From the Diabetes Center, Metabolic Research Unit and
Department of Medicine, University of California, San Francisco,
California 94143-0540 and ¶ Departments of Medicine and Cell
Biology, National Science Foundation Center for Biological Timing,
University of Virginia Health Sciences Center,
Charlottesville, Virginia 22908
Received for publication, July 25, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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The structure of a protein defines its
biochemical properties, but the impact of intracellular location and
environment on protein structure remains poorly defined.
CCAAT/enhancer-binding protein The structure of a protein specifies its interactions with itself
and with other factors. X-ray crystallography and NMR characterize the
structure of concentrated, isolated proteins of static composition and
uniform conformation under nonphysiologic conditions. Methods that
investigate protein structure in living cells, where conformation may
vary with localized protein interactions and with dynamic, subcellular
microenvironments, would complement high resolution x-ray
crystallographic and NMR structures.
It is becoming increasingly evident that the nucleus is composed of
distinct functional subdomains at which transcription regulatory
proteins dynamically associate (1-5).
C/EBP The co-concentration of The sequestration of C/EBP We combined fluorescence microscopy and fluorescence resonance energy
transfer (FRET) techniques to define the relative positions of the TA
and bZIP domains in dimers of C/EBP FRET, normalized for the amounts of BFP and GFP present, was much
higher when BFP and GFP were attached to the bZIP domains than to the
TA domains. This indicated that the bZIP domains were closer to each
other than were the TA domains in the C/EBP Expression of C/EBP All C/EBP Immunostaining of C/EBP For the quantification of C/EBP Image Collection
All quantified FRET and co-localization data were collected on
an Olympus IX-70 using Olympus ×40 Plan Apochromat objective (0.95 numerical aperture). The images shown in Fig. 1A were
collected using an Olympus ×100 Plan Apochromat oil immersion
objective (1.40 numerical aperture). Chroma Corp. (Brattleboro, VT)
filters and multi-band pass dichroic mirror (61000v2bs) were
used together with Sutter Instruments (Novato, CA) Antibody-stained slides were imaged by 1) exciting rhodamine with
550-560-nm light and collecting emissions from 580-630 nm, 2)
exciting GFP with 480-495 nm light and collecting emissions at
500-530 nm, and 3) exciting Hoechst 33342 with 365-395-nm light and
collecting emissions from 435-465 nm. Controls showed no fluorescence bleed-through of rhodamine, GFP, or Hoechst between these channels. For
quantitative co-localization analysis (Figs. 1, C and
D), focusing was done using the Hoechst channel so as to
blind data collection to the presence and amounts of expressed
C/EBP Live cell imaging was used to analyze FRET between BFP and GFP-linked
C/EBP Image Analysis
Co-localization--
All image analysis was done using Metamorph
software (Universal Imaging Corp., Downingtown, PA). For the analysis
of C/EBP FRET--
The nuclei of cells expressing BFP- or GFP-tagged
C/EBP
It is critical that the acceptor cross-talk into the FRET channel is
accounted for. We have noticed, to date, a number of attempts by other
laboratories to calculate FRET without taking into account the
substantial contributions of the acceptor itself into the FRET channel.
Simply measuring the ratio of the amount of fluorescence in the FRET
and donor channels with only donor excitation incorporates acceptor
bleed-through into the FRET channel. If not accounted for, this
bleed-through increases the FRET/donor ratio and results in a false
conclusion of interaction. Since the ratios measured are physical
parameters of the fluorophores, correctly calculated FRET measurements
are highly consistent between separate experiments, provided that all
parameters affecting the relative ratios of fluorescence quantification
in the donor, acceptor, and FRET channels are kept constant. This
includes using the same 1) objective lens, 2) dichroic mirror, 3)
excitation/emission filters, 4) camera, and 5) relative integration
times for the different channels.
All bleed-through-corrected measurements were downloaded into Microsoft
Excel files and were expressed as a mean ± S.D. from multiple
nuclei collected from two or more experiments. Statistically significant differences were determined using t tests.
Slopes and y intercepts were calculated using Excel. 95%
confidence intervals in the slopes were calculated using GraphPad (San
Diego, CA) Prism software, using only data from the linear range
(acceptor/donor amounts <5) and setting the line to have the correct
y intercept of 0.47. Data collected over a wider
acceptor/donor range was fit into a single order interaction plot using
GraphPad Prism.
FRET Nanoscopy--
The amount of FRET at each pixel was
determined by applying our calculations directly on each image using
Metamorph software. The backgrounds were determined as above and then
subtracted from each pixel within each image using Metamorph arithmetic
functions. Also subtracted from each pixel were the bleed-through
contributions of BFP and GFP from matched pixels to the appropriate
green, blue, and FRET channels. The bleed-through-corrected images
contain the fluorescence values for BFP, GFP, and FRET, from which were calculated the FRET/donor and acceptor/donor ratios at each pixel in
the image. Since C/EBP
The formula y = mx + b was used
to calculate the extent of FRET (m) at each pixel in every
precisely registered image: y is the FRET/donor ratio at a
single pixel, x is the acceptor/donor ratio at the same
pixel, and b (the y intercept) is the FRET/donor ratio, where acceptor/donor = 0 (i.e. the constant
determined from the donor alone control). The above FRET nanoscopy
analyses were done in a semiautomated fashion by linking the
calculations into a Metamorph journal.
Selection of Pericentromeric Regions
In the FRET analysis of cells co-expressing BFP and GFP-labeled
C/EBP Estimates of Interfluorophore Distances Calculated from
Relative FRET Slopes
FRET efficiency varies with distance according to the
relationship first described by Förster (34): FRET efficiency
(E) = (1 + (r/ro)6) C/EBP
C/EBP
Counterstaining with an antibody against the molecular chaperone Hsp70
demonstrated that GHFT1-5 cells expressing the C/EBP
C/EBP
The similar pericentromeric targeting of endogenous 3T3-L1 cell
C/EBP
Plotting the C/EBP C/EBP
Images were captured in three different fluorescence channels from each
GHFT1-5 cell expressing C/EBP
The spectral cross-talk constants were used to determine whether there
was energy transfer in cells co-expressing C/EBP
The average FRET/donor ratio for 212 cells co-expressing C/EBP Relative Positions of bZIP and Transcription Activation Domains in
C/EBP
If a transfected cell expresses more acceptor (GFP-linked C/EBP
The slopes of the FRET/donor versus acceptor/donor graphs,
measured within the predominantly linear range for each combination of
GFP and BFP-linked C/EBP Identical Kinetics of Interaction between the bZIP and
Transcription Activation Domains--
To distinguish the contributions
of fluorophore distance from interaction kinetics to the different
levels of FRET, we examined data collected over a wide range of
acceptor/donor ratios for the bZIP to bZIP and TA to TA interactions
(Fig. 5A). The ability to collect data over a large range of
acceptor/donor ratios is limited by the necessity of maintaining FRET
data collection parameters the same for all cells (see "Experimental
Procedures"). However, from the data collected, the curves clearly
followed (r2 = 0.94 and 0.75, respectively)
first order interaction kinetics. This single-order interaction
kinetics indicated a simple bimolecular interaction between C/EBP
Extrapolation of the first-order curves showed that FRET between the
bZIP domains saturated at a much higher FRET/donor ratio (7.6 ± 0.5) than did FRET between the TA domains (2.5 ± 0.4). This
showed that there was more FRET between the bZIP domains than between
the TA domains when kinetic differences were minimized at saturation.
