Single Cell Analysis of Transcriptional Activation
Ilona U. Rafalska-Metcalf1, Sara Lawrence Powers1¤, Lucy M. Joo1, Gary LeRoy2, Susan M. Janicki1*
1Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania, United States of America, 2Department of Molecular Biology, Princeton
University, Princeton, New Jersey, United States of America
Background: Gene activation is thought to occur through a series of temporally defined regulatory steps. However, this
process has not been completely evaluated in single living mammalian cells.
Methodology/Principal Findings: To investigate the timing and coordination of gene activation events, we tracked the
recruitment of GCN5 (histone acetyltransferase), RNA polymerase II, Brd2 and Brd4 (acetyl-lysine binding proteins), in
relation to a VP16-transcriptional activator, to a transcription site that can be visualized in single living cells. All accumulated
rapidly with the VP16 activator as did the transcribed RNA. RNA was also detected at significantly more transcription sites in
cells expressing the VP16-activator compared to a p53-activator. After a-amanitin pre-treatment, the VP16-activator, GCN5,
and Brd2 are still recruited to the transcription site but the chromatin does not decondense.
Conclusions/Significance: This study demonstrates that a strong activator can rapidly overcome the condensed chromatin
structure of an inactive transcription site and supercede the expected requirement for regulatory events to proceed in a
temporally defined order. Additionally, activator strength determines the number of cells in which transcription is induced
as well as the extent of chromatin decondensation. As chromatin decondensation is significantly reduced after a-amanitin
pre-treatment, despite the recruitment of transcriptional activation factors, this provides further evidence that transcription
drives large-scale chromatin decondensation.
Citation: Rafalska-Metcalf IU, Powers SL, Joo LM, LeRoy G, Janicki SM (2010) Single Cell Analysis of Transcriptional Activation Dynamics. PLoS ONE 5(4): e10272.
Editor: Edith Heard, Institut Curie, France
Received January 13, 2010; Accepted March 27, 2010; Published April 21, 2010
Copyright: ? 2010 Rafalska-Metcalf et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the following foundations: The Emerald Foundation, The Mallinckrodt Foundation, the Beckman Young Investigators
Program, the V Foundation, and the Wistar Institute Cancer Center Support Grant (P30CA10815) for use of the Genomics/Sequencing Facility. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: Biomatrica, San Diego, California, United States of America
During transcriptional activation, gene specific activators bind
to their response elements and recruit the regulatory factors
needed to initiate efficient RNA synthesis. The basic steps in this
process include chromatin decondensation, pre-initiation complex
assembly, and RNA polymerase II (RNA pol II) elongation
through the gene [1,2]. Transcriptional output is also a product of
activator strength and likely depends on the regulatory factors
each recruits as well as their recruitment timing . Increasing the
number of activator binding sites can also have a synergistic effect
on activation and has been shown to eliminate the need for specific
regulatory steps such as histone acetylation .
However, in all cases, transcriptional activators must overcome
the significant impediment that chromatin structure imposes on
transcription. In order for the pre-initiation complex to form and
RNA synthesis to proceed, the DNA duplex must be opened,
which requires decondensation of high-order chromatin structure
and nucleosome disassembly [5,6]. Interestingly, many elongation
factors are histone chaperones highlighting the importance of
nucleosomal dynamics in this process. For example, FACT
(facilitates chromatin transcription), composed of the HMG-1-like
protein, SSRP1 and p140/Spt16, transiently binds and removes
H2A/H2B dimers from nucleosomes . Additionally, nucleolin is
required for RNA pol I elongation ; Asf1 mediates nucleosome
disassembly at the PHO5 and PHO8 promoters ; and Brd
proteins, discussed more extensively below, function as chaperones
and facilitate elongation on acetylated chromatin templates in a
defined transcription system .
Enzymes that add and remove protein post-translational
modifications (PTMs) are also important transcriptional regulators
because they create and eliminate regulatory factor binding sites
. The histone proteins – particularly their N-terminal tails –
are important targets, but other factors, including transcription
factors, are also modified [12,13]. Specific histone PTMs are
associated with silent and active chromatin. Histone lysine
acetylation is enriched at active chromatin and the bromodomain
(BD) is the protein module that binds acetylated lysines. BDs are
found in histone acetyltransferases (HATs), including GCN5,
PCAF and p300, chromatin remodeling factors, general transcrip-
tion factors and elongation factors [11,14,15].
Members of the BET (bromodomains and extraterminal)
protein family, found in yeast and animals, contain tandem BDs
and are of great interest because of their roles in regulating early
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events in transcriptional activation . In mammals, Brd2, Brd3
and Brd4 are widely expressed and Brdt is testes specific. Brd
proteins also regulate chromatin organization and epigenetic
inheritance and are required for development – functions that all
require their BDs and/or their ability to bind acetylated lysines.
Acetyl-lysine binding is also required for Brd2 and Brd4 to both
interact with and mediate the attachment of episomal viral
genomes to mitotic chromosomes [17,18,19,20,21,22]. In yeast,
Bdf1 prevents heterochromatin spreading by competing with Sir2
for acetylated H4 at euchromatin-heterochromatin boundaries
[23,24]. Brd4 +/2 cells are hypo-acetylated at H3 K14 and H4
K12 suggesting that Brd4 preserves both its own binding sites
as well as global acetylation . When over-expressed, Brd2
increases histone H4 acetylation and gene activation [17,26,27].
Most studies of transcriptional activation indicate that regula-
tory events occur in a temporally defined order [28,29,30].
However, as they were mostly done utilizing molecular genetic
and biochemical methods, in which effects are averaged in cell
populations, it remains to be seen how they are dynamically
regulated in single cells. Transcriptional activators bind to specific
sequence elements in genes and initiate the recruitment of the
molecular machines needed to decondense chromatin and activate
transcription. Unlike activators though, most of these recruitment
events are mediated by a complex array of dynamic protein-
protein and protein-PTM interactions . Although many have
been extensively characterized using biochemistry, it is not well
understood how they are coordinated at transcription sites in
relation to changes in chromatin organization and the initiation of
Advances in imaging technology and the development of
methods that allow gene expression to be tracked in real time in
single cells now make it possible to address these types of questions
[32,33]. In Drosophila salivary gland tissue, polytene chromosomes
provide the signal intensity needed to identify individual genes by
chromosome banding and have been used to study gene activation
in a natural chromatin environment . The RNA pol II
subunit, Rpb3, and heat shock factor (HSF), the hsp70 trans-
criptional activator, rapidly accumulate at the native hsp70 gene
loci after induction [34,35]. As specific loci cannot be similarly
analyzed in mammalian interphase cells, other systems have been
developed in order to do this.
The multi-copy insertion of transcriptional reporter constructs
into single genomic sites in mammalian cells provides the signal
intensity needed to investigate transcriptional dynamics at a
defined site. A cell line with a multi-copy array (,200 copies) of
the mouse mammary tumor virus/Harvey viral ras (MMTV/v-
Ha-ras) reporter has been used to show that the glucocorticoid
receptor (GR) associates dynamically with its binding sites at a
natural promoter . However, as the LTR only has 4 GR
binding sites (,800–1200 total in array) , it is difficult to utilize
this interaction to track factor recruitment at the earliest stages of
activation. Additionally, the mRNA can only be detected using
RNA FISH making it impossible to study how RNA synthesis is
dynamically coordinated with other regulatory events .
To address these issues, arrays of bacterial and bacteriophage
sequence element repeats have been introduced into inducible
transgenes. Fusion of their requisite binding proteins to auto-
fluorescent proteins allows these DNA and RNA elements to be
used to directly visualize gene activity in single mammalian cells
. Multi-copy integration of these transgenes provides the signal
intensity needed for factor accumulation, RNA synthesis and
chromatin decondensation to be clearly monitored throughout the
course of activation. Additionally, the inactive transcription site
can be visualized in intact cells, changes in chromatin architecture
can be correlated to RNA synthesis, and factor recruitment and
regulatory pathway coordination can be investigated.
