Perhaps the most well-studied transcription factors of endogenous genes in living cells are nuclear receptor (NR) regulated. These ligand activated transcription factors constitute the nuclear hormone receptor superfamily and are involved in regulating a vast array of eukaryotic genes. NR transcription is initiated by agonist binding to the receptor, forming either a homodimer or heterodimer complex. The corepressors (histone deacetylases, NR specific corepressors) associated with the dimer are then replaced by coactivators such as histone acetylases (SRC/p160 family or CBP/p300) and histone methylases (CARM-1, PRMT-1). In addition, ATP-coupled chromatin remodeling complexes (SWI/SNF) are recruited. Eventually, the basal transcription machinery is assembled, followed by the initiation of Pol II. After initiation, transcription can be influenced by NR factors such as vitamin D receptor interacting protein and thyroid-associated protein (38
). Thus, NR transcription is an excellent model system for observing the cooperative interactions among enhancers, repressors, transcription factors, and basal transcription components (63
). The view that has emerged from live cell studies utilizing fluorescence techniques such as FRAP, FRET, and FCS is that these NR complexes are highly dynamic: Individual species have dwell times on the order of seconds to minutes. However, these same complexes can result in cycles of transcriptional progression that can last hours or days (56
). NR-regulated transcription is therefore dynamically responsive to changes in agonist concentration and also capable of long-term changes of gene expression.
Live cell studies of NR-regulated transcription can be divided into those that study nuclear dynamics in general and those that focus on a particular locus. The first approach provides information about multiple possible transcription sites within the nucleus in addition to nonspecific interactions. The second approach has the benefit of providing specific information about interactions and dynamics at an active transcription site but usually requires modification of the locus—either multimerization of an endogenous gene (54
) or creation of an artificial locus (85
). The first example of this approach, which has been used by a number of investigators since its inception, was a large tandem array of a mouse mammary tumor virus/Harvey viral ras (MMTV/v-Haras) reporter, which contains about 200 copies of the LTR and thus includes 800 to 1200 binding sites for the GR (54
). This same array has been used for FRAP studies of the GR (6
), the androgen receptor (AR) (50
), and the progesterone receptor (PR) (69
). For each of those receptors, an agonist dependent decrease in receptor mobility (increase in t
1/2) was observed [GR, t
1/2 : 1–1.6 s (55
); AR, t
1/2 : 0.2–3.6 s (50
); PR, t
1/2 : 0.6–3.7 s; (69
)]. A similar agonist-dependent decrease in mobility was also observed for general nuclear bleaching of the estrogen receptor [ER, t
1/2 :0.8–5.9 s (85
)]. These observations demonstrate that the recovery time reflects the interaction of the NR with the locus in a specific fashion. In fact, Schaaf & Cidlowski (73
) demonstrated that higher-affinity ligands result in slower recovery times, and Kino et al. (49
) directly showed a positive correlation between FRAP t
1/2 times and transcriptional activity, with higher transcriptional activity corresponding to longer effective recovery times.
In contrast, other receptors do not show an agonist-dependent increase in t
1/2 for general nuclear recovery. The retinoic acid receptor (RAR), the thyroid hormone receptor (TR), the peroxisome proliferator-activated receptor (PPAR), and the retinoid X receptor (RXR) all have the same recovery time with or without ligand [RAR, t
1/2 : 1.9–2.3 s; TR, t
1/2 : 1.8–1.8 s (53
); PPAR, t
1/2 : 0.13–0.15 s; RXR, t
1/2 : 0.2–0.25 s (29
)]. In the case of PPAR, this lack of measurable difference may reflect some constitutive activity of the receptor (29
In all FRAP experiments, the recovery dynamics will reflect both specific and nonspecific interactions. In the case of transient transfections, in which an excess of receptor may be present, nonspecific interactions are likely to be a significant contribution to the dynamics for both locus-specific recovery and general nuclear recovery. The recovery curve is likely a convolution of more than one kinetic process. In computational models of AR dynamics, the recovery was separated into two distinct kinetic components: a fast component (due to diffusion or transient binding) of 1–5 s and a slow component of 60 s (28
This slow component presumably represents a longer-lived interaction in the vicinity of the gene such as with chromatin or nuclear matrix (28
), although the nature of this interaction is not clear and may vary between receptors.
