-RUNX2, or EGFP-RUNX2361 expression plasmids, 10 µl
Lipofectamine reagent and 20 µl Plus reagent (Life Technologies).
Mitochondria were stained 15-18 hours following transfection with 100 nM of
Mitotracker Red CM-H2XRos dye (Molecular Probes, Eugene, OR) in pre-warmed
completed McCoy's 5A media for 30 minutes at 37°C. The Mitotracker Red dye
was used as a marker for viability before and after capturing images.
Coverslips were then assembled into the FCS-2 closed cell chamber (Bioptechs,
Butler, PA) in which a peristaltic pump (Instech Laboratories Inc, Plymouth
Meeting, PA) was used to perfuse complete L-15 media without phenol red (Life
Technologies) and 10 nM Mitotracker Red dye through the chamber. Cells were
maintained at 37°C using the chamber controller and objective heater
controller (Bioptechs). Time-lapse images were captured every 10-30 seconds
for 20-30 minutes using the Zeiss Axioplan 2 microscope and a 63x Zeiss
Plan-Apochromat objective with a 1.4 N.A. Exposure times for EGFP fusion
proteins were 100-200 milliseconds and for the Mitotracker Red dye were
100-500 milliseconds. Adobe Photoshop, MetaMorph, Microsoft PowerPoint and
Adobe Illustrator software were used to prepare the digital images.
Fluorescence recovery after photobleaching analysis
SaOS-2 cells were plated in T-25 flasks at a density of
1.2x106 cells/flask and cultured until 70% confluency.
Expression plasmids (2 µg) were transiently transfected using 5 µl of
Lipofectamine and 4 µl Plus reagents (Life Technologies). Transfected cells
were incubated at 37°C for 6 hours, trypsinized using 1 ml Trypsin-EDTA
(Life Technologies) and plated in coverslip live cell chambers. Cells were
incubated overnight at 37°C. The Zeiss Axiovert-10 light microscope was
used with a Zeiss 100x Plan-Neofluor N.A. 1.30 lens, adapted with a
Roper Scientific (Trenton, NJ) cooled CCD camera with a ST-133 controller and
an EEV Type 57 back-illuminated frame transfer chip to capture images.
Pre-bleached images were captured using a 200 millisecond exposure time. A
small area of the nucleus was photobleached using a 476.5 nm Argon ion laser
at 100 mW of power for 100 milliseconds. Images of fluorescence recovery were
captured every second for 45 seconds using 200 millisecond exposure times. The
half-time of recovery (t) was determined by
plotting ln (i
-it) vs. time, where
i
is the fluorescent intensity at infinity, it is
the fluorescent intensity in the bleached area at time (t) and then
was calculated as t
=ln 2 * (-1/slope).
The percentage immobile fraction (F) was calculated using the formula:
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The ratio (Ipost/Ipre), where Ipre is the
pre-bleached intensity over the whole cell and Ipost is the
post-bleached intensity of the whole cell, was used to correct for the extent
of photobleaching. ipre is the fluorescent intensity in the
pre-bleached area of the nucleus and b is the y-intercept of the graph ln
(i-it) vs. t. We calculated the recovery
rates for both the entire photobleached area and for specific foci in the
bleached area. Adobe Photoshop, Microsoft PowerPoint and Adobe Illustrator
were used to assemble the digital images. Standard errors were determined as
the standard error of the mean (s.e.m.).
Online supplemental information
QuickTime Movies 1A-D (available at
jcs.biologists.org/supplemental)
show the time-lapse images corresponding to
Fig. 5A-D. Time-lapse images
were captured for Movie 1A, every 20 seconds for 20 minutes; Movie 1B, every
10 seconds for 30 minutes; Movie 1C, every 20 seconds for 30 minutes; and
Movie 1D, every 30 seconds for 30 minutes. QuickTime Movies 2A-D show cells
corresponding to those captured in Fig.
6A-D for FRAP analysis. Movie 2D (EGFP alone) shows sequential
images captured before bleaching and for every second for the first 10 seconds
after photobleaching. Movies 2A-C (EGFP-RUNX fusion proteins) show sequential
images captured before bleaching and every second for 45 seconds after
photobleaching.
