Article

Nature Cell Biology 6, 1094 - 1101 (2004)
Published online: 24 October 2004 | doi:10.1038/ncb1182

Actin is part of pre-initiation complexes and is necessary for transcription by RNA polymerase II

Wilma A. Hofmann1, Ljuba Stojiljkovic1, Beata Fuchsova1, Gabriela M. Vargas1, Evangelos Mavrommatis1, Vlada Philimonenko2, Katarina Kysela2, James A. Goodrich3, James L. Lessard4, Thomas J. Hope5, Pavel Hozak2 & Primal de Lanerolle1

Actin is abundant in the nucleus and has been implicated in transcription; however, the nature of this involvement has not been established. Here we demonstrate that beta-actin is critically involved in transcription because antibodies directed against beta-actin, but not muscle actin, inhibited transcription in vivo and in vitro. Chromatin immunoprecipitation assays demonstrated the recruitment of actin to the promoter region of the interferon-gamma-inducible MHC2TA gene as well as the interferon-alpha-inducible G1P3 gene. Further investigation revealed that actin and RNA polymerase II co-localize in vivo and also co-purify. We employed an in vitro system with purified nuclear components to demonstrate that antibodies to beta-actin block the initiation of transcription. This assay also demonstrates that beta-actin stimulates transcription by RNA polymerase II. Finally, DNA-binding experiments established the presence of beta-actin in pre-initiation complexes and also showed that the depletion of actin prevented the formation of pre-initiation complexes. Together, these data suggest a fundamental role for actin in the initiation of transcription by RNA polymerase II.

The presence of actin in the nucleus is well established1, 2; however, in contrast to its cytoplasmic functions, the role of actin in the nucleus remains unclear. It has been proposed that nuclear actin engages in processing and transporting RNA. Actin associates with small nuclear ribonucleoproteins (snRNPs), which have a major role in mRNA processing3, 4, and plays a direct part in the nuclear export of retroviral RNA and cellular proteins5, 6. Actin also forms complexes with heterogeneous nuclear ribonucleoproteins (hnRNPs) that bind to and accompany mRNA from the site of transcription to the cytoplasm7, 8. Furthermore, nuclear actin and actin-related proteins have been found in association with chromatin-remodelling and histone acetyl transferase complexes, suggesting a role for actin in chromatin remodelling8. It was recently reported that nuclear actin co-localizes with protein 4.1, linking actin to nuclear assembly processes9.

A number of studies have suggested a role for actin in transcription. A protein that co-purifies with RNA polymerase II from the slime mold Physarum polycephalum was identified as actin, although no functional role for this interaction was established10. Microinjecting anti-actin antibodies into the nuclei of Xenopus oocytes blocks chromosome condensation11, and microinjecting antibodies directed against actin or actin-binding proteins inhibits transcription of protein-coding genes in lampbrush chromosomes12. A protein resembling actin was identified as a transcriptional activator in nuclear extracts and it was suggested that this protein has a role in the pre-initiation stage of transcription13. In addition, actin is one of the cellular factors required for transcription of the viral genes from human respiratory syncytial virus (RSV)14, and an interaction between actin and hrp65 — a component of an hnRNP complex — is necessary for transcription from Balbiani rings by RNA polymerase II in Chironomus tentants15. Recently, actin was linked to transcription of ribosomal genes by RNA polymerase I16.

In this study, we have investigated the role of actin in RNA polymerase II transcription in mammalian cells. Both in vivo and in vitro transcription assays in the presence of anti-actin antibodies showed that RNA polymerase II transcription is indeed dependent on actin. Chromatin immunoprecipitation (ChIP) assays demonstrated the presence of actin at the promoter region of two different inducible genes; other experiments showed that actin stimulates transcription. We have also found that actin is part of the pre-initiation complex (PIC) and that depleting actin prevents their formation. Finally, in vitro transcription assays showed that depletion of actin blocks the initiation of transcription.

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Results

Antibodies to beta-actin inhibit transcription in vivo

We examined the importance of actin for RNA polymerase II-mediated transcription in mammalian cells in vivo. We microinjected antibodies to either muscle actin (HUC 1-1) or non-muscle beta-actin, together with rhodamine-labelled dextran, into the nuclei of HeLa cells. After a 1 h incubation, the cells were gently permeabilized and transcripts were labelled with BrUTP in vivo. Injecting the non-reactive HUC 1-1 antibody had no effect on transcription by RNA polymerase II in vivo (88% of injected cells showed nascent transcripts; Fig. 1). In contrast, the injection of antibodies to beta-actin clearly inhibited transcription. Only 14% of cells injected with the anti-beta-actin antibody showed nascent transcripts in the nucleoplasm (Fig. 1).

Figure 1: Antibodies to bold beta-actin inhibit RNA polymerase II transcription in vivo.

Figure 1 : Antibodies to |[beta]|-actin inhibit RNA polymerase II transcription in vivo.