In addition to receptor dynamics, several studies have addressed the kinetic behavior of coactivators involved in NR-regulated transcription. Becker et al. (6
) observed the receptor coactivator GRIP1 (glucocorticoid receptor interacting protein 1) at the active MMTV array and measured a recovery time that was indiscernible from the GR t
1/2 (5 s), suggesting that the binding and release of these proteins may be coupled. CBP and SRC-1 (ER coactivators) have t
1/2 times of 4 s and 8 s, respectively (85
); BRM and BRG1, subunits of the SWI/SNF chromatin remodeling complex, have t
1/2 times of 2 s and 4 s, respectively (45
Taken together, the remarkable aspect of these data is that these recovery times are all less than or equal to 11 s (). Consider, for example, a typical NR transcription complex: NR t
1/2 = 5 s, SRC1 t
1/2 = 8 s, CBP t
1/2 = 4 s (85
), BRM t
1/2 = 2 s, BRG1 t
1/2 = 4 s (45
), and GRIP1 t
1/2 = 5 s (6
). The only molecular species that has a dwell time on the order of minutes is the elongating polymerase (t
1/2 5 min) (6
). How might these transient interactions lead to transcriptional cycles that are observed in the timescale of hours? One idea that has been proposed is that of a transcriptional ratchet, in which permanent changes—methylation, acetylation, phosphorylation—accumulate at a transcription site as a result of the transient interactions described above (56
). There are several suggestive directions about how such long-lived interactions might occur. SRC1 recovery becomes progressively slower at longer times after stimulation of ER with estradiol (t
1/2 = 8.0–30.2 s) (85
); chromatin decondensation seems to depend on polymerase elongation (59
). Live cell experiments that follow the change in dynamics over an induction period are likely to be informative as well.
Imaging a Single Gene
Imaging the transcription of a single gene is potentially a powerful approach because it obviates the averaging inherent in gene array studies. This way, the behavior of individual transcription units can be quantified and their variability assessed. However, this has been difficult to achieve because of technical challenges: specifically detecting the desired locus and then observing the small numbers of factors involved in transcribing a single gene.
When a major challenge must be overcome, the tool of choice in vivo is fluorescence microscopy. Although a single fluorescent protein molecule can be detected when immobilized on a surface, it is difficult to resolve in the context of a living cell, where it undergoes fast diffusion or transport and where the fluorescent background can be high. So far, only a few experiments have managed to provide direct observation of gene expression at the single gene level.
A series of recent experiments demonstrated that it is possible to detect single protein products resulting from the expression of a single gene in live bacteria (15
). From the distribution of proteins synthesized over time, it is then possible to test different models of transcription. In the first experiment (15
), the reporter was a β-galactosidase protein, which produces a fluorescent product upon hydrolysis of a synthetic substrate. Hydrolysis of a large number of substrate molecules by a single enzyme provides the signal amplification necessary to observe a single protein. By observing discrete values in the rate of hydrolysis, the authors could indeed resolve single protein numbers. In subsequent experiments, the reporter was a fluorescent protein fused to a membrane protein (18
). When bound to the membrane, the protein is slowly diffusing and it is therefore possible to accumulate enough fluorescence to resolve a single protein.