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Results |
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We determined whether EGFP-RUNX fusion proteins localize to sites of active transcription. Nascent transcripts were labeled with BrUTP in SaOS-2 cells transfected with EGFP-RUNX1 and EGFP-RUNX2. Using confocal microscopy we found that the majority of both the EGFP-RUNX1 and EGFP-RUNX2 foci colocalize with sites of BrUTP incorporation (Fig. 3A,B). Matching red and green peaks in the same positions of the line scans (Fig. 3) demonstrate points of colocalization between EGFP-RUNX1 or EGFP-RUNX2 and BrUTP labeling. These results indicate that active transcription occurs at a significant subset of the punctate RUNX foci.
|
RUNX1 and RUNX2 are targeted to common subnuclear domains
RUNX1 and RUNX2 have analogous subnuclear targeting signals in their
C-termini that are highly conserved. Therefore, in situ immunofluorescence
microscopy was used to assess whether RUNX1 and RUNX2 are directed to the same
subnuclear domains. We also examined, as a control, whether RUNX2 proteins
with different epitope tags are localized in the same subnuclear foci. EGFP-
and Xpress (XPR)-tagged RUNX2 proteins
(Fig. 4A) or EGFP-RUNX1 and
XPR-RUNX2 proteins (Fig. 4B)
were co-expressed in SaOS-2 cells and their subnuclear distribution in WC and
NMIF preparations was monitored. XPR-tagged RUNX proteins were visualized
using a Texas-red-conjugated secondary antibody, and this signal was compared
with the intrinsic green fluorescence of EGFP-RUNX proteins. Randomly selected
transfected cells (50) were quantified using a dual band pass fluorescence
filter (Chroma Technology Corp., Brattleboro, VT, #51006) and evaluated for
the extent of signal overlap. The analysis included only those cells that
exhibited comparable fluorescence intensities. The results show that RUNX2
proteins with two different epitope tags are extensively colocalized in WC
preparations (40% of cells: >90% signal overlap; 60% of cells: 60-90%
signal overlap) and NMIF preparations (30% of cells: >90% signal overlap;
70% of cells: 60-90% signal overlap). The extent of colocalization of RUNX1
and RUNX2 proteins is very similar to that observed for RUNX2 proteins
carrying two distinct tags (compare Fig. 4A
with B). All cells in which both proteins were expressed displayed
extensive or complete signal overlap. Taken together, these results
demonstrate that RUNX1 and RUNX2 are targeted to common subnuclear domains,
which is consistent with the amino-acid sequence similarities of their
targeting signals (Zeng et al.,
1997; Zaidi et al.,
2001
).
|
Intranuclear targeting of RUNX2 in living cells is dependent on the
C-terminal domain
To evaluate whether RUNX1 and RUNX2 are localized to punctate foci in
living cells, we examined the subnuclear organization of EGFP-RUNX1 and
EGFP-RUNX2 fusion proteins in SaOS-2 cells using time-lapse microscopy. For
comparison, the subnuclear distribution of EGFP alone was analyzed. To assess
movement of the foci, we captured time-lapse images every 10-30 seconds for
20-30 minutes using exposure times of 100 or 200 milliseconds. Only those
cells that exhibited a significant signal above background were analyzed. The
results show that cells expressing EGFP alone produce a diffuse fluorescence
signal with comparable intensity in the cytoplasm and nucleus
(Fig. 5A; Movie 1A, available
at
jcs.biologists.org/supplemental).
The movement of EGFP proteins is most clearly observed in the time-lapse video
micrographs (see online supplemental information). In cells expressing
EGFP-RUNX1 or EGFP-RUNX2, we observe punctate domains in the nucleus
(Fig. 5B,C), which are very
similar to those in fixed preparations
(Fig. 2). Furthermore, these
foci remain stationary within the nuclear space throughout the 30 minute time
of observation (Fig. 5B,C;
Movies 1B,C, available at
jcs.biologists.org/supplemental).
In the timelapse movies of cells expressing EGFP-RUNX1 and EGFP-RUNX2 the foci
appear to move slightly. However, this apparent movement is limited relative
to the diameter of the nucleus and may represent either changes in the shape
of the foci and/or nuclei or movement of cells relative to the plane of focus.
These results show that the punctate foci observed in fixed cells are bona
fide subnuclear domains that can be visualized in living cells and that these
punctate domains are relatively stable in the nucleus over time.
