Visualization of Pit-1 Transcription Factor Interactions in the Living Cell Nucleus by Fluorescence Resonance Energy Transfer Microscopy
Richard N. Day
Departments of Medicine and Cell Biology National Science
Foundation Center for Biological Timing University of Virginia
Health Sciences Center Charlottesville, Virginia 22908
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ABSTRACT
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The pituitary-specific transcription factor Pit-1
forms dimers when interacting with specific DNA elements and has been
shown to associate with several other nuclear proteins. Recently,
techniques have become available that allow visualization of
protein-protein interactions as they occur in single living cells. In
this study, the technique of fluorescence resonance energy transfer
(FRET) microscopy was used to visualize the physical interactions of
Pit-1 proteins fused to spectral variants of the jellyfish green
fluorescent protein (GFP) that emit green or blue light [blue
fluorescent protein (BFP)]. An optimized imaging system was used to
discriminate fluorescence signals from single cells coexpressing the
BFP- and GFP-fusion proteins, and the contribution of spectral overlap
to background fluorescence detected in the FRET images was established.
Energy transfer signals from living cells expressing a fusion protein
in which GFP was tethered to BFP by short protein linker was used to
demonstrate acquisition of FRET signals. Genetic vectors encoding GFP-
and BFP-Pit-1 proteins were prepared, and biological function of the
fusion proteins was confirmed. FRET microscopy of HeLa cells
coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which
required the two fluorophores to be separated by less than 100 Å.
Biochemical studies previously demonstrated that Pit-1 physically
interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of
cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer
between these fusion proteins, a result consistent with their
association in the nucleus of these living cells. In contrast, there
was no evidence for energy transfer between the BFP-Pit-1 and an
estrogen receptor-GFP fusion proteins. It is likely that the FRET
imaging approach described here can be applied to many different
protein-partner pairs in a variety of cellular contexts.
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INTRODUCTION
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Determining when and where specific protein partners associate
with one another within the cell is of fundamental importance to
understanding biological processes. The relative proximity of two
proteins labeled with different fluorophores can be determined by
conventional fluorescence microscopy to the scale of approximately 0.2
µm (2000 Å), the limit of optical resolution for the light
microscope. Resolving the relative proximities of proteins beyond the
optical limit of the microscope, however, is necessary to reveal the
physical interactions between protein partners. For the light
microscope, this degree of resolution can only be realized through the
use of fluorescence resonance energy transfer (FRET; Refs. 1, 2, 3, 4). FRET
is a quantum mechanical process by which radiationless transfer of
excitation energy can occur from a donor fluorophore to an appropriate
acceptor fluorophore. Energy transfer requires that the donor
fluorophore emission spectrum overlap with the absorption spectrum for
the acceptor fluorophore. Because the efficiency of energy transfer
varies inversely with the sixth power of the distance separating the
two fluorophores, FRET can only occur when the distance separating the
two fluorophores is less than approximately 0.01 µm (100 Å; Refs.
1, 2, 3, 4). Thus, FRET microscopy is an extraordinarily sensitive method for
determining the relative proximity of labeled protein partners.
A major obstacle to the application of FRET microscopy to living cells
has been a lack of suitable methods for specifically labeling
intracellular proteins with donor and acceptor fluorophores. Recent
studies using expression of the jellyfish green fluorescent protein
(GFP) in a variety of cell types have proven this unique protein to be
a versatile molecular reporter (5, 6, 7, 8, 9). GFP retains its characteristic
fluorescence when fused to other protein sequences, allowing
fluorescence microscopy to be used to visualize the spatiotemporal
dynamics of GFP-fusion protein localization in intact living cells
(9, 10, 11, 12). Mutant forms of GFP with emission in both the green and the
blue spectrum [blue fluorescent protein (BFP)] have been engineered
(5, 6, 8). These different color fluorophores provide a general method
for simultaneously labeling two different proteins within the same
living cell (9). Importantly, the excitation and emission spectra for
the mutant GFP and BFP proteins are suitable for FRET, making this
unique noninvasive imaging approach more generally applicable (8, 13, 14, 15, 16, 17).
We reported previously the optimization of a fluorescence microscope
imaging system for the acquisition of green, blue, and energy transfer
fluorescence signals from single living cells expressing GFP- and
BFP-fusion proteins (15, 16, 17). In the present study, the FRET approach
is applied to visualize protein-protein interactions involving the
pituitary-specific transcription factor Pit-1. The Pit-1 protein
dimerizes when associated with specific DNA elements of target genes.
