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. Author manuscript; available in PMC: 2011 Dec 22.
Published in final edited form as: Mol Cell. 2010 Dec 22;40(6):965–975. doi: 10.1016/j.molcel.2010.11.022

Recruitment Timing and Dynamics of Transcription Factors at the Hsp70 Loci in Living Cells

Katie L Zobeck 1,3, Martin S Buckley 1,3, Warren R Zipfel 2, John T Lis 1,*
PMCID: PMC3021954  NIHMSID: NIHMS259094  PMID: 21172661

Summary

Chromatin immunoprecipitation (ChIP) studies provide snapshots of factors on chromatin in cell populations. Here, we use live cell imaging to examine at high temporal resolution the recruitment and dynamics of transcription factors to the inducible Hsp70 loci in individual Drosophila salivary gland nuclei. Recruitment of the master regulator, HSF, is first detected within 20 sec of gene activation and the timing of its recruitment resolves from RNA polymerase II and P-TEFb, and these factors resolve from Spt6 and Topo I. Remarkably, the recruitment of each factor is highly synchronous between different cells. In addition, Fluorescence Recovery after Photobleaching (FRAP) analyses show that the entry and exit of multiple factors are progressively constrained upon gene activation, suggesting the gradual formation of a transcription compartment. Furthermore, we demonstrate that PolyADP-Ribose (PAR) Polymerase activity is required to maintain the transcription compartment. We propose that PAR polymers locally retain factors in a transcription compartment.

Introduction

A variety of cellular signals trigger the recruitment of transcription factors (TFs) to specific target genes allowing for a myriad of biochemical processes that are required for high level transcription. Our knowledge of these processes has been guided mainly by biochemical techniques, including chromatin immunoprecipitation (ChIP), that assay when, where, and to what levels TFs are associated with specific regions of the DNA. These spatial and temporal resolution assays have provided important constraints for models of transcription mechanisms in vivo (Farnham, 2009; Saunders et al., 2006). However, most of these studies are restricted to assaying static images of chromatin-protein interactions in a population of cells. Recent advances in live-cell imaging technology allow for the real time imaging of TFs at specific loci in single cells, providing new insights into gene regulation (Hager et al., 2009).

Drosophila salivary glands offer a unique platform to carry out live-cell imaging studies of transcription (Lis, 2007). Salivary gland nuclei contain polytene chromosomes, comprised of thousands of aligned DNA strands showing packing similar to interphase diploid nuclei (Beermann, 1972). This natural amplification allows for the imaging of the localization and dynamics of TFs at specific native loci in single cells (Yao et al., 2006; Yao et al., 2007; Yao et al., 2008).

Heat shock (HS) results in decondensation of transcriptionally activated chromatin at the Hsp70 loci that are visualized as a discrete pair of puffs on polytene chromosomes (Ritossa, 1962). These two cytological loci, 87A and 87C, are 3D structures that contain 2 and 4 copies of Hsp70, respectively, as well as intervening DNA and a large spectrum of nuclear proteins. Studies of the distribution of specific proteins before and after HS have provided key insights into their function at the Hsp70 loci (Fuda et al., 2009; Gilmour, 2009; Saunders et al., 2006). Importantly, many of the proteins identified as involved in Hsp70 gene regulation have corresponding roles at other genes and in a variety of organisms, indicating their functions during transcription are evolutionarily conserved (Saunders et al., 2006).

HS induction leads to the recruitment of the transcription activator, heat shock factor (HSF), which trimerizes and binds to the regulatory regions of HS genes (Guertin and Lis, 2010; Lis and Wu, 1993; Perisic et al., 1989). Both fixed cell analyses (Westwood et al., 1991) and more recent live cell imaging have shown that HSF-eGFP resides in the nucleoplasm and is recruited to many chromosomal sites, including the Hsp70 loci upon HS (Yao et al., 2006). Fluorescence Recovery after Photobleaching (FRAP) for transcription activators of different genes have shown a dynamic associations with the chromatin (Hager et al., 2009). In contrast, upon HS, HSF stably associates with active Hsp70 loci to form a platform for programming many rounds of RNA polymerase II (Pol II) transcription before dissociating from the locus (Yao et al., 2006).

Interestingly, even before HSF binds, Pol II is transcriptionally engaged but paused at the 5′ end of the Hsp70 gene. After HS, the paused Pol II escapes into active elongation and additional Pol II is recruited to the gene (Rougvie and Lis, 1988). Live cell imaging studies of Pol II have provided insights into the molecular dynamics of a transcribing locus. Intriguingly, eGFP-Pol II reaches maximum fluorescence intensity at the Hsp70 loci after its association with chromatin reaches saturation as measured by ChIP (Yao et al., 2007). Additionally, FRAP assays show a dramatic change in the local dynamics of eGFP-Pol II at Hsp70 loci after activation. During the early stages of HS (20 min HS), eGFP-Pol II recovers completely over the course of 2 min after FRAP, at a rate consistent with the elongation rate; however, after 40 – 60 min of HS, little or no recovery is observed, even though transcription continues (Yao et al., 2007). Together these studies provide evidence for the accumulation of Pol II at transcriptionally active loci followed by progressive retention and recycling of Pol II over the course of activation. The accumulation and progressive retention of Pol II at the Hsp70 loci has been termed a transcription compartment and is postulated to provide a mechanism for the recycling of Pol II to allow continued rounds of transcription (Yao et al., 2007). It has been hypothesized that PolyADP-Ribose (PAR) polymers form the HS puff structure and may be a means to locally retain factors at the loci (Tulin and Spradling, 2003). Interestingly, previous studies have shown that PAR Polymerase (PARP), an enzyme that catalyzes the formation of PAR polymers, is required for HS puff formation (Tulin and Spradling, 2003), and PARP is involved in the rapid loss of nucleosomes at Hsp70 genes upon activation (Petesch and Lis, 2008). However, the importance PARP activity for the local retention of factors in a transcription compartment has not been tested.