In contrast, the curves reached saturation at the same rate: the
acceptor/donor ratios at half the maximal FRET/donor levels were not
statistically different (42 ± 4 and 43 ± 11). These similar
kd values demonstrated that the interactions between
the bZIP domains and between the TA domains were kinetically identical. Identical kd values are expected if the
interaction between the fluorophores is governed only by the
dimerization of C/EBP Rotational Constraints within the C/EBP Local Variations in C/EBP
BFP- and GFP-linked C/EBP
With accurate image registration, we were able to determine local
variations in C/EBP Unique Conformations of C/EBP
Comparing the extent of FRET at, and away from, the pericentromeric
regions (Table II) showed that the
conformations of C/EBP
The different pericentromeric conformation of the bZIP domain was
confirmed by statistically decreased extents of FRET (p = 0.001) between the bZIP and TA domains at the pericentromeric regions
(Table II). In contrast, the extent of FRET between the fluorophore-tagged TA domains was not statistically different at and
away from the pericentromeric regions. The extents of FRET between the
bZIP and TA domains remained asymmetric at both the pericentromeric and
remaining regions of the nucleus, indicating that the torsional
constraints on the dimer were present at both locations. The simplest
interpretation is that the bZIP domains were further apart and
interacting less well at pericentromeric regions and that the TA
domains have similar orientations and torsional constraints at and away
from pericentromeric regions (Fig. 5B).
Incubation with Phorbol Ester Alters C/EBP
We investigated the effects of PMA induction on C/EBP C/EBP C/EBP C/EBP
The lower extent of FRET at pericentromeric chromatin may have resulted
from a skewing of the FRET measurement by nearby pairs of C/EBP Interdomain Distances Suggested by FRET--
The distance
separating the fluorophores will affect the extent of FRET. To provide
an indication of the magnitude in the variations that are detected in
readily understood terms, we have calculated what the different extents
of FRET would connote, assuming that they arose solely as a function of
altered distance. Comparing the relative extents of FRET observed
between different C/EBP
It should be remembered that FRET is actually measured between the
attached fluorophores, which serve as surrogates for the positions of
the specific domains of C/EBP PMA Incubation Alters C/EBP
PMA may also influence C/EBP FRET Nanoscopy Complements Structural Analyses--
FRET nanoscopy
will be very useful for determining the structural parameters of many
interacting molecules at specific intracellular locations. The
structural details of C/EBP
The structures measured by FRET also may be affected by the
requirements of packing of GFP into C/EBP
FRET nanoscopy is a powerful tool with which to investigate the
structural parameters of interacting molecules at localized sites
within living cells. We have already applied FRET nanoscopy to
investigate interactions between other molecules.3 In some
instances, FRET nanoscopy has permitted us to observe interactions that
are limited to a small percentage of sites within the cell. Certainly,
techniques such as two-hybrid interactions also may be employed to
investigate such interactions, but the extent of FRET uniquely measures
the degree to which seemingly similar interactions are different (37).
FRET nanoscopy also correlates those interaction nuances with
spatial, and potentially temporal, considerations in living cells. We
envisage that FRET nanoscopy will become a central technique with which
biochemical interactions and structural parameters may be measured
directly in the physiologic environment with unprecedented accuracy and detail.
(C/EBP
) is a master regulator of
transcription and cellular proliferation that concentrates and is kept
inactive at transcriptionally quiescent, pericentromeric regions in
mouse cell nuclei. C/EBP
dimer structure was measured in
living cells from the amounts of fluorescence energy transferred
between derivatives of the green fluorescent protein attached to
different C/EBP
domains. Comparing the levels of fluorescence
resonance energy transfer at pericentromeric and nonpericentromeric
regions of the nucleus indicated that the DNA binding domains of
C/EBP
dimers were further apart and interacted more poorly at
pericentromeric heterochromatin than in the more euchromatic regions of
the nucleus. In contrast, the position and interactions of the
transcriptional activation domains were similar throughout the nucleus.
Phorbol ester treatment caused a shift in the position of the
transcriptional activation domain relative to the DNA binding domain.
Thus, C/EBP
conformation varies with intranuclear location and with
cellular environment. These "fluorescence resonance energy
transfer nanoscopy" techniques will be broadly applicable for
associating conformational and kinetic variations to
subcompartment-specific actions of C/EBP
or any protein in the
dynamic intracellular environment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 is a transcription
factor that regulates gene expression and cellular proliferation
through distinct mechanisms (6-9). Within mouse cell nuclei, C/EBP
accumulates at one of those subnuclear domains, the pericentromeric
heterochromatin (10, 11), by binding to
-satellite DNA repeats
(10) 2 that concentrate
around the centromere (12, 13). In non-murine cells, the homologous
repetitive element and C/EBP
are less concentrated. Although most
transcription factors do not concentrate at pericentromeric heterochromatin, some factors involved in lymphocyte and erythrocyte development transiently associate with microscopically detectable, mouse pericentromeric heterochromatin at specific differentiation stages (14-17). This suggests that regulated compartmentalization of
transcription factors may be functionally important (2).
-satellite DNA and C/EBP
at
microscopically detectable pericentromeric heterochromatin permits the study of pericentromeric targeting of C/EBP
. Point and deletion mutations of C/EBP
that block pericentromeric targeting still block
cellular proliferation (18), indicating that pericentromeric localization is not required for the antiproliferative effects of
C/EBP
. In contrast, transcriptional activity is regulated by
pericentromeric location. A C/EBP
mutant that no longer binds
-satellite DNA but that retains normal binding to some promoter binding sites also no longer concentrates at the pericentromeric subdomains of the nucleus. This altered specificity mutation releases C/EBP
from sequestration at the transcriptionally quiescent (11) pericentromeric subdomain, which results in a substantial elevation in
C/EBP
activation of promoter activity.2 More naturally,
in mouse pituitary cell cultures, C/EBP
is redistributed to the
euchromatin upon expression of the pituitary-specific transcription factor Pit-1 (19, 20), whereas a Pit-1 mutation identified in human
patients with combined pituitary hormone deficiency (21, 22) is
defective in the redistribution of C/EBP
(19). Thus, the
sequestration of C/EBP
at transcriptionally inactive heterochromatin is functionally significant and regulated.
at pericentromeric heterochromatin may be
accompanied by an alteration in C/EBP
structure. We investigated
whether C/EBP
structure was different when localized at
pericentromeric heterochromatin and at euchromatin in mouse pituitary
progenitor cells. C/EBP
is a member of the bZIP family of
transcription factors, which are characterized by a conserved, carboxyl-terminal dimerization and DNA binding domain (23-25). The
bZIP domain forms a dimeric
-helical coiled-coil that binds DNA (26,
27). Transcription activation (TA) functions are present in more
amino-terminal domains of C/EBP
(28-30). However, the relative
positions of the bZIP and TA domains in the C/EBP
dimer are unknown
under any in vitro or in vivo condition.
