Transcriptional induction of BAC transgenes

A major motivation for moving to this third generation, BAC transgene approach was to analyze changes in large-scale chromatin structure associated with transcriptional activation in a system that more closely approximated the activation of native genes at their normal chromosomal locations. Multi-copy plasmid based transgenes are subject both to strong position effects and to transgene silencing effects which increase with gene copy number.

We used the Hsp70 BAC multi-copy insertion cell line to carefully compare the level of expression and transcriptional induction of the endogenous gene versus the BAC transgene. To induce Hsp70 expression, we incubated cells at 42^oC for 30 min. Transcript levels were measured by qRT-PCR using species-specific primers before and after heat shock. Hsp70 transcription increased ~140-fold for the endogenous genes versus ~200-fold for the transgenes. Normalized by gene copy number, Hsp70 transgene transcript levels were 6–7-fold lower than endogenous Hsp70 transcript levels before heat shock and 3–4-fold lower after heat shock (Table II). Thus, Hsp70 BAC transgene transcription levels were within several fold of the endogenous Hsp70 genes and very similar to the endogenous genes in overall fold induction after heat shock.

DHFR expression varied similarly to literature values reported for chromosomal DHFR amplified genes. CHO DG44 cells are clonally derived CHO cells containing x-ray induced deletions of both endogenous DHFR genes (Urlaub et al., 1986), precluding the comparison of DHFR transgene versus endogenous DHFR transcript levels in the same cells. DHFR expression is present throughout the cell cycle of cycling cells (Feder et al., 1989) but decreases after cell cycle arrest. We blocked cells in a G1 arrest using either serum starvation (Chang et al., 2001) or DMSO treatment (Fiore et al., 2002). In both cases G1 arrest was verified by flow cytometry (unpublished data). G1 arrest led to a 3–4-fold decrease in BAC DHFR transcripts levels, as measured by qRT-PCR (Table II), as compared with a reported ~10-fold reduction in DHFR transcripts in serum starved versus serum stimulated cells (Ljungman and Hanawalt, 1995; Iwanaga et al., 2004). For the MT gene cluster, we observed 4–50-fold increases in MT2A transgene expression with 2–16 h Zn induction, respectively (Table II). This increase is comparable to reported increases for the endogenous MT2A gene in human prostate epithelial cells and in CHO cells after gene amplification (Albrecht et al., 2008; Singh et al., 2008). Differences in primer efficiency prevented an accurate normalization to endogenous gene expression levels.

An overall large-scale chromatin decondensation accompanies transcriptional induction, but active gene loci remain highly condensed

We directly compared changes in large-scale chromatin structure before and after transcriptional induction for each of the three BAC transgene cell lines. Induction conditions suitable for one transgene served as additional negative controls for the cell lines carrying the other two BAC transgenes.

Interestingly, all three cell lines showed similar levels of large-scale chromatin condensation for the three different BAC transgene arrays, both in the uninduced and induced states (Fig. 2, Table I). Using light microscopy, we measured changes in the number of GFP visible dots as well as changes in the contour length over a curvilinear path linking all of these GFP spots.

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Figure 2.

Large-scale chromatin decondensation accompanies gene activation. (A) GFP-lac repressor fluorescence before (top) and after (bottom) gene induction for the DHFR (057L22), Hsp70, and MT BACs. After induction, a more extended “string of dots” is observed. Bar, 2 µm. (B) Histograms showing mean contour lengths (left) or GFP spot number (right) for each BAC cell line at normal growth conditions (CTRL) (DHFR gene induced) and after heat shock (Hsp70 genes induced), Zn addition (MT genes induced), or DMSO induced G1 arrest (DHFR repressed) (see text). Increases in contour length and spot number are specific for the induction conditions appropriate for the particular BAC transgene. (C) Histograms showing contour length under CTRL or induced conditions for Hsp70_5 and MT1_1_4 cell lines with and without transcriptional inhibition. Chromatin decondensation accompanying heat shock is blocked by α-amanitin and significantly reduced by DRB (see text). (B and C) Histogram data points are based on > 100 cells (SEM error bars).

