FACT facilitates chromatin transcription by RNA polymerases I and III

Abstract

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes.

Figure 1

Pol Iα can transcribe through nucleosomal DNA in vitro. (A) Diagram of the reconstituted mono-nucleosomal template. A 3′-end Cy5-labelled 178-bp DNA fragment containing the MMTV nucleosome positioning sequence A and an additional 31 bp was reconstituted with recombinant histone octamers and analysed by 5% native polyacrylamide gel electrophoresis and Cy5 imaging (lane 1; N, mono-nucleosomal DNA; RF, residual nucleosome-free DNA). The presence of the nucleosome protects the AvaII site from digestion; AvaII digestion yields 122- and 56-bp DNA fragments (at the bottom of the gel) from the residual nucleosome-free DNA (lane 2), as revealed following SYPRO Green staining. (B) Mono-nucleosomal (N, lane 1) or nucleosome-free (F, lane 2) DNA templates were incubated with Pol Iα in a transcription reaction mix. Radiolabelled full-length transcripts (178 nt) were analysed on a 7.5 M urea 11% polyacrylamide gel and quantified with the aid of a phosphorimager. Transcript levels are expressed relative to the levels of transcripts detected in the nucleosomal transcription reactions (set at 1.0; rel. transcr.). (C) The 178 nt transcripts generated in Pol Iα transcription reactions with a mono-nucleosomal template are not due to transcription of residual nucleosome-free DNA in that reaction. Pol Iα transcription reactions (as in (B)) through a mono-nucleosomal (N, lanes 1 and 2) or nucleosome-free (F, lanes 3 and 4) DNA template were performed in the absence (lanes 2 and 4) or presence (lanes 1 and 3) of AvaII. Transcripts were analysed as in (B); full-length transcripts of 178 nt and smaller transcripts of 122 and 56 nt, produced from AvaII-digested DNA templates, are indicated on the right of the gel image. RNA size markers are shown on the left of the gel image. Bands marked with an asterisk (^*) are nonspecific background signals, largely independent of the level of Pol I transcription. (D) Diagram of the reconstituted poly-nucleosomal template. A linear DNA fragment of 2496 bp containing twelve 5S nucleosome-positioning sequences was reconstituted into chromatin (Poly-N) and a sample was analysed on a native 2% polyacrylamide–1% agarose composite gel, stained with SYPRO Green and scanned for fluorescence (left panel; RF indicates the residual nucleosome-free DNA following the reconstitution reaction). The Poly-N DNA, untreated (lane 2, right panel) or limit-digested with MNase (lane 3, right panel), was analysed on a 1.2% agarose gel and ethidium bromide-stained. A DNA size marker (100 bp ladder) was run in lane 1 (right panel). (E) Pol Iα transcription reactions with poly-nucleosomal (Poly-N, lane 1) or an equivalent amount of nucleosome-free (F, lane 2) DNA template (0.3 μg). Radiolabelled transcripts were analysed on an 8% denaturing polyacrylamide gel and detected by phosphorimaging. The full-length transcript signal is indicated. (F) Pol Iα transcription reactions with poly-nucleosomal (Poly-N, lanes 1 and 3) or non-nucleosomal (F, lanes 2 and 4) DNA templates (0.3 μg), included either NTPs (lanes 1 and 2) or an NTP mixture with a non-hydrolysable analogue of ATP, AMP-PNP, substituted for ATP (lanes 3 and 4). Radiolabelled transcripts (2496 nt) were analysed on a 1.2% formaldehyde-agarose gel and detected by phosphorimaging. Transcript levels were quantified and expressed as in (B).

Figure 2

Pol I transcription of a mono-nucleosomal template is inhibited by crosslinking of the histones within the nucleosome. (A) Crosslinking of the histones in the octamer of the mono-nucleosomal template with the homo-bifunctional crosslinker bis(sulphosuccinimidyl) suberate (BS³). Histones from the BS³-treated (lane 2) and untreated (lane 3) mono-nucleosomal templates were analysed on a 4–12% gradient denaturing protein gel, SYPRO Ruby-stained. The positions of free histones and crosslinked histone octamers are marked. Lane 1 contains a protein size marker. (B) Duplicate transcription reactions contained the mono-nucleosomal 178 bp DNA template (0.3 μg) pretreated with BS³ (+, lanes 1 and 2), inactivated BS³ (+ⁱ lanes 3 and 4) or crosslinking buffer alone (−, lanes 5 and 6), or untreated (control C, lanes 7 and 8). Radiolabelled transcripts were analysed by 7.5 M urea 11% polyacrylamide gel electrophoresis and phosphorimaging. The position of the full-length transcript (178 nt) from a representative experiment is indicated (as determined using a radiolabelled RNA size marker). The graph shows the results of duplicate reactions from two independent experiments; average transcript (178 nt) levels are expressed in arbitrary units (AU) and the ranges are indicated.