Moreover, cooperative interactions between Pit-1 and other classes of
transcription factors were shown to be important for hormone and growth
factor regulation of Pit-1-dependent gene expression. For example a
strong synergy between Pit-1 and members of the ets
transcription factor family was shown to be involved in regulation of
PRL gene transcription (18, 19), and biochemical studies showed a
physical association of Pit-1 and Ets-1 (20). In addition, cooperative
interactions between the Pit-1 protein and the estrogen receptor were
also shown to be important in regulation of the PRL gene transcription
(21, 22), and a physical association of these two proteins was
demonstrated (23, 24). The studies presented here use GFP- and
BFP-fusion proteins and the FRET technique to visualize homologous
interactions involving the Pit-1 protein and heterologous interactions
between Pit-1 and the Ets-1 or estrogen receptor proteins in the living
cell nucleus.
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RESULTS
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The GFP-Fusion Proteins Are Biologically Functional
Genetic vectors encoding GFP- and BFP-fusion proteins were used in
transient cotransfection of HeLa cells to achieve cellular expression
of the protein partners. In each case, expression of the fusion
proteins was confirmed by Western blotting (see individual figures).
The expression vectors for GFP-Pit-1 and BFP Pit-1 encoded proteins
with the fluorophores tethered to the amino-terminal end of Pit-1 by a
five-amino acid (AA) linker (see Materials and Methods and
Ref. 15). Several different methods were used to demonstrate that these
fusion proteins were functional. First, Western blotting was used to
demonstrate that the expected 60-kDa GFP-Pit-1 was synthesized in
transfected cells (inset, Fig. 1
). Second, the GFP-Pit-1 protein was
shown to bind to both the PRL gene 3P Pit-1 DNA element
(inset Fig. 1
) and 1P element (not shown) using the gel
mobility shift assay (EMSA). The specificity of this DNA-protein
complex was demonstrated by immunoclearing of the complex from the EMSA
reaction by addition of an antibody specific for Pit-1
(inset, Fig. 1
). Further, competition studies with unlabeled
oligonucleotides confirmed the fusion protein-bound DNA with
appropriate binding specificity, and the fusion protein did not bind to
an unrelated interferon response element (data not shown). Importantly,
the results shown in Fig. 1
demonstrated that expression of GFP-Pit-1
induced transcription from a cotransfected PRL promoter-luciferase
reporter gene to a similar extent as the wild-type Pit-1 protein.
Moreover, the GFP-Pit-1 protein was also shown to synergize with a
GFP-Ets-1 protein in activation of the PRL gene promoter (Fig. 1
).
These results confirmed that the GFP-Pit-1 fusion protein retained the
characteristics of specific DNA binding, transcriptional activation,
and cooperative interactions with Ets-1.

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Figure 1. GFP-Pit-1 and GFP-Ets-1 Fusion Proteins Are
Biologically Functional
Representative experiment demonstrating transcriptional activation of
the -306 rat PRL promoter luciferase reporter gene by cotransfection
with CMV Pit-1 or CMV GFP-Pit-1 expression vectors. Much greater than
additive luciferase activity was observed when CMV GFP-Pit-1 and CMV
GFP-Ets-1 were cotransfected than when they are transfected separately
(note scale break). Results are triplicate transfections plotted as
fold activation over the empty vector control ± SE.
Inset, Western blot analysis of GFP-Pit-1 protein in
extracts from transiently transfected cells probed with an antibody
directed against GFP (lane 1). Numbers indicate size
marker migration EMSA demonstrating binding of proteins from whole-cell
extracts prepared from Rat-1 cells transfected with the CMV GFP-Pit-1
plasmid to a duplex 32P-labeled 3P PRL Pit-1 site probe
(lane 2). DNA-protein complex (arrowhead) was not
observed for extracts pretreated with a polyclonal antibody directed
against Pit-1 (lane 3).