at different subregions of
living cell nuclei. The TA and bZIP domains of full-length C/EBP
were labeled with the autofluorescent green and blue fluorescent proteins (GFP and BFP) and expressed in living cells. Like endogenous C/EBP
, fluorescent protein-labeled C/EBP
was constitutively nuclear and accumulated at pericentromeric heterochromatin (10, 11,
31). Since FRET only occurs if BFP and GFP are less than 80 Å apart
(32, 33) and decreases to the sixth power of the distance separating
the fluorophores (34), the amount of energy transferred from BFP to GFP
indicated the relative interactions between the fluorophore-tagged TA
and bZIP domains at each site within the cell (34-37).
dimer or that the
interactions between the bZIP domains were kinetically more favorable
(36). Bimolecular interaction plots of the amount of FRET measured
against the amounts of BFP and GFP-tagged C/EBP
present indicated
identical kinetics for interactions between the bZIP and between the TA
domains. FRET, measured at each of thousands of subregions throughout
each nucleus, showed that the bZIP domains were further apart or
interacted less well in C/EBP
dimers concentrated at the
pericentromeric subregions than in dimers at the remaining euchromatic
regions of the nucleus. In contrast, the spatial relationship between
the TA and bZIP domains was identical at the pericentromeric and
noncentromeric locations but was altered by incubating the cells with a
phorbol ester. Thus, we provide the first characterization of the
conformational state of a gene- and cell cycle-regulatory factor at
localized positions within living nuclei.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Fusions with GFP and BFP in
GHFT1-5 Cells
fusion proteins were expressed under the control of
the human cytomegalovirus after transfection by electroporation, as
previously described, into pituitary progenitor GHFT1-5 cells (11). The
transfected cells were plated on number 1 borosilicate glass coverslips
and grown 40-48 h post-transfection before imaging. Transfected cells
grown for 24 h were treated with 10
8 M
PMA (Sigma) or control Me2SO drug vehicle for 1 day
prior to imaging or to extract preparation for promoter activation
studies (38). The growth hormone promoter and the C/EBP
-sensitive
promoter containing the growth hormone TATA box (
33 to +8 relative to the transcription start site) and a single growth hormone C/EBP
binding site (
239 to
219) have been previously characterized (11,
38, 39).
co-localization with Hoechst
33342-stained DNA in 3T3-L1 cells, differentiated 3T3-L1 cells, and
transfected GHFT1-5 cells, those cells were washed with
phosphate-buffered saline (PBS), fixed for 5 min with methanol, treated
for 5 min with 0.05% Triton X-100 in PBS, blocked by incubation with
5% horse serum in PBS, and then incubated for 1 h with a 1:500
dilution of the C-18, anti-C/EBP
primary antibody (goat) from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA) (sc-9314) in 0.5% horse
serum in PBS. Slides were washed three times with PBS and incubated for
1 h with a 1:500 dilution of donkey, anti-goat,
rhodamine-conjugated secondary antibody from Santa Cruz Biotechnology
(sc-2094). Following washing with PBS, slides were incubated for 5 min
with 0.2 µg/ml Hoechst 33342 and then washed three times with PBS.
For the 3T3-L1 cell images shown in Fig. 1B, the 14AA
anti-C/EBP
primary antibody (rabbit) from Santa Cruz Biotechnology
(sc-61) was used in conjunction with the donkey, anti-rabbit
rhodamine-conjugated secondary antibody from Santa Cruz Biotechnology
(sc-2095).
-10 excitation
and emission filter wheels, controlled by Universal Imaging Corp.
(Downingtown, PA) Metamorph data acquisition software. The single,
immobile, multi-band pass dichroic mirror in combination with mobile
excitation and emission filters and the use of chromatically corrected
objectives maximized the image registration required for pixel-by-pixel
co-localization analysis and FRET nanoscopy. An Opti-Quip (Highland
Hills, NY) model 1962 long term stabilizer was used to keep light
intensity constant for accurate quantitative data collection.
. Once data collection parameters were established that ensured
no saturation in any pixels, all integration times, camera gain, and
pixel binning were kept constant to permit accurate quantitative
comparisons of fluorescence amounts between different cell nuclei.
. Images were always collected in the following order: 1)
acceptor (excitation filter 480-495 nm/emission filter 500-530 nm, 2)
donor (365-395 nm/435-465 nm), 3) FRET (365-395 nm/500-530 nm). The
acceptor and donor channels yielded no bleed-through between BFP and
GFP fluorescence (Fig. 2). It is particularly important to collect the
FRET channel after the donor channel to eliminate any possibility of
false FRET signals arising from decreased donor emissions that would
occur if BFP were photobleached during an initial collection in the
FRET channel. Images were collected by focusing only for the
GFP-labeled C/EBP
using the acceptor filter combination. This
prevented, until data collection, the excitation of BFP, which is more
prone than other GFP derivatives to photobleaching (31). Practically,
we had little difficulty with BFP photobleaching as was indicated by
the reproducibility of our constants obtained from the control cells
expressing only C/EBP
fusions with BFP (Fig. 2).
co-localization with Hoechst, a region of no fluorescence
adjacent to the cell was used to determine the average background level of fluorescence in each of the rhodamine, GFP, and Hoechst channels. The background amount was then subtracted from each pixel in each channel. Nuclei were identified as regions of contiguous pixels containing higher than background levels of blue fluorescence in the
Hoechst channel. The selected region was transferred to the matched
images collected in the rhodamine and GFP channels. For co-localization
analysis of cells expressing C/EBP
fused to either BFP or GFP,
nuclei were similarly identified as regions of contiguous pixels
containing significantly higher than background levels of GFP
fluorescence. Correlation coefficients were calculated by comparing the
amounts of fluorescence measured in each matched pixel of two different
channels using the Metamorph "correlation plot" application.
were identified from background-subtracted BFP or GFP
fluorescent images as described above. The amounts of nuclear
fluorescence collected from the acceptor (GFP) or donor (BFP) in each
channel were expressed as ratios relative to the amount of fluorescence collected in the respective acceptor or donor channels (see Fig. 2).
These spectral cross-talk ratios were used to calculate the contributions of BFP and GFP to each channel in cells co-expressing BFP- and GFP-labeled C/EBP
. Briefly, from the amount of acceptor fluorescence in each co-expressing cell was subtracted the minimal fluorescence contamination of C/EBP
-BFP to the acceptor channel (0.0013× donor fluorescence amount). The minimal contribution of
C/EBP
-GFP to the donor channel (0.0040 times the BFP-corrected acceptor fluorescence) was similarly subtracted. Although these contaminations were negligible in the current experiments, they can be
significant with other fluorophore or filter
combinations3 and must be
accounted for. For calculating FRET and FRET efficiency from the
nucleus, the fluorescence contribution of the acceptor C/EBP
-GFP
then was subtracted from the background-subtracted FRET channel (0.0882 times the BFP-corrected acceptor fluorescence). The amount of remaining
fluorescence in the FRET channel then was divided by the amount of
remaining fluorescence in the donor channel. If the resulting ratio is
the same as the ratio obtained from cells containing only donor
(0.4719), then there was no FRET. If higher than 0.4719, energy was
transferred from the donor to the acceptor.
should assume the same relative intranuclear distribution regardless of whether it is linked to BFP- or GFP, the
high correlation coefficients observed for BFP- and GFP-linked C/EBP
expressed in the same cell confirmed the precise image registration
required for this analysis.