Transcriptional activation of the Hsp70 genes by HS results in the recruitment of numerous TFs including P-TEFb, Spt6 and Topoisomerase I (Topo I) (Andrulis et al., 2000; Gilmour et al., 1986; Kaplan et al., 2000; Lis et al., 2000). P-TEFb is a kinase responsible for phosphorylating the C-terminal domain (CTD) of Pol II to permit Pol II’s escape into active elongation (Marshall et al., 1996). P-TEFb is composed of CycT and Cdk9 and localizes to the 5′ end as well as the entire transcribed region of Hsp70 after HS (Boehm et al., 2003; Peterlin and Price, 2006). Spt6 is a nucleosome chaperone (Bortvin and Winston, 1996), known to be recruited to the nucleosome-containing region of the gene upon activation and is notably absent from the 5′ end of the gene, which is free of nucleosomes (Ni et al., 2008; Saunders et al., 2003). Topo I is recruited to remove supercoils generated by transcription (Wang, 2002) and is strongly recruited to the Hsp70 loci upon activation (Fleischmann et al., 1984). It has been identified as a hyperphosphorylated CTD interacting protein (Carty, 2002) and, like Spt6, associates with the nucleosome-containing region as opposed to the 5′ end (Gilmour et al., 1986; Kroeger and Rowe, 1992). Even though these factors bind to different regions of the Hsp70 gene, ChIP studies done after different lengths of HS, show they are all rapidly recruited upon activation (Boehm et al., 2003; Ni et al., 2008); however these studies are not of sufficient temporal resolution to resolve their order of recruitment.

In this study, we examined the recruitment of the transcription activator HSF, RNA polymerase Pol II, and the elongation factors P-TEFb, Spt6 and Topo I to the native Hsp70 loci with unprecedented temporal resolution in living cells. Our studies resolve the recruitment timing of these factors, providing temporal information that sets limits for their functional roles, and identify a strikingly synchronous recruitment among different cells of these factors upon activation. We also assess the fate of histone H2B and PARP at the Hsp70 loci after activation and chromatin decondensation. Then using FRAP, we test the possibility that these factors are progressively retained in a transcription compartment that forms over extended gene activation. Finally, we test the importance of PARP activity for retention of Pol II in the transcription compartment.

Results

Localization of TFs with Pol II at Active Transcription Loci in Living Cells

Activation of the Hsp70 genes leads to the decondensation of the chromatin at the Hsp70 loci (87A&C) and concomitant recruitment of Pol II to these sites. In both fixed chromosome spreads and intact living nuclei, these sites are the only doublet of Pol II “puffs” within HS nuclei and unambiguously identify the Hsp70 loci (Jamrich et al., 1977; Yao et al., 2006). ChIP and immunostaining of polytene squashes have also shown that many TFs colocalize at the Hsp70 loci after HS (Lis, 2007). We selected key factors involved in different aspects of the transcription elongation; P-TEFb, Spt6, and Topo I, and examined their localization patterns and recruitment to the Hsp70 loci in vivo. To do this, we generated Drosophila transgenic lines that express a fluorescent protein (FP, either eGFP or mRFP)-tagged TF and the complementary FP-tagged on the Rpb3 subunit of Pol II, within third instar larva salivary glands. Laser scanning confocal microscopy (LSCM) was used to identify their location before and after HS activation in living cells.

LSCM shows that FP-tagged P-TEFb, Spt6 and Topo I are primarily localized within the nucleus before and after HS. More specifically, we observed colocalization between all three factors and FP-tagged Pol II at developmentally regulated loci in non-heat shocked (NHS) nuclei (Figures 1A–1C and S1A–S1C). After a 20 min HS, the factors are depleted from the developmental loci and now colocalize with Pol II at the HS loci including the two Hsp70 loci (Figures 1D–1F and S1D–S1F). These colocalization results concur with previous immunofluorescence studies on squashed polytene chromosomes (Andrulis et al., 2000; Fleischmann et al., 1984; Kaplan et al., 2000; Lis et al., 2000) and confirm the recruitment of these factors to the Hsp70 loci upon HS in living cells. Notably, these TFs are not detected at the Hsp70 loci before HS (note the absence of signal for the Hsp70 loci during NHS), unlike Pol II, which is present at these sites at low levels before HS. Additionally, consistent with what was seen previously in squashed polytene chromosomes (Fleischmann et al., 1984; Muller et al., 1985), we observe a strong recruitment of eGFP-Topo I to the nucleolus upon HS in living salivary glands.