HeLa cells were microinjected into the nuclei with antibodies directed against muscle actin as control (HUC 1-1; top) or with antibodies directed against beta-actin (bottom) together with rhodamine-labelled dextran to identify the injected cells. One hour after microinjection, cells were permeabilized and incubated with BrUTP. The nascent transcripts were visualized with an Alexa Fluor 488 conjugated antibody directed against BrdU (left). Middle, rhodamine fluorescence, which identifies injected nuclei; right, merged image of Alexa Fluor 488, rhodamine and DAPI (to identify the nuclei). Microinjection of the HUC 1-1 antibody had no effect on the formation of nascent transcripts in the nucleoplasm. In contrast, microinjecting anti-beta-actin antibodies greatly decreased Alexa Fluor 488 fluorescence in the nucleoplasm, indicating inhibition of transcription by RNA polymerase II. Scale bars represent 10 mum.

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To verify that this inhibition of transcription by antibodies to beta-actin is through a direct effect and not an indirect effect such as chromatin remodelling, we performed in vitro transcription assays. In this experiment, HeLa cell nuclear extracts that contain RNA polymerase II and all transcription factors were incubated with antibodies to beta-actin or with the control HUC 1-1 antibody. Transcription was initiated by adding rNTPs and a negatively supercoiled plasmid DNA template containing the adenovirus major late promoter (AdMLP) fused to a 380-base-pair (bp) G-less cassette. Antibodies directed against beta-actin, but not the control HUC 1-1 antibody, efficiently inhibited in vitro transcription (Fig. 2). This confirms that actin is indeed directly involved in transcription.

Figure 2: Antibodies to bold beta-actin inhibit RNA polymerase II transcription in vitro.

Figure 2 : Antibodies to |[beta]|-actin inhibit RNA polymerase II transcription in vitro.

A HeLa nuclear extract was incubated with 2 mug each of HUC 1-1 antibody or anti-beta-actin antibody. The control represents samples that were incubated with buffer only. After incubation for 30 min, transcription was initiated by adding the DNA template and rNTPs. The transcripts were isolated, separated by urea–PAGE on a 6% gel and analysed by autoradiography. In the presence of the HUC 1-1 antibodies, the 390-nucleotide (nt) RNA product was transcribed. In contrast, the beta-actin antibodies significantly inhibited transcription. This experiment was repeated three times and the mean fractional activities are shown at the bottom. The migration of the 390-nt transcription product is shown.

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beta-Actin co-purifies and co-localizes with RNA polymerase II

Next, we purified RNA polymerase II from HeLa cell nuclei using established techniques (see Methods section). Our goal in these experiments was to analyse the role of actin in transcription by adding actin to an RNA polymerase II fraction that was free of actin. However, when probing with the broad specificity C4 antibody, actin was discovered in the purified RNA polymerase II fraction (P11 eluant) and in all pooled fractions from the various columns (Fig. 3a). These fractions also contained the large subunit of RNA polymerase II, suggesting an important interaction between actin and the RNA polymerase II complex. Next, the purified RNA polymerase II was probed with a panel of antibodies to identify the specific actin isoform. The actin in the RNA polymerase II fraction was recognized by the broad specificity C4 and 56-4 antibodies17 and a monoclonal anti-peptide antibody to beta-actin18; it was not recognized by the HUC 1-1 or B4 antibodies that recognize muscle actins17, 19 (Fig. 3b). Thus, beta-actin seems to be the major actin sub-type associated with RNA polymerase II.

Figure 3: bold beta-actin is associated with RNA polymerase II.

Figure 3 : |[beta]|-actin is associated with RNA polymerase II.

(a) Co-purification of actin with RNA polymerase II. RNA polymerase II was purified from HeLa cells using multiple columns as described34. An aliquot of the pre-column fraction (PC), the flow-through fraction containing the unbound protein (FT) and the eluted fraction (E) from each column was analysed by western blotting with an antibody to the large subunit of RNA polymerase II and the C4 antibody to actin. The eluted fraction from each column contains strong bands representing RNA polymerase II and actin. The migration of the relevant molecular weight markers is shown to the left. (b) Identification of beta-actin in association with RNA polymerase II. The purified RNA polymerase II fraction (P11 eluant) was analysed by western blotting with different anti-actin antibodies as indicated. The broad-specificity C4 and 56-4 antibodies produced a strong signal, whereas two antibodies directed against muscle actin, B4 and HUC 1-1 produced no signal. Subsequent experiments using an anti-peptide monoclonal antibody to beta-actin identified this actin in association with the RNA polymerase II. The migration of the relevant molecular mass standard is shown on the left.