, there was no counterstaining with the blue fluorescent Hoechst 33342, normally used to identify pericentromeric regions. Because of the high correlation of C/EBP
location with Hoechst 33342 staining (Fig. 1), pericentromeric regions within the nuclei were
identified as pixels containing more than 1.2 times the average amount
of background-subtracted GFP-tagged C/EBP
fluorescence. Visually and
quantitatively, this corresponded well to pericentromeric regions
identified by staining with the blue fluorescent Hoechst 33342 (40).
FRET and the extent of FRET measurements were compared at and away from
the marked pericentromeric regions using the co-localization
application of Metamorph. The proportions of pixels showing specific
extents of FRET were calculated using Metamorph by measuring the number
of pixels of progressively higher slope using sequential threshold values.
1,
where r is the distance between two fluorophores and
ro is the distance between two fluorophores at which
energy transfer is 50% efficient. Other factors, including the
interaction kinetics and the rotational orientation of the fluorophore,
contribute to FRET efficiency and are included in more expansive
equations (34, 36). The relative slopes from the linear portions of the
FRET/donor versus acceptor/donor graphs are a close
surrogate measurement of the relative FRET efficiencies (37).
Determination of the actual FRET efficiency would depend upon equipment
calibrations that equate fluorescence amounts with the amounts of each
molecule. However, we can compare the relative FRET efficiencies for
two different interactions as the ratio of the slopes
E1 and E2,
where r2 and r1
are the BFP to GFP distances under the two conditions being examined;
ro for FRET from BFP to GFP is 41.4 Å (33). By
assuming hypothetical values for r2 anywhere between 40 and 100 Å, we calculated the value of
r1. We then determined the differences between
r2 and r1 distances over
a range of values for r2. This indicated the
range in average distances separating the fluorophores used to label
the proteins under the two different experimental conditions.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Localizes to Pericentromeric
Heterochromatin--
C/EBP
, fused at either its amino
(GFP-C/EBP
) or carboxyl (C/EBP
-GFP) terminus with GFP, was
transiently expressed in mouse pituitary progenitor GHFT1-5 cells.
GHFT1-5 progenitor cells contain no endogenous C/EBP
(11, 38) and
have been used extensively in our laboratories to study the effects of
C/EBP
expression on pituitary gene transcription and cellular
proliferation (11, 18, 38, 40, 41). Western blotting showed the
expressed fusion proteins to be of the appropriate molecular weight
(11, 18, 40) and were expressed, on average, at a level comparable with
that of the pituitary-specific transcription factor Pit-1 (19), which
is present at low levels within GHFT1-5 cells (11, 38, 42). The
expression level of the GFP-C/EBP
and C/EBP
-GFP proteins also was
similar to that of endogenous C/EBP
in other cell types (discussed below).
-GFP (Fig. 1A) (18),
GFP-C/EBP
(11), and unfused C/EBP
(40) expressed in GHFT1-5 cells
all concentrated at discrete locations within the cell nucleus. In all
instances, the regions of concentrated C/EBP
coincided with AT-rich
DNA stained by the blue fluorescent dye Hoechst 33342 (Fig.
1A) (11, 18, 40). The Hoechst 33342-stained structures are
common to mouse cell lines and represent pericentromeric
heterochromatin (12, 13). We previously confirmed in GHFT1-5 cells that
the Hoechst 33342-stained structures surround foci that stained with
antibodies against the centromeric kinetochore (11). We also determined
that the Hoechst 33342-stained structures are relatively devoid of
bromo-UTP-labeled, nascent transcripts (11). Therefore, C/EBP
expressed ectopically in pituitary progenitor GHFT1-5 cells
concentrated at a distinct intranuclear structure, the pericentromeric
heterochromatin.
View larger version (45K):
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Fig. 1.
C/EBP co-localizes
with Hoechst 33342-stained DNA. A, C/EBP
-GFP
expressed after transient transfection into mouse GHFT1-5
pituitary progenitor cells. A parallel image of GHFT1-5 cells
sham-transfected with the expression vector lacking the
C/EBP
-GFP fusion protein is shown as a control. B,
immunofluorescence imaging of endogenous C/EBP
expressed in
mouse 3T3-L1 cells 7 days after initiating adipocyte differentiation by
incubation with insulin, dexamethasone, and isobutylmethylxanthine. A
parallel image of 3T3-L1 cells not induced is shown as a control. See
"Experimental Procedures" for antibody staining and fluorescence
detection procedures. C, comparison of the levels of
background-subtracted, rhodamine, and Hoechst 33342 fluorescence
collected in matched pixels from a single differentiated 3T3-L1 cell
nucleus. D, correlation coefficients derived from
pixel-by-pixel comparison of anti-C/EBP
and Hoechst 33342 fluorescence for multiple cell nuclei were calculated and
plotted against the average anti-C/EBP
fluorescence amount in each
pixel.
fusions with
GFP and GHFT1-5 cells not expressing C/EBP
both had low amounts and
identical intranuclear distributions of Hsp70 (data not shown). This
demonstrated that cells expressing C/EBP
remained healthy and did
not recognize the accumulation of ectopically expressed C/EBP
in
pericentromeric heterochromatin as a folding defect to be corrected by
co-concentrating Hsp70 at those sites. Together with our data
demonstrating that C/EBP
sequestration at pericentromeric
heterochromatin is functionally significant and regulated
(18-20),3 this strongly argued that the observed
subnuclear distribution of C/EBP
was not a consequence of protein
aggregation and precipitation into inclusion bodies, as is sometimes
observed for other cellular factors (43-45).
point mutants that were expressed to the same level as
C/EBP
failed to concentrate at the pericentromeric
heterochromatin.2 This further suggested that C/EBP
localization to pericentromeric heterochromatin cells was not an
artifact of C/EBP
overexpression in GHFT1-5 cells. Indeed,
endogenous C/EBP
expressed in mouse 3T3-L1 cells upon chemical
induction of adipocyte differentiation (46, 47) and detected by
anti-C/EBP
antibody staining also targeted to pericentromeric
heterochromatin (Fig. 1B) (10). Two different anti-C/EBP
antibodies (see "Experimental Procedures"), counterstained with
rhodamine-linked secondary antibodies, showed the same co-localization
of red (rhodamine fluorescence) and blue (Hoechst 33342 fluorescence)
in many induced 3T3-L1 cells. No C/EBP
was detected in uninduced
3T3-L1 cells (Fig. 1B). Therefore, C/EBP
expressed in
GHFT1-5 cells takes up the same intranuclear distribution of endogenous
C/EBP
in another murine cell type.