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Characterizing Spectral Overlap
FRET microscopy requires an imaging system that can discriminate
fluorescence signals with overlapping spectra. We previously reported
the use of GFP- and BFP-fusion proteins to characterize and optimize
excitation and emission filters for detection of blue and green
fluorescence signals from individual living cells (15, 16, 17). In the
present study, the extent of blue-fluorescence overlap into the filter
set used to detect energy transfer (the acceptor filter set) was
quantified. This is important, since this signal overlap contributes to
the background signal against which energy transfer signals will be
compared. HeLa cells were transfected with the expression vector
encoding BFP-Pit-1, and the expression of the expected 60-kDa protein
was verified by Western blotting (Fig. 2A
). Cells expressing the BFP-Pit-1
fusion protein were then identified by nuclear-localized blue
fluorescence (Fig. 2C
). Images of cells expressing BFP-Pit-1 were then
obtained under constant conditions of neutral density (nd) and
integration time with each of the three filter combinations used in
these studies (Fig. 2
, BD; see Materials and Methods for
filter characteristics). After background subtraction, a mosaic of the
donor (BFP-Pit-1) and acceptor (overlap) images was generated and a
look-up table was applied to the gray scale image to quantify signal
intensity (Fig. 2
, C and D; white indicates highest fluorescence
signal). The gray level intensity of the donor and acceptor
fluorescence signals across the profile of the nucleus was then
determined for each image (position of the profiles is indicated by
lines in Fig. 2
, C and D), and the results were plotted (Fig. 2
, E and
F). The results shown in Fig. 2
, E and F, quantify the intensity of the
BFP fluorescence detected by both the donor and acceptor filter sets,
demonstrating the extent of BFP-fluorescence signal overlap into the
acceptor (FRET) image.

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Figure 2. Characterization of BFP Fluorescence Overlap in the
Acceptor Image
HeLa cells were transiently transfected with the CMV BFP-Pit-1 vector
and expression of the fusion protein was detected by Western blot (A).
Images of a cell expressing the fusion protein were obtained using a nd
1.0 filter and 1-sec integration with the GFP filter set (panel B; bar
indicates 10 µm), or 5-sec integration with the BFP filter set (C)
and the acceptor filter set (D). The donor and acceptor images were
combined in a single mosaic image (panels C and D), and a look-up
table was applied to facilitate comparison of fluorescence signal
intensity (calibration bar indicates signal level with
white being highest intensity). A profile was taken
across the cell nucleus at the position indicated by the lines in
panels C and D, and the gray level intensity for this profile was
plotted for both the donor and acceptor images (panels E and F).
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Since the FRET technique requires discrimination of green and blue
fluorescence signals, imaging of cells expressing colocalized BFP- and
GFP-fusion proteins that would not be expected to physically interact
was used to characterize further the extent of fluorescence signal
overlap detected with the acceptor filter set. HeLa cells were
cotransfected with the vector encoding BFP-Pit-1 and a vector encoding
GFP with a nuclear localization signal (GFP-NLS; see Materials
and Methods for sequence). Western blotting was used to
demonstrate that both the 29-kDa GFP-NLS and 60-kDa BFP-Pit-1 fusion
proteins were expressed in transiently transfected cells (Fig. 3
A). As noted above, the BFP-Pit-1
protein was exclusively localized to the nucleus, and coexpression with
the GFP-NLS resulted in both green and blue fluorophores in the nuclear
compartment (Fig. 3
, B and C). Background subtracted donor (BFP-Pit-1)
and acceptor images were acquired under constant conditions of nd and
image acquisition time, and the same look-up table was applied to the
mosaic image to show fluorescence intensity (Fig. 3
, C and D).
The gray level intensity for nuclear fluorescence at the indicated
positions across the profile of the nuclei from these two cells was
then determined. The results shown in Fig. 3
, E and F, demonstrated the
extent of combined GFP- and BFP-signal overlap detected with the
acceptor filter set. For the cells shown in Fig. 3
, the acceptor signal
represents approximately 45% of the donor signal. Together, these
results demonstrated the contribution of both GFP- and BFP-fusion
protein fluorescence signal overlap into the acceptor image and
established the baseline fluorescence against which FRET signals were
compared.

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Figure 3. Discrimination of Fluorescence Signals from Cells
Expressing Noninteracting GFP- and BFP-Fusion Proteins
HeLa cells were transiently transfected with the CMV GFP-NLS or CMV
BFP-Pit-1 vectors, and expression of the fusion proteins was detected
by Western blot (panel A, lanes 1 and 2, respectively). Images of HeLa
cells coexpressing these proteins were then obtained using the GFP
filter set (panel B; bar indicates 10 µm), BFP filter
set (C), and the acceptor filter set (D), and the mosaic image of donor
and acceptor fluorescence (panels C and D) was acquired as described in
the legend of Fig. 2 . The gray level intensity profile across the
nuclei of the two cells was taken at the position indicated by the
lines in panels C and D and was plotted for both the donor and acceptor
images (panels E and F).
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Visualizing FRET from Single Living Cells
We and others demonstrated previously that energy transfer signals
can be acquired from living cells expressing a fusion protein in which
GFP is tethered directly to BFP through a protein linker (13, 14, 15, 17).