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The interaction of actin with RNA polymerase II was subsequently analysed in situ by performing immuno-electron microscopy with antibodies directed against actin and the large subunit of RNA polymerase II (see Supplementary Information, Fig. S1). Double immunogold labelling demonstrated the co-localization of actin and RNA polymerase II. Because most extra-nucleolar transcripts are concentrated in foci with mean diameters of about 80 nm20, we evaluated particles that were within 200 nm of each other. A statistical analysis21 showed that this co-localization is highly significant at distances ranging from 30–130 nm between particles (P < 0.01). Similar immunolocalization experiments demonstrated significant co-localization of nuclear actin and TBP at distances ranging from 30–60 nm (P < 0.05; see Supplementary Information, Fig. S1).

beta-Actin is required for transcription by RNA polymerase II

Because actin seems to specifically associate with RNA polymerase II, in vitro transcription assays were performed using purified RNA polymerase II22 and recombinant transcription factors to examine the importance of actin in transcription. The purified RNA polymerase II is apparently free of transcription factors because it has minimal activity when any of the transcription factors are deleted from the assay. In this experiment, recombinant TBP, TFIIB, TFIIF and RNA polymerase II were incubated with different proteins before adding the DNA template containing the AdMLP fused to the 380-nucleotide G-less cassette. Transcription was then initiated by adding nucleotides to the reaction mixture. The addition of the non-reactive HUC 1-1 or B4 antibodies had no effect on transcription (Fig. 4); however, addition of the beta-actin, C4 and 56-4 antibodies inhibited transcription.

Figure 4: Antibodies to bold beta-actin and NEM-modified S1 inhibit transcription in a purified system.

Figure 4 : Antibodies to |[beta]|-actin and NEM-modified S1 inhibit transcription in a purified system.

(a) Schematic representation of the transcription assay. Recombinant transcription factors TBP, TFIIF, TFIIB and purified RNA polymerase II were incubated with 2 mug each of the indicated antibodies or 7 mug of NEM-modified S1 (NEM-S1) for 20 min. The AdMLP template was added and the reaction mixture was incubated for another 10 min. The transcription reaction was then initiated by adding NTPs. The reaction was terminated as required and the transcripts were purified and analysed as described in the Methods section. (b) The effect of actin antibodies and NEM-S1 on transcription. The same actin antibodies used in Fig. 3a were incubated with an aliquot of the purified RNA polymerase II22 and assayed as shown in a. Note that the antibodies that recognize actin in Fig. 3a (C4, 56-4 and anti beta-actin) inhibit transcription. In contrast, antibodies to muscle actin that do not recognize actin in Fig. 3a (B4 and HUC 1-1) had no effect on transcription. NEM-S1, which binds specifically and tightly to actin23, also inhibited transcription. To demonstrate the specificity of this effect, 2 mug of the beta-actin antibody (which gives maximal inhibition) was pre-incubated with 0.5 mug of purified beta-actin or buffer on ice for 30 min before incubation with the purified RNA polymerase II and recombinant transcription factors. The purified beta-actin prevented the beta-actin antibody from inhibiting transcription (last lane). The control represents samples incubated with buffer only. Each experiment was repeated 3–5 times. (c) Adding beta-actin stimulates transcription by RNA polymerase II. HPLC-purified RNA polymerase II24 was incubated with varying amounts of purified beta-actin for 30 mins on ice before initiation of the transcription assay (a). Note that 4 ng of exogenous beta-actin stimulates transcript accumulation by almost eightfold and that there is no further increase in the presence of 10 ng of beta-actin.

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Five additional experiments were performed to establish that actin is involved in transcription. First, the effect of the N-ethylmaleimide-modified S1 subunit of skeletal muscle myosin II (NEM-S1) on transcription was investigated. NEM-S1 binds tightly to actin to form rigor-like complexes and it has been used as a highly specific probe for actin23. Like antibodies to beta-actin, NEM-S1 also inhibited transcription, reinforcing a specific role for actin in transcription (Fig. 4b). Second, incubating the beta-actin antibody with purified beta-actin abolished antibody inhibition of RNA polymerase II activity (Fig. 4b). Third, incubating the purified RNA polymerase II with varying concentrations of the anti-beta-actin antibody (see Supplementary Information, Fig. S2a) induced partial inhibition of transcription by the lower concentrations of the antibody. Fourth, repeating this inhibition experiment with the reactive C4 antibody showed that it too inhibited transcription in a dose-dependent manner (see Supplementary Information, Fig. S2b). Fifth, addition of upto 6 mug of the non-reactive HUC 1-1 antibody had no effect on RNA polymerase II activity (see Supplementary Information, Fig. S2c).

Lastly, the effect on transcription of adding beta-actin to the purified RNA polymerase II was determined. Adding actin to the purified RNA polymerase II described above had no effect on transcription because of the abundance of actin (data not shown). Therefore, we purified RNA polymerase II as described24. The RNA polymerase II purified in this manner contains a small but detectable amount of beta-actin. This actin is still required for activity because antibodies to beta-actin inhibited activity (data not shown). Most importantly, adding purified beta-actin stimulated transcript accumulation by a maximum of about eightfold with 4 ng of beta-actin (Fig. 4c).

Actin is recruited to the promoter of the MHC2TA and G1P3 genes

A ChIP assay was used to discover when actin associates with the transcription complex. HeLa cells were treated with interferon gamma (INF-gamma) for 6 h and prepared for ChIP analysis. The DNA–protein complexes were immunoprecipitated with antibodies to beta-actin, RNA polymerase II and acetylated histone H4. The crosslinks were reversed and the DNA that co-purified with the immunocomplexes was amplified with primers specific for a region of the INF-gamma-inducible MHC2TA promoter IV. As expected, neither actin nor RNA polymerase II or acetylated histone H4 was associated with the MHC2TA promoter IV without INF-gamma induction; however, after induction, histone H4 became acetylated25 and RNA polymerase II and actin were recruited to the promoter region (Fig. 5a).

