Fig. 1. SAP130 is a component of the TFTC complex. (A) A 50 µg aliquot of HeLa cell nuclear extract (NE; lane 1), 15 µl of immuno purified and eluted SAP130 from HeLa NE [E(IPSAP130); lane 2], 15 µl of immunopurified TFTC (lane 3) and TFIIDβ (lane 4) were separated by SDS–PAGE, blotted on a nitrocellulose filter and examined for the presence of the indicated proteins using the corresponding antibodies. (B) TFTC (lane 1, and see C) was re-immunoprecipitated (IP) with either an anti-TAF_II135 mAb (lane 2) or an anti-hSPT3 mAb (lane 3), the mAb–protein G-bound proteins were washed extensively, separated by SDS–PAGE, blotted on a nitrocellulose filter and examined for the presence of the indicated proteins by using the corresponding antibodies. (C) A 15 µl aliquot of TFTC was separated by SDS–PAGE and analysed by silver staining.

Fig. 2. The SAP130 contained in the TFTC is not involved in splicing. (A) A 500 µg aliquot of NE was immunodepleted with an anti-TAF_II30 (ID TAF_II30) antibody. Proteins were analysed by western blotting using the indicated antibodies. The efficiency of the anti-TAF_II30 immunodepletion and that of the SAP130 depletion was calculated by using a Bio-Rad GS700 Imaging Densitometer. (B) The extracts tested in (A) were used for in vitro splicing assays with a short version of the adenovirus E1A gene, which is spliced only in the 13S mRNA. The pre-mRNA (Transcript) and the correctly spliced transcript (mRNA) are indicated. Where indicated, 100 ng of SR proteins and 150 ng of TFTC were added to the reactions. Res: control immunodepletion with protein G–Sepharose alone.

Fig. 3. SAP130 and TFTC preferentially bind to UV-damaged DNA. A ³²P-labelled DNA fragment was UV irradiated at different doses as indicated, incubated with increasing amounts of either immunopurified SAP130 (A) or TFTC (B) and tested for retention on nitrocellulose filters. The amounts (µl) of SAP130 and TFTC used in this assay were previously normalized in Figure 1A. Graphs represent the amount of labelled DNA retained on the filters in arbitrary units (AU). AUs were obtained after phosphoimager scanning of the nitrocellulose filters and by deducting the buffer-only background from every TFTC- or SAP130-containing sample. Similar and comparable results (± 5%) were obtained in at least three independent experiments. (C) UV-irradiated DNA inhibits TFTC-mediated Pol II transcription in vitro. TFTC and TFIIDβ were pre-incubated for 10 min with 25 ng of templates, containing the AdMLP or the β-globin (Glob) promoter, in the absence or presence (100 ng) of either UV-irradiated (compet. +UV) or non-UV-irradiated (compet. –UV) competitor DNA fragments. Then the heat-treated HeLa whole-cell extract (WCE, 45°C) was added for 20 min and transcription initiated. The positions of the correctly initiated transcripts from the AdMLP (+1) and the Glob (+1) promoters, determined by quantitative S1 mapping, are indicated. (D) Nucleosomal arrays were assembled as in Figure 5A and the binding of TFTC (10 µl) to the non-irradiated (–) or UV-C-irradiated (2.5 J/cm²) templates was analysed by western blotting with the indicated antibodies. (E) Preferential recruitment of TFTC components and the repair damage recognition protein XP-A from a repair-competent HeLa NE onto bead-linked UV-damaged DNA (2.5 J/cm²) (UV-C), but not bead-linked DNA containing single-stranded breaks (DNase I). The different bead-linked DNA fragments were incubated with the repair-competent HeLa NE (50 µg), washed, and bound proteins were analysed by western blotting. Chromatin assembly factor-1 (CAF-1 p60) is recruited during the repair of both types of damage.

Fig. 4. TFTC preferentially acetylates mononucleosomes assembled on UV-damaged DNA templates. (A) A bead-linked 220 bp DNA fragment irradiated with increasing UV-C doses was reconstituted either with (nucleosome) or without (naked DNA) human histone octamers. Bead-linked nucleosomes (or naked DNA) were then either (B) digested with MNase, transfered to a nylon membrane and hybridized with a ³²P-labelled probe, or (C) incubated with 200 ng of TFTC or 5 ng of p300 in the presence of [³H]acetylCoA, washed and counted for incorporation of radioactivity in histones. The graph represents the average of two independent experiments performed in duplicate and is normalized for the loss of DNA due to UV irradiation. Grey column, p300; white column, TFTC.

Fig. 5. TFTC preferentially acetylates nucleosomal arrays assembled on UV-damaged DNA templates. (A) A bead-linked DNA fragment, with five copies of the 208 bp 5S rDNA nucleosome positioning sequence that allows the formation of regular nucleosomal arrays, was irradiated with increasing UV-C doses (as indicated) and reconstituted with purified human histone octamers. Bead-linked nucleosomal arrays were either digested with MNase, separated on an agarose gel and stained with ethidium bromide (asterisks indicate nucleosome boundaries), or incubated with 200 ng of TFTC in the presence (+) or absence (–) of acetyl-CoA and washed. (B) Acetylated histones were analysed by western blotting using antibodies recognizing acetylated histones. Nucleosomes assembled on the different DNA fragments were verified with antibodies recognizing non-acetylated histone H4 or acetylated histone H3. Lane 1, magnetic only beads. (C) The graph represents the acetylation of histone H3 in four independent experiments after normalization for DNA and histone content in the UV-irradiated samples. The standard deviation is given by error bars.

Fig. 6. Histone H3 and H4 acetylation is increased in HeLa cells after UV irradiation. HeLa cells were either untreated (–), UV irradiated (+UV) or treated with sodium butyrate (+NaBu). Cells were then fixed and subjected to immunofluorescence detection. In each column, the right-hand panels show the Hoechst DNA staining (blue), and the left-hand panels correspond to immunodetection with specific antibodies (red), as indicated. A mouse IgG fraction was used as a control in (A), (C) and (E).