In this study, we use such a system  to examine the
recruitment of transcriptional regulatory factors to a heterochro-
matic transcription site during activation. GCN5, RNA pol II
and Brd4 are all rapidly recruited with a VP16-transcriptional
activator. In contrast, Brd2, which requires its BDs for recruit-
ment, lags behind the activator by ,2 minutes. Interestingly,
RNA accumulates at the site simultaneously with the VP16-
activator indicating that a strong activator can rapidly overcome a
condensed chromatin environment and bypass the need for the
expected ordered recruitment of regulatory factors before trans-
cription can commence. As RNA accumulates at the transcription
site in significantly more cells expressing the VP16-activator
compared to a p53-activator, this demonstrates the importance of
activator strength in this process. Additionally, transcriptional
activation factors are still recruited to the transcription site in a-
amanitin pre-treated cells. However, chromatin decondensation is
significantly decreased compared to control cells providing further
evidence for the essential role of transcription in driving large-scale
changes in chromatin architecture [40,41].
Description of the experimental system
In order to study the dynamics of gene expression in living cells,
we previously developed a cell line (2-6-3) in which we can
simultaneously visualize a transcription site and the RNA and
protein produced from it in real time in single cells . It is
derived from the human osteosarcoma cell line, U2OS, and
contains a stable multi-copy chromatinized array of an inducible
transgene (,200 copies integrated into a euchromatic region of
chromosome 1). The transgene contains lac operator repeats (256
copies), which allow it to be visualized in both the inactive and
active states when lac repressor fused to an auto-fluorescent
protein, Cherry  or YFP, is expressed (Figs. 1A and1B, panels
b and e).
Transcription is induced using the Tet-On/Tet-Off system.
When tetracycline-regulated activators bind to the tetracycline
response elements (TREs; 96 copies), transcription is driven from
the CMV minimal promoter (Fig. 1A). In previous studies, we used
the reverse tet transcriptional activator (rtTA; reverse tet repressor
fused to VP16), which binds to the TREs in the presence of
doxycycline. However, rtTA localizes throughout the nucleus and
cytoplasm and is prone to transcriptional leakiness. In order to
achieve tighter transcriptional regulation and to use the activator/
DNA interaction to visualize the transcription site, we fused the
tetracycline transcriptional activator (tTA; tet repressor fused to
VP16), to Cherry and the hormone binding domain of the
estrogen receptor (ER)  to create the tamoxifen-inducible
Cherry-tTA-ER (Fig. 1A). Before activation, Cherry-tTA-ER is
sequestered in the cytoplasm and cannot be detected at the
transcription site, marked by YFP-lac repressor confirming that
the system is not leaky (Fig. 1B, panels a–c; Table S1). After the
addition of tamoxifen, a population of Cherry-tTA-ER enters the
nucleus, accumulates at the transcription site, and co-localizes with
YFP-lac repressor in 85.263.6% of transfected cells (Fig. 1B,
panels d and j; Fig.1C; Table S1). The majority of Cherry-tTA-
ER, however, remains in the cytoplasm after activation.
In the inactive state, the transcription site is highly condensed
and, as previously reported, is enriched for the heterochromatic
histone modification, H3 tri-meK9, and proteins (HP1s) . To
determine the effect of transcriptional activation on the conden-
sation state of the transcription site chromatin, we measured and
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compared the pixel area of the inactive (marked by Cherry-lac
repressor) and active (marked by Cherry-tTA-ER) sites (Fig. 1D).
The active site occupies ,6 times the area of the inactive site
demonstrating that the chromatin significantly decondenses during
activation (Fig. 1D).
The transcribed RNA encodes cyan fluorescent protein (CFP)
fused to a peroxisomal targeting signal (SKL), 24 repeats of the
MS2 bacteriophage translational operator and a splicing cassette
and the 39 UTR from the rabbit b-globin gene. This RNA can be
visualized by expressing the YFP-MS2 binding protein, which
binds to the stem loop structure of the MS2 translational oper-
ator as a dimer (Fig. 1A). 3 hours after activation, YFP-MS2
accumulates at the transcription site in 88.361.8% of cells (Fig. 1B,
panel k; Fig. 1C; Table S1). There are also a greater number of
pixels with high CFP signal intensity in cells activated for 3 hours
compared to inactive cells indicating that CFP-SKL is highly
expressed and confirming that the mRNA is processed, exported
and translated  (Fig. 1E).
To determine the timing of transcriptional activation, we
monitored the recruitment of Cherry-tTA-ER to the transcription
site using time-lapse imaging (Fig. 2A; Movie S1). For this analysis,
cells expressing Cherry-tTA-ER at equivalent intensity levels were
selected for imaging. The levels of this factor at the transcription
site were determined and fit to a logistical model and a good
agreement was seen between the data and the fit curve. Using this
model, the start time of accumulation was defined as the point
when the signal intensity at the transcription site reached 5% of
the total value between the normalized initial baseline (0% signal)
and the final total accumulation (100% signal). At this threshold,
the signal intensities are above background levels but not yet in the
regions of rapid accumulation where varying accumulation rates
would distort initial time measurements. Under these parameters,
Cherry-tTA-ER is recruited 7.561.0 min after the addition of
tamoxifen (Fig. 2B; Table S2).
Histone acetyltransferases are strongly and rapidly
recruited to the transcription site
To evaluate the changes that occur to the transcription site
chromatin during activation, we immunostained inactive and
transcriptionally activated cells using antibodies against histone H4
acetyl-K5, histone H4 acetyl-K12, and histone H3 acetyl-K9,
which are binding sites for the acetyl-lysine binding, Brd2 and
Brd4 proteins [10,17,44,45,46]. Figure 3A and Table S1 show
that they are only enriched at active transcription sites. These
antibodies were also used for chromatin immunoprecipitation
(ChIP) to determine the sequence elements at which they are
enriched (primer pairs Fig. 3B). All of the acetyl-lysine modifica-
tions were strongly and specifically enriched at the promoter after
activation (Fig. 3C).
Since we detected strong accumulation of histone lysine
acetylation at the active site (Fig. 3), we also wanted to identify
the histone acetyltransferases (HATs) recruited to it. YFP-tagged
GCN5, PCAF and p300 were not enriched at the inactive site,
marked by Cherry-lac repressor (Fig. S1A; Table S1). However, all
were strongly recruited and co-localized with Cherry-tTA-ER
upon activation (Fig. 4A; Table S1).
YFP-GCN5 was enriched at the transcription site in 79.564.5%
of activator-expressing cells (Fig. 1C and S1B). To determine its
recruitment timing in relation to Cherry-tTA-ER, we collected
time-lapse images of cells expressing these factors during
activation. Cherry-tTA-ER and GCN5 reached the 5% accumu-
lation threshold 6.961.0 and 7.061.1 min after the addition of
tamoxifen, respectively (Fig. 4B; Movie S2; Table S2). As
described above, the 5% threshold was set as the start point of
accumulation. To validate this parameter value, the times at the
2.5 and 10% thresholds were also examined and shown to exhibit
identical trends (Table S2). Consistency across a range of values
for this parameter is within the error of the logistic fit and the
limits of our time resolution and supports the conclusion that
GCN5 is recruited with the activator.
