Tracking FACT and the RNA Polymerase II Elongation Complex Through Chromatin in Vivo

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Science  22 Aug 2003:
Vol. 301, Issue 5636, pp. 1094-1096
DOI: 10.1126/science.1085712


RNA polymerase II (Pol II) transcription through nucleosomes is facilitated in vitro by the protein complex FACT (Facilitates Chromatin Transcription). Here we show that FACT is associated with actively transcribed Pol II genes on Drosophila polytene chromosomes. FACT displays kinetics of recruitment and of chromosome tracking in vivo similar to Pol II and elongation factors Spt5 and Spt6. Interestingly, FACT does not colocalize with Pol III–transcribed genes, which are known to undergo nucleosome transfer rather than disassembly in vitro. Our observations are consistent with FACT being restricted to transcription that involves nucleosome disassembly mechanisms.

Nucleosomes are inhibitory to transcription (1). One mechanism of inhibition is by blocking the path of the elongating RNA polymerase. In eukaryotes, three different RNA polymerases exist: Pol I, II, and III. Studies in vitro suggest that at least two polymerases, Pol III and II, have distinct mechanisms by which they transcribe through nucleosomes. Pol III is able to transcribe chromatin under physiological conditions in a purified system (2), whereas Pol II transcription requires nucleosome disassembly (3).

The Drosophila heat shock gene hsp70 has promoter and leader regions that are free of nucleosomes (4), and a promoter-proximal, paused polymerase (5) that prime it for rapid activation (6). Upon hsp70 gene induction by thermal stress, Pol II encounters nucleosomes downstream of +130 (7, 8). The machinery that enables Pol II to transcribe through these nucleosomes has yet to be characterized. The nucleosome remodeling complexes SWI/SNF and NURF do not appear to play a role during hsp70 transcription elongation (9, 10). Prime candidates that remain include FACT (Facilitates Chromatin Transcription) and elongation factors Spt5 and Spt6. FACT is a complex that comprises Spt16 and SSRP1 (11) and facilitates Pol II elongation through chromatin in vitro (12). Spt6 has histone chaperone activity (13), and Spt5 and Spt6 show considerable colocalization with the elongating Pol II on Drosophila polytene chromosomes (14, 15). Also Spt5, Spt6, and Spt16 have mutant phenotypes similar to those of histone genes (16).

To examine whether FACT is positioned to regulate chromatin structure during transcription in vivo, we performed comparative immunofluorescence analysis on Drosophila polytene chromosomes. Immunofluorescence staining reveals the global distribution of FACT (Spt16) relative to the hyperphosphorylated, elongating Pol IIo (Fig. 1). Antibodies against either FACT or Pol IIo label many sites on chromosomes prepared from unstressed, developing, third instar larvae, several of which correspond to major transcriptionally active loci [e.g., ecdysone puffs at 74E and 75B (Fig. 1)]. Merging the FACT and Pol IIo images reveals a striking coincidence of FACT with Pol IIo labeled sites. FACT is also at the nucleolus (Fig. 1), the site of Pol I transcription. In contrast, FACT is absent from the tandem cluster of 5S ribosomal RNA (rRNA) genes at 56F1-2 that are transcribed by Pol III and known to be strongly stained by antibodies to Pol III–specific transcription factors (17).

Fig. 1.

FACT colocalizes with Pol II on Drosophila polytene chromosomes. Global view of phosphorylated Pol II (H14 antibody, green) and FACT (Spt16 antibody, red) localization on Drosophila polytene chromosomes under non–heat shock conditions (Non HS) and after a 20-min heat shock (HS). White arrows locate the ecdysone puffs at 74 and 75; the blue arrow identifies the 5S-rDNA locus at 56F1-2; purple arrows indicate the endogenous heat shock loci hsp70 87Aand 87C and the LacZ-hsp70 transgene at 59B; and orange arrows show the nucleolar organizer (NO). Hoechst staining of DNAis used for chromosomal mapping. Chromosomes were prepared from the Z243 Drosophila strain as previously described (32, 33).

