Metabolic Research Unit and Department of Medicine (F.S., X.W., C.T., R.S.), University of California, San Francisco, California 94143-0540; Departments of Medicine and Cell Biology (J.F.E., R.N.D.), National Science Foundation Center for Biological Timing, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; and Department of Physiology (R.E., O.A.M.), University of Michigan Medical School, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Fred Schaufele, University of California, San Francisco, California 94143-0540. E-mail: freds{at}metabolic.ucsf.edu
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ABSTRACT |
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![]() |
INTRODUCTION |
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GH is one of the most powerful regulators of energy metabolism.
Our previous studies have implicated C/EBP as an activator of
pituitary-specific GH gene expression (11, 12). C/EBP
is present in GH-secreting pituitary cell lines, but absent from
immortalized pituitary GHFT15 cells, which do not express GH. The
GHFT15 cell line was derived by targeted transformation of
embryonic pituitary cells and has characteristics of the progenitor
for the GH-secreting, pituitary somatotrope cell lineage
(13). Expression of exogenous C/EBP
in GHFT15 cells
leads to activation of a cotransfected GH gene promoter
(12) and blockage of proliferation (Liu, W.,
W. Hyun, R. N. Day, and F. Schaufele,
submitted). This suggested that C/EBP
might play a role in
somatotrope cell differentiation, analogous to its role in the
regulation of gene expression and proliferation during adipocyte cell
differentiation (7, 14).
Recently, it was shown that, during adipocyte cell differentiation,
C/EBP became localized to specific regions of the cell nucleus that
stained preferentially with DNA binding dyes that associated with
markers for centromeres (15). Here, we demonstrate that
C/EBP
, when expressed as a fusion protein with GFP, also localizes
to intranuclear sites associated with pericentromeric chromatin in
pituitary progenitor GHFT15 cells. We extend these observations to
demonstrate that the CREB binding protein (CBP), which we show to
enhance C/EBP
gene regulatory activity in GHFT15 cells, does not
localize to pericentromeric chromatin in these pituitary cells. The
paradox of differing intranuclear locations for cooperating C/EBP
and CBP was resolved by finding that C/EBP
expression caused CBP to
translocate to the pericentromeric chromatin and colocalize with
C/EBP
. Similarly, the basal factor TATA-binding protein (TBP) was
recruited to these intranuclear domains upon GFP-C/EBP
expression.
C/EBP
truncated of its transcriptional activation functions still
targeted to the Hoechst-stained chromosomal domains, but was incapable
of reorganizing either CBP or TBP to these nuclear domains. Thus,
C/EBP
regulates the spatial positions of critical coregulatory
factors within the nucleus. This alteration in the concentration of
specific regulatory complexes at particular subnuclear structures may
constitute a new means by which a transcription factor directs changes
in patterns of gene expression.
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RESULTS |
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CBP Enhancement of C/EBP-Dependent Transcription
The CREB-binding protein (CBP) is a coactivator of Pit-1
(23) and some C/EBP family members other than C/EBP
(24, 25). The CBP-related protein, p300, physically
interacts with C/EBPß (24) and functionally interacts
with both C/EBP
(22) and C/EBPß (24). We
found CBP to be present in nuclear extracts of GHFT15 cells at a
level comparable to that in the GH-secreting GC cells (Fig. 1A
).
Adenovirus 12S E1a is an effective inhibitor of CBP coactivator
function (26, 27). To investigate the potential role of
CBP as a coactivator for C/EBP
-dependent transcription from the GH
promoter, we initially determined the effect of E1a coexpression on
activation of the full-length (-237 to +8) rGH promoter and of the
C/EBP-TATA promoter. Coexpression of the E1a protein blocked
C/EBP
-dependent transcription of the full-length rGH promoter (Fig. 2A
) and the minimal C/EBP-TATA promoter
(data not shown). Western blots confirmed that E1a expression did not
affect the expression from the cotransfected C/EBP
vector
(inset, Fig. 2A
). This result suggested that an
E1a-sensitive coactivator, such as endogenous CBP/p300, enhanced
C/EBP
transcriptional activity.