Figure 1. Colocalization of P-TEFb, Spt6 and Topo I with Pol II at Developmental and Hsp70 Loci in Living Polytene Nuclei.

Figure 1

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LSCM maximum intensity projections of polytene nuclei co-expressing (A, D) mRFP-P-TEFb (left panel), eGFP-Pol II (middle panel), (B, E) eGFP-Spt6 (left panel) and mRFP-Pol II (middle panel) and (C, F) eGFP-Topo I (left panel) and mRFP-Pol II (middle panel). (A–C) were imaged under NHS conditions and (D–F) are the same nuclei imaged 20 min after HS. Merge presented in right panel. Arrows highlight the position of the Hsp70 loci. Scale bars are 10 μm. See also Figure S1.

Differences in the Timing and Rates of Recruitment among TFs

Figure 1 shows that mRFP-P-TEFb, eGFP-Spt6 and eGFP-Topo I are recruited to the Hsp70 loci before 20 min HS. However, the precise timing and rate of this recruitment could differ, distinguishing two possible mechanisms, whereby TFs are simultaneously corecruited upon activation or where they are recruited in ordered, temporally and kinetically distinct steps. To achieve sufficient temporal resolution, we used spinning disk confocal microscopy (SDCM) because it provides faster data acquisition than LSCM. 3D image stacks were collected in both green and red channels during NHS, using a 40x oil immersion objective maintained at room temperature. HS was activated by swapping in a matched objective heated to 36°C. The large heat capacity of the lens causes a nearly instantaneous HS (Yao et al., 2007) when the immersion medium contacts the cover slip (t = 0 sec). Then, after adjusting for any changes in the focal plane, we obtained a time series consisting of 2-channel 3D image stacks for 20 min at time intervals of 20–30 sec (Figure 2A and Experimental Procedures). As previously seen (Yao et al., 2007), activation of the Hsp70 genes does not result in a relocation of the Hsp70 loci.

Figure 2. Recruitment Timing of TFs.

Figure 2

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(A) SDCM was used to obtain 3D images of FP-tagged TFs with the complementary FP-tagged Pol II. First, Drosophila salivary glands were imaged using a room temperature objective, then an objective pre-heated to 36°C was swapped in, causing a nearly instantaneous HS. 3D time series were then obtained continuously over 20 min.

(B–C) Normalized TF fluorescence intensities (F.I.) were averaged using 10 sec windows (B) using mean F.I. and (C) using total F.I. HSF in red, Pol II in black, P-TEFb in blue, Spt6 in green, Topo I in purple. Number of samples (n) = 10, 32, 10, 12, and 10, respectively.

(D–G) Reproducible differences in recruitment times relative to Pol II were calculated by fitting exponential curves to each set of recruitment data for each factor. (D) HSF (n=10), (E) P-TEFb (n=10), (F) Spt6 (n=12), and (G) Topo I (n=10). Times to reach a specific intensity were calculated and subtracted from the times for Pol II from the same nucleus. Because HSF was not imaged with Pol II, a random set of Pol II curves were used to calculate the paired differences. The red dotted line at Δ t=0 represents the time Pol II reaches the corresponding intensity. Error bars represent the SEM. See also Figure S2.

By imaging pairs of FP-tagged TFs, we are able to measure both the timing and rate of recruitment for both factors in the same nucleus. We measured the recruitment in the fly lines shown in Figure 1 as well as a line expressing HSF-eGFP, the activator of Hsp70 transcription. The recruitment data for each factor was averaged using consecutive 10 sec windows for both the mean intensity (Figure 2B) and the total intensity measurement (Figure 2C). Remarkably, the recruitment of HSF is detectable within 20 sec after HS, while the recruitment of Pol II and the three other TFs occurs considerably later (>100 sec after HS). Notably, HSF mean intensity decreases around 100 sec after HS, while HSF total intensity plateaus at this time. Together, with the observation that the Hsp70 loci increase in volume with Pol II recruitment (Figure S2A), these results suggest that the decrease in HSF mean intensity is due to chromatin decondensation, not its dissociation from the loci.

To quantify reproducible differences in the initial timing of recruitment, we fit paired data sets with exponential curves and calculated the difference in recruitment times (Δt) for the TF and Pol II to reach the same normalized fluorescence intensity. Because HSF was not imaged with Pol II in the same cell, we used a random sample of Pol II curves to calculate the pair-wise differences in timing of recruitment. The time of initial recruitment, relative to Pol II, can be observed as the mean fluorescent intensities approach zero (Figures 2D–2G). We report all measurements averaged over multiple samples along with the standard error of the mean. If the TF is recruited faster than Pol II, the curve will slope to the left; if it is recruited slower it will slope to the right. HSF is recruited 82 ± 5 sec before Pol II. In contrast, P-TEFb is initially recruited around the same time as Pol II (1 ± 6 sec after Pol II), and Spt6 and Topo I are recruited after Pol II (12 ± 4 and 22 ± 5 sec after Pol II, respectively).