RNA synthesis begins rapidly upon transcriptional
We next evaluated the localization of RNA pol II and the
FACT subunit, p140/Spt16, at the transcription site. Using
immunofluorescence (IF), we found that neither co-localized with
the inactive site although accumulations were detected around its
periphery (Fig. 5A, panels a–c and g–i; Table S1). Similar results
were obtained in YFP-RNA pol II expressing cells (Fig. S2,
panels a–d). However, both RNA pol II and p140/Spt16 were
strongly recruited to the active transcription site marked by
Cherry-tTA-ER (Fig. 5A, panels d–f and j–l; Fig. S2, panels e–h;
In order to determine the timing of RNA pol II recruitment, we
collected time-lapse images of cells co-expressing YFP-RNA pol II
and Cherry-tTA-ER during activation. Cherry-tTA-ER accumu-
lated 6.361.8 min and YFP-RNA-pol II accumulated 6.56
2.1 min after tamoxifen addition, indicating that RNA pol II is
Figure 1. Description of the live-cell gene expression system. (A) Schematic diagram of the inducible transgene drawn to scale. The
interaction of Cherry-lac repressor with the lac operator repeats allows the transgene to be visualized in both the inactive and active states. Cherry-
tTA-ER (Cherry-tetracycline transcriptional activator-estrogen receptor) binds to the tetracycline response element (TRE) repeats in the presence of
tamoxifen and activates transcription from the CMV minimal promoter. The transcribed RNA includes CFP fused to a peroxisomal targeting signal
(CFP-SKL), 24 MS2 translational operators (MS2 repeats) and a splicing unit and the 39 UTR from the rabbit b-globin gene. The RNA is visualized
through the interaction of YFP-MS2 coat protein dimers with the MS2 translational operators (stem loop structures). YFP-tagged proteins co-
expressed with either Cherry-lac repressor or Cherry-tTA-ER can be evaluated for recruitment to the inactive and active transcription site, respectively.
Repeat elements in the transgene are shaded gray; the length of each is shown in parentheses. (B) Localization of the proteins that mark the inactive
and active transcription site. Cherry-tTA-ER is sequestered in the cytoplasm (panels a and g) until the addition of tamoxifen induces its entry into the
nucleus where it both activates transcription and marks the active site (panels d and j). YFP-lac repressor constitutively binds to the lac operator
repeats, and therefore, marks both the inactive (panel b) and active (panel e) transcription site. YFP-MS2 is diffusely distributed throughout the
nucleus before activation (panel h). It accumulates at the transcription site and becomes particulate throughout the nucleus after activation (panel k).
Scale bar represents 5 mm. Scale bar in the enlarged inset represents 1 mm. (C) Graph represents the percentage of transfected cells with
accumulation of the regulatory factors at the transcription site 3 hr after activation. 100 cells were analyzed from 3 independent experiments.
Average values and SEM (in the form of error bars) are presented in the graphs. (D) Measurement of the pixel area of the inactive transcription site
(marked by Cherry-lac repressor) and VP16-activated transcription sites (marked by Cherry-tTA-ER) 3 hrs after activation. Values represent the
averages of 10 cells. SEM (in the form of error bars) and a p value is presented in the graph. (E) Frequency histogram showing the distribution of the
blue pixel intensity levels (blue bars) as a measure of the CFP-SKL protein in inactive and activated cells. Black bars represent the background signal.
The x-axis is the average fluorescence pixel intensity in each bin on a scale from 0 to 1 and divided into bin sizes of 0.02; the y-axis is the number of
pixels in each bin, on a logarithmic scale. The bar beneath the histogram shows the intensity range. 10 independent cells were analyzed.
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rapidly recruited upon activator binding (Fig. 5B; Movie S3; Table
In order to determine when mRNA synthesis begins, we
measured YFP-MS2 levels (6.761.3 min) at the transcription
site in relation to Cherry-tTA-ER (6.661.1 min) (Fig. 6;
Movie S4). As the timing was almost identical (Fig. 6B), this
indicates that transcription is rapidly induced upon activator
Figure 2. Real-time analysis of the recruitment of the VP16-transcriptional activator. (A) Still images from a time series of 2-6-3 cells
expressing Cherry-tTA-ER during activation. Selected images taken from Movie S1 show the accumulation of the VP16 activator at the transcription
site. Scale bar is equal to 5 mm. (B) Quantification of Cherry-tTA-ER recruitment to the transcription site during activation. Tamoxifen was added to
the media immediately after the first time point (,0 min). Images were collected every 1 min for ,40 min. Measured intensities were normalized to
the high and low plateau values and fitted to a logistic fit (solid black line). The initial accumulation time (blue X and arrow) is the point when the
fitted curve reaches 5% of the total accumulation. The graph is the average of 6 cells imaged from 4 different coverslips on 3 different days. Error bars
represent the standard deviation.
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Analysis of Brd2 and Brd4 recruitment during activation
As the active transcription site is enriched with histone lysine
acetylation and GCN5 rapidly accumulates upon activation, we
also wanted to evaluate the recruitment of acetyl-lysine binding
factors with roles in regulating transcriptional activation. Brd4
(Fig. 7 and S1B) and Brd2 (Fig. 8 and S1B) are double BD proteins
that interact with histone H4 acetylated at K5 and K12 – PTMs
enriched at the active site (Fig. 3) [10,17,44,45,46]. Neither was
enriched at the inactive site (Fig. 7A, panels a–c and Fig. 8A,
panels a–c; Table S1), but both were strongly recruited upon
activation in 74.363.3% and 78.265.5% of transfected cells,
respectively (Fig. 7A, panels d–f and Fig 8A, panels d–f; Fig. 1C;
Table S1). Immunofluorescence (IF) staining with an antibody
against Brd4 showed that the endogenous factor accumulates at
the active site in a pattern similar to YFP-Brd4 (Fig. 7A, panels g–i;
We next evaluated the recruitment timing of Brd4 and Brd2 in
relation to Cherry-tTA-ER. Brd4 accumulated rapidly with the
activator (Fig. 7B; Movie S5; Table S2) but, interestingly, Brd2
lagged behind by ,2 minutes (Fig. 8B; Movie S6; Table S2). To
Figure 3. Immunofluorescence and ChIP analyses of histone acetyl-lysine modifications at the transcription site. (A) Histone H4
acetyl-K12, histone H4 acetyl-K5 and histone H3 acetyl-K9 levels at the transcription site were visualized by immunostaining. H4 acetyl-K12 (panels a–
c), H4 acetyl-K5 (panels g–i) and H3 acetyl-K9 (panels m–o) are not enriched at the inactive site, marked by Cherry-lac repressor. H4 acetyl-K12
(panels d–f), H4 acetyl-K5 (panels j–l) and H3 acetyl-K9 (panels p–r) are enriched at the active site, marked by Cherry-tTA-ER. Transcription was
activated for 3 hr by the addition of tamoxifen. Scale bar is equal to 5 mm. Scale bar in enlarged inset is equal to 1 mm. (B) Schematic representation
of the inducible transgene shows the location of primers and probes used for real-time PCR in the ChIP assay. (C) The presence of the histone acetyl-
lysine modifications along the transgene in transcriptionally inactive and active cells was analyzed using ChIP. Results were obtained from 3
independent experiments. Average values and SEM (in the form of error bars) are presented in the graphs.
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investigate the role of acetyl-lysine binding on Brd2 recruitment,
we evaluated a construct with mutations that prevent this
interaction. Tyrosine (Y) 113 in BD1 and Y386 in BD2 in Brd2
mediate the interaction with acetylated lysines; Y to phenylalanine
(F) conversions inhibit it [10,17]. YFP-Brd2 BD(1+2) YRF does
not accumulate at the active site indicating that this interaction is
mediated by acetyl-lysine binding (Fig. 8A, panels g–i; Table S1).