Figure 5: Recruitment of actin to the MHC2TA promoter IV and the G1P3 promoter.

Figure 5 : Recruitment of actin to the MHC2TA promoter IV and the G1P3 promoter.

Genomic DNA was prepared from HeLa cells before (-) or after (+) induction with INF-gamma (a) or INF-alpha (b). ChIP assays were performed with antibodies to beta-actin, RNA polymerase II, acetylated histone-H4 or non-specific IgG as a control. The DNA was analysed with primers for the MHC2TA promoter IV (a) or the G1P3 promoter (b). Actin, RNA polymerase II and acetylated histone-H4 are not present at the MHC2TA promoter IV or the G1P3 promoter before activation (-). After induction, however, actin and RNA polymerase II are recruited to the promoter region (+) and the histone H4 is acetylated. Input represents a 1:10 dilution series of the genomic DNA used.

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INF-gamma-induced chromatin remodelling of the MHC2TA promoter IV is BRG1 dependent26; furthermore, actin can be found associated with BRG1 (ref. 27). Therefore, it is possible that the presence of actin at the promoter region of this gene results from the recruitment of the BRG1 complex to the promoter. To investigate this possibility, we analysed the promoter region of the INF-alpha-inducible gene G1P3 because the activation of this gene is independent of BRG1 activity28. HeLa cells were treated with INF-alpha for 16 h before ChIP analysis with primers specific for the G1P3 promoter region. Without induction, antibodies to actin, RNA polymerase II or acetylated histone H4 failed to precipitate DNA containing the promoter region of G1P3 (Fig. 5b). However, after induction by INF-alpha, histone H4 was acetylated and both actin and RNA polymerase II were recruited to the promoter region of the G1P3 gene. Taken together, the data in Fig. 5 demonstrate that actin is required at a step involving the initiation of transcription that is independent of chromatin remodelling by BRG1.

Actin is required for the initiation of transcription

The role of actin in transcription initiation was investigated next by using a limiting set of nucleotides containing UTP, alpha-32P-CTP and the dinucleotide ApC. The DNA template used in these experiments is designed so that it contains an adenine at position +1, whereas the next adenine is at position +16 (ref. 29). When ApC is used as a priming nucleotide instead of ATP, the polymerase pauses after it has formed an initiation complex and synthesized a 15-nucleotide-long transcript. To analyse the requirements for actin at this step of transcription, recombinant transcription factors and the purified RNA polymerase II were incubated with the anti-beta-actin, C4 or HUC 1-1 antibodies. Transcription was then initiated by adding the DNA template, ApC, UTP and alpha-32P-CTP and the synthesis of the 15-nucleotide transcript was monitored. The beta-actin and C4 antibodies inhibited transcription of the 15-nucleotide RNA product, whereas the control HUC 1-1 antibody had no effect (Fig. 6). These results demonstrate that beta-actin is critically involved in transcription initiation.

Figure 6: Antibodies to bold beta-actin inhibit transcription initiation.

Figure 6 : Antibodies to |[beta]|-actin inhibit transcription initiation.

This assay was performed as shown in Fig. 4a with two exceptions: first, ApC was substituted for ATP and the transcription products were analysed on a 20% acrylamide gel to resolve the 15-nt transcripts; second, the DNA template, recombinant transcription factors and purified polymerase II were incubated with 2 mug, each, of the HUC 1-1, anti-beta-actin or C4 antibodies. The control represents samples that are incubated with buffer only. Transcription upto position +15 was initiated by the addition of ApC, a-32P-CTP and UTP. The anti-beta-actin or C4 antibodies inhibited transcription initiation, whereas the control antibody HUC 1-1 had no effect. The 15-nt RNA product is indicated; RNA size markers are shown in the left lane.

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beta-Actin is required for the formation of pre-initiation complexes

Next, PICs were assembled on a biotinylated DNA template containing the AdML promoter immobilized on streptavidin-coated beads. PICs were assembled by incubating the template with a HeLa nuclear extract in the presence or absence of sarcosyl. Sarcosyl at a concentration of 0.05% inhibits PIC formation by preventing the binding of TBP to the TATA box30. The assembled PICs were washed, eluted from the beads and analysed by resolving on an SDS–PAGE gel and immunobloting with antibodies against TBP, RNA polymerase II and actin (Fig. 7a). TBP, the large subunit of RNA polymerase II, as well as beta-actin, were found in the PICs assembled in the absence of sarcosyl. In contrast, neither TBP, the large subunit of RNA polymerase II nor actin was found in the presence of sarcosyl. Because sarcosyl prevents the binding of TBP to the TATA box, the data demonstrate a specific interaction between actin and the PIC that requires the binding of TBP to the TATA box.

Figure 7: Actin is required for PIC formation.

Figure 7 : Actin is required for PIC formation.