The recruitment data also provide the rates of recruitment to the Hsp70 loci (Figure S2B), as assessed by fitting the data to a single exponential curve. The rate of HSF recruitment is much faster than the rate for either Pol II or the three elongation factors, this was also observed in Figure 2D by the curve sloping to the left. Additionally, the rates of recruitment for P-TEFb and Topo I are significantly slower than Pol II (Figures S2B, 2E and 2G). Spt6, however, has a rate that is indistinguishable from Pol II (Figures S2B and 2F), suggesting a proportional recruitment of these two factors. These differences in timing and rates of recruitment for the different TFs demonstrate a sequential and independent recruitment of these factors to the Hsp70 loci rather than a concerted co-recruitment of factors, supporting the view that each factor has its own mechanism and cue for recruitment.

Synchrony in the Recruitment of TFs to Hsp70 Loci

Boettiger et. al. (2009) identified two patterns of developmental gene activation within Drosophila embryos: a stochastic activation of transcription, where the first detection of transcripts in different cells occurred over a range of 15–20 min, and a synchronous activation of transcription, where transcripts in cells are detected within a 3 min range. This synchronous activation is hypothesized to facilitate the homogeneous expression of genes vital for proper development. Similarly, the coordinated and rapid activation of HS genes may be required to survive HS (O’Brien and Lis, 1993). Our time-lapse recruitment data provide a way to assess whether the recruitment of FP-tagged HSF, Pol II, P-TEFb, Spt6 and Topo I to the Hsp70 loci occurs in a synchronous or stochastic manner in individual salivary gland nuclei. To assess the recruitment method, we compared the recruitment curves from nuclei within the same salivary gland as well as different salivary glands.

In all nuclei, HSF is recruited to the Hsp70 loci rapidly after HS and the initial recruitment is already observed by the first time point after HS (Figures 3A and S3A). Both the recruitment rates and times show little variation, reported here as standard errors, for nuclei in the same or from different glands, with the recruitment of HSF occurring 17 ± 1 sec after HS. These data indicate rapid and highly synchronous recruitment. The recruitment times of Pol II following HS also show little variation, 103 ± 2 sec (Figure 3B). Additionally, the recruitment times of the three TFs, P-TEFb, Spt6 and Topo I, are similarly restricted, 95 ± 3, 115 ± 2, and 132 ± 4 sec, respectively (Figures 3C–3E and S3). Therefore, all five factors are recruited to Hsp70 in a synchronous manner.

Figure 3. Synchrony in the Recruitment of TFs to Hsp70 loci Among Nuclei of the Same Gland or in Different Glands.

Figure 3

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(A–E) Representative SDCM recruitment images and corresponding mean F.I. recruitment plots for (A) mRFP-HSF, (B) Pol II (mRFP and eGFP), (C) mRFP-P-TEFb, (D) eGFP-Spt6 and (E) eGFP-Topo I. The 1st image for each factor is the NHS image, 2nd image is the time point before recruitment, while the rest of the images are spaced to depict the recruitment kinetics of the factors (note: HSF is recruited by the first time point after HS). Scale bars are 10 μm. Plots show normalized mean F.I. of factors’ recruitment in nuclei of the same gland (same color lines) and nuclei from different glands (different color lines) over 20 min HS. Each graph represents recruitment data from three or more glands containing 2 or more nuclei from each gland. Red arrows mark the location of the Hsp70 loci. See also Figure S3.

Effect of HS Activation on the Localization of Histone H2B and PARP

Upon HS, dramatic structural changes occur at the Hsp70 loci, which is visible in polytene chromosomes as the formation of a transcriptionally active puff (Ritossa, 1962). We have observed that the decondensation of the chromatin led to the observed decrease in mean fluorescence intensity of HSF, while the total fluorescence intensity remained constant (Figures 2B and 2C). Because HS activation leads to nucleosome loss at the Hsp70 gene, via a rapid and transcription-independent and a slower transcription-dependent loss (Petesch and Lis, 2008), we wanted to address whether histones remain associated with the Hsp70 loci during HS.

To address this possibility, we measured both the mean and total fluorescence intensity of mRFP-H2B at the Hsp70 loci. We observed that the mean fluorescence intensity decreases over time at the Hsp70 loci (Figures 4A and 4B). However, to address whether histones are 1) physically lost from the loci, or 2) stay associated with the loci but decrease in intensity due to chromatin decondensation, we measured the total intensity of H2B at the Hsp70 loci using a constant volume defined by the maximum size of the Hsp70 loci. Interestingly, the total fluorescence intensity remains constant, suggesting that H2B remains associated with loci after HS activation and chromatin decondensation (Figure 4C), even though in this time frame, nucleosome structure is disrupted at the Hsp70 loci (Petesch and Lis, 2008).

Figure 4. Association of H2B and PARP with Hsp70 Loci after Decondensation.

Figure 4

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(A and D) Representative time course images illustrating the localization of (A) mRFP-H2B and (D) PARP-eGFP to the Hsp70 loci after HS. Top panel shows the localization of the factor, while the bottom panel shows a merge between the factor (green) and Pol II (red). The 1st image (0 sec) of each panel is the NHS image, 2nd image is the time point before additional Pol II recruitment and consecutive images are spaced out over the course of HS. Arrows indicate the Hsp70 loci, and a progressive decrease in mRFP-H2B or PARP-eGFP intensity can be seen in these images. Scale bars are 10 μm.