In the uninduced state, all three cell lines showed an average of 2.5–3 GFP dots per nucleus (maximum 6) with an average contour length of ~0.5–0.75 µm (maximum 1.5 µm). After induction, the average number of GFP spots increases to 4–4.5 (maximum 10) and the average contour length increased 1.4–2.9-fold to ~1.0–1.5 µm (maximum 3.5 µm) (Table I, Fig. S2). Relative to the linear DNA compaction ratio of 30-nm chromatin fibers, the mean compaction ratio for uninduced BAC transgenes was ~25–50 fold higher (Table I). An ~1.5–3 fold average decondensation accompanied gene induction, with the minimum observed compaction (175) still several fold higher than 30-nm chromatin fibers. Uninduced and induced chromatin compaction ratios were similar for the three different transgenes even though transcription levels varied significantly (Table II). The above data does not exclude the possibility that the transcribed genes might be forming more highly decondensed DNA loops protruding outwards from a highly condensed, large-scale chromatin “backbone” containing the lac operator repeats. Lac operator repeats were located 64, 25, 37, 4, and 35 kb away from the DHFR, HspA1A, HspA1B, MT1A, and MT2A genes, respectively (Fig. 1 B). However, for the DHFR BAC, the lac operator repeat is inserted within the Msh3 gene, expressed from the same divergent promoter as DHFR and showing similar cell cycle expression (Farnham and Schimke, 1986; Schilling and Farnham, 1995). For the MT BAC, the lac operator repeat is inserted within a cluster of MT1 genes, ubiquitously expressed in various tissues (Hidalgo and Carrasco, 1998). Thus, in both these BACs, the lac operator repeat is inserted within regions likely to be transcriptionally active.

However, to more rigorously test this possibility, we used two-color, 3D DNA FISH on DHFR and Hsp70 BAC transgene arrays to localize the signal from the lac operator repeats relative to the BAC FISH signal. The lac operator FISH probe produced a spot-like pattern very similar to GFP-lac repressor binding in the same cell lines (Fig. 2 A), frequently forming a linear trajectory. To best visualize the spatial relationship between lac operator and BAC FISH signals, we focused on cells with highly extended BAC transgene arrays with a large number of lac operator spots (Fig. 3). Lac operator probe (red) spots generally coincided and were co-linear with similar FISH signals from the BAC probe (green) with no evidence for looping of BAC sequences a significant distance away from a linear backbone (Fig. 3).

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Figure 3.

No evidence for extended looping of BAC sequences from a linear backbone. (A–C) 3D DNA FISH shows co-linear signals from the Lac operator (red) and BAC (green) probes. DNA is stained by DAPI (gray). (A) DHFR D10 cells. (B) DHFR-110-5-E4 cell with lac operator repeats inserted into the DHFR gene fifth intron. (C) Hsp70_5 cell 30 min after heat shock. Middle columns (deconvolved data) and right columns (raw data) show 2x enlarged views (Bars, 2 µm). (D) EM of DHFR D10 clone by in vivo immunogold staining of EGFP-LacI shows the BAC transgenes are compacted indistinguishable from surrounding genomic DNA, with no obvious looping structure from the transgene backbone. Left and middle: serial sections (150 nm); right: 500-nm section (Bar, 500 nm).

We also examined a different stable cell line (110–5-E4) created from the shorter DHFR BAC (560M7-K8.32-C-5, Fig. 1 B) in which the lac operator repeat is inserted directly into the fifth DHFR intron. The configuration of GFP spots in this cell line looked very similar to that observed in the DHFR D10 BAC cell line, frequently lining up in a linear trajectory (Fig. 3 B). Co-linear lac operator and BAC FISH signals for the Hsp70_5 cell line were also observed (Fig. 3 C).

Using a new, in vivo immunogold labeling procedure, we also visualized by EM the location of the lac repressor stained operator repeats relative to chromatin in DHFR D10 cells. Consistent with the 3D FISH results, lac repressor spots localized within condensed, large-scale chromatin structures (Fig. 3 D).