(a) Actin is present in PICs. A biotinylated DNA template containing the AdML promoter was immobilized on streptavidin–Sepharose and incubated with a HeLa cell nuclear extract for 30 min at 30 °C in the presence (+) or absence (-) of 0.05% sarcosyl. The beads were then washed and the eluted complexes were resolved on an SDS–PAGE gel and analysed by immunoblotting with antibodies to RNA polymerase II (RNAPII), TFIID (TBP) or beta-actin. PICs formed in the absence of sarcosyl contain actin. In contrast, sarcosyl prevented the binding of TBP to the TATA box and the formation of PICs. The absence of actin in the presence of sarcosyl shows that TBP is required for actin to bind to the DNA and eliminates the possibility of a non-specific interaction between actin and DNA or the Sepharose beads. (b) Antibodies to beta-actin prevent association of RNA polymerase II with PICs. HeLa cell nuclear extracts were incubated with antibodies to beta-actin or with the control HUC 1-1 antibody before assembling PICs on the DNA template. Analysis of the DNA-bound complexes showed that antibodies to beta-actin markedly decreased the presence of actin and RNA polymerase II in the PICs, whereas the HUC 1-1 antibody had no effect on PIC assembly. Note that the binding of TBP to the DNA was not affected by either antibody. HeLa cell nuclear extract (NE) was used as positive control. (c) Co-immunoprecipitation of RNA polymerase II, TBP and actin. Western blot analyses of proteins immunoprecipitated using antibodies to actin and TBP demonstrated the presence of RNA polymerase II and TBP when actin was immunoprecipitated. Similarly, actin and RNA polymerase II were present when TBP was immunoprecipitated. Neither actin, TBP nor RNA polymerase II were present in immunoprecipitates using the HUC 1-1 antibody. The migration of the relevant molecular mass standard is shown on the left.

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Having demonstrated that beta-actin binds to the DNA as part of the PIC, the importance of actin in the formation of PICs was analysed next. For this, PICs were assembled as described above with the exception that the HeLa nuclear extract was incubated with anti-beta-actin or HUC 1-1 antibodies before adding the immobilized DNA template. Analysis of the promoter-bound proteins showed that neither antibody affected the binding of TBP to the AdML promoter (Fig. 7b). Nor did the HUC 1-1 antibody affect the binding of the large subunit of RNA polymerase II or beta-actin to the promoter. The anti-beta-actin antibodies, however, significantly decreased the amount of actin and RNA polymerase II bound to the promoter. Thus, the beta-actin antibodies inhibited PIC formation, suggesting that an interaction with actin is necessary for the integration of RNA polymerase II into PICs.

Lastly, we immunoprecipitated actin or TBP from nuclear extracts and analysed the immunoprecipitates by resolving on an SDS–PAGE gel and western blotting. The blots were probed with antibodies to the large subunit of RNA polymerase II and TBP. Because the molecular weights of the heavy chain of the precipitating antibody and actin are similar, the binding of the secondary antibody to the precipitating antibody could lead to a false positive. To eliminate this possibility, a biotinylated beta-actin antibody and streptavidin-conjugated horseradish peroxidase were used to probe for actin. RNA polymerase II and TBP co-immunoprecipitated with actin, and actin and RNA polymerase II co-immunoorecipitated with TBP (Fig. 7c). These data further support important interactions among actin, RNA polymerase II and TBP.

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Discussion

Transcription of DNA into mRNA is a complex, highly regulated process that involves chromatin remodelling, the formation of PICs and the binding of transcription factors to regulatory regions of DNA. Ultimately, large transcriptional complexes consisting of RNA polymerase II and other proteins translocate relative to equally large DNA molecules, thereby remodelling chromatin and synthesizing mRNA as transcription proceeds. Even though nuclear actin has been connected to many of these transcriptional events, no clear role has yet been established. In this study, we show for the first time that nuclear beta-actin is involved in transcription by RNA polymerase II in mammalian cells by being an integral part of the PIC.

This conclusion is based on the following data: first, antibodies to beta-actin inhibited the synthesis of nascent transcripts, in vivo (Fig. 1) and transcription by a purified, reconstituted transcription assay in vitro (Figs 2 and 4); second, adding actin to a highly purified RNA polymerase II fraction stimulated transcription (Fig. 4); third, actin co-purified and co-localized with RNA polymerase II (Fig. 3); fourth, a ChIP assay showed that actin is recruited to the promoter region of transcribing genes in vivo, suggesting that actin becomes involved at an early step of transcription (Fig. 5); fifth, antibodies to beta-actin inhibited the production of a 15-nucleotide transcript that is a pre-requisite for the commitment to elongation (Fig. 6); and finally, actin was found to be a component of PICs and depletion of actin prevented the formation of PICs (Fig. 7).