(B and E) Mean F.I. plots of (B) mRFP-H2B and (E) PARP-eGFP, using Pol II as an indicator for the volume of the Hsp70 loci. Lines of the same color are from the same gland, while different colors are from different glands. 2–3 glands containing 2–4 nuclei each are plotted. (C and F) Total F.I. plots using a constant volume for (C) mRFP-H2B and (F) PARP-eGFP. Lines of the same color are from the same gland, while different colors are from different glands. Fluorescence intensity of 2–4 nuclei each from 2–3 glands are plotted for each factor.

Decondensation of the HS loci as well as the loss of nucleosome structure at Hsp70 genes upon activation is dependent on PARP (Petesch and Lis, 2008), an enzyme that catalyzes the polymerization of ADP-ribose units from NAD+ (Kraus and Lis, 2003). In Drosophila, polytene squashes have shown that both PARP and PAR localize to the Hsp70 loci upon activation (Tulin and Spradling, 2003). Therefore, using the same methods as for H2B, we measured the mean fluorescence intensity of PARP-eGFP over the course of HS and observed a decrease in intensity, similar to H2B (Figures 4D–4E). However, measuring the total intensity of PARP (Figure 4F) revealed no large changes in total PARP intensity, suggesting that PARP, like H2B, remains associated with the Hsp70 loci at the same level before and after decondensation.

TFs Progressively Retained in Transcription Compartment over Time Course of HS

The FP-tagged TFs provide a means of not only examining their recruitment, but also their dynamics of association with the Hsp70 loci. For example, 10 min after HS, FRAP of eGFP-Pol II shows a complete and linear recovery of fluorescence that reflects the entry of new fluorescent Pol II onto the gene. In contrast, continued HS, causes the progressive decrease in both the rate and total recovery of Pol II, even though transcription remains robust. This observation has been attributed to the progressive retention of Pol II in a transcription compartment that allows the recycling of Pol II for continued rounds of transcription (Yao et al., 2007). To address the possibility that this compartment is a more general feature of active transcription, we examined 1) whether there is an accumulation of FP-tagged factors beyond the amount needed to maximally bind the genes, and 2) if the dynamics of representative TFs, P-TEFb, Spt6 and Topo I, also change with the duration of HS.

First, we have compared the time for maximum recruitment to the Hsp70 loci (Figure S3) and to the Hsp70 genes (Figure S4A–S4C). Our high resolution recruitment assay and salivary gland ChIP of Pol II on Hsp70 (Figures S3 and S4A) agrees with the previous report that Pol II reaches maximum intensity at the loci after maximum binding has occurred on the gene (Boehm et al., 2003; Yao et al., 2007). We observe maximum levels at ~3 min HS for chromatin by ChIP (Figure SX) and ~8 min HS at the locus by live cell imaging (Figure S3B). Salivary gland ChIP experiments for the TFs P-TEFb and Spt6 also show maximal gene occupancy by ~3 min (Figure S4B and S4C), while they continue to accumulate at the Hsp70 loci achieving maximum total intensity at 10 and 8 min, respectively (Figures S3C and S3D). This suggests that there is an accumulation of TFs at the Hsp70 loci beyond what is required to saturate the DNA.

Second, we examined whether the dynamics of these factors change with the duration of HS using FRAP. All FRAP experiments were done during recruitment equilibrium, where mean and total intensities of the TF are constant over time (Figures 5 and S4D–S4F). At 10 min HS, shortly after reaching recruitment equilibrium, the FRAP profile provides critical information regarding the dynamics of association between the factor and the Hsp70 loci. The ½ max (τ1/2;) recovery time can be used to identify whether recovery is limited by the dissociation of the pre-existing bleached factors from their targets of interaction, i.e. a binding event, or if recovery is limited to diffusion (Sprague and McNally, 2005). For mRFP-P-TEFb, eGFP-Spt6 and eGFP-Topo I, the estimated τ1/2; for diffusion is less than 0.030 sec, while the observed τ1/2; for these factors are 30, 40, and 10 sec, respectively (Figures 5B, 5D and 5F). This result indicates that these TFs are in fact binding at the Hsp70 loci.

Figure 5. FRAP Dynamics of TFs at the Hsp70 Loci Change After Length of HS.

Figure 5

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(A, C, and E) FRAP of the three TFs after different lengths of HS: (A) mRFP-P-TEFb, (C) eGFP-Spt6 and (E) eGFP-Topo I. Panels show representative FRAP images. The top panel was bleached after 10 min HS, middle panel was bleached after 20 min HS and lower panel was bleached after 40 min HS (C) or 60 min HS (A and E). All scale bars equal 10 μm.

(B, D and F) Plots of Normalized F.I. at the Hsp70 loci. (B) mRFP-P-TEFb, (D) eGFP-Spt6 and (F) eGFP-Topo I. Bleaching resulted in a decrease of 40–60% initial F.I.; however, these plots are normalized to both initial F.I. and bleach depth. Red, 10 min HS; Green, 20 min HS; Blue, 40 min HS; Purple, 60 min HS (n=11, 10, 12, 10 for P-TEFb, respectively; n=13, 6, 6 for Spt6, respectively; n=13, 20, 17, 16 for Topo I, respectively). Error bars represent the SEM. See also Figure S4.