Colocalization of FACT with many Pol II–transcribed genes suggests that FACT is intimately involved in Pol II transcription. This point is further strengthened by the observation that, after a 20-min heat shock, Pol IIo and FACT redistribute to and concentrate at heat shock loci (Fig. 1). Pol II is recruited to heat shock loci within seconds, and the first Pol II molecule progresses through the gene in less than 2 min (6, 18). If FACT facilitates Pol II transcription through nucleosomes at hsp70, then it must be recruited with corresponding rapidity. We examined the fluorescence staining of FACT during a time course after heat shock at the transgenic sites 9D and 61A (19), which contain just one copy of the hsp70 gene in a known orientation (20). Figure 2 (panels 1 and 2) shows the distribution of the promoter-restricted heat shock transcription factor, HSF (21), relative to both FACT subunits, SSRP1 and Spt16, at 9D (the same results were obtained at 61A). HSF and FACT are strongly recruited to the transgenic loci within 2.5 min of heat shock. Even at this early time, FACT resolves from the promoter-associated HSF. The merged image shows a red edge of FACT staining emerging to the right of HSF, indicating that FACT localizes further downstream than HSF. Spt5 and Spt6, factors known to associate with the entire transcription unit, are also recruited within 2.5 min to these heat shock loci (Fig. 2, panels 3 and 4). In contrast to HSF, the staining of Spt6 and Spt5 completely overlaps with that of FACT. As activation continues (see the 10-min heat shock time point), the chromatin at 9D decondenses further creating a chromosomal “puff,” and the differential staining of HSF and FACT becomes more apparent, whereas Spt6 still completely overlaps with FACT, and Spt5 mostly overlaps with FACT. Thus, FACT, Spt6, and Spt5 are recruited rapidly to hsp70 upon heat shock, and they associate with the same decondensed regions of the puff.

Fig. 2.

FACT is rapidly recruited to the body of the hsp70 transcription unit. Fluorescent staining with antibodies to the FACT subunits (red) is compared to HSF (green) in panels 1 and 2 (from left) or to Spt6 or Spt5 (also green) in panels 3 and 4, respectively, during the time course: non–heat shock (Non HS) and 2.5 and 10 min after heat shock. Shown is the LacZ-hsp70 transgene at 9D in Drosophila strain Bg9 (19). White arrows indicate the direction of transcription (20). Merged images are shown. DNAis Hoechst stained (purple).

To examine at higher resolution the temporal and spatial distributions of Pol II, FACT, Spt6, and Spt5 on hsp70, we performed cross-linking and chromatin immunoprecipitation (ChIP) experiments at various times after an instantaneous heat shock. A short (1 min) cross-linking time provided snapshots of the process of transcription elongation. Figure 3A shows the location of primer sets used to quantify the association of specific proteins along the hsp70 gene. The rapid activation of hsp70 is evident from the detectable recruitment of Pol II to the leader region after a 75-s heat shock (Fig. 3B). FACT is rapidly recruited to and enriched in the first region of hsp70 that is packaged into nucleosomes at this early time. FACT associates with the 3′ end of the gene after 150 s (Fig. 3C), the same time Pol II is first detected at the 3′ region (Fig. 3B). After a 5-min heat shock, FACT is enriched in the open reading frame (ORF), and the level of FACT in the 3′ region is at least eightfold higher than any level of FACT detected upstream (–154). This upstream region is constitutively nucleosome-free (4), and the level of FACT on it does not increase upon heat shock. These data are consistent with FACT and the Pol II elongation complex cotranslocating along hsp70, starting at the first nucleosome.

Fig. 3.