Our data also indicate a strong interaction between actin and RNA polymerase II. Immunoblots of the protein eluted from the various columns revealed the presence of actin and the large subunit of RNA polymerase II in each of these fractions (Fig. 3a). Although there is more actin in the flow-through fractions from the DEAE Sephadex and P11 columns, there is an abundance of actin and RNA polymerase II in all of the eluted fractions. The apparent association of actin and RNA polymerase II through multiple column steps suggests a significant interaction between these two proteins. Actin was also present in the RNA polymerase II purified using HPLC24. These data, along with the immunolocalization of RNA polymerase II and actin (see Supplementary Information, Fig. S1), the ChIP assay (Fig. 5) and the PIC assay (Fig. 7), suggest a strong and specific interaction between actin and RNA polymerase II.

As for a mechanism, the data suggest that actin may be involved in recruiting RNA polymerase II to the PIC and/or that it functions as a bridge between the polymerase and the other constituents of the PIC. This notion is supported by three experiments. First, the ChIP assays show that actin is recruited to genes that are poised to start transcribing. Second, because sarcosyl inhibits PIC formation by preventing the binding of TBP to the TATA box, the absence of actin in the presence of sarcosyl (Fig. 7a) demonstrates that PIC formation is required for the association of actin with promoter DNA. These two experiments also belie the idea that actin non-specifically interacts with the promoter region; if this were the case, actin would have been found at the promoter in the non-induced genes and in the presence of sarcosyl. Third, antibodies to beta-actin prevent PIC formation. One plausible explanation for this is that immunodepleting actin also depletes the RNA polymerase II because of a specific interaction between the two proteins, a conclusion that is supported by the co-purification of actin with RNA polymerase II (Fig. 3a). A second possibility is that the binding of the antibodies to actin prevents a protein–protein interaction that is necessary for the binding of the RNA polymerase II to the PIC. In either case, our data demonstrate the central importance of beta-actin in the formation of PICs.

The presence of actin in PICs and its role in PIC formation is of considerable interest in light of recent studies showing that actin and actin-related proteins (ARPs) are associated with components of chromatin remodelling complexes8. The organization of chromatin presents a barrier to transcription and it has been assumed that ATP-dependent remodelling of chromatin is a pre-requisite for PIC formation. Among other roles, it has been suggested that actin, as a component of the SWI/SNF-like BAF chromatin-remodelling complex, could enhance the ATPase activity of one of the subunits of the BAF complex27. Based on these data, it could be predicted that the presence of actin at the pre-initiation stage of transcription is because of its role in chromatin remodelling. That is, the observed inhibition of transcription by the actin antibodies could be due to an interaction with the actin in the BAF complex.

Our data exclude this possibility. Antibodies to beta-actin inhibited transcription by a nuclear extract in an in vitro transcription assay on naked DNA that does not require chromatin remodelling. Furthermore, by using an in vitro transcription assay that only includes purified transcription factors, we could exclude the presence of chromatin-remodelling factors like BRG1 with which actin could interact. Therefore we can conclude that the observed effect of antibodies directed against actin results from a direct involvement of actin in PIC formation, rather than through a secondary effect based on an involvement of actin in chromatin remodelling.

The observation that exogenous beta-actin stimulates transcription also warrants comment. This effect could result from actin promoting PIC formation, the stabilization of PICs, stimulating re-initiation or another aspect of transcription. We recently identified myosin IC (NMI), a member of the myosin superfamily of actin-based motors, in the nucleus31, 32. This has raised another intriguing role for actin in transcription. NMI and RNA polymerase II co-localize and antibodies directed against NMI inhibit transcription by RNA polymerase II in vitro32. Because all myosins are actin-dependent ATP hydrolases33, a possible role for NMI in transcription is predicted to involve actin. For instance, actin and NMI could function as a molecular motor, especially during transcription elongation. Because actin stimulates transcription, it is tempting to speculate that actin and NMI are involved in transcription elongation. At the same time, this study shows that nuclear actin has a functional role at the pre-initiation stage of transcription and that it is necessary for integrating RNA polymerase II into the PIC. Consequently, our data suggest that actin is involved in multiple stages of the transcription process.

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Methods

Antibodies.

The monoclonal antibody to beta-actin (Sigma, St. Louis, MO) is an anti-peptide antibody that specifically recognizes only the beta-actin isoform18. HUC 1-1 is a monoclonal antibody that binds to an epitope shared by all muscle actins without recognizing non-muscle actins19. C4 reacts with an epitope present in muscle and non-muscle actin17. B4 shows a selective reactivity for smooth muscle beta-actin with some cross reactivity towards the other muscle actins but no binding to cytoplasmic actins17. The 56-4 antibody reacts with both non-muscle and muscle actins (J.L.L., unpublished observation). The HUC 1-1 and anti-beta-actin antibodies were affinity purified on Protein A-Sepharose before use. The purified anti-beta-actin antibody was biotinylated using the EZ-Link Sulfo-NHS-Biotinylation kit from Pierce (Rockford, IL) following the manufacturer's protocol. A streptavidin–horseradish peroxidase conjugate used to detect the biotinylated antibody was purchased from Amersham Pharmacia (Piscataway, NJ). The 8WG16 monoclonal antibody to the C-terminal domain of RNA polymerase II and the antibody specific for TFIID (TBP) were obtained from BABCO (Richmond, CA) or Santa Cruz Biochemical (Santa Cruz, CA), respectively. The anti-acetyl-histone H4 antibody (ChIP grade) was acquired from Upstate Biotechnology (Lake Placid, NY). The peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies were purchased from Vector Laboratories (Burlingame, NH) or from Research Diagnostics (Flanders, NJ) respectively.