Large-scale chromatin decondensation is blocked or significantly reduced after transcriptional inhibition

To investigate whether transcription is required for decondensation, we treated cells carrying the Hsp70 or MT BACs with two different transcriptional inhibitors: α-amanitin and 5,6-dichloro-1-β-d-ribobenzimidazole (DRB). α-Amanitin specifically inactivates and targets the RNA polymerase II largest subunit for rapid degradation (Nguyen et al., 1996), whereas the transcription inhibitory effects of DRB may be related to interference with the activity of the CTD kinase and therefore transcriptional elongation (Dubois et al., 1994).

With α-amanitin treatment, both the Hsp70 and MT transgene arrays after transcriptional induction became slightly more condensed (shorter contour lengths) than that observed in noninduced control cells (>99% confidence level). This may be related to inhibition of constitutive transcription levels (Fig. 2 C), although we cannot exclude general effects induced by global transcriptional inhibition on nuclear and/or chromosome structure. In DRB treated cells, the Hsp70 transgene array showed a statistically significant decondensation (>99% confidence level) after heat shock, but the magnitude of this decondensation was significantly reduced relative to cells not treated with DRB (>99% confidence level). For the MT transgene array, no statistically significant difference in contour lengths was observed between noninduced control cells versus Zn induced DRB treated cells. Hsp70 is known to have a poised polymerase (Price, 2008). The differential decondensation observed with these two inhibitors therefore might be related to the presence or absence of RNA polymerase at the Hsp70 promoter, with DRB treatment still leaving a poised polymerase at the Hsp70 promoter and allowing initiation of transcripts after heat shock.

Transcriptional activation occurs for most BAC copies but gene and BAC sequences remain co-linear

On average the BAC transgenes are expressed within several fold of the endogenous genes (Table II). This could occur either through a reduced transcription rate at each transgene copy or through a normal transcription rate at a small fraction of BAC transgenes. In the latter case, our previous observations would not be informative with regard to the relationship between active transgene expression and large-scale chromatin compaction.

Using RNA FISH, we asked roughly what fraction of BAC transgene copies were active within the transgene arrays, focusing on DHFR and Hsp70 BAC cell lines. RNA FISH signals using the entire BAC as a probe were found adjacent and/or partially overlapping most GFP spots in both DHFR BAC transgene cell lines (Fig. 4, A and B). Similarly, RNA FISH signals, detected using the HspA1A gene as a probe, also were close to or overlapping with most GFP spots at early times (5–10 min) after heat shock in the Hsp70 BAC transgene cell line (Fig. 4 C).

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Figure 4.

Most BAC transgene copies are transcriptionally activated with gene sequences co-linear with BAC sequences. (A–C) RNA FISH (red) shows DHFR BAC and Hsp70 gene transcripts relative to GFP-lac repressor (green) after gene induction. DNA is stained by DAPI (gray, left column). (A and B) Log phase cells with DHFR BAC transgenes showing RNA FISH using the entire BAC as probe. (A) D10 cell line with 057L22-K-8.32-C29 DHFR BAC containing lac operator repeats inserted 64 kb upstream of the DHFR gene. (B) 110–5-E4 cell line with 560 M7-K-8.32_C5 DHFR BAC containing lac operator repeats inserted into the DHFR gene fifth intron. (C) Hsp70_5 cell line 5 (top) or 10 (bottom) mins after heat shock induction using Hsp70 transcribed region as RNA FISH probe. RNA FISH signals are adjacent or partially overlapping with GFP signals in all three cell lines. (D–F) DNA FISH reveals actual localization of transcribed gene sequences relative to overall BAC sequence. Co-localization is much more extensive than that observed with RNA FISH and co-linear with the linear BAC signals. (D and E) Pooled PCR probes covering most of DHFR gene (red), total BAC sequence (green) in D10 (D) or 110–5-E4 (E) cells. (F) Hsp70_5 cell line 30 min after heat shock. Approximately 2 kb Hsp70 gene probe (red) localized used tyramide amplification method, relative to directly labeled BAC probe (green). Bar, 2 µm.