To determine whether the C-terminus of RUNX2 is necessary for the targeting
of RUNX proteins to punctate foci in living cells, we analyzed a RUNX2
deletion mutant lacking the C-terminus (EGFP-RUNX2361). This fusion
protein displays a diffuse fluorescence signal throughout the cytoplasm and
nucleus (Fig. 5D; Movie 1D),
which is similar to EGFP alone (compare
Fig. 5A and
Fig. 5D; Movies 1A,D). However,
the fluorescence intensity of EGFP-RUNX2
361 was greater in the nucleus
than in the cytoplasm (Fig.
5D). Moreover, the subnuclear distribution of EGFP-RUNX2
361
is very different from that of wild-type RUNX1 and RUNX2. Thus, our results
indicate that the C-terminus is required for localization of RUNX2 into
punctate subnuclear domains.
Deletion of the C-terminus of RUNX2 increases the intranuclear
mobility of RUNX2 proteins
The relative mobility of the EGFP-RUNX fusion proteins was determined by
using FRAP analysis. SaOS-2 cells were transiently transfected with
EGFP-RUNX1, EGFP-RUNX2, EGFP-RUNX2361 or EGFP alone. A defined area in
the nucleus of cells expressing each of these proteins was photobleached with
a laser beam for 100 milliseconds. Recovery of the fluorescence signal in the
entire bleached area was determined by capturing sequential images following
photobleaching (Fig. 6). The
estimated half-time of recovery of EGFP-RUNX1 and EGFP-RUNX2 proteins,
respectively, is calculated to be 10.2±0.6 (n=5) and
10.7±1.1 (n=10) seconds and the mean percent immobile fraction
is calculated to be 20.0±2.8% and 26.1±7.1%. These findings were
reproduced in independent experiments (n=15). We observed that the
punctate domains containing EGFP-RUNX1 and EGFP-RUNX2 proteins recovered after
photobleaching with a similar morphology as before photobleaching and in the
same location (Fig. 6A,B; see
Movies 2A,B, available at
jcs.biologist.org/supplemental).
Additionally, we determined the dynamic exchange of RUNX factors at
specific foci in the photobleached area (boxed areas in
Fig. 6A and 6B show examples).
For EGFP-RUNX1 and EGFP-RUNX2 the mean half-time of recovery of the foci was
similar to that of the entire photobleached area. The mean percent immobile
fraction for the foci is 29.7±4.5% for EGFP-RUNX1 and 32.1±7.9%
for EGFP-RUNX2. Thus, these results indicate that RUNX proteins undergo
dynamic exchange at the stationary subnuclear punctate domains. RUNX2 proteins
that have the C-terminus deleted (EGFP-RUNX2361) exhibit a mobility
comparable to that of EGFP alone. EGFP and EGFP-RUNX2
361 proteins
(Fig. 6C and D; see Movies
2C,D) are completely mobile in comparison with EGFP-RUNX1 and EGFP-RUNX2
proteins (see Fig. 6A,B). The
relative recovery curves of EGFP-RUNX1, EGFP-RUNX2, EGFP-RUNX2
361 and
EGFP proteins are shown in Fig.
6E. The estimated half-time of recovery is <600 milliseconds
for EGFP-RUNX2
361 and <400 milliseconds for EGFP alone. Both
EGFP-RUNX2
361 and EGFP are almost completely recovered within 1 second
of photobleaching. The increased mobility of RUNX2
361 compared with
full-length proteins suggests that deletion of the C-terminal domain perturbs
the association of RUNX proteins with subnuclear foci in living cells. One
plausible interpretation of our finding is that the C-terminus together with
its interacting proteins contributes to the stabilization of RUNX subnuclear
foci.
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Discussion |
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The homologous targeting signals present in the C-termini of RUNX proteins
(Zeng et al., 1997;
Zaidi et al., 2001
) may direct
these factors to common subnuclear domains. The C-terminal truncation of
RUNX2, which removes the intranuclear targeting signal, results in a lethal
phenotype in vivo, suggesting that the C-termini of RUNX proteins are
essential for functional activity (Choi et
al., 2001
). Interactions of the C-termini of RUNX factors with
co-repressors and co-activators are important for regulation of transcription
(Hanai et al., 1999
;
Javed et al., 2000
;
Lutterbach and Hiebert, 2000
).