Figure 3

FACT associates with purified Pol Iα and with Pol I, II and III complexes from HeLa cell nuclear extract. (A) Mono-S fraction of Pol Iα, purified from HeLa cell nuclear extracts in a multistep process as detailed in the Materials and methods, was separated into its subunits and associated proteins on a 4–12% gradient denaturing protein gel and SYPRO-Ruby stained (see also Panov et al, 2006b). The bands representing the SSRP1 (structure-specific recognition protein, ∼80 kDa) and Spt16 (∼130 kDa) subunits of FACT, as identified by mass spectrometric analysis, and the positions of nine of the core Pol I subunits (following the nomenclature in Panov et al, 2006b) are indicated. (B) Pol Iα gradient salt-eluted fractions from the Mono-S column were analysed for Pol I transcription activity in a nonspecific transcription assay (graph, fractions 15–29; inclusion of 0.1 mg/ml α-amanitin does not affect RNA synthesis activity; Miller et al, 2001) and by immunoblotting (fractions 18–26) using antibodies specific for SSRP1 (upper panel) and for Pol I subunit hRPA19 (lower panel). (C) Antibodies specific for SSRP1 or control IgG were used for immunoprecipitation of Pol Iα (peak activity from the Mono-S column) and the immunoprecipitates (IP, beads) and supernatants (Sup, following IP) were analysed for Pol I transcription activity in a nonspecific transcription assay. Transcript levels from two independent experiments were quantified, expressed as a percentage of maximum, set at 100%, and plotted as the average and range. (D) Antibodies specific for Spt16 (lanes 1 and 2) or control IgG (lanes 3 and 4) were used for immunoprecipitation from Pol Iα (as above) and the immunoprecipitates (IP, lanes 2 and 4) and supernatants (Sup, lanes 1 and 3) were analysed by immunoblotting using antibodies specific for Pol I subunit hPAF53. (E) Pol I was immunoprecipitated through the Flag epitope from nuclear extracts of HeLa cells expressing Flag-tagged Pol I subunit CAST. Flag antibody immunoprecipitates (Flag-IP, lane 3), the supernatants (10%) of the immunoprecipitation (Sup, lane 2) and control immunoprecipitates (IgG-IP, lane 1) were immunoblotted with antibodies specific for Spt16 and Pol I subunit PAF53. (F) Pol Iβ was immunoprecipitated through hRRN3 from a chromatography fraction containing both Pol Iα and Iβ (0.2 M KCl DEAE fraction; Miller et al, 2001). hRRN3-antibody immunoprecipitates (lane 4), the supernatant (10%) left following immunoprecipitation (lane 2), control immunoprecipitation with IgG (lane 1) and a control fraction (C) of purified Pol Iα (containing FACT; lane 3) were immunoblotted with antibodies specific for Spt16 and human Pol I subunits RPA190 and PAF53. (G) Immunoprecipitation of FACT and associated RNA polymerases from HeLa cell nuclear extracts. Immunoprecipitation (IP) was performed with the SSRP1 mouse monoclonal (10D1) antibody (lane 3) or with control mouse IgG (lane 2). Immunocomplexes were boiled in SDS sample buffer and subsequently analysed by SDS–PAGE and immunoblotting using antibodies specific for FACT subunits SSRP1 and hSpt16, Pol I subunit CAST, Pol II largest subunit CTD4H8 and Pol III subunit RPC5. In lane 1, 1.5% of the input (In) nuclear extract was loaded.

Figure 4

SSRP1-specific antibodies selectively inhibit nucleosomal transcription by Pol Iα. Pol Iα was pre-incubated with SSRP1 antibodies (1 μg, sc-25382; lanes 1 and 2), with control rabbit IgGs (lanes 3 and 4) or in buffer alone (lanes 5 and 6) prior to incubation with mono-nucleosomal templates (N; lanes 2, 4 and 6) or nucleosome-free templates (F; lanes 1, 3 and 5) in a transcription reaction mix. Radiolabelled transcripts were analysed by 7.5 M urea 11% polyacrylamide gel electrophoresis and phosphorimaging. The two RNA bands around 178 nt are the products of bi-directional end-to-end transcription; due to the asymmetrical nature of the nucleosomal template, Pol I favours one DNA-end over the other. The 178-nt transcripts levels of two independent experiments were quantified (in arbitrary units) with the aid of a phosphorimager. The data of duplicate samples are presented as the average and range. Note the two scales for the Y axis; transcription signals (in arbitrary units) from the nucleosomal template (N, black bars) are approximately 10 times lower than those from the equivalent nucleosome-free DNA template (F, white bars).

Figure 5

Subcellular localization of FACT and Pol I. Indirect immunofluorescence analysis was performed on HeLa cells to observe localization of endogenous SSRP1 (red) and CAST/hPAF49 (green). Before immunostaining, HeLa cells were cultured in the presence of 50 ng/ml actinomycin D for 30 min to inhibit Pol I activity (+Act D) or in its absence (−Act D). Nuclear DNA was stained by DAPI (blue). Images were captured by laser scanning confocal microscopy and single sections are shown (scale bar is 10 μm).