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Transcriptionally Active Fusion of C/EBP with GFP
Using GFP as a label for the ER expressed in living cells, we
(29) and others (30) recently demonstrated
that expression of the ER dramatically affected the intranuclear
organization of the coactivator proteins GRIP1 and SRC-1. Here, we
studied whether C/EBP expression similarly affected the intranuclear
redistribution of the coactivator CBP. Initially, C/EBP
fusions with
GFP were used to identify the intranuclear location of C/EBP
in
living cells. Expression vectors were constructed in which the cDNA for
GFP was fused to either the amino terminus or the carboxy terminus of
the cDNA for C/EBP
(GFP-C/EBP
or C/EBP
-GFP, respectively).
Western analysis showed that the expressed C/EBP
-GFP and
GFP-C/EBP
were of the size expected for full-length GFP-C/EBP
(Fig. 3
). The C/EBP
-GFP fusion was
transcriptionally active at the C/EBP-TATA promoter in GHFT15 cells,
whereas the GFP-C/EBP
fusion was comparatively inactive (Fig. 3
).
Transfection of the C/EBP
-GFP expression vector into GHFT15 cells
resulted in a 12.01 ± 3.54 fold activation of the cotransfected
C/EBP-TATA promoter, compared with a 2.28 ± 1.37 fold promoter
activation by expression of GFP-C/EBP
. On average, C/EBP
-GFP
was 42.00 ± 14.59% as effective in activating the C/EBP-TATA
promoter as similarly expressed, unfused C/EBP
in parallel
experiments. Despite this transcriptional difference, the C/EBP
-GFP
and the GFP-C/EBP
fusions, as well as ectopically expressed and
antibody-stained C/EBP
, all behaved similarly in the subsequent
experiments described in this report.
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Targeting of C/EBP to Pericentromeric Chromatin
The H33342-stained foci have been previously described in other
mouse cell-types as tracts of satellite DNA repeats located at
centromeric regions of interphase chromosomes (31, 32, 33). We
found that the H33342-stained chromatin was associated with
the centromeres of interphase chromosomes in the GHFT15
cell nucleus (Fig. 5A). Nontransfected
GHFT15 cells were fixed, and immunohistochemical staining was
performed using a serum containing a human autoantibody that reacts
with centromeric kinetochore proteins (34). The
kinetochore signal was visualized with tetramethylrhodamine
isothiocyanate-conjugated secondary antibody. Dual-color imaging
of the fixed cells counterstained with H33342 showed a pair of
kinetochores were typically associated with each stained chomatin
focus. Thus, the H33342-stained chromatin surrounds the centromeres of
the interphase GHFT15 cell nucleus.
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It was previously speculated that the pericentromeric targeting of
C/EBP in 3T3-L1 cells induced to differentiate into adipocytes was
due to the presence of C/EBP
binding sites within the repeated DNA
sequences that comprise the bulk of pericentromeric chromatin
(15). We examined whether the DNA binding domain of
C/EBP
was sufficient for targeting to pericentromeric chromatin in
GHFT15 cells. A GFP-C/EBP
fusion was constructed in which only
amino acids 245358 of C/EBP
were retained. This encompassed the
entire "bZIP" DNA binding domain located between amino acids
278344 of C/EBP
. By itself, this isolated bZIP region targeted
specifically to H33342-stained DNA (Fig. 4C
), as did a second fusion
protein in which GFP was appended to the carboxy terminus of C/EBP
amino acids 259358 (data not shown). Moreover, C/EBP
deleted of
the leucine zipper component of the DNA binding domain no longer
concentrated at the H33342-stained chromatin (Liu, W., W.
Hyun, R. N. Day, and F. Schaufele, submitted). Thus,
an intact DNA binding domain, which is critical for gene-specific
transcriptional activation, is both sufficient and necessary for
C/EBP
targeting to pericentromeric chromatin.
The Intranuclear Distribution of CBP and C/EBP Are Distinct
Although DNA binding was sufficient for pericentromeric targeting,
it was not sufficient for transcriptional activation. This implied that
activities beyond DNA binding and/or pericentromeric targeting were
required for C/EBP activity. We therefore examined the intranuclear
position of CBP, which we had determined to enhance C/EBP
activation
(Fig. 2
). In striking contrast to GFP-C/EBP
, CBP expressed in
GHFT15 cells as a fusion to GFP was distributed throughout the
nucleus (Fig. 6A
), similar to that
previously shown in immunohistochemical staining of
endogenous CBP in HEp-2 nuclei (37). Moreover,
GFP-CBP was excluded from the pericentromeric chromatin
preferentially labeled with H33342 (Fig. 6A
, overlay). We
then determined that endogenous CBP also was excluded from
H33342-labeled pericentromeric chromatin; fixed GHFT15 cells were
stained with a primary antibody directed against mouse CBP and a
secondary antibody labeled with TRITC (Fig. 6B
). Thus, the intranuclear
localization of the expressed GFP-CBP accurately reflected the
distribution of its endogenous counterpart, and both were absent from
the pericentromeric sites to which GFP-C/EBP
was preferentially
localized.