This same approach was taken to characterize the acquisition of energy
transfer signals under the constant conditions used in the present
study. HeLa cells were transfected with an expression vector encoding
GFP coupled to BFP through a three-AA linker and expression of the
56-kDa protein was confirmed by Western blotting (Fig. 4A
). Cells expressing the fusion protein
were identified by green fluorescence throughout the cell (Fig. 4B
).
Images of these cells were then acquired using the donor (BFP) and
acceptor filter combinations, and the same look-up table was applied to
the background-subtracted mosaic image to show the fluorescence signal
intensity (Fig. 4
, B and C). The gray level intensity across the
profile of the two cells shown in Fig. 4
was determined at the
positions indicated. In contrast to the results from the previous
experiment with colocalized, but noninteracting fluorophores, the
results shown in Fig. 4
, E and F, demonstrated that the gray level
intensity for the acceptor image was approximately 2-fold greater than
the signal measured in the donor image. This result showed energy
transfer from BFP to GFP.

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Figure 4. FRET Microscopy of Cells Expressing GFP Fused
Directly to BFP by a Three-AA Linker
HeLa cells were transiently transfected with the CMV GFP-three-AA-BFP
vector, and expression of the protein was detected by Western blot (A).
Images of two cells expressing the GFP-three-AA-BFP protein were
obtained using the GFP filter set (panel B; bar
indicates 10 µm), BFP filter set (C), and the acceptor filter set
(D), and a mosaic image of donor and acceptor fluorescence (panels C
and D) was acquired as described in the legend of Fig. 2 . The gray
level intensity profile across the two cells was taken at the position
indicated in panels C and D and was plotted for both the donor and
acceptor images (panels E and F).
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Visualizing the Physical Association of Pit-1 Proteins
The FRET imaging approach described above was then applied to
cells coexpressing the GFP- and BFP-Pit-1 fusion proteins. HeLa cells
were cotransfected with vectors encoding the GFP-Pit-1 and BFP-Pit-1
fusion proteins, and expression of the fusion proteins was confirmed by
Western blotting (Fig. 5A
). Cells
coexpressing the BFP-Pit-1 and GFP-Pit-1 proteins were first identified
by green fluorescence (Fig. 5B
). Images of these same cells were then
obtained under constant conditions of nd and integration time using the
donor and acceptor filter sets. A mosaic image showing the background
subtracted donor (BFP-Pit-1) and acceptor images was obtained, and the
same look-up table was applied to indicate fluorescence intensity (Fig. 5
, C and D). The gray level intensity across the profile of the two
nuclei shown in Fig. 5
at the positions indicated was then plotted
(Fig. 5
, E and F). The results demonstrated that the acceptor signal
exceeded the donor signal by approximately 2-fold, an indication of
energy transfer from BFP-Pit-1 to GFP-Pit-1. This required that the
fluorophores be separated by less than approximately 100 Å, as would
be the case for physically associated Pit-1 fusion proteins.

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Figure 5. FRET Microscopy of Cells Expressing GFP- and
BFP-Pit-1 Proteins
HeLa cells were transfected with vectors encoding GFP-Pit-1 or
BFP-Pit-1, and expression of the proteins was detected by Western blot
(panel A, lanes 1 and 2, respectively). Images of HeLa cells
cotransfected with these vectors were then obtained obtained using the
GFP filter set (panel B; bar indicates 10 µm), BFP
filter set (C), and the acceptor filter set (D), and a mosaic image of
donor and acceptor fluorescence (panels C and D) was acquired as
described in the legend of Fig. 2 . The gray level intensity profile
across the two cell nuclei were taken at the position indicated in
panels C and D and was then plotted for both the donor and acceptor
images (panels E and F).
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FRET Imaging of Pit-1 and Ets-1 or the Estrogen Receptor
This imaging technique was then used to examine HeLa cells
coexpressing the GFP-Ets-1 and BFP-Pit-1 fusion proteins. Cooperative
interactions between the Pit-1 and Ets-1 proteins in the
transcriptional activation of the PRL gene promoter were reported
(18, 19, 20), and the GFP-Ets-1 protein used in the current study was shown
to synergize with GFP-Pit-1 in transcriptional activation of a PRL
promoter-luciferase reporter gene (Fig. 1
). Western blotting of protein
extracts from transiently transfected HeLa cells confirmed that both
the 80- kDa GFP-Ets-1 and the 60-kDa BFP-Pit-1 fusion proteins were
expressed (Fig. 6A
). Cells expressing the
GFP-Ets-1 protein were identified by green fluorescence, and
coexpression of the BFP-Pit-1 protein was verified by presence of
nuclear blue fluorescence (Fig. 6B
). Images of cells coexpressing the
two fusion proteins were then obtained under constant conditions of nd
and integration time using the donor and acceptor filter sets. A mosaic
image showing the background subtracted donor (BFP-Pit-1) and acceptor
(GFP-Ets-1) images was obtained as described above (Fig. 6
, C and D).