Therefore, the time difference detected between activator and
Brd2 recruitment provides a measure of the time required for the
site to become hyper-acetylated.
Interestingly, Brd2 decreased the timing of activator recruit-
ment by ,2 min (Table S2). This is consistent with reports that
Brd2 over expression enhances transcriptional activation and
global histone acetylation [17,26,27]. Perhaps it makes the TRE
Figure 4. Analysis of histone acetyltransferases dynamics during activation. (A) YFP-GCN5 (panels a–c), YFP-PCAF (panels d–f), and YFP-
p300 (panels g–i) are strongly recruited to the active transcription site, marked by Cherry-tTA-ER, 3 hr post induction. Transcription was induced by
the addition of tamoxifen. Scale bar represents 5 mm. Scale bar in the enlarged inset represents 1 mm. (B) Quantification of Cherry-tTA-ER (top panel,
solid red circles) and YFP-GCN5 (lower panel, solid green circles) accumulation at the transcription site during activation. Tamoxifen, was added to the
media immediately after the first time point (,0 min). Images were collected every 1.5 min for ,40 min. Measured intensities were normalized to
the high and low plateau values and fitted to a logistic fit (solid black line). The initial accumulation time (blue Xs and arrows) is the point when the
fitted curve reaches 5% of the total accumulation. The graphs are the average of 7 cells imaged from 4 different coverslips on 4 different days. Error
bars represent the standard deviation.
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Figure 5. Analysis of RNA pol II and FACTp140 at the transcription site. (A) RNA pol II (panels a–c) and the FACT subunit p140/Spt16 (panels
g–i) surround but do not co-localize with the inactive transcription site, marked by Cherry-lac repressor. RNA pol II and p140/Spt16 are strongly
enriched and co-localize with the active transcription site, marked by Cherry-tTA-ER (panels d–f and j–l). Transcription was induced by the addition of
tamoxifen and cells were fixed and stained 3 hr later. Scale bar represents 5 mm. Scale bar in enlarged inset represents 1 mm. (B) Quantification of
Cherry-tTA-ER (top panel, solid red circles) and YFP-RNA pol II (lower panel, solid green circles) accumulations at the transcription site during
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binding sites more accessible to the activator. Interestingly, YFP-
Brd4 did not have a similar effect on activator accumulation
(Table S2) although its expression stimulates transcription of some
cellular genes .
Activator Strength Regulates the Degree of Activation
As RNA levels increase simultaneously with Cherry-tTA-ER
(Fig. 6B), and Brd2 accumulation is delayed compared to the
activator (Fig 8B), this suggests that the VP16 activator can rapidly
overcome the heterochromatic repression at the site and initiate
transcription before the chromatin is hyper-acetylated. However,
as there are 96 activator-binding sites upstream of the CMV
promoter (Fig. 1A), it is also possible that the rapid and strong
activation is due to the number of VP16 molecules recruited.
To distinguish between these two possibilities, we compared
cells expressing Cherry-tTA-ER (VP16 activator) to cells express-
ing Cherry-TetR-p53-ER, a construct in which the VP16 acidic
activation domain was replaced by the first 70 amino acids of p53,
the p53 transactivation domain (p53TAD)(Fig. 9A) . Western
blotting of flag-tagged versions of these constructs showed that
they are comparably expressed (Fig. 9B). Similar to the VP16
activator, the p53 activator induced the accumulation of YFP-
MS2 at the transcription site (Fig. 9C, panels a–c) as well as the
recruitment of GCN5 and Brd2 (Fig. 9C, panels d–i).
In cells activated for 3 hours, Cherry-tTA-ER and YFP-MS2
accumulated at the transcription site in 87.663.4% and
84.764.2% of cells, respectively (Fig. 9D). In contrast, Cherry-
TetR-p53-ER and the RNA were detected at the site in only
32.963.0 and 36.266.3% of cells, respectively (Fig. 9D). Similar
results were seen when GCN5 and Brd2 recruitment were
evaluated (Fig. 9E). These results, therefore, suggest that activator
strength, not binding site number, regulates the percentage of cells
in which a gene is activated.
Interestingly, the number of cells activated by VP16 was
approximately the same at both the 30 min and 3 hour time
points suggesting that the decision to activate is made early and
that the number does not increase over time (Fig. 9D). The lower
rate of p53 activator induced accumulation is likely due to its lower
ability to access the TRE binding sites in the condensed inactive
transcription site. Although the p53 activator cannot be detected
at the site 15 min after activation, accumulations of YFP-MS2 are
detected in 12.762.2% of cells indicating that it is a more sensitive
detector than the activator.
Additionally, the VP16 activator induced a significantly higher
degree of chromatin decondensation than the p53-activator, as
determined by measuring the pixel area of the transcription site
(Fig. 9C, panels a, d and g, and 9F). The area occupied by the p53-
activator bound transcription site was ,1.4 times larger than the
inactive site bound by Cherry-lac repressor, but significantly
smaller than the VP16-activated site (Fig. 9F), which is ,6 times
the size of the inactive site. CFP-SKL levels in p53-activated cells
were also lower than in VP16-activated cells demonstrating that
activator strength also affects the quantity of protein synthesized
(Fig. 9G). Taken together, these results indicate that the extent of
activation and the ability to overcome a condensed chromatin
configuration is due largely to activator strength and not to the
number of binding sites upstream of the promoter.
Transcription is essential for chromatin decondensation
To evaluate the role of transcription in chromatin decondensa-
tion and regulatory factor recruitment, we pre-treated cells
expressing the VP16-activator and YFP-tagged regulatory factors
with a-amanitin, added tamoxifen to the media, and evaluated
factor recruitment to the transcription site 3 hr later. The lack of
YFP-MS2 enrichment confirmed that transcription was inhibited
(Fig. 10A, panels g–i, and 10B; Table S1). Interestingly, Cherry-
tTA-ER, YFP-GCN5 and YFP-Brd2 were still recruited (Fig. 10A,
panels a–f, and 10B). The recruitment of Brd2, which requires it
BDs for association (Fig 8A, panels g–i), indicates that the
chromatin is acetylated (Fig. 10A, panels d–f). Interestingly, the
transcription site in the a-amanitin pre-treated cells did not
decondense to the same extent as in untreated cells (,1.6 times the
area of the inactive site) (Fig 9F). This indicates that, although
VP16 can still bind to the site and recruit transcriptional
regulatory factors, transcription is required to induce large-scale
In this study, we evaluated the recruitment of transcriptional
activation factors to a transcription site composed of a multi-copy
array of an inducible reporter construct, which allows DNA, RNA
and protein to be simultaneously visualized in single living cells.
Transcription is initiated when a tetracycline-regulated activator
binds to TRE repeats upstream of the CMV minimal promoter in
the transgene. In order to track factor recruitment in relation to
the activator, we fused tTA (pTet-Off) to both Cherry and the ER
hormone binding domain  to make the tamoxifen-inducible
VP16 activator, Cherry-tTA-ER. The addition of Cherry allows
the construct to be used to visualize the active transcription site.
The ER domain retains the activator in the cytoplasm until
tamoxifen triggers its entry into the nucleus . This cytoplasmic
retention serves to both reduce transcriptional leakiness and
provides a distinct start point from which to evaluate activation.
Cherry-tTA-ER accumulates at the site ,7 min after activa-
tion. This reflects the time required for it to enter the nucleus,
diffuse through the nucleoplasm, and gain access to its binding
sites. It is also consistent with previous reports that the
glucocorticoid receptor, which also resides in the cytoplasm prior
to activation, is detected at the MMTV array within 10 minutes of
hormone addition . Heat shock factor (HSF), which is in the
nucleus before activation, is highly enriched at the hsp 70 loci
5 minutes after heat shock .