High-resolution ChIP analysis of the FACT, Spt6, and Spt5 distributions on hsp70. (A) Schematic showing location of the hsp70 primer sets used during real-time polymerase chain reaction (PCR) amplification of the immunoprecipitated material and the position of the first nucleosome on the ORF. (B to E) Charts showing the percent of starting material immunoprecipitated in different regions of hsp70 by an antibody to (B) the largest subunit of Pol II (RpII215), (C) the SSRP1 subunit of FACT, (D) the C-terminal domain of Spt6, and (E) the N-terminal domain of Spt5, after various periods of heat shock. The background primer set anneals to a region with no annotated genes, 32 kb downstream of the third copy of hsp70 at 87C. Standard error bars are shown; N is indicated on the key. The number on the x axis corresponds to the gene region analyzed (A). Note that the modest heat shock–induced recruitment of FACT to the leader region (+58) in (C) may simply be a consequence of FACT's association with sonicated fragments that cover the most 5′ nucleosomes and the leader region. For each heat shock period, at least two ChIP assays were performed on each of at least two separate formaldehyde cross-linked Drosophila Kc cell extracts. Extracts were prepared, and ChIP assays were performed as outlined previously (18, 33).

Spt6, like FACT, is rapidly recruited to the leader and 5′ regions of hsp70 and is first detectable at the 3′ end 150 s after heat shock. The greater occupancy of Spt6 in the nucleosome-occupied coding region is consistent with Spt6 being involved in the modulation of the chromatin structure. Overall, our kinetic data place FACT and Spt6 at the correct time and place to contribute to cotranscriptional nucleosome disassembly on hsp70 in vivo. Although we have not detected a reduction in hsp70 RNA when FACT or Spt6 protein levels are depleted a few fold by RNA interference (RNAi) treatments of Kc cells, these proteins are abundant and may be in excess, especially during heat shock when the general reduction of transcription of most genes presumably increases the availability of elongation factors (16).

Another factor implicated in the control of transcription through chromatin, Spt5, is also recruited to hsp70 upon heat shock and tracks along the gene with kinetics similar to Pol II, FACT, and Spt6. In contrast to FACT and Spt6, the level of Spt5 associated with hsp70 is less at the 3′ end of the gene than at the leader region after a 5-min heat shock (Fig. 3E and Fig. 2). As we have shown previously (14), even before heat shock we detect a strong signal for Spt5 in the region of the paused polymerase (leader region). Spt5 is known to have a role with the NELF complex (22) in restricting Pol II's elongation early in the transcription cycle and in stimulating the mRNA 5′-capping machinery (23, 24), activities that require its association with the leader region. Thus, in addition to a positive role in elongation (14, 15, 25), Spt5 appears to have a role that is both spatially and temporally separate from that of FACT and Spt6.

Coimmunoprecipitation in Drosophila nuclear extracts provides support for physical associations of Spt5, Spt6, FACT, and elongationally active Pol II (fig. S1). These results are consistent with those from yeast that support the idea that multiple Spt5 complexes exist, one of which is an elongation complex that includes Spt5, Spt6, and FACT (26). Other elongation factors, the Paf1 complex and the chromodomain adenosine triphosphatase (ATPase), Chd1, also show physical and genetic interactions with FACT (27, 28), indicating that transcription elongation through chromatin in vivo involves a sophisticated molecular machine.

Nucleosome reassembly after transcription-induced disassembly is essential for the integrity of chromatin structure. A link between Spt6 and nucleosome reassembly is known (13), and recent genetic evidence suggests a similar role for FACT (29). Our data demonstrate that upon hsp70 induction, FACT and Spt6 are strongly recruited to regions of hsp70 occupied by nucleosomes. Spt6 has been shown to interact with H3 and H4 (13), and FACT with H2A and H2B (11). In the accompanying paper (30), it is shown that the SSRP1 subunit contacts the H3·H4 tetramer of the disassembled nucleosome, whilst Spt16 maintains interaction with the displaced H2A·H2B dimer. It is appealing to speculate that chromatin reassembly is facilitated by Spt6 stabilizing the nucleosomes via interaction with H3 and H4 and by FACT maintaining a stable interaction with both the remodeled nucleosome and the displaced H2A·H2B dimer (29, 31). Whether disassembly or reassembly is the most critical function of FACT in vivo remains an intriguing question.

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Materials and Methods

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