The gray level intensity across the profile of the two nuclei shown in
Fig. 6
at the positions indicated was then determined (Fig. 6
, E and
F). The results demonstrated that the fluorescence intensity in the
acceptor image was greater than that for the donor image. The acceptor
signal was above the base fluorescence established in the control
experiments (Figs. 2
and 3
), an indication that the Ets-1 and Pit-1
fusion proteins were in close association.

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Figure 6. FRET Microscopy of Cells Coexpressing the GFP-Ets-1
and BFP-Pit-1 Proteins
HeLa cells were transfected with vectors encoding BFP-Pit-1 or
GFP-Ets-1, and expression of the proteins was detected by Western blot
(panel A, lanes 1 and 2, respectively). Images of HeLa cells
cotransfected with these vectors were then obtained using the GFP
filter set (panel B; bar indicates 10 µm), BFP filter
set (C), and the acceptor filter set (D). The mosaic image of donor and
acceptor fluorescence (panels C and D) was acquired as described in the
legend of Fig. 2 . The gray level intensity profile across the two cell
nuclei was taken at the position indicated in panels C and D. The
upper graph obtained for both the donor and acceptor
images (panels E and F) was from the nucleus on the
right.
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The FRET imaging approach was also applied to cells expressing Pit-1
and the estrogen receptor to determine whether a physical association
between these two proteins could be detected. HeLa cells were
cotransfected with an expression vector encoding the human estrogen
receptor with GFP fused at the carboxy terminus [human estrogen
receptor (hER)-GFP] and the BFP-Pit-1 fusion protein.
Functional cooperativity between Pit-1 and the hER-GFP fusion protein
was observed in cotransfection studies using a 3-kb rat PRL
promoter/enhancer luciferase reporter gene (data not shown). Expression
of the expected 91-kDa hER-GFP fusion protein was verified by Western
blotting (Fig. 7A
). Cells expressing the
hER-GFP protein were identified by nuclear localized green
fluorescence, and coexpressed BFP-Pit-1 protein in the same cells was
then confirmed by nuclear blue fluorescence (Fig. 7B
). Images of cells
coexpressing the two fusion proteins were acquired, and the mosaic
image showing the background subtracted donor (BFP-Pit-1) and acceptor
(hER-GFP) images was obtained as described above (Fig. 7
, C and D). The
profile of gray level intensity across the two nuclei shown in Fig. 7
, E and F, demonstrated that the fluorescence signal in the acceptor
image represented less than 45% of the donor signal. These results
were similar to those obtained for the colocalized, but noninteracting
proteins shown in Fig. 3
, implying that the fluorophores labeling the
estrogen receptor and Pit-1 were too far apart for FRET to occur.

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Figure 7. FRET Microscopy of Cells Coexpressing the hER-GFP
and BFP-Pit-1 Proteins
HeLa cells maintained in medium supplemented with serum containing
endogenous estrogens were transfected with vectors encoding BFP-Pit-1
or hER-GFP, and expression of the proteins was detected by Western blot
(panel A, lanes 1 and 2, respectively). Images of HeLa cells
cotransfected with these vectors were then obtained using the GFP
filter set (panel B; bar indicates 10 µm), BFP filter
set (C), and the acceptor filter set (D). The mosaic image of donor and
acceptor fluorescence (panels C and D) was acquired as described in the
legend of Fig. 2 . The gray level intensity profile across the two cell
nuclei was taken at the position indicated in panels C and D and was
then plotted for both the donor and acceptor images (panels E and F).
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DISCUSSION
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The molecular cloning of GFP (25) and its expression in a variety
of cell types (7, 9, 10, 26) demonstrated the potential of this unique
protein as a biological marker. It is the use of GFP as fluorescent tag
to visualize dynamic cellular events, however, that have proven it to
be a valuable tool for the cell biologist (12, 27, 28, 29). Modifications
of the GFP protein sequence have yielded variant forms with increased
brightness and differing spectral characteristics (5, 6, 8). Some of
the spectral variants of GFP were shown to be suitable as donor and
acceptor for FRET microscopy (8, 30). Studies by Heim and Tsien (8)
determined that direct transfer of excitation energy from BFP Y66H to
GFP S65T would be 50% efficient over a distance of 40 Å, and recent
studies have taken advantage of FRET between these fluorophores to
visualize dynamic events within the living cell. For example, FRET
microscopy was used to monitor spatio-temporal changes in intracellular
free calcium from single cells expressing a fusion protein containing
BFP coupled to GFP by a calmodulin-binding peptide (14, 30). These
studies illustrate the strength of the combined use of the GFPs and
FRET microscopy to visualize the interactions of protein partners on
the scale of ångstroms.