and C/EBP
-GFP or GFP-C/EBP
ectopically expressed in
GHFT1-5 cells also was confirmed by parallel staining with the same
anti-C/EBP
antibody and the subsequent imaging of differentiated 3T3-L1 cells and transfected GHFT1-5 cells under identical collection parameters. For these comparisons, co-localization was quantified by
plotting the amount of background-subtracted red fluorescence (C/EBP
, C/EBP
-GFP, or GFP-C/EBP
) in each of the thousands of pixels of each image against the amount of blue fluorescence in the
corresponding pixel of the matched Hoechst 33342 image (Fig. 1C). Correlation coefficients were calculated that described
the degree by which C/EBP
and Hoechst fluorescence at each pixel varied from a perfect correlation of 1.00. Overall, the correlation coefficients averaged 0.78 ± 0.14 for endogenous C/EBP
expressed in differentiated 3T3-L1 cells (calculated from 205 separate
nuclei), 0.69 ± 0.16 for GFP-C/EBP
expressed in GHFT1-5 cells
(n = 94 nuclei), and 0.70 ± 0.13 for C/EBP
-GFP
expressed in GHFT1-5 cells (n = 89 nuclei). The
slightly lower correlations for C/EBP
expressed in GHFT1-5 cells
probably are due to the low levels of Pit-1 in these cells; we have
observed Pit-1 to interact directly with (20) and relocate (19)
C/EBP
from the heterochromatic to the euchromatic regions of the
cell nucleus. By comparison, the correlation coefficients measured for
red C/EBP
fluorescence and green GFP-C/EBP
or C/EBP
-GFP
fluorescence, which should correlate well, since the red and green
signals both originate from C/EBP
, were 0.85 ± 0.07 or
0.88 ± 0.06, respectively.
to Hoechst 33342 correlation coefficients
calculated for each nucleus against the average rhodamine (C/EBP
) fluorescence intensity of the same nucleus (Fig. 1D)
demonstrated that C/EBP
co-localization with Hoechst 33342-stained
DNA did not vary with the C/EBP
expression level. Low level
rhodamine fluorescence from GHFT1-5 cells sham-transfected with the
empty expression vector or from undifferentiated 3T3-L1 cells did not correlate with Hoechst 33342 fluorescence (Fig. 1D).
Similarly, rhodamine fluorescence from equivalently expressed, mutant
C/EBP
proteins that failed to target to pericentromeric
heterochromatin showed correlation coefficients with Hoechst 33342 fluorescence of zero.2 Finally, C/EBP
was determined to
co-localize strongly with Hoechst 33342-stained DNA within the nuclei
of most induced 3T3-L1 cells (94.1% of the nuclei that express
C/EBP
have a correlation coefficient of >0.5) and of GHFT1-5 cells
expressing GFP-C/EBP
(88.3%) or C/EBP
-GFP (89.9%). This
analysis also demonstrated that the levels of ectopically expressed
GFP-C/EBP
and C/EBP
-GFP studied in our experiments were variable
but were globally similar to that of endogenous C/EBP
in 3T3-L1
cells. Therefore, C/EBP
concentrated at pericentromeric
heterochromatin when expressed to physiologic levels in GHFT1-5 cells.
Dimerization in Living Cells--
C/EBP
is
believed to act primarily as a dimer (26, 27), although the extent of
dimerization in the physiologic environment is unknown. To measure
dimerization in living cells, we fused the cDNA for BFP to the
carboxyl terminus of the cDNA for C/EBP
and quantified if any
fluorescence energy was transferred from the donor (C/EBP
-BFP)
to the acceptor (C/EBP
-GFP) when co-expressed (31, 35-37).
C/EBP
-GFP (11, 40) and C/EBP
-BFP3 activated a
C/EBP
-responsive promoter when expressed in GHFT1-5 cells.
-GFP or C/EBP
-BFP alone or in
combination (Fig. 2A): 1) the
GFP-specific "acceptor" channel (excitation with light of 480-495
nm; emission collected from 500-530 nm), 2) the BFP-specific
"donor" channel (excitation 365-395; emission 435-465), and 3)
the "FRET" channel (excitation 365-395; emission 500-530). The
fluorescence contribution of C/EBP
-GFP to the donor channel was
quantified from cells expressing only C/EBP
-GFP as a statistically
insignificant 0.0040 ± 0.0145 of the background-subtracted
fluorescence detected in the acceptor channel (n = 97 cells). Similarly, the contribution of C/EBP
-BFP to the acceptor
channel was 0.0013 ± 0.0058 that detected in the donor
channel (n = 108 cells). To the FRET channel,
C/EBP
-GFP consistently contributed 0.0882 ± 0.0151 the
amount of fluorescence measured in the acceptor channel, whereas
C/EBP
-BFP contributed 0.4719 ± 0.0151 the amount of
fluorescence measured in the donor channel. These "spectral
cross-talk" ratios are constants that reflect the spectral properties
of BFP and GFP and the physical properties of the detection equipment.
As such, the ratios are the same in cells expressing low and high
amounts of C/EBP
-GFP or C/EBP
-BFP (Fig. 2B) and do not
vary from experiment to experiment when using the same detection
equipment.
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Fig. 2.
FRET between BFP and GFP attached to the
carboxyl-terminal bZIP domain in full-length C/EBP
and expressed in GHFT1-5 pituitary progenitor cells.
A, representative images of nuclei in which C/EBP
-GFP and
C/EBP
-BFP were expressed alone or were co-expressed. The amounts of
nuclear fluorescence were quantified in the green, blue, or FRET
channels (shown) and then corrected (see "Experimental Procedures")
for background fluorescence and acceptor, donor, and FRET from acceptor
fluorescence using bleed-through ratios determined from the control
cells (see numbers in images). Energy
transfer resulted in an increased FRET emission at the expense of donor
emission (FRET/donor) in the co-expressing cells relative to the cells
containing donor alone (see Table I). Each channel was presented with
identical fluorescence scaling to facilitate the visual comparison of
intensity. B, graphing the bleed-through ratios against the
average intensity fluorescence in each cell showed that the ratios were
consistently measured over a wide range of fluorescence amounts. Only
cells containing fluorescence amounts within this region of accurate
measurement were processed for FRET determination.
-GFP and
C/EBP
-BFP (34, 36, 37). Background-subtracted donor, acceptor, and
FRET signals were quantified from multiple cells co-expressing the
labeled proteins. The contribution of acceptor to the FRET channel was
calculated using the cross-talk ratio and subtracted from the signal in
the FRET channel (see "Experimental Procedures"). The remaining
fluorescence in the FRET channel contained the contribution of the
donor C/EBP
-BFP (0.4719 of the amount of corrected blue
fluorescence) plus any sensitized emissions resulting from the transfer
of energy from BFP to GFP. If there were no energy transfer between
C/EBP
-BFP and C/EBP
-GFP, the FRET/donor ratios would remain at
the 0.4719 ± 0.0151 value determined for C/EBP
-BFP alone. If
there were energy transfer in cells co-expressing C/EBP
-BFP and
C/EBP
-GFP, the amount of fluorescence in the FRET channel would
increase, and the amount of C/EBP
-BFP fluorescence in the blue
channel would decrease, so the FRET/donor ratio would increase.