We assumed the increased separation of RNA FISH versus GFP-lac repressor signals, as compared with lac operator versus BAC DNA FISH signals (Fig. 3), reflected transcript accumulation away from the actual sites of transcription (for review see Johnson et al., 2000). Indeed dual DNA FISH showed excellent colocalization between DHFR gene and total BAC probes (Fig. 4, D and E). The short, ~2-kb Hsp70 gene sequences were detected using tyramide signal amplification (TSA) (Fig. 4 F). Again increased colocalization was observed in most regions relative to the RNA FISH results. A minority of Hsp70 signals did not overlap with BAC hybridization, but because these were co-linear with nearby BAC signals we interpret their lack of overlap with gaps created by incomplete BAC hybridization, compounded by the much higher detection sensitivity of the TSA Hsp70 gene detection (Fig. 4 F). However, we cannot exclude a more extensive decondensation in the neighborhood of some transcribed Hsp70 genes.

Our previous measurements (Fig. 2) suggested an average of 2–3 BAC transgene copies per GFP spot for induced transgenes. Some GFP spots have lower intensity than others, suggesting a variable number of BAC copies, and therefore a differential overall large-scale chromatin compaction per spot. However, RNA FISH signals were observed independent of the brightness of adjacent GFP spots (Fig. 4). Therefore, transcription activation occurs for most individual BAC copies independent of differential large-scale chromatin compaction. RNA FISH reveals Hsp70 transgene expression begins in >90% of cells within 5 min of heat shock whereas large-scale chromatin decondensation increases from 10–30 min after heat shock (Fig. S3).

Hsp70 transgenes associate with nuclear speckles after heat shock

Transcriptionally active BAC transgenes showed a several fold average decondensation of large-scale chromatin structure, with the more highly decondensed examples showing extended, linear conformations for the DHFR and MT genes (Figs. 2–4). In contrast, at 30 min after heat shock a significant fraction of Hsp70 transgenes showed a unique, semi-circular conformation not observed for DHFR or MT BAC transgenes (Fig. 2 A). Moreover, whereas RNA FISH showed good colocalization of GFP spots with Hsp70 transcripts at short times after heat shock (Fig. 4 C), at later times Hsp70 transcripts accumulated within a DAPI poor hole encircled by the GFP spots (Fig. 5 A).

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Figure 5.

Hsp70 transgenes associate with nuclear speckles after heat shock. (A) Hsp70 transcripts, detected by RNA FISH (red), accumulate within a DAPI (grayscale) poor region encircled by GFP-LacI signals (green) marking the Hsp70 transgene locations. (B and C) Close association of Hsp70 BAC transgenes, marked by EGFP-LacI (green), with nuclear speckles immunostained either with ASF/SF2 (B) or SC35 (C) antibodies (red) in Hsp70_5 cells 30 min after heat shock (bottom panel) as compared with nonheat shocked control cells (top panel). Grayscale shows DNA staining (DAPI). Bar (A–C), 2 µm. (D) In vivo immunogold staining of EGFP-LacI marks transgene location (arrows) associated with IGC (nuclear speckle). Enlarged area (right) shows that transgenes appear at the periphery of the dense cluster of granules defining the IGC (arrowheads). Bar, 1 µm. (E) Comparison of the diameter of the nearest nuclear speckle (average of longest and shortest speckle axes, green) versus transgene association index (red, see text) at different times after heat shock. Transgene association index increases from 50–90% during heat shock with no major change in average size of nearest speckle.

This impression was confirmed by a strong association of the Hsp70 BAC transgenes, but not DHFR or MT transgenes, with SC-35 or ASF/SF2 immunostained speckles (Fig. 5, B and C). Analysis of several independently derived cell lines from each of the three BACs revealed this speckle association in all of the Hsp70 BAC transgene cell lines but none of the DHFR or MT BAC cell lines, indicating that this association was specific to the Hsp70 transgene rather than the chromosome integration site (unpublished data). In vivo immunogold labeling of the Hsp70 BAC transgene revealed localization of the lac operator tag to the periphery of interchromatin granule clusters (IGC)—specifically to the amorphous/fibrillar material surrounding the dense central, granular core (Fig. 5 D). Combined immunofluorescence and RNA FISH confirmed that Hsp70 transcript accumulation was occurring within SC-35 stained speckles at later times after heat shock (unpublished data).