Previously, we showed that this transcription site is heterochro-
matic in the inactive state . Consistent with those results, we
show here by IF and ChIP that histone H3 K9- and H4 K5- and
K12- acetylation are not enriched at the inactive site but that they
become specifically enriched at the promoter after activation.
Therefore, although this transcription site is composed of a
repetitive array of a transgene, the sequence elements within it are
regulated as they are in single-copy genes.
To investigate the timing of the events regulating transcriptional
activation, we tracked the recruitment of GCN5, RNA pol II, Brd4
and Brd2 in relation to the VP16 activator, Cherry-tTA-ER. We
wantedto determine whether these factors are recruitedin a specific
activation. Tamoxifen, was added to the media immediately after the first time point (,0 min). Images were collected every 1.5 min for ,40 min.
Due to the low intensity of the YFP-RNA pol II signal, a gamma correction of 2 was applied to each image in both channels to distinguish factor
accumulation from background at the early time points. Measured intensities were normalized to the high and low plateau values and fitted to a
logistic fit (solid black line). The initial accumulation time (blue Xs and arrows) is the point when the fitted curve reaches 5% of the total accumulation.
The set of graphs are the average of 4 cells imaged from 4 different coverslips on 3 different days. Error bars represent the standard deviation.
PLoS ONE | www.plosone.org9April 2010 | Volume 5 | Issue 4 | e10272
order and how their recruitment correlates with the initiation of
RNA synthesis. GCN5, RNA pol II and Brd4 accumulated
coincidently with the activator. Interestingly, Brd2 lagged behind
Cherry-tTA-ERby,2 min.SinceBrd2 withmutated BDsdoesnot
associate with the site, the timing of its recruitment provides a
measure of the time required for the site to become hyper-
acetylated. RNA levels also increase simultaneously with the
activator suggesting that when activated by the VP16 activator,
transcription can proceed before the site is hyper-acetylated.
In general, this assay utilizes transient expression of auto-
fluorescently tagged regulatory factors to analyze transcriptional
dynamics, although in this study, YFP-RNA pol II and YFP-MS2
were stably expressed. Factor over expression can sometimes
introduce effects into a system that can skew results. However, in
this study, only YFP-Brd2 detectably increased the rate of
activator accumulation at the transcription site (Table S2). This
suggests that increased Brd2 expression changes global chromatin
structure in such a way that the activator can more rapidly access
the TRE binding sites. Yet despite the faster rate of activator
binding, Brd2 recruitment is delayed (Table S2) indicating that it
requires additional activator-induced regulatory steps to accumu-
late. Since the Brd2 double BD mutant is not recruited, this
suggests that lysine acetylation is the required event. Interestingly,
transient expression of GCN5 and Brd4 did not significantly
increase the rate of Cherry-tTA-ER accumulation compared to
when it was expressed alone demonstrating that factor over
expression does not always affect this system (Table S2). However,
when it does, it can reveal interesting new information about
regulatory factor dynamics.
Importantly, this analysis also allowed us to identify a difference
between Brd2 and Brd4, which likely reflects their differential
functions. Although highly homologous in their BDs, they are less
conserved in their other domains and have been found in different
biochemical complexes . For example, Brd4, not Brd2, is a
component of select Mediator complexes  and is in a complex
with P-TEFb, which it recruits to transcription sites where it
regulates the transition from initiation to elongation by phosphor-
ylating the RNA pol II C-terminal domain (CTD) at serine 2
[44,45]. The rapid recruitment of Brd4 to the transcription site in
this system is consistent with its role in regulating early events in
transcriptional activation. Interestingly, using fluorescent recovery
after photobleaching (FRAP) assays, Brd4 was found to have
increased chromatin binding during telophase when nuclear
factors are beginning to re-associate with the chromatin indicating
that these factors also function differently when transcription
reinitiates after cell division .
Overall, our results indicate that the VP16 activator, Cherry-
tTA-ER, induces rapid transcriptional activation and that most of
the examined factors accumulate simultaneously with it. As most
nuclear factors move rapidly throughout the nucleus by diffusion,
this suggests a mechanism for how co-accumulation occurs .
In contrast, the rapid initiation of RNA synthesis at the site,
coincident with VP16 activator accumulation, is surprising given
that transcriptional initiation on naked DNA [51,52] and the
establishment of productive transcriptional complexes in living
cells are very inefficient . However, experiments performed by
Darzacq and colleagues in the very cell line used in this study
determined the maximum rate of RNA pol II elongation without
pausing to be 4.3 kb min21. At this rate, the entire 3.3 kb
transcription unit could be transcribed in 46 seconds, Therefore,
at least two RNA molecules could be produced from one unit
within the 1.5 min imaging interval used in this study. If more
than one transgene is initially transcribed by multiple polymerases,
a significant pool of RNA would rapidly accumulate at the site.
Figure 6. Real-time analysis of mRNA synthesis. (A) Still images
from a time series of 2-6-3 cells expressing Cherry-tTA-ER and YFP-MS2.
Selected images from Movie S4 show their accumulation at the
transcription site during activation. Scale bar represents 5 mm. Scale bar
in enlarged inset represents 1 mm. (B) Quantification of Cherry-tTA-ER
(top panel, solid red circles) and YFP-MS2 (lower panel, solid green
circles) accumulations at the transcription site during activation.
Tamoxifen was added to the media immediately after the first time
point (,0 min). Images were collected every 1.5 min for ,40 min.
Measured intensities were normalized to the high and low plateau
values and fitted to a logistic fit (solid black line). The initial
accumulation (blue Xs and arrows) is when the fitted curve reaches
5% of the total accumulation. The set of graphs represents the average
of 8 cells imaged from 6 different coverslips on 4 different days. Error
bars represent the standard deviation.
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Additionally, each RNA has 24 MS2 repeats, which are sufficient
for the detection of a single RNA, making it within our detection
limits to visualize even a few RNA molecules at these early time
It is also possible that transcription begins so quickly because
VP16 is a very strong activator. Unlike many cellular activators,
which initiate transcription through a cascade of regulatory factor
recruitment events regulated by the progressive addition of histone
Figure 7. Analysis of Brd4 recruitment to the transcription site. (A) YFP-Brd4 is not enriched at the inactive transcription site marked by
Cherry-lac repressor (panels a–c). YFP-Brd4 and endogenous Brd4, immunostained with antibody Brd4CA, are strongly recruited to the active site
marked by Cherry-tTA-ER (panels d–f and g–i, respectively). Transcription was induced for 3 hr by the addition of tamoxifen. Scale bar represents
5 mm. Scale bar in enlarged inset represents 1 mm. (B) Quantification of Cherry-tTA-ER (top panel, solid red circles) and YFP-Brd4 (lower panel, solid
green circles) accumulations at the transcription site during activation. Tamoxifen was added to the media immediately after the first time point
(,0 min). Images were collected every 1.5 min for ,40 min. Measured intensities were normalized to the high and low plateau values and fitted to a
logistic fit (solid black line). The initial accumulation (blue Xs and arrows) is when the fitted curve reaches 5% of the total accumulation. The set of
graphs represents the average of 6 cells imaged from 4 different coverslips on 4 different days. Error bars represent the standard deviation.
PLoS ONE | www.plosone.org11 April 2010 | Volume 5 | Issue 4 | e10272
PTMs , VP16, itself, may serve as a platform for their direct
recruitment. For example, VP16 interacts with the SAGA
complex, of which GCN5 is a component [56,57] and it directly
recruits P-TEFb [58,59]. As such, it may be able to bypass
expected intermediate regulatory steps, such as chromatin hyper-
acetylation, en route to the initiation of RNA synthesis.