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To determine whether C/EBP expression had an effect on the
intranuclear position of critical coregulatory factors including CBP,
we first tagged C/EBP
with the spectrally distinct blue color
variant of GFP (BFP) (39, 40, 41). We then specifically
detected the intranuclear positions of BFP-tagged C/EBP
and
GFP-tagged CBP expressed in the same cell by using BFP- and
GFP-specific excitation and emission filter sets (see Materials
and Methods). When expressed in GHFT15 cells, BFP-C/EBP
(Fig. 7A
, top left panel) and
C/EBP
-BFP (not shown) assumed the same distinctive intranuclear
distribution of GFP-C/EBP
described above. This was confirmed by
coexpressing BFP-C/EBP
and GFP-C/EBP
in the same cells and
observing that their intranuclear distributions overlapped (data not
shown).
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Expression of GFP-C/EBP also altered the subnuclear localization of
the endogenous CBP protein. GHFT15 cells expressing either
GFP-C/EBP
(Fig. 7B
, upper panels) or GFP-C/EBP
154
(Fig. 7B
, lower panels) were fixed and stained using an
antibody directed against CBP. Dual-color imaging showed the
antibody-labeled endogenous CBP colocalized with the full-length
GFP-C/EBP
. Endogenous CBP did not localize to these subnuclear sites
in cells expressing the mutant GFP-C/EBP
154 protein. Together, these
results showed that the expression of exogenous C/EBP
in GHFT15
cells caused a trans-activation domain-dependent recruitment
of CBP to specific subnuclear sites.
The failure of the transcriptionally inactive 154 mutant of C/EBP
to recruit CBP may suggest a role for CBP recruitment in
transcriptional activation. Indeed, the expression of BFP-C/EBP
is
also associated with an enhanced concentration of a GFP fusion with TBP
at the location of BFP-C/EBP
(Fig. 7C
, upper panels). The
concentration of GFP-TBP was not seen with the transcriptionally
inactive BFP-C/EBP
154 (Fig. 7C
, lower panels). However,
the sites of active transcription in GHFT15 cell nuclei, detected by
Br-UTP labeling (see Fig. 5B
), were as absent from pericentromeric
chromatin after C/EBP
-GFP expression as they were in the absence of
C/EBP
(data not shown). Because Br-UTP labeling of nascent
transcripts measures global transcription rather than
C/EBP
-regulated transcription, the transcriptional consequences of
the change in the intranuclear distribution of CBP and TBP upon
C/EBP
expression may require mapping the intranuclear locations of
transcripts, specifically activated or repressed upon C/EBP
expression, relative to the locations of C/EBP
and pericentromeric
chromatin.
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DISCUSSION |
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The GFP-C/EBP and C/EBP
-GFP fusions, which differed in their
ability to activate transcription of a C/EBP
-sensitive reporter
gene, and the transcriptionally defective C/EBP
154 mutant all
localized specifically at pericentromeric chromatin. This indicated
that pericentromeric targeting of C/EBP
was not sufficient for
transcriptional activation. Indeed, the DNA binding domain of C/EBP
by itself was sufficient for pericentromeric targeting (Fig. 4C
) but
not transcriptional activation (22 and our unpublished
data). In contrast, the disruption of CBP and TBP recruitment to the
intranuclear location of C/EBP
(Fig. 7
) by the transcriptionally
defective C/EBP
154 mutant suggested that the C/EBP
-mediated,
intranuclear relocation of CBP may be associated with transcriptional
activation. However, C/EBP
expression is not associated with a
global enhancement of nascent, Br-UTP-labeled transcripts at
pericentromeric chromatin (Fig. 5B
and data not shown) although the low
abundance of genes within centromeric DNA (42) may have
precluded our ability to detect transcription activation by a global
labeling of transcripts. At a minimum, the data strongly suggest that
C/EBP
organizes CBP and TBP into macromolecular complexes that are
readily visible because of the distinct intranuclear localization
pattern of C/EBP
. It is not yet known whether CBP and TBP
recruitment are linked or are separate, unrelated consequences of
C/EBP
expression.