Accumulation of transcripts within SC-35 speckles from genes associated with these speckles has been described previously (Johnson et al., 2000; Smith et al., 2007). This association of Hsp70 BAC transgenes with nuclear speckles/IGCs mirrors the previously reported association of the endogenous Hsp70 gene with nuclear speckles, with speckle association increasing from 10% to 90% after heat shock (Jolly et al., 1999). We defined the association index as the fraction of GFP spots in close contact with the edge of an SC-35–labeled speckle. In this cell line, the association index increased from 47% to 85% between 0 and 30 min after heat shock, whereas the average size of the nearest speckle remained relatively stable (Fig. 5 E), suggesting that Hsp70 transgenes might simply move closer to preexisting speckles.

Dynamics of Hsp70 transgene and nuclear speckle association

To address more directly how Hsp70 transgenes associate with nuclear speckles after heat shock, we performed live-cell imaging. Previous live-cell imaging of GFP-ASF/SF2 dynamics during activation of a viral transgene suggested recruitment of splicing factors to the viral transgene through a local protrusion from a nearby nuclear speckle (Misteli et al., 1997); in this study the location of the viral transgene was revealed only at the end of the observation period after fixation and FISH. Increased speckle association of Hsp70 transgenes after heat shock could occur through several distinct processes: (1) nucleation of new speckles near the transgene; (2) enlargement of a preexisting, nearby speckle; (3) transgene movement toward a speckle; (4) speckle movement or protrusion toward the transgene.

To distinguish among these possibilities, we developed a stable cell line coexpressing mRFP-ASF/SF2 and GFP-dimer lac repressor (Hsp70_5_P9_4). The mRFP fluorescence pattern was nearly identical to the immunofluorescence staining using anti-SC35 or anti-ASF/SF2 antibodies. Western blot analysis showed expression levels of mRFP-ASF/SF2 were approximately one fourth endogenous ASF/SF2 protein levels (unpublished data). Live-cell imaging revealed examples of the first three possibilities outlined above. Most frequently we observed nucleation of new splicing speckles adjacent to transgenes or enlargement of a preexisting nearby speckle, but in rare cases long-range movement of transgenes toward SC-35 speckles was observed (Fig. 6).

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Figure 6.

Live imaging shows three possible transgene/speckle dynamic events during heat shock. (A) Long-range movement of Hsp70 BAC transgenes (green) toward a ASF/SF2 speckle (red) occurred within a 5-min heat shock period (Video 1). (B) Enlargement of preexisting speckle together with decondensation and increased association and encircling of BAC transgenes around speckle exterior (Video 2). (C) Nucleation of a new ASF/SF2 speckle adjacent to the Hsp70 BAC transgenes several minutes after heat shock; newly nucleated speckle then merges with preexisting adjacent speckle forming a larger speckle (Video 3). Bar, 5 µm.

Previous work examining chromosome movement in response to transcription factor tethering had revealed a pronounced photosensitivity of long-range chromosome movement (Chuang et al., 2006). Due to technical limitations of our microscope system and the expression levels of the GFP and mRFP fusion proteins, exposure times required in these Hsp70/speckle live experiments exceeded the threshold at which inhibition of long-range chromosome movement previously was observed. In fact, the few examples of long-range chromosome movement observed here involved movement within the first few time points. It is therefore possible that long-range movements occur more frequently; experiments beyond the scope of the current study are now in progress to investigate this possibility.

Seen frequently is a relatively early movement of the neighboring GFP spots, initially not in contact with an adjacent, preexisting speckle, to the speckle periphery (Fig. 6 A; Video 1). GFP spots appear to encircle the edge of the speckle as the BAC transgene array decondenses (Fig. 6 B; Video 2). Interestingly, in all live experiments GFP spots showed a strong tendency to remain associated with the speckle periphery, versus becoming engulfed within a growing speckle. In one example in which two speckles merged, GFP spots that appeared transiently within the merged speckle moved back to the periphery of the merged speckle (Fig. 6 C; Video 3).