Figure 8. Analysis of Brd2 recruitment to the transcription site. (A) YFP-Brd2 is not enriched at the inactive transcription site marked by
Cherry-lac repressor (panels a–c). YFP-Brd2 is strongly recruited to the active site marked by Cherry-tTA-ER (panels d–f). YFP-Brd2 BD(1+2) YRF in
which tyrosine (Y)113 and Y136 are mutated to phenylalanines (F) is not recruited to the active transcription site (panels g–i). Transcription was
induced for 3 hr by the addition of tamoxifen. Scale bar represents 5 mm. Scale bar in enlarged inset represents 1 mm. (B) Quantification of Cherry-
tTA-ER (top panel, solid red circles) and YFP-Brd2 (lower panel, solid green circles) accumulations at the transcription site during activation. Tamoxifen
was added to the media immediately after the first time point (,0 min). Images were collected every 1.5 min for ,40 min. Measured intensities were
normalized to the high and low plateau values and fitted to a logistic fit (solid black line). The initial accumulation time (blue Xs and arrows) is the
point when the fitted curve reaches 5% of the total accumulation. The set of graphs represents the average of 5 cells imaged from 3 different
coverslips on 3 different days. Error bars represent the standard deviation.
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PLoS ONE | www.plosone.org13 April 2010 | Volume 5 | Issue 4 | e10272
Transcriptional activation rates are also strongly influenced by
the number of activator binding sites in a reporter. Increasing the
number of NF-kB enhancers bypasses the need for histone
acetylation and induces the direct recruitment of transcriptional
regulatory complexes . As the transcription unit evaluated in
this study has 96 activator binding sites, we compared activation
dynamics in cells expressing the VP16-activator to a weaker p53-
activator. In p53-activated cells, the RNA, the p53 activator,
GCN5 and Brd2 accumulate at only ,35% of the transcription
site compared to ,90% in VP16-activated cells 3 hrs after
induction. This indicates that activator strength rather than
binding site number has a much stronger effect on the activation
process. Although we cannot definitively say whether the
activation is slower in p53-expressing cells because the low signal
intensity of this construct at the site and the low percentage of cells
activated by it prohibit time-lapse imaging, this analysis provides
direct insight into the effects of activator strength on RNA
synthesis and chromatin organization at the single cell level.
As most investigations of transcriptional activation have relied
on techniques such as chromatin immunoprecipitation (ChIP),
nuclear run-on and RNase-protection, in which measurements are
taken at widely spaced time points and effects are averaged in cell
populations, it has been difficult to determine how transcription is
regulated in single cells. Our results show that the number of
VP16-activated cells is approximately the same at both the
30 minutes and 3 hours time points indicating that the decision to
activate is made early. Although the number of activated cells
increases between these time points in p53-activated cells, this is
most likely due to detection limits at the early time points. This is
also suggested by the fact that the RNA, but not the activator, can
be detected 15 min after p53 mediated activation demonstrating
that it is the more sensitive detector. This sensitivity difference
could also explain why the RNA is detected so rapidly after VP16-
activator recruitment in the time-course imaging (Fig. 6).
Our evaluation of transcriptional activation factor recruitment
and the condensation state of the transcription site in cells pre-
treatment with a-amanitin provides further evidence for the
essential role of transcription in driving large-scale chromatin
decondensation [1,40,41]. Although the VP16 activator, GCN5
and Brd2 are still recruited, the chromatin does not significantly
decondense. The fact that Brd2, which requires its BDs for
association with the site, is recruited confirms that the site is
acetylated and demonstrates that the presence of this PTM is not
sufficient for the chromatin to decondense.
Much of what we know about how genes are activated comes
from genomic, biochemical and genetic analyses. However,
chromatin is a highly dynamic regulator of gene expression. As
changes in chromatin architecture occur within minutes of
activation, it is difficult to truly understand the nature of these
events when measurements are taken at widely spaced time points
and by averaging effects in cell populations. Live cell imaging now
makes it possible to monitor transcriptional activation in single
cells. In this study, we present one of the first delineations of the
timing of transcriptional activation events in single mammalian
cells at the earliest stages of activation. Future analyses will allow
us to place other regulatory events into this temporal context
and to further explore how activation events are coordinately
Materials and Methods
Monomeric YFP (mYFP) and monomeric Cherry (mCherry)
 (gift of R. Tsien) C1, C2, and C3 expression vectors, were
made by replacing EGFP in the respective pEGFP vectors
(Clontech) with mYFP or mCherry (NheI/BsrGI). To make
tTA-ER, the tetracycline transcriptional activator (tTA from pTet-
Off; Clontech) was cloned in frame with the estrogen receptor
hormone binding domain (ER), which is responsive to both b-
estrodiol and tamoxifen and contains the Gly400Val mutation
, in the pBabePuro:hbER vector (BamHI/EcoRI)(gift of A.
Capobianco). mCherry-tTA-ER was made by inserting tTA-ER
into mCherry-C3 (KpnI/ApaI). Lentiviral expression plasmid,
pLU-mYFP-tTA-ER, was constructed by inserting mYFP-tTA-ER
(NheI/BamHI) into a modified pLU vector (gift of A. Ivanov)
(XbaI/BamHI). To make mCherry-TetR-p53-ER, TetR was
PCRed from pTet-Off and cloned into pCMV-mCherry-C3
(XhoI/HindIII). The ER hormone domain was PCRed and
cloned into pCMV-mCherry-TetR-C3 (SacII/BamHI) to make
pCMV-mCherry-TetR-ER-C3. The p53 TAD (aa 1–70) was
PCRed and cloned into pCMV-mCherry-TetR-ER-C3 (EcoRI/
KpnI). Flag-mCherry-tTA-ER and Flag-mCherry-TetR-p53-ER
were made by PCRing mCherry-tTA-ER and mCherry-TetR-
p53-ER and cloning them into p3xFLAG-CMV-10 (NotI/
BamHI). pSV2-mCherry-lac repressor was constructed by replac-
ing EYFP in pSV2-EYFP-lac repressor  with mCherry. Mouse
GCN5 was cloned into pEYFP-C1 (KpnI/ApaI). Mouse PCAF
was cloned into EYFP-C2 (HindIII/Apa1). Human p300 (gift of
R. Marmorstein) was cloned into mYFP-C3 (XhoI/Hind III).
mYFP-Brd2-C1, mYFP-Brd2 BD(1+2)-Y/F-C1, and mYFP-Brd4-
C1 (all are mouse) were made by replacing EGFP with mYFP
(AgeI/BsrGI) in constructs provided by K. Ozato [17,18].
The U2OS derived cell line, 2-6-3 , was cultured in high
glucose Glutamax media (Invitrogen) with 10% tetracycline free
Figure 9. Single cell analysis of transcriptional activation induced by VP16 and p53 activators. (A) Schematic representation of the VP16
and p53 activators. (B) Western blot analysis of Flag-tagged versions of the p53 and VP16 activators showing that they are expressed at comparable
levels in 2-6-3 cells. (C) YFP-MS2 (panels a–c), YFP-GCN5 (panels d–f), and YFP-Brd2 (panels g–i) are enriched at the transcription site, marked by
Cherry-tTetR-p53-ER. Scale bar represents 5 mm. Scale bar in enlarged inset represents 1 mm. (D) Analysis of the percentage of cells with the activators
and YFP-MS2 enriched at the transcription site 15 min, 30 min, and 3 hr after activation. 100 cells were analyzed from 3 independent experiments;
SEMs (in the form of error bars) are presented in the graphs. (E) Analysis of the percentage of cells with accumulations of YFP-GCN5 and YFP-Brd2 at
the transcription site 3 hr after activation. 100 cells were analyzed from 3 independent experiments; SEMs (in the form of error bars) are presented in
the graphs. (F) Measurement of the pixel area of the transcription site in the inactive state (marked by Cherry-lac repressor) and after 3 hrs activation
by the VP16 activator (Cherry-tTA-ER with and without a-amanitin pre-treatment) and the p53 activator (Cherry-TetR-p53-ER). Inactive and VP16-
activated transcription site data is the same as in Fig. 1D. Area values are averages of 10 cells. SEM (in the form of error bars) and p values are
presented in the graph. (G) Frequency histogram showing the distribution of the blue pixel intensity levels (blue bars) as a measure of the CFP-SKL
protein in cells activated for 3 hrs by the VP16 and p53 activators. Black bars represent the background signal. The x-axis is the average fluorescence
pixel intensity in each bin on a scale from 0 to 1 and divided into bin sizes of 0.02; the y-axis is the number of pixels in each bin, on a logarithmic
scale. The bar beneath the histogram shows the intensity range. Measurements are from 5 independent experiments. Data for the inactive and VP16-
activated cells is from the graph in Fig. 1E.