Because P-TEFb, Spt6 and Topo I are Pol II interacting proteins (Fleischmann et al., 1984; Ni et al., 2008; Yoh et al., 2007), we also compared their recovery curves to Pol II to see if any of these three factors were stably binding to Pol II. If a stable interaction exists, we expect the FRAP dynamics to mimic that of Pol II. At early times of HS, 10–20 min, Pol II recovers from photobleaching linearly for 2 min. This recovery corresponds to the time it takes bleached Pol II molecules to complete transcription elongation, and for new fluorescent Pol II to refill the gene (Yao et al., 2007). The recovery curves for the three TFs are all exponential, indicating that these three TFs are not stably bound to Pol II, but instead interact transiently with Pol II or the Hsp70 loci.

Prolonged HS also impacted the FRAP dynamics of the three TFs at the Hsp70 loci. The most dramatic effect was seen with Spt6, which recovers to 50% initial intensity after a 10 min HS. However after a 20 min HS, less recovery is observed and after 40 min HS no recovery is observed after FRAP (Figures 5C and 5D). Figure S4 confirms Spt6 is present at similar intensities at all of these time points. Together these results indicate that Spt6 becomes more stably associated with the Hsp70 loci with increasing HS time, and these findings are consistent with this factor, which is required for maximum transcription elongation rates (Ardehali et al., 2009), being reused and recycled within the compartment.

In contrast to Spt6, Topo I and P-TEFb remain dynamic during prolonged HS. However, increasing HS time still causes a progressive decrease in the mobile fraction of Topo I and P-TEFb, such that the immobile fractions of both Topo I and P-TEFb reach 60% of the total population in the 60 min HS sample (Figures 5A, 5B, 5E and 5F). The increase in the immobile fraction observed for these two factors could be explained in a similar manner as Spt6, whereby the immobile fraction consists of an increasing number of molecules that can be recycled for use in transcription.

Together our salivary gland ChIP results and FRAP dynamic studies suggest that the transcription compartment is a general feature of active transcription, and is not limited to Pol II. Notably, our FRAP results emphasize that the compartmentalization process is not simply a mechanism by which TFs become completely retained at the loci after HS, but suggest differences in a TF’s function, size, or affinity for the loci, may play a role in the ability of a given TF to diffuse into or out of the compartment.

Role of PARP in Compartment Formation

Previous studies have shown that PAR chains accumulate at the Hsp70 loci upon activation, and that the enzymatic activity of PARP, the enzyme that catalyzes PAR chain formation, is required for the structure of HS puff (Tulin and Spradling, 2003). Our study extends these finding to show that PARP continues to associate with the Hsp70 loci upon activation. Based on these findings, we hypothesized that PARP catalytic activity is responsible for the retention of factors in the transcription compartment. To address this hypothesis, we tested the effect of specifically inhibiting PARP catalytic activity, using the drug PJ34 on the compartmentalization of eGFP-Pol II, the defining factor of the transcription compartment (Yao et al., 2007), during late HS. The experimental setup is illustrated in Figure 6A. We heat shocked the glands for 35 min and then perfused 3 μM PJ34, or media only, over the glands for 5 min after which we acquired FRAP curves. Perfusion does not affect the compartment behavior of Pol II when compared to a 40 min HS no perfusion control (Figures 6B and 6D). Strikingly, PJ34 perfusion after 40 min HS has a drastic effect on the FRAP dynamics of Pol II, which now completely recovers over the course of 120 sec (Figures 6B and 6C). This recovery is remarkably similar to the 10 min HS FRAP behavior of Pol II, indicating that PARP inhibition leads to a condition resembling the pre-compartment state. This result indicates that the catalytic activity of PARP is required for retaining Pol II in a compartment.

Figure 6. PARP Catalytic Activity is Required for the Maintenance of the Transcription Compartment.

Figure 6

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(A) Schematic of PJ34 perfusion protocol. Media only or 3μM PJ34 was perfused over the gland for 5 min starting 35 min after HS. FRAP of eGFP-Pol II at the Hsp70 loci was initiated as soon as perfusion stopped.

(B–D) FRAP of eGFP-Pol II in PJ34 perfused glands and controls. (B) FRAP curves of eGFP-Pol II perfused with PJ34 (red, n=3) at 40 min HS or with Media only at 40 min HS (blue, n=4) and no perfusion controls at 10 min HS (gray squares, n=4) and 40 min HS (gray circles, n=3). Error bars represent SEM. Representative images of (C) eGFP-Pol II after PJ34 perfusion and (D) after Media only perfusion. Scale bars = 5μm.

(E) Model for the progressive formation of transcription compartment during the time course of HS. PARylated proteins accumulate at the Hsp70 loci over HS activation. The accumulation of PAR restricts the ability of proteins to diffuse into and out of the compartment, with individual factors behaving differently, with Spt6 and Pol II unable to exchange with the surrounding nucleoplasm by 40 min HS. Treatment with PJ34 a PARP inhibitor at 40 min HS, reduces the amount of PAR present and restores the ability of Pol II to exchange with the nucleoplasm. Light shaded arrows represent the potential dynamics of the TFs after PJ34 treatment.