Highly Specific, Intranuclear Marshalling of CBP by C/EBP
We refer to the alteration in the intranuclear location of
transcription coregulatory factors induced by a transcription factor as
intranuclear marshalling (29). The specificity of the
intranuclear marshalling of CBP and TBP by C/EBP was illustrated by
investigating the consequences of C/EBP
expression on the
intranuclear locations of a number of other transcription factors and
cofactors (data not shown). Most of these factors did not localize at
pericentromeric chromatin, and their intranuclear positions were not
affected by C/EBP
expression. For instance, the coactivator GRIP1,
which distributes throughout the nucleus (29) and,
like CBP, contains histone acetyltransferase activity, was
not affected by the expression of BFP-C/EBP
(data not shown).
However, GRIP1 was recruited to the subnuclear location occupied by the
ER, but only if the cells were treated with estrogens
(29). The ER did not recruit CBP to its intranuclear
location, and the intranuclear distribution of the ER was not affected
by C/EBP
coexpression. Similarly, GFP fusions to the basal factor
TFIIB or the Sin3A component of some histone deacetylase complexes
distributed independently of coexpressed C/EBP
in GHFT15 cells
(not shown). The C/EBP
- and ER-induced sequestrations of different
histone acetyltransferase-containing factors to different regions of
the cell nucleus may dramatically affect the balance of acetylation
activities at discrete locations within the nucleus. Indeed, we have
determined that the expression of C/EBP
is associated with an
increase in the amount of acetylated histone H3 present in
pericentromeric chromatin relative to the amount of acetylated histone
H3 outside of pericentromeric chromatin (our unpublished data).
Despite highly specific marshaling of CBP to pericentromeric chromatin
by C/EBP, in vitro studies of CBP interactions with
column-attached C/EBP
, coimmunoprecipitation studies in cellular
extracts, and fluorescence resonance energy transfer studies in living
cells have, to date, failed to reliably detect any evidence of a strong
physical interaction between CBP and C/EBP
. The intracellular
complexes detected by intranuclear marshalling may therefore reflect an
association of CBP and C/EBP
involving other factors within the
complex. We found that GRIP1, which is known to interact with CBP
(43, 44), bound in vitro to column-attached
C/EBP
(our unpublished data). However, we saw no evidence of
transcriptional coactivation or intranuclear marshaling by C/EBP
and
GRIP1. Thus, the intranuclear marshaling of CBP by C/EBP
correlated
better with the observed functional interactions of C/EBP
than did
in vitro interaction assays. This may be because
intranuclear marshalling and functional studies are conducted under the
same cellular environments.
Pericentromeric Organization and Regulation of Gene Expression
Given the potential contribution of nuclear architecture to gene
expression, there have been very few studies of the spatial
organization of transcription-regulatory factors within the nucleus
(29, 30, 36, 45, 46, 47, 48, 49). Specific intranuclear locations for
transcription factors and coregulatory factors may allow productive
interactions only between colocalized transcription-regulatory factors
and gene sets. Perhaps as important, the formation of complexes between
factors, cofactors, and genes sequestered in different compartments may
be restricted. The role that C/EBP association with pericentromeric
chromatin may play in any of the differentiative effects of C/EBP
remains to be defined. However, pericentromeric targeting of C/EBP
in GHFT15 cells required the bZIP domain of C/EBP
(our unpublished
data) essential for C/EBP
dimerization and DNA binding, suggesting
that pericentromeric targeting is associated with at least one activity
important to transcriptional regulation.
We have found that expression of the transcription factor Pit-1, an
important coregulator of pituitary differentiation (17),
leads to a highly selective marshaling of C/EBP, and associated CBP,
away from pericentromeric chromatin in GHFT15 cells
(Enwright III, J. F., M. Kawecki, F. Schaufele,
and R. N. Day, submitted). Thus, C/EBP
targeting to
pericentromeric chromatin may be an intermediate step in
differentiation of GH-secreting cell types. In contrast, C/EBP
remains targeted to the pericentromeric chromatin in differentiated
adipocytes. The different final locations of C/EBP
and associated
CBP relative to pericentromeric chromatin may contribute to the
cell-specific differences in the complement of genes expressed in these
two different cell types. It will be important to identify the genes
differentially expressed or repressed in both cell types and to compare
the activity and locations of those genes relative to pericentromeric
chromatin.