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Figure 10. Analysis of the effects of a-amanitin on regulatory factor recruitment. Cells transfected with the indicated factors were pre-
treated with a-amanitin for 2 hrs then incubated with tamoxifen for 3 hrs. (A) YFP-GCN5 (panels a–c) and YFP-Brd2 (panels d–f) were enriched at the
transcription site marked by Cherry-tTA-ER. YFP-MS2 was not enriched at the transcription site after a-amanitin treatment (panels g–i) but did
accumulate at the transcription site in untreated cells (panels j–l). Scale bar represents 5 mm. Scale bar in enlarged inset represents 1 mm. (B) Graph
shows the percentage of cells with accumulations of each factor at the transcription site after a-amanitin pre-treatment followed by a 3 hr incubation
with tamoxifen. 100 cells were analyzed from 3 independent experiments. SEM (in the form of error bars) are presented in the graphs.
PLoS ONE | www.plosone.org15 April 2010 | Volume 5 | Issue 4 | e10272
FBS (BD Biosciences), 1% pen/strep and 100 mg/ml hygromycin
B. YFP-RNA pol II and YFP-MS2  were stably expressed in 2-
6-3 cells by selecting in 40 mg/ml G418. Transfections were done
using FuGENE 6 (Roche) according to the manufacturer’s
protocol or electroporation as previously described . Cells
transfected with Cherry-tTA-ER were activated with 1 mM
tamoxifen (Sigma). For a-amanitin analysis, the 2-6-3 and 2-6-3
YFP-MS2 stable cell lines  were transfected overnight with the
activator and/or other factors, pre-treated with 100 mg/ml a-
amanitin for 2 hrs, and fixed after a 3 hr incubation with
For IF, 2-6-3 cells were transfected overnight with SV2-Cherry-
lac repressor or Cherry-tTA-ER. Cells were first pre-extracted
with 0.5% Triton X-100 in CSK buffer (10 mM PIPES, pH 7.0,
100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 with freshly
added protease inhibitors) for 5–10 min and then fixed in 3%
formaldehyde in 16 PBS for 15 min, both steps at room
temperature (RT). Cells were blocked with 3% BSA in 16 PBS
for 1 hour at RT and incubated with 1u Abs diluted in blocking
buffer. The following Abs were used: RNA pol II 4H8 (Covance,
1:500), FACTp140 (BD, 1:100), histone H4 acetyl-K12 (Abcam,
1:100), histone H4 acetyl-K5 (Upstate, 1:2000), histone H3 acetyl-
K9 (Abcam, 1:200) and Brd4CA  (gift of J. You; 1:80,000). 2u
antibodies were Alexa Fluor AF488 conjugated (Invitrogen) and
diluted 1:3000 in 16PBS.
For time-lapse imaging, 2-6-3 cells were plated on 40 mm
coverslips (Bioptechs Inc., Butler, PA) and transfected overnight
with Cherry-tTA-ER alone or co-transfected with YFP-tagged
factors; Cherry-tTA-ER was transfected overnight into 2-6-3 cells
stably expressing YFP-RNA pol II and YFP-MS2. Coverslips
were placed in an FCS2 live-cell chamber (Bioptechs Inc., Butler,
PA) with Leibovitz’s L-15 medium (Invitrogen) and activation
was induced by prefusing ,3 ml of medium containing 1 mM
tamoxifen into the chamber. The chamber and objective lens
were maintained at 37uC using heating units (Bioptechs Inc.,
Butler, PA). The microscope was enclosed in the Incubator BL
connected to heating and temperature control units (PeCon
GmbH, Erbach, Germany) maintained at 37uC. Images were
acquired using a Leica DMI 6000 B inverted automated
microscope with HCX PL APO 100x/1.4020.70 oil objective
lens using a 457/488/514nm 30mW Argon-Ion laser for YFP, a
561nm/25mW diode laser for mCherry and a 442nm/70mW
diode laser for CFP imaging. To avoid cellular toxicity, the laser
powers were decreased to 25% for YFP imaging and 20% for
mCherry imaging. In time-course experiments, images were
captured using a Hamamatsu electron multiplier CCD digital
camera with a 5126512 chip. A Marzhauser point-visiting stage
and a Z-drive controlled by COMPIX SimplePCI software were
used to collect images at multiple X-Y points. A Yokogawa CSU-
10 real-time spinning disk confocal attachment with Nipkow and
microlens disks was used to collect stacks of 6 images (0.5 mm
increments) every 1 or 1.5 min. Exposure time and gain settings
were maintained at specific values for each factor studied (for
mCherry tagged factors, 0.3 sec exposure time/gain 200; for
YFP-GCN5 0.3 sec exposure time/gain 150; for all other YFP-
tagged factors 0.3 sec exposure time/gain 200). Cells with
intensity levels registering in the middle of the dynamic range
of the camera were selected for imaging in order to ensure that
the cells to be analyzed expressed equivalent levels of each factor.
For every factor, time course imaging was done on multiple days
and on multiple coverslips with 3 cells typically tracked on
each coverslip. Cells which went our of focus during imag-
ing, developed a saturated signal at the transcription site,
bleached, or showed any kind of aberrant physiological features
were not included in the computational analysis. On average, 25–
30 cells were imagined for a given factor and ,10 could be
included in the final analysis. Graphs were obtained according to
the procedure described below only for cells showing a clear
signal to noise ratio of factor accumulation at the transcription
For fixed images, cells were plated on 1.5 coverslips, transfected
overnight, activated as indicated, and fixed for 15 min in 3% PFA
in 16 PBS. Coverslips were mounted in antifade fluorescence
mounting medium (1 mg/ml p-phenylenediamine, 90% glycerol
in PBS, pH 8.0–9.0 adjusted with Sodium Carbonate/Bicarbon-
ate Buffer, pH 9.2) . Image stacks were taken (0.4 mm
increments) using Hamamatsu ORCA-AG camera (161 binning;
134461024 pixels). Maximum projections were obtained using
COMPIX SimplePCI software. Image contrast adjustments were
performed using COMPIX SimplePCI and Adobe Photoshop
Image analysis was done using custom software written in
Matlab (The Mathworks). In each time-lapse series, the transcrip-
tion site was manually selected in the frame when the
accumulation of the activator could first be clearly detected
(,10 min after tamoxifen addition). The intensity (arbitrary units)
at a point inside the transcription site was compared to the
intensity of an outside but nearby point and a threshold, calculated
from the intensity difference, was used to define the transcription
site boundary. At each time point, activator and factor levels were
determined by summing the intensities of all pixels within this
boundary and subtracting the background intensity levels. The
values in images acquired at earlier time points (before ,10 min)
was determined by applying this first defined boundary to the site.