-BFP
and C/EBP
-GFP was 1.5750 (Table I),
indicating energy transfer and C/EBP
dimerization. As controls, the
FRET/donor ratios of cells co-expressing C/EBP
-BFP and GFP-p300 or
GFP-CBP, both of which co-localize with C/EBP
-BFP at pericentromeric
chromatin (11), were measured as 0.4777 ± 0.0334 (n = 23) for p300 and 0.4568 ± 0.0237 (n = 19) for CBP (Table I). Thus, C/EBP
interacts specifically with other C/EBP
molecules in living cells.
FRET/donor and slope values for indicated donor alone
controls and combinations of GFP- and BFP-linked C/EBP
(mean ± S.D. of n cells)
Dimers--
Expression vectors also were
constructed in which GFP and BFP were fused to the TA domains at the
amino terminus of C/EBP
(GFP-C/EBP
and BFPC/EBP
). The
four possible pairwise combinations of C/EBP
tagged with BFP or GFP
at its bZIP or TA domains (Fig. 3,
A-D) were co-expressed in GHFT1-5 cells. All four
combinations showed FRET/donor ratios greater than 0.47 (Table I),
confirming an in vivo interaction between C/EBP
proteins.
There was no indication of FRET between any combination of C/EBP
-BFP
or BFP-C/EBP
and the GFP-p300 or GFP-CBP (Table I). Thus, specific
interactions were observed between the carboxyl terminal bZIP domains,
between the amino-terminal TA domains, and between the bZIP and TA
domains in pituitary progenitor GHFT1-5 cells. Although the
interactions observed may occur in dimers or in higher order multimers,
the term "dimer" is used hereafter for simplicity and because
kinetic considerations, discussed below, indicated a bimolecular
interaction.
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Fig. 3.
Combinations of BFP- and GFP-labeled
C/EBP that were co-expressed to determine the
conformation of C/EBP
dimers in living
cells. The different combinations depicted (A-D) are
presented in the same order in Figs. 4, 6, and 8,
A-D.
)
than donor (BFP-linked C/EBP
), a greater proportion of BFP-linked
C/EBP
will dimerize with GFP-linked C/EBP
than with another
BFP-linked C/EBP
. Thus, FRET increases with increasing amounts of
acceptor relative to donor (35, 37). This was observed when the
FRET/donor ratio was plotted against the acceptor/donor ratio from
multiple cells for each combination of GFP-linked C/EBP
with
BFP-linked C/EBP
(Fig. 4,
A-D). These curves flatten toward a plateau provided enough
acceptor is present to saturate interactions with donor (Fig.
5A).
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Fig. 4.
The extent of energy transfer, normalized for
the amount of acceptor and donor, varied with the domains in
C/EBP to which BFP and GFP were attached.
FRET/donor ratios increased proportionally with the amount of acceptor
relative to donor in cells co-expressing full-length C/EBP
fusions
of BFP and GFP attached to different domains in C/EBP
(A-D). The relationship of FRET/donor against
acceptor/donor was quasilinear at lower acceptor/donor levels. The
slope of the graphs represented the extent of FRET at normalized
acceptor/donor levels for each of the four combinations of bZIP- and
TA-tagged C/EBP
. There was no FRET between BFP-tagged C/EBP
and
either CBP or p300 tagged with GFP (× in the graphs). 95%
confidence intervals in the slopes of these graphs, calculated for
acceptor/donor levels <5 and the y intercept forced through
0.47, are presented in Table I.
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Fig. 5.
Relative positions of the transcriptional
activation (TA) and helical bZIP domains of
C/EBP in living cells. A, the
FRET/donor ratio saturated at a higher level for interactions between
the bZIP domains than for interactions between the TA domains. This
indicated differences in the position and orientation of the
fluorophores attached to the TA and bZIP domains in C/EBP
dimers.
The kd values determined from the interaction curves
were identical. B, model of the C/EBP
dimer structure in
living cells. The bZIP domains were closer together
(double-headed arrow) than were the TA domains. The bZIP
domains also were slightly further apart, and interacting less well, at
pericentromeric heterochromatin than in the transcriptionally active
regions of the nucleus. The TA domain is under a rotational constraint
(curved arrow) that is altered upon incubation of
the cells with phorbol 12-myristate 13-acetate.
(Table I), reflect the relative "extent
of FRET" for each combination at equivalent acceptor/donor ratios.
The extent of FRET observed for C/EBP
tagged with BFP and GFP at
their bZIP domains was significantly higher than the extents of FRET
measured for interactions between the TA domains (p = 1 × 10
20) or between the TA and bZIP domains
(p = 5 × 10
25 and p = 5 × 10
34). This indicated either that the bZIP
domains were closer together than were the TA domains in the C/EBP
dimer or that the bZIP interactions were kinetically more stable
(higher on rate and/or lower off rate).
monomers or between two interacting units.
itself. Thus, the different extents of FRET
arose primarily from nonkinetic considerations, which would include
differences in the distances separating the fluorophores and/or
differences in the rotational orientation of fluorophore dipoles that
do not radially emit energy (34, 36).
Dimer--
The extent of FRET between the bZIP and TA domains varied
significantly (p = 0.0005) if the locations of the
donor BFP and acceptor GFP at the TA and bZIP domains were reversed
(slopes of 0.059-0.077 and 0.097-0.110; Table I). Since the average
distance between the domains and the kinetics of their interactions
would not be changed by swapping the fluorophores, the extent of FRET for the two TA and bZIP combinations should, at first glance, be the
same. However, energy transfer from many fluorophores is not radial, so
the extent of energy transfer also depends upon the orientation of
nonradial fluorophores (34, 36). Symmetry in the TA to bZIP and bZIP to
TA FRET would be observed only if both the donor and acceptor
fluorophores were rotating freely in space. The asymmetry in the extent
of FRET detected for bZIP to TA domain interactions indicated that one
or both of the bZIP or TA domains were somewhat constrained in the
C/EBP
dimer in living cells. Thus, the different level of FRET
observed between the bZIP and between the TA domains potentially
includes some contribution from rotational constraints on the
fluorophores as well as from different domain distances.
Dimer Conformation within
the Nucleus--
The relative positions and rotations of the bZIP and
TA domains, determined using the total fluorescence from each nucleus, represent the average conformation of C/EBP
in the nucleus (Fig. 5B). However, the type of C/EBP
dimers formed and the
ability to from dimers may vary with subnuclear location. To analyze
C/EBP
dimerization at different sites within the nucleus, the extent of FRET was calculated at each pixel from the background-subtracted and
bleed-through-corrected acceptor, donor, and FRET images (usually 5,000-10,000 pixels/nucleus for ×40 magnification) (Fig.
6, A-D). This required that
pixels measured in the separate acceptor, donor, and FRET channels be
perfectly matched.
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Fig. 6.
Representative background-subtracted and
bleed-through-corrected acceptor, donor, and FRET images from cells
co-expressing BFP and GFP fusions with full-length C/EBP .