Cell culture and establishment of BAC cell lines

CHO DG44 cells with a double deletion of the endogenous DHFR locus (Urlaub et al., 1986) were incubated at 37°C with 5% CO₂ in Ham's F12 media (Invitrogen) plus 10% FBS (Hyclone). CHO DG44 cells (Clone 7) stably expressing EGFP-dimer lac repressor (Tumbar et al., 1999) were used for all BAC transfections. Circular BAC DNA was transfected using Fugene6 (Roche) according to the manufacturer's directions. Stable cell clones containing DHFR BACs were selected in F12 media without thymidine and hypoxanthine (Invitrogen) and with 10% dialyzed FBS (Hyclone). The Hsp70 and MT BAC transgene cell lines were selected in 200 µg/ml G418 in F12 media supplemented with 10% FBS. Stable transformants were subcloned by serial dilution.

Mitotic chromosome examination

Logarithmically growing cells were arrested with 200–600 ng/ml nocodazole in fresh media for 2 h. Mitotic cells were collected by shake-off, centrifuged in media onto poly-l-lysine–coated coverslips at 4°C, fixed in 1.6% formaldehyde (Polysciences) in PBS buffer for 30 min at RT, and stained with DAPI. Methanol/acetic acid–fixed mitotic chromosome spreads were prepared according to standard cytogenetic procedures (Beatty and Scherer, 2002).

DNA probe preparation

BACs, pSV2-DHFR-8.32 (lac operator 256mer repeat) (Robinett et al., 1996), and HSPA1A and DHFR gene PCR products (primers in Table S3) were biotin labeled by nick translation (BioNick kit; Invitrogen) according to the manufacturer's instructions. Alternatively, the biotin-labeled nucleotide mix in the kit was substituted with the digoxigenin nucleotide mix (0.5 mM dATP, dGTP, and dCTP, 0.375 mM dTTP (Invitrogen), 0.125 mM DIG-dUTP (Roche), 0.5 M Tris-HCl pH 8.0, 0.5 mg/ml BSA (Sigma-Aldrich), and 50 mM MgCl₂) for digoxigenin labeling.

3D DNA FISH

Cells on coverslips were fixed using freshly prepared 4% paraformaldehyde in calcium, magnesium-free PBS (CMF-PBS) for 10 min at RT followed by permeabilization in CMF-PBS using 0.5% Triton X-100. Coverslips were placed in 20% glycerol/CMF-PBS for 30 min, subjected to four freeze-thaw cycles using liquid nitrogen, then immersed in 0.1M HCl for 7.5 min before storing in 50% deionized formamide (Sigma-Aldrich)/2xSSC as described elsewhere (Solevei et al., 2002). 2 µl of hybridization mix (50 ng/µl probe concentration in 50% deionized formamide, 10% dextran sulfate, 2X SSC) was applied before mounting coverslips to glass slides using rubber cement. Denaturation at 75°C for 2 min on a heat block was followed by hybridization overnight at 37°C in a humidified chamber, followed by washes in 0.4X SSC at 70°C for 2 min and SSCT (2xSSC/0.2% Tween 20) at room temperature. Detection used Strepavidin-Alexa 594 (1:200; Invitrogen) or anti-DIG-fluorescein antibodies (1:200; Roche) in 4% BSA/SSCT.

RNA FISH

RNA FISH and RNA FISH combined with immunostaining procedures were performed as previously described (Chaumeil et al., 2004) with using Streptavidin-Alexa 594 (1:200; Invitrogen) in 1% BSA/4xSSC/2 mM vanadyl ribonucleoside complex (VRC; New England Biolabs, Inc.).

Transgene copy number analysis

BAC transgene copy number estimates were determined for three cell lines (DHFR D10, HSP70_5, and MT1_1_4) using both quantitative, real-time PCR (qRT-PCR) and chromatin fiber FISH. Genomic DNA was isolated using phenol/chloroform extraction. qRT-PCR was done using a Power SYBR green PCR Master mix (Applied Biosystems) and the iCycler machine (Bio-Rad Laboratories). Copy number was calculated using the absolute quantitation method (Ginzinger, 2002) with known concentrations of purified BAC DNA used to generate a standard curve. (Table S2 lists primers used). Stretched chromatin fibers were prepared as described elsewhere (Henry and Heng, 2002) and FISH was performed as described above.