Our results presented here together with previous data suggest that RUNX
proteins assemble into macromolecular complexes with co-regulatory proteins at
nuclear-matrix-associated sites to regulate gene transcription
(Berezney and Wei, 1998
;
Lutterbach and Hiebert, 2000
;
Stein et al., 2000a
;
Javed et al., 2000
;
Zeng et al., 1997
;
Zeng et al., 1998
;
Zaidi et al., 2001
). The
C-terminal segment of RUNX proteins appears to reduce the mobility of these
proteins by mediating association with nuclear architecture, perhaps by
supporting in situ formation of complexes. We propose that the functional
activity of RUNX proteins at subnuclear foci may critically depend on the
spatial-temporal availability of co-factors.
Foci that contain RUNX transcription factors remain stationary within the
nuclear space, but are dynamic structures with which RUNX proteins
continuously associate and disassociate. Our results suggest that
immobilization of these subnuclear domains within the nuclear space may
reflect association with the nuclear matrix. Previous commentaries have argued
that some subnuclear structures could be artifacts resulting from the fixation
and/or extraction procedures (Pederson,
2000), as opposed to functional compartments that support gene
expression (Penman, 1995
;
Stein et al., 2000b
;
Wei et al., 1998
;
Cook, 1999
;
Stenoien et al., 2000
;
Nickerson, 2001
). Here, we
show that these RUNX transcription factor domains are observed in both fixed
and living cells and that a subset of these foci represent active sites of
transcription (as revealed by BrUTP labeling). Thus, our findings demonstrate
that the RUNX domains are functional subnuclear structures.
Our observation that RUNX proteins continuously and rapidly shuttle into
and out of the dynamic, yet spatially stable foci may reflect a mechanism for
the organization and reversible formation of transcriptional complexes in
situ. The movement of RUNX transcription factors into these stationary domains
occurs within the same time scale as the movement of the splicing factor ASF
into splicing factor domains (Phair and
Misteli, 2000; Kruhlak et al.,
2000
). It has been well established that processing of gene
transcripts occurs within specific domains (SC-35 `speckles') in the nucleus,
which reflects the spatial compartmentalization of the splicing machinery
(Xing et al., 1993
;
Spector, 1993
;
Phair and Misteli, 2000
;
Kruhlak et al., 2000
). Our
data indicate that tissue-specific transcription factors are similarly
compartmentalized within the nucleus in living cells. Thus, we conclude that
the dynamic association of RUNX factors to stationary subnuclear foci through
a common C-terminal signal provides a biological mechanism for the formation
of essential tissue-related and gene-specific regulatory complexes.
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Acknowledgments |
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Footnotes |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berezney, R. and Wei, X. (1998). The new paradigm: integrating genomic function and nuclear architecture. J. Cell Biochem. Suppl. 30-31,238 -242.
Berezney, R., Dubey, D. D. and Huberman, J. A. (2000). Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci. Chromosoma 108,471 -484.[CrossRef][Medline]
Bidwell, J. P., Fey, E. G., van Wijnen, A. J., Penman, S., Stein, J. L., Lian, J. B. and Stein, G. S. (1994). Nuclear matrix proteins distinguish normal diploid osteoblasts from osteosarcoma cells. Cancer Res. 54,28 -32.[Abstract]
Choi, J.-Y., Pratap, J., Javed, A., Zaidi, S. K., Xing, L.,
Balint, E., Dalamangas, S., Boyce, B., van Wijnen, A. J., Lian, J. B., Stein,
J. L., Jones, S. N. and Stein, G. S. (2001). Subnuclear
targeting of Runx/Cbfa/AML factors is essential for tissue-specific
differentiation during embryonic development. Proc. Natl. Acad.
Sci. USA 98,8650
-8655.
Cook, P. R. (1999). The organization of
replication and transcription. Science
284,1790
-1795.
Ellenberg, J., Siggia, E. D., Moreira, J. E., Smith, C. L.,
Presley, J. F., Worman, H. J. and Lippincott-Schwartz, J.
(1997). Nuclear membrane dynamics and reassembly in living cells:
targeting of an inner nuclear membrane protein in interphase and mitosis.
J. Cell Biol. 138,1193
-1206.
Hanai, J., Chen, L. F., Kanno, T., Ohtani-Fujita, N., Kim, W.