Images presented contain similar amounts of acceptor (GFP) and
donor (BFP) fluorescence for each co-expression combination
(A-D). Fluorescence scaling was identical for each channel
to facilitate visual comparisons of intensity. FRET normalized for
acceptor and donor amounts (FRET Extent) was
calculated at matched pixels from each image (see "Experimental
Procedures").
should concentrate at the same
locations in each nucleus. There was a linear correlation in the amounts of background- and cross-talk-corrected fluorescence emitted from BFP- and GFP-linked C/EBP
in each of thousands of pixels from
each nucleus co-expressing both fusion proteins (correlation coefficients of 0.86 ± 0.07, n = 374 cells). This
correlation demonstrated that the amount of BFP and GFP fluorescence in
each channel emitted by a small number of C/EBP
molecules (roughly estimated to average 5-10 molecules/pixel) was the same, and
accurately measured, in matched pixels.
conformation by calculating (see "Experimental Procedures") the extent of FRET at each pixel within an image (Fig.
6, A-D, FRET Extent). The proportion
of pixels containing specific extents of FRET was determined in each
cell nucleus and then averaged from multiple nuclei (Fig.
7A). For each of the four
different interactions mapped, the extent of FRET distributed around a
peak that was very similar to the average extent of FRET measured from
all the pixels in the cell (Fig. 7B). This suggested that
the conformation of C/EBP
within the nucleus was variable around a
single preferred conformation. Note the wider distribution in the
extent of FRET for interactions between the bZIP domains, which
suggests a broader variation in this interaction at different locations
in the cell (see below).
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Fig. 7.
Variations in C/EBP
dimer conformation. A, the proportion of pixels
within the nuclei containing particular extents of FRET (see Fig. 6)
were averaged from multiple cells co-expressing full-length C/EBP
fusions of BFP and GFP attached to different domains in C/EBP
(see
Fig. 3, A-D). B, extent of FRET averaged from
all of the pixels of the same nuclei. The average extent of FRET was
similar to the extent of FRET with the highest proportion of pixels,
indicating a relatively normal variation in FRET efficiency for each of
the four aspects of C/EBP
dimer conformation investigated.
Dimers at
Pericentromeric Regions--
The pixel-by-pixel analysis identified a
distribution in C/EBP
conformation but did not associate those
variations with any subnuclear structures. We therefore compared
C/EBP
conformation at and away from pericentromeric regions by
measuring the extent of FRET at and away from pericentromeric regions
for each combination of BFP- and GFP-tagged C/EBP
. Since Hoechst
33342-stained pericentromeric regions corresponded to the regions of
concentrated C/EBP
(Fig. 1), pericentromeric heterochromatin was
marked in cells co-expressing BFP and GFP-tagged C/EBP
as pixels
containing more than 1.2 times the average fluorescence intensity of
GFP-linked C/EBP
within each nucleus (40). On average, those marked
pixels had fluorescence intensities 1.74 ± 0.28 times more than
in the remaining pixels (n = 374 cells).
were different in these subnuclear domains.
The extent of FRET between the bZIP domains at pericentromeric regions
was a statistically significant (p = 1 × 10
23) 0.8954 times that measured away from
pericentromeric regions. This showed that the bZIP domains of C/EBP
dimers were in closer contact away from the pericentromeric chromatin.
Comparison of kd and the amount of FRET at
acceptor/donor saturation calculated at, and away from, pericentromeric
chromatin indicated that the bZIP regions both interacted less well and
were further apart at the pericentromeric chromatin.
Relative extents of FRET at, and away from, Pericentromeric regions
(areas of concentrated C/EBP; mean ± S.D. of n
cells)
Dimer
Conformation--
Physiological environment also may affect C/EBP
dimer conformation. Previously, we found that incubation of pituitary
progenitor GHFT1-5 cells with PMA and forskolin enhanced C/EBP
activation of the full-length rat growth hormone promoter (38). We
first determined that a growth hormone promoter deleted of all
sequences except those surrounding the C/EBP
binding site (
239 to
209) and the TATA box (
33 to +8) responded to PMA, and not to
forskolin, in a C/EBP
-dependent fashion (data not
shown). This suggested that C/EBP
or co-regulatory factors that
cooperate with C/EBP
were a direct target of phorbol ester activation.
conformation
by comparing the extent of FRET for all combinations of GFP- and
BFP-linked C/EBP
in sham-treated cells and in cells treated with
10
8 M PMA (Figs.
8, A-D). Pixel-by-pixel
analysis showed the interactions between the bZIP domains to be
identical in sham- and PMA-treated cells (Fig. 8A), even
when compared for the different interactions at and away from the
pericentromeric regions (not shown). This suggested that the overall
contact and distances between the bZIP domains were similar regardless
of PMA incubation. In contrast, PMA incubation resulted in a
statistically significant decrease in the extent of FRET between the
BFP-tagged TA and GFP-tagged bZIP domains (Fig. 8C,
p = 0.03) and between the BFP-tagged TA and GFP-tagged
TA domains (Fig. 8D, p = 0.04). For both TA
to bZIP and TA to TA FRET, the decreased extent of FRET was
particularly prominent at the pericentromeric regions
(p = 0.01 for both) but less consistent away from the
pericentromeric region (p = 0.06 for both). This
suggested that the PMA-induced alteration in conformation was more
uniform for C/EBP
dimers at the pericentromeric regions than for
dimers away from the pericentromeric regions. The asymmetric effect of
PMA incubation on the extent of FRET from TA to bZIP (Fig.
8C), but not from bZIP to TA (Fig. 8B) or from
bZIP to bZIP, indicated that the change in conformation induced by PMA
included a torsional rotation of the TA domains relative to each other and to the rest of the dimer (Fig. 5B).
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Fig. 8.
Incubation with the phorbol ester PMA alters
the rotational position of the TA domain relative to the rest of the
C/EBP dimer. PMA incubation had no effect
on the extent of FRET measured for BFP and GFP fused to the bZIP end
(A) or BFP fused to the bZIP end and GFP fused to the TA end
(B). In contrast, PMA incubation decreased significantly the
extent of FRET between GFP fused to the bZIP end and BFP fused to the
TA end (C) and between BFP and GFP fused to the TA ends
(D). The asymmetric effect of PMA on TA to bZIP
(C) and TA to TA (D) FRET indicated that the
effect of PMA incubation on C/EBP
dimer conformation included an
alteration in the torsional constraints of the donor fluorophore around
the TA domain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Forms Dimers in Living Cells--
FRET between
spectral derivatives of the green fluorescent protein fused to C/EBP
defined that C/EBP
is a dimer in living cells (Fig. 2). For BFP and
GFP, FRET is 50% efficient when the donor and acceptor are 41.4 Å apart (33) and falls very rapidly, to the sixth power, as the
fluorophores are separated (33, 34, 36). Thus, energy transfer
demonstrated that the fluorophores, and therefore C/EBP
, were well
within 80 Å of each other within the cell. These interactions were
very specific. No FRET was observed between C/EBP
and the
co-activators CBP and p300 (Table I) although GFP-tagged CBP and p300
co-localized with C/EBP
when expressed in GHFT1-5 cells (11). Both
CBP and p300 enhanced transcriptional activation by C/EBP
(11, 48)
but have never been observed to directly interact with C/EBP
(11,
48). However, C/EBP
expression does cause CBP (11) and
p3003 to redistribute to the intranuclear location of
C/EBP
, suggesting that C/EBP
forms some sort of complex with CBP
and p300 within the cell.