Discussion

Recruitment Timing and Rates for TFs During HS

The Drosophila Hsp70 genes provide a unique system with which to examine the recruitment of TFs to specific chromosomal loci. They are rapidly and robustly activated upon HS and are located at two cytological loci, whose appearance provides a diagnostic doublet for identifying the Hsp70 loci in living cells. By measuring the intensity of TFs at the Hsp70 loci over the course of HS, we have been able to distinguish differences in the initial timing and rates of recruitment of TFs.

We have previously shown, in a population of cells, that HSF appeared to be recruited to the Hsp70 genes before Pol II (Boehm et al., 2003), however our present study shows this more convincingly at high temporal resolution and also demonstrates a sequential recruitment of the other factors P-TEFb, Spt6 and Topo I to the Hsp70 loci rather than a concerted co-recruitment of a pre-assembled transcription complex. In single cells, we observe that HSF is recruited 80 sec before Pol II suggesting that other co-activators or modifications are required for Pol II recruitment. P-TEFb is first detected at Hsp70 loci when Pol II also begins to increase, which is consistent with the P-TEFb kinase’s role in promoting active elongation (Zhou et al., 2000). The recruitment of Spt6 follows 10 sec after Pol II, which is consistent with the known role of Spt6 as a chromatin remodeler and the time for Pol II to reach the first nucleosome. Then, Topo I, which interacts with the phosphorylated CTD of Pol II (Carty, 2002), is recruited 20 sec after Pol II and P-TEFb. Interestingly, this raises the possibility that Pol II phosphorylation is not sufficient to recruit of Topo I, but rather that Topo I recruitment may require transcription-driven accumulation of supercoiled DNA (Madden et al., 1995). Finally, a model has been recently proposed where genes are recruited to nuclear sites called transcription factories, which contain the TFs required for transcription and its regulation (Carter et al., 2008). The distinct kinetics observed here for the five TFs and the failure to see movement of the Hsp70 loci within the nucleus in polytene nuclei upon activation (here and Yao et al. 2007) are inconsistent with Hsp70 genes being recruited to a preformed transcription factory. Furthermore, we do not observe an increase in colocalization of the HS loci in diploid nuclei after HS (Yao et al., 2007).

Synchronous Recruitment of TFs

This study provides a single cell analysis with the temporal resolution to address whether TFs are synchronously recruited to Hsp70 loci. Our study revealed that HSF, Pol II, and three TFs (P-TEFb, Spt6 and Topo I) are each synchronously recruited to the Hsp70 loci. We observed a standard error for each TF’s recruitment timing to be less than 6 sec. Interestingly, genes that have paused Pol II may in general be synchronously activated. Boettiger and Levine examined numerous development genes in early embryos at the level of RNA accumulation in cells and observed that genes containing a paused Pol II were synchronously activated (range of 3 min), while genes without a paused Pol II were stochastically activated (range of 20 min) (Boettiger and Levine, 2009).

We propose that the paused Pol II, present on Hsp70 genes, may facilitate the synchronous recruitment of HSF by maintaining an open chromatin state at the promoter to provide the activator, HSF, with accessible binding sites. It has been shown that HSF binding to Hsp70 transgenes depends on an open chromatin state that in turn requires GAGA factor and paused Pol II (Shopland et al., 1995). More generally, the paused/stalled Pol II at promoters has been shown to be important in keeping nucleosomes off promoters (Gilchrist et al., 2008). The open chromatin state may increase the probability of activator binding its target sequence (Tsukiyama et al., 1994). In addition, other factors may also gain more efficient access to genes that have a promoter paused Pol II. The pre-assembled general TF complex at these promoters should facilitate binding and initiation of newly recruited Pol II following activation (Lebedeva et al., 2005). Synchronous recruitment of Pol II might then promote the synchronous recruitment of the other TFs and synchronous transcript accumulation. Importantly, these studies provide insights into a mechanism that ensures the rapid and uniform induction of the HS response that is vital to the viability of organisms under stress conditions.

Transcription Compartment

The transcription compartment has previously been defined by Pol II’s progressive retention and recycling over extended times of gene activation (Yao et al., 2007). By comparing the times of maximum recruitment for the Hsp70 loci and the Hsp70 genes, we were able to show there is an accumulation of molecules at the loci beyond what can bind DNA. Then, using FRAP, we showed three TFs, CycT, Spt6, and Topo I, are also progressively retained at the Hsp70 loci. These findings suggest that the transcription compartment is not limited to Pol II, but also involves the retention, and perhaps recycling, of other components of the transcription machinery.

Moreover, our results set further limitations on the structure of the compartment. In the case of Pol II (Yao et al., 2007) and Spt6 (Figure 5D), the FRAP recoveries progressively decrease until no recovery is observed. In contrast, a portion of P-TEFb and Topo I continue to exchange with the nucleoplasm even after an hour of HS, but over time, an increasing percentage of molecules remain stably associated with the locus. These different behaviors suggest that the transcription compartment is not a completely closed entity formed over time, but rather suggests either an affinity-based retention or the formation of a porous barrier.