These experiments studied reporter genes under the control of the Lac repressor. In this classic system, two operator sequences on the DNA can be bound by a tetramer repressor. Upon full induction, the repressor unbinds the DNA and the cell fully expresses the lac
genes downstream. In the absence of inducer, protein is produced in infrequent bursts (0.5–1 per cell cycle) in which a few (2
) proteins are produced. The distribution of the number of proteins produced per burst is consistent with a model in which a burst results from the transcription of a single mRNA molecule, finally yielding a few proteins. In the regime of moderate inducer concentration, both low-expressing cells (0–10 proteins) and high-expressing ones (hundreds of proteins) are observed. This bimodal distribution results from the presence of frequent, small bursts (similar to the noninduced state) and infrequent, large bursts of protein production. The authors proposed a model in which the small bursts consist of a partial dissociation of the repressor from one of the operator sequences; one RNA molecule is transcribed typically before the repressor binds back the operator sequence. In contrast, the large bursts correspond to full dissociation of the repressor from both operator sequences. In this case, many mRNA molecules are transcribed before another repressor binds the DNA, leading to the production of a large number of proteins.
A similar detection approach was used to study transcription factor dynamics in Escherichia coli
at the single molecule level (27
). Lac repressor (Lac I) molecules fused to a fluorescent protein could be detected when bound to their promoter sequence by imaging for long periods of time (1 s) to average out the background of freely diffusing molecules. This made it possible to measure the kinetics of association of Lac I to its promoter in vivo. The authors also used short light excitation pulses to characterize the diffusion of the repressor as well as its nonspecific binding to DNA. From these results emerged a picture of Lac I dynamics: Searching for its target sequence, the protein spends 87% of its time in short events (<5 ms), where it is nonspecifically bound to DNA and undergoes 1D diffusion while it scans the DNA. These short events are separated by periods where the repressor diffuses in three dimensions between different DNA segments.
It is also possible to directly visualize mRNA molecules using a technique that exploits the high affinity between RNA stem loops and the bacteriophage MS2 coat protein (7
). By introducing repeats of the stem loop coding sequence in the desired gene, and expressing the MS2 coat protein fused to a fluorescent protein, one can detect single mRNA molecules. This technique has been used in numerous studies to characterize single mRNA motion and localization in E. coli
), yeast (5
), mammalian cells (33
), and Drosophila
Golding et al. (35
) used this technique to study transcription in E. coli
by utilizing an inducible reporter gene under the control of the Plac/ara
promoter. By measuring the distribution of mRNA molecules per cell, the authors tested two models for transcription. The simplest model, in which transcription events were randomly initiated according to a Poisson process (with a constant probability per unit time), could not fit the data; a more elaborate model, in which the gene can switch between an “off” state (no transcription takes place) and an “on” state (transcription is randomly initiated) successfully described the data. The gene stays “on” for typically 6 min, during which it produces approximately two transcripts. In contrast, the “off ” state lasts much longer (37 min), which results in a burst-like transcription behavior, even in full-induction conditions.
Bursts of transcription have also been observed on dscA
, an endogenous developmental gene in the social amoeba Dictyostelium discoideum
). Although the authors could not detect single mRNA particles, they could resolve the sites of transcription because of the high fluorescence accumulated by the multiple nascent mRNAs. As differentiation occurred, they could observe the dscA
gene switch between the “on” and “off” states, which displayed similar lifetimes (5.2 and 5.8 min, respectively), in contrast with the E. coli
result. Over the course of development, variation was only observed in the fraction of the population expressing the gene, but the lifetime of the “on” and “off” states remained constant. In addition, the authors observed transcriptional memory, as a gene was more likely to enter the “on” state if it had been transcribing before than when it was undergoing de novo transcription.
In spite of their quantitative differences, both studies could be modeled the same way, using a simple two-state system. The nature of the event(s) that dictates the transition between the “on” and “off” states has yet to be discovered, but it could consist of DNA conformational change and/or chromatin remodeling, binding (or release) of an activator (or repressor), or transcription pausing/ reinitiation.
These studies demonstrate the potential of single-molecule techniques in studying transcription in vivo and open avenues for future research. Upcoming directions could involve expanding these observations to different systems and/or trying to combine imaging of different factors at a given locus.