Historically, pericentromeric chromatin has been viewed as being devoid
of expressed genes. More recent evidence suggests that these regions
are actively involved in gene regulation (35). A few genes
are even embedded within the centromeric DNA of Arabidopsis
thaliana, the multicellular organism for which genome sequencing
is most complete in the centromeric regions (42). This
shows that the centromeres are not completely transcriptionally
inert. Some centromere-associated factors, such as the
zinc-finger protein Ikaros/Lyf-1, may play a role in silencing
particular genes during lymphocyte activation (50, 51).
The centromere also facilitates the initiation of chromatin
condensation and decondensation and positions chromosome territories
within the interphase nucleus (52) and may therefore play
a structural role in both gene activation and repression. The
transcriptional regulator ATRX, in association with the
chromatin-binding protein HP1, interacts with the SWI/SNF complex at
pericentromeric chromatin (53). The Polycomb group complex
that, like the SWI/SNF complex, regulates higher order chromatin
structure in Drosophila (54) also associates
with centromeric chromatin in human cell lines (55). Thus,
the centromere may be an important nexus at the interface of
intranuclear architecture and gene regulation. The C/EBP-induced
concentration of specific coregulatory factors at the centromere, or
away from the centromere in the presence of Pit-1, may provide a
molecular and cellular basis for regulating the transcriptional
regulatory complexes available to these sites.
C/EBP Assembles Nucleoprotein Complexes
The intranuclear marshalling of CBP and TBP by the C/EBP
activation domain (Fig. 7
) is consistent with the results reported by
others that the amino-terminal region of some C/EBP family members is
involved in interactions with TBP and CBP (20, 24). Our
finding that critical factors including CBP and TBP did not localize to
pericentromeric chromatin unless C/EBP
was coexpressed suggests
that, although stable, these assemblies are not permanent structures
(36, 45). This supports the view that certain
architectural proteins can nucleate the assembly of
transcription-coregulatory complexes within the nucleus (46, 56, 57, 58, 59).
Beyond simple recruitment, it is intriguing to speculate that the
marshaling of CBP and TBP specifically to pericentromeric sites could
globally influence gene expression by permitting CBP and TBP access to
factors and genes that target to pericentromeric DNA. Alternatively,
CBP sequestration at pericentromeric chromatin may restrict CBP access
to factors and genes present in nonpericentromeric locations. At a
minimum, the marshaling activity of C/EBP demonstrates that C/EBP
promotes the assembly of specific multiprotein complexes, and it is
conceivable that the relocation of these complexes to specific
chromatin compartments may dramatically affect the cohort of genes
expressed in a cell. Thus, the recruitment of CBP by C/EBP
to
pericentromeric regions in pituitary presomatotrope and in preadipocyte
cells might reflect a general mechanism by which the cell controls the
progression of specific programs of gene expression. Together, these
results demonstrate that specific protein domains play critical roles
in the assembly of cooperating factors at certain subnuclear sites. The
remodeling of nuclear structure and organization are likely to be key
components in the flow of regulatory information controlling cell
type-specific gene expression in response to environmental cues or
developmental programs.
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MATERIALS AND METHODS |
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Unlike wild-type E1a, E1a containing the RG2 mutant, which specifically
disrupts E1a interaction with CBP (26, 27), did not block
C/EBP transcriptional activation (data not shown), suggesting that
CBP was the target of E1a-mediated inhibition. However, Western blots
using three different commercially available E1a antibodies showed that
this mutant protein was not present in nuclear extracts prepared from
transfected GHFT15 cells. An informal survey of the literature shows
that most publications have not included controls for expression of the
RG2 mutant. We caution against future interpretations based upon this
commonly used reagent in the absence of this expression control.