Y., Guo, W.-H., Imamura, T., Ishidou, Y., Fukuchi, M., Shi, M. J., Stavnezer,
J., Kawabata, M., Miyazono, K. and Ito, Y. (1999).
Interaction and functional cooperation of PEBP2/CBF with smads. Synergistic
induction of the immunoglobulin germline c promoter. J.
Biol. Chem. 274,31577
-31582.
Javed, A., Barnes, G. L., Jassanya, B. O., Stein, J. L.,
Gerstenfeld, L., Lian, J. B. and Stein, G. S. (2001).
runt homology domain transcription factors (Runx, Cbfa, and AML)
mediate repression of the bone sialoprotein promoter: evidence for promoter
context-dependent activity of Cbfa proteins. Mol. Cell.
Biol. 21,2891
-2905.
Javed, A., Guo, B., Hiebert, S., Choi, J.-Y., Green, J., Zhao,
S.-C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A.
J. and Stein, G. S. (2000). Groucho/TLE/R-Esp proteins
associate with the nuclear matrix and repress RUNX
(CBF/AML/PEBP2
) dependent activation of tissue-specific gene
transcription. J. Cell Sci.
113,2221
-2231.
Kanda, T., Sullivan, K. F. and Wahl, G. M. (1998). Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 8,377 -385.[Medline]
Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y.-H., Inada, M., Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S. and Kishimoto, T. (1997). Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89,755 -764.[Medline]
Kruhlak, M. J., Lever, M. A., Fischle, W., Verdin, E.,
Bazett-Jones, D. P. and Hendzel, M. J. (2000). Reduced
mobility of the alternate splicing factor (ASF) through the nucleoplasm and
steady state speckle compartments. J. Cell Biol.
150, 41-51.
Lamond, A. I. and Earnshaw, W. C. (1998).
Structure and function in the nucleus. Science
280,547
-553.
Lutterbach, B. and Hiebert, S. W. (2000). Role of the transcription factor AML-1 in acute leukemia and hematopoietic differentiation. Gene 245,223 -235.[CrossRef][Medline]
McNeil, S., Zeng, C., Harrington, K. S., Hiebert, S., Lian, J.
B., Stein, J. L., van Wijnen, A. J. and Stein, G. S. (1999).
The t(8;21) chromosomal translocation in acute myelogenous leukemia modifies
intranuclear targeting of the AML1/CBFalpha2 transcription factor.
Proc. Natl. Acad. Sci. USA
96,14882
-14887.
Meyers, S., Lenny, N., Sun, W.-H. and Hiebert, S. W. (1996). AML-2 is a potential target for transcriptional regulation by the t(8;21) and t(12;21) fusion proteins in acute leukemia. Oncogene 13,303 -312.[Medline]
Misteli, T. and Spector, D. L. (1998). The cellular organization of gene expression. Curr. Opin. Cell Biol. 10,323 -331.[CrossRef][Medline]
Nickerson, J. A. (2001). Experimental
observations of a nuclear matrix. J. Cell Sci.
114,463
-474.
Nickerson, J. A., Blencowe, B. J. and Penman, S. (1995). The architectural organization of nuclear metabolism. In Structural and Functional Organization of the Nuclear Matrix (eds. R. Berezney and K. W. Jeon), pp.67 -123. New York: Academic Press.
North, T., Gu, T. L., Stacy, T., Wang, Q., Howard, L., Binder,
M., Marin-Padilla, M. and Speck, N. A. (1999). Cbfa2 is
required for the formation of intra-aortic hematopoietic clusters.
Development 126,2563
-2575.
Okuda, T., van Deursen, J., Hiebert, S. W., Grosveld, G. and Downing, J. R. (1996). AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84,321 -330.[Medline]
Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Gilmour, K. C., Rosewell, I. R., Stamp, G. W. H., Beddington, R. S. P., Mundlos, S., Olsen, B. R., Selby, P. B. and Owen, M. J. (1997). Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89,765 -771.[Medline]
Pederson, T. (2000). Half a century of
"the nuclear matrix". Mol. Biol. Cell
11,799
-805.