In the present study, FRET microscopy was used to visualize both
homologous and heterologous protein-protein interactions involving the
transcription factor Pit-1. The Pit-1 protein is a homeodomain
transcription factor that is expressed exclusively in the anterior
pituitary, where it functions as an important determinant of
pituitary-specific gene expression (32). Pit-1 binds to DNA elements
within the promoters of several different pituitary genes, including
the PRL and GH genes (32, 33, 34, 35). Biochemical studies showed that the
Pit-1 protein exists as a monomer in solution, but that it binds to
most DNA elements as a dimer (23, 35, 36). The Pit-1 protein contacts
DNA through the carboxy-terminal POU-specific domain and homeodomain,
and the dimerization contacts that form between monomers also require
segments within these two domains (36). In the present study, Pit-1
proteins tagged with the GFPs were visualized in the living cell
nucleus. The GFP-Pit-1 fusion protein retained both DNA binding
specificity and the ability to transcriptionally activate the PRL gene
promoter. When FRET microscopy was used to visualize the coexpressed
BFP-Pit-1 and GFP-Pit-1 proteins, energy transfer was observed. This
required that the fluorophores attached to the Pit-1 proteins be
separated by less than approximately 100 Å. Due to the flexibility of
Pit-1 protein structure, it is not possible to know the precise spatial
positioning of the fluorophores relative to one another. This
flexibility, however, might be expected to provide dynamic averaging of
the relative orientations of donor and acceptor (37). Because the FRET
signals obtained from cells expressing the Pit-1 proteins (Fig. 5
) were
similar to signals from cells expressing the GFP-three-AA-BFP fusion
protein (Fig. 4
), the results suggested that the GFP- and
BFP-fluorophores fused to the Pit-1 proteins were in physical
contact.
Since HeLa cells do not express the genes that are transcriptionally
activated by Pit-1, it is unlikely that the observed FRET signals
originated from dimerized Pit-1 proteins bound to specific DNA
elements. It is possible that nonspecific binding of the Pit-1 fusion
proteins to genomic DNA could give rise to the observed protein-protein
interactions. However, there was no evidence for energy transfer from
the BFP-Pit-1 protein to the hER-GFP protein (Fig. 7
), demonstrating
that nuclear colocalization of two DNA-binding proteins was not
sufficient for FRET to occur. Further, this observation also argued
against energy transfer resulting from nonspecific protein-protein
interactions due to high concentrations of the donor and acceptor
fusion proteins in the nuclear compartment. The actual number of fusion
proteins expressed in the cells used in the present study was not
determined, but it was recently shown that as few as 10,000 GFP
molecules could be detected in single cells in culture (38). Since the
fusion proteins used in this study were spatially localized to the
nuclear compartment, still fewer GFP molecules would be required for
detection. Thus, detection of GFP-fusion proteins does not require
expression to levels greatly exceeding the normal physiological range
for cellular proteins, and nonspecific interactions between fusion
proteins was an unlikely source of energy transfer signals.
It is clear that binding to specific DNA elements leads to dimerization
of Pit-1 proteins (36). It was therefore somewhat surprising that FRET
imaging should reveal Pit-1 protein-protein interactions in the absence
of specific DNA contacts in the HeLa cell nucleus. It is possible that
the Pit-1 fusion proteins are assembling as part of larger complexes of
nuclear proteins, and a number of transcription factors have been shown
to cooperatively interact with Pit-1. In transfection studies in
nonpituitary cells, cooperative interactions between Pit-1 and other
non-cell type-specific transcription factors was shown to be required
for transcriptional activation of the PRL promoter. These studies lead
to the demonstration of a strong synergy between Pit-1 and members of
the ets transcription factor family (18, 19), and
biochemical studies showed that Pit-1 and Ets-1 could physically
associate (20). In the present study, the results of FRET imaging of
Pit-1 and Ets-1 proteins provided evidence for energy transfer between
the fluorophores (Fig. 6
), indicating that these proteins were in
physical contact or close association in the cell nucleus. The
relatively weak FRET signals obtained with Pit-1 and Ets-1 may indicate
that the fluorophores were separated by a distance close to the limit
necessary for energy transfer.
