tifications (5, 9, 10, 23). To verify that the transcription rate of the tagged mRNA reflected that
of the untagged allele, we measured the amount
of Ctgf mRNA in the GT:ctgf cell line (21) and
obtained a relative transcription rate of 88 T 3%
as compared to the tagged Ctgf allele. Hence, the
insertion of a luciferase cassette did not markedly
affect the Ctgf transcription rate.
Our model could also reconstruct the most
probable temporal sequence of protein accumulation, mRNA accumulation, and gene activity
states (Fig. 2A and fig. S9A). From this, we
computed the distributions of time intervals
during which the gene remained “on” and “off,”
respectively. The “on” intervals followed exponential distributions, suggesting a first-order off-switching of gene transcription (Fig. 2B and fig.
S9C). In contrast, the “off” intervals showed a
local maximum that was best described by
assuming two sequential exponential processes,
indicating a refractory period in the “off” state
before the gene can be switched on again (Fig.
2C and figs. S9D and S10A). The burst frequency and size of the circadian genes Bmal1a/b,
Dbp, and Per2::luc oscillate during a circadian
cycle (fig. S11), and we observed a clear phase
advance of the former as compared to the latter in
the Bmal1a/b and Dbp cell lines (fig. S11).
Therefore, burst size and frequency can be uncoupled during up- and down-regulation of a
We next investigated the role of promoter
architecture, previously reported to affect on- and
off-switching of gene activity in yeast (24). To
keep the genomic environment invariable, we
used NIH-3T3 Flp-In cells to engineer cell lines
carrying a single copy of a different artificial con-
struct at the same genomic locus (21). To drive
NLS-luc expression, the transgenes contain one or
two CCAAT boxes (25) of different affinities for
the ubiquitously expressed NF-Y transcriptional
activator upstream of a TATA box, (21) (table S1).
We generated and analyzed a total of eight different
cell lines in which the overall strength of the
artificial promoters correlated with both the
number of CCAAT boxes and their affinity for
NF-Y (fig. S12). Indeed, using two CCAAT boxes
instead of one, or choosing CCAAT boxes of
higher affinity for NF-Y, increased the mean
burst sizes (Fig. 3C, x axis). This reflected mainly
an increase in the number of transcripts produced
during the “on” times, because the duration of
“on” times was largely unaffected (Fig. 3A). In
addition, the decrease in the duration of “off”
intervals (Fig. 3B and fig. S10A) resulted in a
higher percentage of activity time in the presence
of two CCAAT boxes (fig. S10B).
Fig. 4. Influence of TSA treatment on
transcriptional bursting. (A) Distribution of “on” intervals; black lines show
exponential fits. (B) Distribution of “off”
intervals. Black lines show best fits to
“two-step” model (21). Black vertical
dotted lines show medians of distributions. (C) Mean burst sizes versus
percentage of time during which the
gene is active. Ellipses represent contours
of mean burst sizes T 2SD.
Our findings show that the bursting kinetics
of single-allele mammalian genes measured in individual cells at high temporal resolution are highly gene-specific. Cis-acting regulatory elements
play a dominant role in shaping transcriptional
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with time-lapse imaging, P. Salmon and D. Trono for
providing lentiviral constructs, A. Oates for critical
reading of the manuscript, and N. Roggli for the artwork.
The computations were performed at Vital-IT
( www.vital-it.ch). The U.S.’s laboratory was supported
by the Swiss National Science Foundation (SNF
31-113565, SNF 31-128656/1, and the NCCR program
grant Frontiers in Genetics), the European Research
Council (ERC- 2009-AdG 20090506), the State of
Geneva, and the Louis Jeantet Foundation of Medicine.
F.N.’s laboratory was supported by the Swiss National
Science Foundation (SNF grants 3100A0-113617 and
31-130714) and the Ecole Polytechnique Fédérale de
Supporting Online Material
Materials and Methods
Tables S1 to S3
Figs. S1 to S20
Movies S1 to S3
10 December 2010; accepted 4 March 2011
Published online 17 March 2011;