Affinity-based retention and porous barrier models are not mutually exclusive. PAR, which accumulates at the Hsp70 and developmental loci in response to gene activation (Tulin and Spradling, 2003), could be the foundation of the porous barrier, but could also be responsible for the affinity-based retention of factors with strong nucleic acid affinity, as has been documented for histones (Althaus et al., 1994). To investigate these models, we assayed the role of PAR in compartment formation by using PJ34 a specific inhibitor of PARP catalytic activity (Garcia Soriano et al., 2001) and show that the dynamics of Pol II at the Hsp70 loci, 40 min after HS, return to the early HS dynamics upon PJ34 treatment. Inhibition of PARP prevents the polymerization of more PAR, which the PAR glycohydrolase (PARG) can now rapidly degrade (D’Amours et al., 1999). Thus, we suggest that PARG activity reduces the overall amount of PAR at the Hsp70 loci and allows a re-mobilization of Pol II to the 10 min HS behavior. We hypothesize that the other factors will similarly be re-mobilized, and a thorough study of these factors and the formation and properties of the compartment requires a future concentrated study.

Based on our results, we propose the model in (Figure 6E) where extended HS activation leads to the accumulation of PAR chains at the Hsp70 loci. Early on (10 min HS), the number or size of these chains are small enough that many proteins can easily diffuse into and out of the area. Some factors, like Spt6, however may already be partially immobilized because of their affinity towards nucleic acids. Over time (20 min HS and then 40 min HS), the number of PAR chains increase at the Hsp70 loci reducing the ability of some factors (Topo I and CycT) to freely diffuse with the nucleoplasm and fully preventing the exchange of Spt6 and Pol II with the nucleoplasm, although they are able to be reused for continued rounds of transcription (Yao et al., 2007).

Finally, our study addresses whether histones remain associated with the Hsp70 loci after activation and chromatin decondensation, and the results indicate surprisingly that H2B remains associated, even though upon activation the nucleosomes rapidly dissociate from the Hsp70 genes (Petesch and Lis, 2008). Therefore, we suggest that the early transcription compartment may be involved in retaining H2B at the Hsp70 loci. Additionally, our observation that PARP, also remains associated with the Hsp70 loci suggests that PARP might be a component of the compartment. However, further experiments are needed to test these hypotheses.

In conclusion, our kinetic analysis of TF recruitment and dynamics by live cell imaging provides insights into the overall mechanics and architecture of the transcription loci. Future development of imaging technologies should provide the ability to examine diploid cells, and thereby address the conservation of these mechanisms in different cell types and organisms.

Experimental Procedures

Spinning Disk Microscopy

Drosophila salivary glands, from the crosses described in the Supplemental Experimental Procedures, were dissected from third instar larva as previously described (Yao et al., 2008) and were transferred, immediately with medium to a MatTek glass-bottomed culture dish (P35G-1.0-14-C) and a glass cover-slip was placed on top to reduce evaporation and movement of the glands. A Carl Zeiss Cell Observer SD system with the Yokogawa CSU-X1 spinning disk unit and a Hamamatsu C9100-13 EMCCD was used to obtain confocal 3D stacks with 2-channels and time intervals of 10–30 sec. Two identical Plan-Apochromat 40x/1.3 Oil Iris objectives were used; one was maintained at room temperature and one was heated to 36°C using a Bioptechs Objective Heater. A 40 μm pre-activation z-stack, with 1 μm sections, was taken using the room temperature objective in both channels alternating channels every slice. Then the 36°C objective was moved into position; HS times were started at the moment the objective contacted the slide. A xyzt series with 40 z-sections was obtained after readjusting the focal position. Time intervals were between 10–30 sec and the time series lasted 20 min (Figure 2A). Images were taken at a resolution of 512×512 pixels, using 16-bit color depth. Details regarding data analysis can be found in the Supplemental Experimental Procedures.

Laser Scanning Confocal Microscopy and Multiphoton Microscopy

Dissected glands were transferred with medium to a Bioptechs FCS3 Closed Chamber System with a 0.2 mm spacer. We used an upright confocal/multi-photon microscope system (Carl Zeiss LSM510 META). NHS images were obtained with a C-Apochromat 63x, 1.2 NA, water immersion objective. For HS, an identical objective was swapped in for the room temperature objective the same manner as described above.

FRAP

Dissected glands were imaged using MPM as described above. Perfusion of 3μM PJ34 or Media alone into the FCS3 chamber occurred as depicted in Figure 6A. We used a circular ROI limited to the dimensions of the Hsp70 loci. eGFP samples were photobleached with a Mai Tai laser (Spectrum Physics) at 910 nm with a power of 15–20 mW (measured after the objective). mRFP photobleaching used the same laser at 800 nm using 40–50 mW. These settings photobleached the samples to 40–60% initial intensity. Images were corrected for acquisition photobleaching by monitoring a small nuclear region. FRAP curves were normalized for pre-bleach images to equal one, and first image after the bleach equal to 0. Recovery times were obtained by fitting the FRAP data to f(t)=A×(1-Ceq×e−koff×t) (Sprague et al., 2004).

Supplementary Material

01

Acknowledgments

We thank Nina Allen and Scott Olenych from the David H. Murdock Research Institute for use of the Carl Zeiss Cell Observer SD system, and Tudor Marian for his generosity in writing the MatLab programs. This work was partly performed in the Developmental Resources for Biophysical Imaging Opto-Electronics and was supported by an NIH grant GM25232 to J.T.L., an NSF grant CHE-0242328 to W.W. Webb & J.T.L., and an NIH grant GM087003 to M.S.B.

Footnotes

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