Antibody staining

Cells were permeabilized in 0.1% Triton X-100/PBS* (CMF-PBS, 0.1 mM EDTA, and 5 mM MgCl₂) for 30 s before fixation in 1.6% formaldehyde/PBS* for 15 min at RT, blocked in 5% normal goat serum (Sigma-Aldrich)/PBS* for 1 h at RT followed by primary antibody incubation (mouse anti-ASF/SF2; Invitrogen), 1:200, or anti-SC35 (Sigma-Aldrich), 1:200, at 4°C overnight, three washes for 5 min, and secondary antibody (goat anti–mouse Texas red; The Jackson Laboratory), 1:1,000, for 4 h at RT, all in 0.1% Triton X-100/PBS*.

RNA extraction and RT-PCR

Total RNA was isolated using the RNeasy mini kit (QIAGEN) and on-column RNase-free DNase set (QIAGEN) according to the manufacturer's instructions. First-strand cDNA synthesis (ImProm-II kit; Promega) was performed using an oligo(dT) primer (Sigma-Aldrich). The comparative 2^−ΔΔCt method (Wong and Medrano, 2005) was used to measure relative expression levels. Table S2 lists primers used.

Transcription inhibition

Transcription was inhibited by incubating cells in 100 µg/ml α-amanitin (Sigma-Aldrich) for 3.5 h or 50 µg/ml DRB (Sigma-Aldrich) for 4.5 h in complete medium at 37°C before heat shock treatment for 30 min in the Hsp70_5 cell line. For the MT cell line, α-amanitin was added 2 h and DRB 3 h before the beginning of a 2-h zinc induction. Subsequently, cells were fixed in 1.6% formaldehyde/PBS for 30 min at RT, followed by three washes in PBS; the fixation procedure is the same as that without drug treatment (Fig. 2 A). In vivo labeling of RNA synthesis for confirmation of transcription inhibition used BrUTP incorporation as described elsewhere (Memedula and Belmont, 2003).

Microscopy and live observation

All samples after fixation were stained in 0.2 µg/ml DAPI and mounted in Prolong Antifade (Invitrogen). 3D optical section datasets of fixed cells were collected using a 60x objective (NA 1.4) on an inverted light microscope (IMT-2; Olympus) and deconvolved using an iterative algorithm as described previously (Tumbar et al., 1999). Live-cells maintained at 37°C in a closed chamber system (FCS2; Bioptechs) were imaged with a 63x objective (NA 1.40) using a Zeiss Axiovert 100M microscope equipped with a Roper CoolSNAP HQ slow-scan CCD camera (Roper Scientific).

Contour measurements were made from single optical sections from cells in which all GFP spots were visible and all spots in focus within a range of no more than two optical sections (0.2 µm depth). In fact, this applied to nearly all cells in the population, suggesting a nonrandom, polarized orientation of chromatin within the nuclei. In nearly all cases, at most one GFP spot was not in focus within the image, with the out of focus spot usually at one end or the other of the contour path. This allowed us to quickly make measurements from hundreds of cells per data point, while obtaining accurate estimates of contour lengths.

EM immunogold labeling used a new procedure (Kireev et al., 2008) in which primary mouse monoclonal IgG1κ anti-GFP or anti-Lac repressor antibodies or Fab' fragments labeled covalently with Mono-Sulfo-NHS-Nanogold (Nanoprobes) were microinjected into cells (Fab') or cell nuclei (IgG) together with 1.5 µg/µl Texas red–conjugated dextran (70 kD; Sigma-Aldrich). After microinjection cells were incubated at 37°C for 1 h before a brief detergent permeabilization in 0.1% Triton X-100, then fixed and processed for EM as described elsewhere (Kireev et al., 2008). TEM used a Phillips CM200 microscope using 120 kv for thin sections and 200 kv for thick sections, with images acquired using a TVIPS 2Kx2K Peltier-cooled CCD camera and software (Tietz Video and Image Processing Systems GmbH).

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. DHFR BACs were a gift from the laboratory of Edith Heard. pRSETB was a gift from the laboratory of Roger Tsien.

This work was supported by grant nos. GM58460 and GM42516 from the National Institute of General Medical Sciences awarded to A.S. Belmont.