Penman, S. (1995). Rethinking cell structure. Proc. Natl. Acad. Sci. USA 92,5251 -5257.[Abstract]
Phair, R. D. and Misteli, T. (2000). High mobility of proteins in the mammalian cell nucleus. Nature 404,604 -609.[CrossRef][Medline]
Schepmoes, G., Breen, E., Owen, T. A., Aronow, M. A., Stein, G. S. and Lian, J. B. (1991). Influence of dexamethasone on the vitamin D-mediated regulation of osteocalcin gene expression. J. Cell. Biochem. 47,184 -196.[Medline]
Spector, D. L. (1993). Nuclear organization of pre-mRNA processing. Curr. Opin. Cell Biol. 5, 442-447.[Medline]
Stein, G. S., Montecino, M., van Wijnen, A. J., Stein, J. L. and
Lian, J. B. (2000a). Nuclear structure gene
expression interrelationships: implications for aberrant gene expression in
cancer. Cancer Res. 60,2067
-2076.
Stein, G. S., van Wijnen, A. J., Stein, J. L., Lian, J. B.,
Montecino, M., Choi, J.-Y., Zaidi, K. and Javed, A. (2000b).
Intranuclear trafficking of transcription factors: implications for biological
control. J. Cell Sci.
113,2527
-2533.
Stenoien, D. L., Simeoni, S., Sharp, Z. D. and Mancini, M. A. (2000). Subnuclear dynamics and transcription factor function. J. Cell Biochem. Suppl. 35, 99-106.
Stewart, M., Terry, A., Hu, M., O'Hara, M., Blyth, K., Baxter,
E., Cameron, E., Onions, D. E. and Neil, J. C. (1997).
Proviral insertions induce the expression of bone-specific isoforms of
PEBP2alphaA (CBFA1): evidence for a new myc collaborating oncogene.
Proc. Natl. Acad. Sci. USA
94,8646
-8651.
Tang, L., Guo, B., Javed, A., Choi, J.-Y., Hiebert, S., Lian, J.
B., van Wijnen, A. J., Stein, J. L., Stein, G. S. and Zhou, G. W.
(1999). Crystal structure of the nuclear matrix targeting signal
of the transcription factor AML-1/PEBP2B/CBF
2. J.
Biol. Chem. 274,33580
-33586.
Towler, D. A., Bennett, C. D. and Rodan, G. A. (1994). Activity of the rat osteocalcini basal promoter in osteoblastic cells is dependent upon homeodomain and CP1 binding motifs. Mol. Endocrinol. 8,614 -624.[Abstract]
Wang, Q., Stacy, T., Binder, M., Marin-Padilla, M., Sharpe, A.
H. and Speck, N. A. (1996). Disruption of the Cbfa2 gene
causes necrosis and hemorrhaging in the central nervous system and blocks
definitive hematopoiesis. Proc. Natl. Acad. Sci. USA
93,3444
-3449.
Wei, X., Samarabandu, J., Devdhar, R. S., Siegel, A. J.,
Acharya, R. and Berezney, R. (1998). Segregation of
transcription and replication sites into higher order domains.
Science 281,1502
-1505.
Xing, Y., Johnson, C. V., Dobner, P. R. and Lawrence, J. B. (1993). Higher level organization of individual gene transcription and splicing. Science 259,1326 -1330.[Medline]
Zaidi, S. K., Javed, A., Choi, J.-Y., van Wijnen, A. J., Stein, J. L., Lian, J. B. and Stein, G. S. (2001). A specific targeting signal directs Runx2/Cbfa1 to subnuclear domains and contributes to transactivation of the osteocalcin gene. J. Cell Sci. 114, 3102.
Zeng, C., van Wijnen, A. J., Stein, J. L., Meyers, S., Sun, W.,
Shopland, L., Lawrence, J. B., Penman, S., Lian, J. B., Stein, G. S. and
Hiebert, S. W. (1997). Identification of a nuclear matrix
targeting signal in the leukemia and bone-related AML/CBF transcription
factors. Proc. Natl. Acad. Sci. USA
94,6746
-6751.
Zeng, C., McNeil, S., Pockwinse, S., Nickerson, J. A., Shopland,
L., Lawrence, J. B., Penman, S., Hiebert, S. W., Lian, J. B., van Wijnen, A.
J., Stein, J. L. and Stein, G. S. (1998). Intranuclear
targeting of AML/CBF regulatory factors to nuclear matrix-associated
transcriptional domains. Proc. Natl. Acad. Sci. USA
95,1585
-1589.