Microinjection experiments.

HeLa cells grown in DMEM supplemented with 10% FBS were trypsinized and 20,000 cells were seeded onto 20-mm diameter, fibronectin-coated glass coverslips (BD Biosciences, Palo Alto, CA). The cells were grown overnight in 0.5% FBS in DMEM and nuclei were microinjected with an Eppendorf automated microinjection system linked to an Olympus IX70 microscope. Injections were performed at 37 °C on a heated stage. The antibodies were dialysed in PBS, concentrated and combined with rhodamine-labelled dextran. The dextran was used to identify injected nuclei. Because of constant discharge from the pipette, there was usually some dextran in the cytoplasm. The final concentrations were: HUC 1-1 antibody, 6.8 mg ml1; beta-actin antibody, 4.6 mg ml1; and rhodamine-dextran, 5 mg ml1. About 50 fl were injected into each nucleus and about 20 nuclei were injected for each coverslip in 30 min. The microinjected cultures were then incubated for 1 h at 37 °C. The cells were washed twice in PBS containing 1 mM Mg2+ at room temperature and twice in ice-cold 100 mM potassium acetate, 30 mM KCl, 10 mM Na2HPO4, 1 mM MgCl2, 1 mM Na2ATP, 1 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 50 mg ml-1 bovine serum albumin (BSA), 5 U ml-1 RNAGuard (Pharmacia) at pH 7.2 (collectively termed 'PB+'). Cells were permeabilized by incubating for 5 min in ice-cold PB+ containing 0.25 mg ml-1 saponin (Sigma). The cells were then washed twice in ice-cold PB+ and incubated in 100 mM KCl, 5 mM MgCl2, 0.5 mM EGTA, 25% Glycerol, 1 mM PMSF, 2 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.02 mM UTP, 0.2 mM Br-UTP, 1 U ml-1 RNAGuard and 50 mM Tris-HCl at pH 7.4 for 10 min at 35 °C. Cells were rinsed twice with ice-cold PB+ and fixed and permeabilized as described29. Nascent transcripts were visualized with an Alexa Fluor 488 conjugated monoclonal antibody to BrdU (Molecular Probes, Eugene, OR). The cells were also treated with 1 mug ml-1 DAPI (Sigma) to stain DNA. The coverslips were mounted in ProLong Antifade (Molecular Probes) and examined using an Olympus IX70 microscope equipped with a Cooke Sensicam. Slidebook software from Intelligent Imaging Innovations (Denver, CO) was used for the capture and deconvolution of the images.

Purification of RNA Polymerase II.

RNA polymerase II was purified from isolated HeLa cell nuclei34 using one of two methods. The first method used ammonium sulfate precipitation and sequential DEAE cellulose, DEAE-Sephadex A-25 and phosphocellulose column chromatography22. The second method included an HPLC step24. Only the experiment in Fig. 4c used HPLC-purified RNA polymerase II, and reaction mixtures using this highly purified enzyme were incubated for 6 min after the addition of nucleotides. The presence of RNA polymerase II in column fractions was determined by protein immunoblotting. The purified RNA polymerase II was aliquoted and stored at -80 °C in 150 mM ammonium sulphate, 2 mM DTT, 0.1 mM EDTA, 25% glycerol, 0.2 mM PMSF and 50 mM Tris-HCl at pH 7.9.

In vitro transcription assays.

In vitro transcription assays with nuclear extract were performed using the HeLaScribe nuclear extract in vitro transcription system (Promega, Madison, WI) following the manufacturer's protocol, with one exception: the DNA template used was negatively supercoiled plasmid DNA containing the adenovirus major late promoter (AdMLP) fused to a 380-bp G-less cassette29.

The in vitro transcription assays using purified transcription factors were performed as described29, with minor modifications. Reactions were performed at 30 °C and each reaction contained 5 ng of TBP, 10 ng of TFIIB, 6 ng of TFIIF, 70 ng of RNA polymerase II, 1 nM template DNA and 625 muM UTP, 625 muM alpha-32P-CTP (5 muCi per reaction) and 625 muM ApC or ATP. Nuclear extract or the recombinant transcription factors with purified RNA polymerase II and template DNA were incubated with 2 mug of the respective anti-actin-antibody (or less in Figs 4c and Supplementary Information, Fig. S2) or with 7 mug NEM-modified S1 for 30 min on ice. Nucleotides were then added to initiate transcription. After incubation for 20 min, the transcription products were extracted and separated by 6% (to monitor the 390-nucleotide transcript) or 18% (to monitor the 15-nucleotide RNA product) polyacrylamide, 7 M urea denaturing gel electrophoresis and visualized using a PhosphorImager (Molecular Dynamics, Amersham). The Decade marker system (Ambion, Austin, TX) was used to size RNAs. In all experiments, the densities of the bands representing the transcription products were quantified using ImageQuant software (Molecular Dynamics) and expressed as a fraction of the band in the control lane. The mean fractional activity is shown at the bottom of each lane of the relevant figures.