Previous studies also demonstrated that cooperative interactions
involving Pit-1 and the estrogen receptor were important in regulation
of the PRL gene transcription (21, 22, 23, 24). As was the case for Ets-1,
biochemical studies demonstrated that physical interactions also
occurred between Pit-1 and the estrogen receptor (24). In contrast to
the results obtained with Ets-1 and Pit-1 in the present study, FRET
imaging provided no evidence for energy transfer between the
fluorophores attached to Pit-1 and the estrogen receptor (Fig. 7
).
Given the extremely restricted distance over which FRET can occur, it
is conceivable that Pit-1 and estrogen receptor proteins were in
physical contact, but that the fluorophores were separated by more than
100 Å or obstructed in some way. It was recently shown that the
coactivator protein RIP140 participates in the molecular interactions
between Pit-1 and the estrogen receptor (39), and this protein could
potentially act as a spacer between the fusion proteins.
The results from this experiment also illustrated an important
limitation of the FRET techninque; while positive results demonstrate
physical interactions between protein partners, a negative result
provides no information about protein-protein interactions. The results
presented here demonstrated that Pit-1 proteins were in physical
contact in these living HeLa cell nuclei, and also implicated
that Pit-1 and Ets-1 were in close association. It is possible that
these proteins come in contact as part of complexes involving other
nuclear proteins. In support of this view, we reported previously the
use of a digital deconvolution computer algorithm to process images of
GFP-Pit-1 acquired at different optical sections through the cell
nucleus (40). The resulting high-resolution images demonstrated an
ordered pattern of Pit-1 localization within the nuclear compartment
that may indicate inclusion of the fusion proteins within subnuclear
structures (41). Taken together, the results presented here illustrate
the utility of FRET microscopy to visualize interactions between
protein partners tagged with the GFPs in the living cell.
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MATERIALS AND METHODS
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Materials
Construction of some of the GFP- and BFP-fusion protein
expression vector DNAs was described previously (15, 16). The sequences
encoding GFP (S65T; Ref. 6), and BFP (Y66H, Y145F; Ref. 5) were
modified for optimal mammalian cell expression through human codon
usage (15, 42). A plasmid vector using the strong cytomegalovirus
promoter (pCMV) was used for expression of all the fluorescent fusion
proteins (43). The sequence encoding Pit-1(29) and cEts-1 (44) were
obtained by PCR amplification with the proofreading KlenTaq polymerase
mix (CLONTECH Laboratories, Inc., Palo Alto, CA) using the cloned DNA
sequences as templates. Introduction of unique restriction endonuclease
sites into the amplified DNA facilitated in-reading frame
insertion of these sequences at the extreme 3'-end of the coding
sequence for GFP and BFP. The sequence encoding the human estrogen
receptor AA 1576 linked to GFP(S65T) was resected from the
pNEFGFPS65T vector (kindly provided by Dr. D. Picard, Universite de
Geneve, Geneva, Switzerland) and inserted into the pCMV vector. The
pCMV GFP-three-AA-BFP vector encoding GFP fused to BFP by a three-AA
linker was prepared by ligation of the BFP cDNA into the GFP vector,
placing the BFP coding sequence in the reading frame of GFP. The vector
encoding GFP with NLS was prepared by insertion of a duplex
oligonucleotide sequence encoding the SV40 NLS (45) protein sequence
LYPKKKRKGVEDQYK at the 3'-terminus of the GFP coding sequence. For
transfection, large-scale recovery of expression vector plasmid DNAs
was performed by double-banded CsCl gradient centrifugation, and the
plasmid DNAs were verified by restriction enzyme analysis and direct
sequence analysis.
Cell Culture and Transfection
HeLa cells or Rat 1 cells were maintained as a monolayer in a
1:1 mixture of phenol red-free Hams F12-DMEM containing 10% newborn
calf serum. The cells were harvested and transfected with the indicated
genetic vector(s) by electroporation as described previously (46). For
the luciferase reporter gene experiments, the total amount of DNA was
kept constant using empty vector DNA. After electroporation, the cells
were immediately diluted in medium and used to inoculate culture dishes
for analysis of luciferase reporter gene activity, or for preparation
of cell extracts for Western blot analysis or EMSA. Luciferase assays
were performed as described previously (46). For imaging of GFP- and
BFP-fusion protein expression, transfected cells were used to inoculate
culture dishes containing 25-mm glass cover slips. These cultures were
maintained at 33 C in a humidified 5% CO2 incubator for
24 h before fluorescence microscopy for optimized protein
expression (38).