Figure 6

FACT is present at the chromatin of genes transcribed by all three nuclear RNA polymerases. (A) Schematic representation of the rDNA repeat region indicating the positions of the PCR primers used in chromatin immunoprecipitation (ChIP) analysis. (B) ChIP assays were performed on formaldehyde crosslinked chromatin from HeLa cells using antibodies specific for FACT subunit SSRP1 (monoclonal 10D1) or control mouse antibodies (IgG). Primer sets used in the quantitative real-time PCR for the Pol I-transcribed rDNA repeat were as follows: the ribosomal DNA promoter (promoter, 1), 5′-ETS (2), 18S (3) or 28S (4) gene region, or the non-transcribed intergenic spacer (IGS; 5) of the rDNA repeat (indicated in (A)). PCR primers also included those that detect the γ-actin gene transcribed by Pol II and four Pol III-transcribed genes (5S rRNA, U6 snRNA, tRNA^Tyr and 7SL RNA). The bar graph shows the relative levels of bound DNA in the SSRP1 immunoprecipitates, as determined by quantitative RT–PCR; results from three independent ChIP experiments combined are expressed as a percentage of input chromatin (and standard deviation), normalized to control IgG samples.

Figure 7

RNAi-mediated downregulation of FACT expression in cells leads to a decrease in the synthesis of RNAs from Pol I- and Pol III-transcribed genes. (A) Whole-cell extracts (20 μg) of HeLa cells transfected with various amounts of SSRP1-specific siRNAs (sc-37877; lanes 1–4) or with control siRNAs (control sc-36869, lane 5) were immunoblotted using antibodies specific for FACT subunit SSRP1 (top panel) or Pol I subunit PAF53 (bottom panel). (B) HeLa cells were transfected with 20 nM of SSRP1-specific siRNA (SSRP1, lanes 3 and 4) or control siRNA (control, lanes 1 and 2) as in (A), then total RNA was extracted from the cells at 24 h (lanes 1 and 3) and 48 h (lanes 2 and 4) post-transfections. The levels of 47S pre-rRNA were analysed in a northern blot probed with a ³²P-labelled DNA probe complementary to a region from +81 to +125 relative to the transcription start site (top panel). The levels of 28S and 18S rRNA were assessed in a 0.8% agarose denaturing gel, stained with ethidium bromide (bottom panel). The levels of 47S pre-rRNA (24 h post-transfections) of two independent experiments were quantified by phosphorimaging, normalized to 28S rRNA levels, calculated as a percentage relative to the maximum level (set at 100%) in the control siRNA-treated cells. The data of duplicate samples are presented as the average and range. (C) HeLa cells grown on coverslips were transfected with plasmids expressing control or SSRP1-specific siRNAs (si-SSRP1) and 54 h post-transfection nascent RNA synthesis was visualized through a 10 min BrUTP labelling of cells. Cells were probed with antibodies for SSRP1 (green) and BrdU (red). Within the same microscopy field, a mixture of cells can be seen in the bottom of the siSSRP1 panels; some cells have reduced levels of SSRP1 and BrUTP incorporation (white arrowheads), whereas others appear to have levels unaffected and hence serve as an internal reference. All images were generated at pre-set levels of brightness/contrast and laser intensity, by laser scanning confocal microscopy, and single sections are shown (scale bar is 10 μm). (D) Total RNA was extracted 24 h after HeLa cells were transfected with 20 nM of specific (SSRP1, grey bars) or control siRNA (control, black bars) as in (A). Samples were analysed by S1 nuclease protection with an oligonucleotide probe complementary to +1 to +40 of the 47S pre-rRNA. A representative phosphorimage of the protected 40 nt S1 probe is shown on the left. S1 signals from two experiments were quantified with the aid of a phosphorimager, normalized to 28S rRNA levels (as derived from the ethidium bromide image of the same samples analysed on denaturing agarose gels, see (B)), calculated as a percentage relative to the maximum level (set at 100%) in the control siRNA-treated cells. The data of duplicate samples are presented as the average and range. (E) RNAi-mediated downregulation of FACT expression in HeLa cells reduced pre-rRNA levels. RNAi-mediated downregulation of SSRP1 or hSpt16 expression was assessed by immunoblotting with antibodies for SSRP1 or hSpt16 (lanes 1 and 3, respectively) and compared with controls (lanes 2 and 4). Actin was used as a control for equal loading of the cell extracts. The levels of 47S pre-rRNA in SSRP1 (grey bar) and hSpt16 (white bar) RNAi-treated cells were determined by qRT–PCR from three independent experiments. The results were normalized to 18S rRNA levels of the respective samples, and the mean and standard deviation values have been plotted. (F) The levels of tRNA^Tyr and 7SL RNA from two independent experiments were determined by qRT–PCR in cells treated with SSRP1-RNAi (grey bar) or control RNAi (black bar). The levels were normalized to GAPDH mRNA levels and expressed as a percentage of those detected in control RNAi-treated cells. The data of duplicate samples are presented as the average and range.