The site was tracked and its location, size, and intensity values
were recalculated in all later images. For analysis of YFP-RNA pol
II expressing cells, a gamma correction of 2 was applied to each
image in both the red and green series. Error bars on the graphed
data sets represent one standard deviation across at least three sets
of normalized data. The intensity data were fit by performing a
nonlinear least-squares regression using the Levenberg-Marquart
algorithm in MATLAB with the ‘‘lsqcurvefit’’ function to a logistic
Intensity=(A1–A2)/(1+(x/x0)‘p) + A2,
x0=the center (intensity value halfway between A1 and
The timing of the initial accumulations of factors and the
activator at the transcription site were determined from the
logistic fit. The accumulation start point was defined as the time at
which the intensity increased 5% of the difference between the
initial and final values. The error estimates of the initial accu-
mulation represent a 90% confidence interval on the estimates
of the four model parameters using the function nlparci in
To determine the area of the locus, a point inside and a point
outside but nearby the transcription site was manually selected.
A threshold calculated from the intensity (in arbitrary units)
PLoS ONE | www.plosone.org16April 2010 | Volume 5 | Issue 4 | e10272
difference between the two points was used to define the boundary
of the transcription site. The ‘‘imreconstruct’’ function in Matlab
was used to obtain the region of the transcription site with pixels
above the threshold value. The number of pixels identified in the
region was then summed for the total locus area of the
Quantitative colocalization analysis of fixed images was
performed using SimplePCI software. The transcription site was
selected manually and Pearson’s correlation coefficient (Rr) values
with background correction for region of interest (ROI) were
calculated. Values from 0.5–1.0 indicate strong colocalization;
values from 21.020.5 indicate a lack of co-localization. 2D
intensity profiles across the transcription site were also obtained for
For ChIP experiments, cells were infected with the lentiviral
expression plasmid, pLU-YFP-tTA-ER for 24 hrs then activated
(1 mM tamoxifen) for 3 hrs. ChIP assays were performed using the
standard Upstate protocol. The following ChIP grade rabbit
polyclonal Abs were used: H4 acetyl-K12 (Abcam), H3 acetyl-K9
(Abcam), histone H4acetyl-K5 (Upstate) and normal rabbit IgG
(Abcam). DNA samples from input (In) and antibody-bound (IP)
were purified using QIAquick PCR purification kit (Qiagen) and
analyzed by real-time PCR using the TaqMan Fast Universal
PCR Master Mix (Applied Biosystems) and Applied Biosystems
7500 Fast Real-Time PCR System. Primers and TaqMan probes
were designed using Applied Biosystems Primer Express 3.0
software (sequences available upon request). Individual PCR
reactions were carried out in triplicates and experiments were
repeated three times. Data quantification was performed by
applying the modified comparative Ctmethod . Values of %
Input were calculated using the following formula IP/In=
inactive and active transcription site and expression levels of
expressed constructs. (A) YFP-GCN5 (panels a–c), YFP-PCAF
(panels d–f), and YFP-p300 (panels g–i), are not enriched at the
inactive transcription site marked by Cherry-lac repressor. Scale
bar represents 5 mm. Scale bar in the enlarged inset represents
1 mm. (B) Western blot showing the levels of the transiently
expressed factors used for time lapse imaging: YFP-Brd4, YFP-
Brd2 and YFP-GCN5. The lower level of Brd4 compared to Brd2
a result of the less efficient transfer of this higher molecular weight
Found at: doi:10.1371/journal.pone.0010272.s001 (0.70 MB
Association of the histone acetyl-transferases with the
and active transcription site. Cells stably expressing YFP-RNA
pol II were transfected with Cherry-lac repressor, to mark the
inactive transcription site (panels a–c). Cherry-tTA-ER marks
the transcription site 3 hrs after activation induced by tamoxifen
(panels e–g). Intensity profile shows that YFP-RNA pol II (green
line) surrounds but does not co-localize with the inactive site (red
line) (panel d). YFP-RNA pol II significantly co-localizes with
Cherry-tTA-ER (panel h). Yellow lines in enlarged insets in c
and g show the path starting at the asterisk through which the
red and green intensities were measured (panels d and h). Scale
bar represents 5 mm. Scale bar in the enlarged inset represents
Found at: doi:10.1371/journal.pone.0010272.s002 (2.89 MB TIF)
Association of YFP-RNA pol II with the inactive
Found at: doi:10.1371/journal.pone.0010272.s003 (0.05 MB
Analysis of factor co-localization with the transcription
series images of activator and regulatory factor accumulation at
the transcription site during activation. The gray shaded column is
the 5% accumulation threshold, which is marked by arrows in the
graphs in the figures.
Found at: doi:10.1371/journal.pone.0010272.s004 (0.03 MB
Summary of the recruitment time analyses from time
cells and transcription was induced by the addition of tamoxifen.
Frames were collected every minute for ,40 min. Movie display
rate is 8 frames per second. Still images from this movie are shown
in Figure 2A.
Found at: doi:10.1371/journal.pone.0010272.s005 (3.41 MB AVI)
Cherry-tTA-ER was transiently transfected into 2-6-3
transfected into 2-6-3 cells and transcription was induced by the
addition of tamoxifen. Frames were collected every 1.5 min for
,40 min. Movie display rate is 8 frames per second.
Found at: doi:10.1371/journal.pone.0010272.s006 (0.91 MB AVI)
Cherry-tTA-ER and YFP-GNC5 were transiently
2-6-3 cell line stably expressing YFP-RNA pol II. Transcription
was induced by the addition of tamoxifen. Frames were collected
every 1.5 min for ,40 min. Movie display rate is 8 frames per
Found at: doi:10.1371/journal.pone.0010272.s007 (1.20 MB AVI)
Cherry-tTA-ER was transiently transfected into a
3 cell line stably expressing YFP-MS2. Transcription was induced
by the addition of tamoxifen. Frames were collected every 1.5 min
for ,40 min. Movie display rate is 8 frames per second. Still
images from this movie are shown in Figure 6.
Found at: doi:10.1371/journal.pone.0010272.s008 (1.04 MB AVI)
Cherry-tTA-ER was transiently transfected into a 2-6-
transfected into 2-6-3 cells and transcription was induced by the
addition of tamoxifen. Frames were collected every 1.5 min for
,40 min. Movie display rate is 8 frames per second.
Found at: doi:10.1371/journal.pone.0010272.s009 (13.33 MB
Cherry-tTA-ER and YFP-Brd4 were transiently
transfected into 2-6-3 cells and transcription was induced by the
addition of tamoxifen. Frames were collected every 1.5 min for
,40 min. Movie display rate is 8 frames per second.
Found at: doi:10.1371/journal.pone.0010272.s010 (0.75 MB AVI)
Cherry-tTA-ER and YFP-Brd2 were transiently
We would like to thank Anthony Capobianco for his suggestion to use the
estrogen receptor hormone-binding domain to regulate the transcriptional
activator and for providing the construct. We thank Jianxin You for
generously providing the Brd4CA antibody. We thank Roger Greenberg,
Niraj Shanbhag, Nadia Dahmane and Gerd Blobel for their critical
comments on the manuscript. We thank Zhong Deng for technical advice
on the ChIP experiments.
Conceived and designed the experiments: IURM GL SJ. Performed the
experiments: IURM LMJ. Analyzed the data: IURM SLP SJ. Contributed
reagents/materials/analysis tools: IURM SLP LMJ GL SJ. Wrote the
paper: IURM SLP SJ.
PLoS ONE | www.plosone.org 17 April 2010 | Volume 5 | Issue 4 | e10272
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PLoS ONE | www.plosone.org18 April 2010 | Volume 5 | Issue 4 | e10272