Dimers Form throughout the Nucleus of Living
Cells--
We combined the angstrom level resolution of FRET with the
resolution of light microscopy (250 nm for green light) to
map C/EBP
dimer structure throughout the cell (Figs. 4-8). By
changing the positions of the fluorophore tags within C/EBP
, this
"FRET nanoscopy" technique was used to define, in living cells, the interactions of the bZIP and TA domains in C/EBP
dimers at localized intranuclear domains and under different cellular conditions. FRET
nanoscopy demonstrated that, in virtually all regions of the nucleus,
C/EBP
was positioned sufficiently close to another C/EBP
molecule
to allow energy to transfer from the attached BFP to GFP. Thus,
intermolecular C/EBP
interactions were spread throughout the cell
nucleus and were not excluded from any subnuclear structure.
Conformation Varies with Subnuclear Location
and with Cellular Environment--
The amounts of FRET at individual
100 × 100-nm regions within each nucleus were also calculated,
taking into account the amounts of donor and acceptor at each localized
site (Figs. 6-8). This "extent of FRET" closely approximates the
FRET efficiency, which is affected by the relative distance between the
attached fluorophores, by the on and off rates of the interacting
C/EBP
, and by constraints on the rotational freedom of asymmetric
donor and acceptor fluorophores imposed by their attachment to C/EBP
(34-36). The pixel-by-pixel measurement of FRET extent therefore
defined not only whether factors interact within a cell but also the
relative quality of that interaction at localized sites. This allowed
us to study the relative positioning and rotational constraints of
different functional domains of C/EBP
(Fig. 5B) and to
define the subcellular conformations of C/EBP
dimers under different
cellular conditions.
molecules, not dimerizing, but co-concentrating and weakly interacting
at the pericentromeric chromatin. We are confident that this was not
the case, since 1) this concentration would have led to a higher extent
of FRET at the pericentromeric chromatin rather than the observed lower
extent of FRET; 2) the lower extent of FRET was only observed for
interactions involving the bZIP domain (interactions between the
equally concentrated TA domains showed no change in interaction); 3) no
FRET was observed for measurements using the similarly concentrated CBP
and p300 indicating the absence of near neighbor effects; and 4)
C/EBP
was only concentrated 1.74 times, on average, at the
pericentromeric chromatin, which is unlikely to be enough to have
resulted in a substantive difference in near neighbor effects. Thus,
the different extents of FRET represent true conformational differences
in the C/EBP
dimers. Local variations in dimer conformation may
reflect the presence or absence of specific C/EBP
-interacting
co-factors at the pericentromeric sites (11, 40) or the consequences to
C/EBP
dimers of interactions with different chromosomal structures.
domains in equations developed by
Förster (34) provides an estimation of the relative
fluorophore-to-fluorophore distance for each combination of expressed
C/EBP
(see "Experimental Procedures"). These calculations would
suggest that the fluorophores attached to the TA domains in the dimers
were spaced, on average, 10.4-14.9 Å further apart than were the
fluorophores attached to the bZIP domains. These calculations also
suggest that the fluorophores attached to the bZIP domains were, on
average, 1.2-1.8 Å further apart at the pericentromeric regions than
elsewhere in the nucleus. Again, this analysis assumes equivalent
interaction kinetics and equivalent rotational constraints at and away
from the pericentromeric chromatin, which is probably not true.
However, the calculations provide an indicator of the sensitivity of
the FRET measurements.
. Since the GFP-derived fluorophores
themselves constitute a complete protein domain, actual distances
between the domains vary with the stearic requirements of GFP. However,
the relative extents of FRET between the donor and acceptor fluorophore
remain a good approximation of the relative conformational parameters,
including interdomain distance.
Dimer Structure--
The
enhancement of C/EBP
-activated transcription upon incubation of
GHFT1-5 cells with PMA was accompanied by an alteration in C/EBP
dimer structure consisting of a change in the position and/or
orientation of the TA relative to the bZIP domain (Figs. 5B
and 8). The effects of PMA on C/EBP
conformation may be related to
PMA activation of protein kinase C, which is known to phosphorylate C/EBP
(49, 50). We have seen an identical PMA-induced alteration in
the conformation of C/EBP
measured in another cell type using different donor and acceptor fluorophores (cyan and yellow fluorescent proteins). This specific change in the position and/or
orientation of the TA domains of C/EBP
is associated with a single
phosphorylation site in
C/EBP
.4
conformation by regulating other
factors that form complexes with C/EBP
(11), by regulating the
C/EBP
-induced alteration in histone acetylation at pericentromeric regions (40) or by regulating dimerization of C/EBP
with other C/EBP
family members (46, 51). PMA incubation also may indirectly change the
environment of the pericentromeric region to affect C/EBP
dimer
conformation. Regardless of the underlying basis, the FRET measurements
show that C/EBP
dimer conformation was altered when the
intracellular environment was changed.
, to date, were limited to the assumption
that the last 80 amino acids (the bZIP domain) of the 358-amino
acid-long C/EBP
were similar to coiled-coil structures identified
for other bZIP domains (26, 27). This predicted that the carboxyl
termini of C/EBP
should be in very close proximity. Indeed, our FRET
measurements demonstrated that the carboxyl termini were considerably
closer to each other in living cells than were the other domain
interactions that were measured.
. Given the close
proximities of the carboxyl terminal
-helices in the predicted
C/EBP
dimers, it was somewhat surprising that the C/EBP
-GFP
fusions remained transcriptionally active (11, 40) and competent to
block cellular proliferation in GHFT1-5 cells (18). The fusion proteins
containing GFP fused to the amino-terminal TA domain of C/EBP
also
remained competent to block cellular proliferation (18) but were
defective in transcriptional activation (40). Packing constraints
imposed by the large GFP fluorophore at the amino terminus even may
have contributed to the rotational constraint necessary to detect the PMA-induced change in TA domain conformation at the pericentromeric regions. Thus, the large size of the GFP fluorophores may have some
unexpected stearic advantages as well as limitations. The development
of smaller fluorophore tags (52, 53) may reduce, but not eliminate, the
stearic consequences of fluorophore tags.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert Fletterick (University of California, San Francisco) for critical reading of the manuscript and Dr. Roland Kwok (University of Michigan, Ann Arbor, MI) for the GFP-p300 expression vector.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK 54345 and grants from the University of California San Francisco Research Evaluation and Allocation Committee and from the University of California San Francisco Committee on Research (to F. S.).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.
§ To whom all correspondence should be addressed. Tel.: 415-476-7086; Fax: 415-564-5813; E-mail: freds@diabetes.ucsf.edu.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M207466200
2 B. Wu and F. Schaufele, unpublished results.
3 F. Schaufele and R. N. Day, unpublished data.
4 S. Ross, B. Wu, F. Schaufele, and O. A. MacDougald, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
C/EBP, CCAAT/enhancer-binding protein
;
TA, transcription activation;
FRET, fluorescence resonance energy transfer;
GFP, green fluorescent protein;
BFP, blue fluorescent protein;
PMA, phorbol 12-myristate 13-acetate;
PBS, phosphate-buffered saline.
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