Immunohistochemistry
Nontransfected mouse pituitary GHFT15 cells, mouse 3T3-L1
cells or GHFT15 cells transfected with the indicated expression
vectors, were cultured on glass cover slips. Cells were maintained in
culture 2448 h, and then fixed by a 5-min incubation in cold methanol
and processed for immunohistochemical detection. Cells expressing only
GFP fusion proteins were not fixed and were viewed live. CBP was
detected using the tyramide signal amplification technique
(61). The fixed cells were incubated with an anti-CBP
primary antibody (1:200 dilution of sc-369, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by a biotinylated
antirabbit secondary antibody and tertiary step using the
Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). The target was detected using a
horseradish peroxidase-catalyzed reaction of tyramide. After the
tyramide reaction, the fixed cells were washed and then stained for 5
min with H33342 at a concentration of 0.2 µg/ml, and the coverslips
were subsequently mounted using Vectashield (Vector Laboratories, Inc., Burlingame, CA). Kinetochore proteins were detected by
incubating the fixed cells with sera containing the human nuclear
centromere autoantibody (1:250 dilution of ANA, Cortex Biochem, San
Leandro, CA), followed by incubation with an antihuman TRITC-conjugated
secondary antibody. Endogenous C/EBP was detected in fixed
adipocytes by incubation with a rabbit polyclonal C/EBP
primary
antibody (1:100 dilution of sc-61, Santa Cruz Biotechnology, Inc.) followed by incubation with an antirabbit
rhodamine-conjugated secondary antibody.
Labeling of nascent mRNA transcripts was performed as previously described (36) except cells were exposed to Br-UTP for 20 min. Briefly, cells that had been plated on cover glasses the previous day were permeabilized with saponin, incubated with an in vitro transcription buffer containing Br-UTP, CTP, GTP, and ATP for 20 min at 33 C, and then fixed in paraformaldehyde. After fixation, cells were washed, and incubated overnight at 4 C with antibromouracil antibody to detect the nascently transcribed mRNA. The next day cells were washed followed by detection with a Texas Red-conjugated secondary antibody. Cells were washed again and stained with H 33342 at a concentration of 0.2 µg/ml, and the coverslips were subsequently mounted using Vectashield (Vector Laboratories, Inc.).
Microscopy and Image Analysis
Pituitary GHFT15 cells were typically transfected with 310
µg of expression plasmid DNA encoding the GFP-fusion proteins. The
transfected cells were inoculated into culture dishes containing no. 1
borosilicate cover glasses. The cells were maintained in culture as
described above, and then subjected to dual color fluorescence
microscopy (39, 40, 41, 62). For experiments involving
staining with H33342, the stain was added to a final concentration of
0.5 µg/ml approximately 20 min before imaging living cells or at 0.2
µg/ml for 5 min to image fixed cells. The fluorescence images were
acquired with either an inverted IX-70 (Olympus Corp.,
Lake Success, NY) or Axioplan microscope (Carl Zeiss,
Thornwood, NY) equipped with a 60x aqueous-immersion or a 63x
oil-immersion objective lens, respectively. The filter combinations
were 485/22 nm excitation and 535/50 nm emission for GFP images; 365/15
nm excitation and 460/50 nm emission for H33342 or BFP images; and
Texas Red or rhodamine filter sets for immunohisochemical staining
(Chroma Technology Corp., Brattelboro, VT). Grayscale images with no
saturated pixels were obtained using a cooled digital interline camera
(Orca-200, Hamamatsu, Bridgewater, NJ). All images were collected at a
similar gray-level intensity by controlling the excitation intensity
using neutral density filtration, and by varying the on-camera
integration time. For the result shown in Fig. 4B, the relative
illumination energy was calculated as the product of integration time
and excitation intensity, with 1 sec at 0.1 excitation equal to 1. ISEE
software (Inovision Corp., Raleigh, NC) or Metamorph software
(Universal Imaging Corp., Downingtown, PA) was used to background
subtract and then convert the digital images to red-green-blue
images. The GFP signal was assigned to the green channel, H33342 or BFP
signals to the blue channel, and the TRITC or rhodamine signals to the
red channel of the red-green-blue digital image. Image files were
processed for presentation using Adobe Photoshop 5.5 or 6.0 (Adobe
Systems, Inc., San Jose, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: BFP, Blue fluorescent protein; Br-UTP, bromo-uridine triphosphate; CBP, CREB binding protein; C/EBP, CCAAT/enhancer binding protein; CREB, cAMP response element binding protein; GFP, green fluorescent protein; GRIP, GR-interacting protein; H33342, Hoechst 33342; TBP, TATA binding protein; TRITC, tetramethylrhodamine isothiocyanate.
Received for publication April 3, 2001. Accepted for publication June 25, 2001.
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REFERENCES |
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