Western Blotting and EMSA
Transiently transfected HeLa cells were lysed at 4 C in
detergent buffer as described previously (46). Samples were
fractionated by SDS-PAGE on 10% gels. Protein standards were run in
adjacent lanes for determination of mol wt. The proteins were
transferred to nitrocellulose for 1 h by electroblotting at
100 V and then detected by Ponceau S staining. The membranes were
blocked with 5% nonfat dried milk in TBS-T buffer [20 mM
Tris-HCl (pH 7.6), 137 mM NaCl, 0.1% Tween-20], washed in
TBS-T, and incubated with GFP antibody (Molecular Probes, Eugene, OR;
1:10,000 final dilution) for 1 h at room temperature. After washes
in TBS-T, the membranes were incubated with a 1:50,000 final dilution
of horseradish peroxidase-conjugated antirabbit Ig (Amersham Corp.,
Arlington Heights, IL). The membranes were washed in TBS-T and
incubated in enhanced chemiluminescence (ECL) reagents (DuPont/NEN,
Boston, MA) for 1 min. The membranes were then exposed to Kodak XAR 5
film (Eastman Kodak, Rochester, NY) for 515 min.
EMSAs were performed on whole cell extracts prepared from transiently
transfected HeLa cells as described previously (46). A duplex
oligonucleotide corresponding to the 3P-PRL Pit-1 binding site
(5'-GGAGGCCTGAATATGAA-TAAGA, Ref. 19) was end labeled using [
-
32P] ATP and T4 polynucleotide kinase and used as probe.
Whole cell extract (20 µg) was added to 15 µl reaction mixtures
assembled on ice. For immunoclearing experiments, 0.75 µl Pit-1
polyclonal antibody was added to the reaction mixtures and incubated
for 1 h at 4 C. The reaction mixtures were transferred to tubes
containing 25,000 to 50,000 cpm of the end labeled probe. The
mixtures were then incubated for 20 min at room temperature and loaded
on prerun 6.0% polyacrylamide gels prepared in running buffer
containing 25 mM Tris-HCl (pH 8.3), 192 mM
glycine, 1 mM EDTA. The gels were run at 150 V, dried, and
autoradiographed overnight using Kodak XAR 5 film.
Fluorescence Microscopy
The FRET imaging system used in these studies is based on a
conventional inverted microscope equipped for epi-fluorescence and
transmitted illumination (IX-70, Universal infinity system; Olympus
America, Inc., Melville, NY). Fluorescence images were acquired using a
60x aqueous-immersion objective lens. The excitation light source was
a 100 W mercury-xenon arc lamp (Hamamatsu Corp., Middlesex, NY) coupled
to excitation and nd filter wheels. The emission filter wheel
was coupled to the output port of the microsocope and then to
the camera. Images were captured using a slow scan, liquid
nitrogen-cooled charge-coupled device camera with a back-thinned,
back-illuminated imaging chip (CH260, Photometrics, Ltd., Tucson, AZ).
The digital image output of the camera was 512 x 512 pixels with
16 bits resolution. For these studies, all images were collected using
a nd 1.0 filter and constant integration times. All fluorescence
signals fell within the range of 10 to 35 K gray level intensity
(before background subtraction), and none of the images had saturated
pixels. The Silicon Graphics, Inc.-based ISEE software (Inovision
Corporation, Raleigh, NC) was then used to obtain the mosaic images and
determine the gray level intensity profiles.
 |
ACKNOWLEDGMENTS
|
---|
The author is grateful to Dr. Ammasi Periasamy of the Advanced
Cellular Imaging Facility at the University of Virginia for expert
assistance in imaging and the technical assistance of Diana Berry and
Margaret Kawecki. The author also wishes to thank Dr. David Brautigan
for helpful discussion and comments. The mutant GFP S65T and Y66H/Y145F
cDNAs were provided by Drs. Roger Tsien and Roger Heim (University of
California, San Diego, CA), and Dr. Jen Sheen (Massachusetts General
Hospital, Boston, MA) provided the mutant GFP with optimized codon
usage.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Richard N. Day, Ph.D., Department of Internal Medicine, Box 578, University of Virginia Health Sciences Center, Charlottesville, Virginia 22903. E-mail:
rnd2v{at}virginia.edu
This study was supported by National Science Foundation (NSF) Grant
DIR-8920162, Center for Biological Timing Technology Development
subproject, and NSF Grant IBN9528526 and NIH Grant
RO1-DK-43701.
Received for publication April 7, 1998.
Revision received April 30, 1998.
Accepted for publication May 27, 1998.
 |
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