Immunoprecipitation experiments.

HeLa cell nuclear extract (100 mug) diluted in 10 volumes of 10 mM Tris-HCl at pH 7.9, 20 mM HEPES at pH 7.9, 8% glycerol, 45 mM KCl, 8 mM MgCl2, 5 mM (NH4)2SO4, 2% PEG, 4.5 mM beta-mercaptoethanol and 0.05 mM EDTA (collectively referred to as 'TB') were incubated with 10 mug of either anti-TFIID (TBP) or anti-beta-actin antibody for 2 h at 4 °C. Protein-A–Sepharose (250 mul of a 50% solution; Pharmacia) was added and the mixture was incubated for another hour at 4 °C. The beads were washed extensively in TB buffer, eluted by boiling in SDS sample buffer and analysed by protein immunoblotting using antibodies to TFIID or the large subunit of RNA polymerase II. Actin was visualized using a biotinylated anti-beta-actin antibody.

Pre-initiation complex formation.

PICs were formed on immobilized linear DNA template as described35, with minor modifications. Briefly, PCR was used to prepare a linear, 5' biotinylated DNA template from a plasmid that contained the adenovirus major late promoter fused to a 390-bp G-less cassette in the background of the pBluescript (KS+) plasmid. The biotinylated template was bound to streptavidin–Sepharose beads (Pharmacia). The DNA-bound beads were washed in TB. PICs were assembled on the immobilized DNA templates by incubating with HeLa nuclear extract in transcription buffer for 45 min at 30 °C in the presence or absence of 0.05% sarcosyl. The beads were washed with 50 volumes of ice-cold TB containing 0.025% sarcosyl. PICs were then eluted by boiling in SDS sample buffer, and were analysed by protein immunoblotting. In the case of antibody treatment, HeLa nuclear extracts were incubated with either buffer, HUC 1-1 antibody (4 mug) or anti-beta-actin antibody (4 mug) for 30 min on ice before incubation with the immobilized DNA template.

Chromatin immunoprecipitation assays.

HeLa cells grown in DMEM supplemented with 10% FBS were left untreated or treated with IFN-gamma for 16 h or with INF-alpha for 6 h before the ChIP assay, which was performed as described36 but with some modifications. Pre-clearing of the chromatin solution after sonication was carried out using protein-G–Sepharose (Amersham) blocked with 1 mg ml-1 of sheared salmon-sperm DNA (Ambion) and 1 mg ml-1 BSA. Immunoprecipitation was performed overnight at 4 °C using either non-specific IgG or antibodies to beta-actin, RNA polymerase II or acetyl histone-H4. Immunoprecipitation, washes and elutions were performed as described26, except that protein G-Sepharose was used. The DNA fragments were purified with the QiaEx II gel extraction kit (Qiagen, Valencia, CA) and PCR performed. The MHC2TA promoter-IV-specific primers were 5'-TTGGACTGAGTTGGAGAG-3' and 5'-GTGACCTTGAGCAAGTAG-3'. The G1P3 promoter-specific primers were 5'-GGTGAAAGGCCTGTGTGCC-3' and 5'-CAGCGAGTAAACGGTTCTCCG-3'.

BIND identifiers.

Eight BIND identifiers (www.bind.ca) are associated with this manuscript: 180058, 180059, 180060, 180061, 180062, 180063, 180067 and 180068.

Note: Supplementary Information is available on the Nature Cell Biology website.



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Acknowledgements

We thank P. Raychaudhury (Department of Biochemistry, University of Illinois at Chicago; UIC) for providing HeLa cells for purifying RNA polymerase II; M. Vigneron (Illkirch, France) for providing antibodies to RNA polymerase II; R. Moss (University of Wisconsin) for providing NEM-S1; M. L. Chen (Research Resources Center, UIC) for assistance with the confocal microscopy; and S. Huang (Northwestern University) for assistance with the in vivo transcription assay. V.F. was supported by the Grant Agency of the Czech Republic (Reg. No 304/03/P118). P.H. was supported by the Grant Agency of the Czech Republic (Reg. No. 304/01/0661 and 2004/04/0108), by the Grant Agency of the Academy of Sciences of the Czech Republic (Reg. No. IAA5039202) and by the Institutional Grant No. AV0Z5039906. Additional support was provided by grants from the US Public Health Service to P.H. (NSF/MSMT Program ME143), J.L.L. (HL57291), J.A.G. (GM55235), T.T.H. (AI 047770) and P.de.L. (NSF INT 9724168 and NIH HL 56489).

Competing interests statement

The authors declare no competing financial interests.

Received 10 September 2004; Accepted 20 September 2004; Published online 24 October 2004.

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  1. Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL 60612, USA.
  2. Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague 142 20, Czech Republic.
  3. Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
  4. Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH 45229, USA.
  5. Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA.

Correspondence to: Primal de Lanerolle1 e-mail: primal@uic.edu

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