Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription - PubMed (original) (raw)
Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription
V Muth et al. EMBO J. 2001.
Abstract
Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.
Figures
Fig. 1. TTF-I interacts with PCAF. (A) Pull-down of cellular HAT activity by TTF-I. A 10 µg aliquot of FLAG-TTF-I bound to 10 µl of M2-agarose beads (lane 3) or M2 beads saturated with the FLAG peptide (lane 4) was incubated with 2 mg of mouse whole-cell extract proteins for 4 h at 4°C in a total volume of 380 µl. After stringent washing, 50% of the beads were assayed for HAT activity using 5 µg of histones and 1 µCi of [3H]acetyl-CoA. In lanes 1 and 2, histone acetylation by recombinant PCAF or cell extract is shown. Histones were separated by 15% SDS–PAGE and visualized by Coomassie Blue staining (left panel) and fluorography (lanes 1–4). (B) Interaction of PCAF with bead-bound TTF-I. A 35 µl aliquot of Ni2+-NTA–agarose saturated with histidine-tagged TTF-I (lane 1) or cyclin A (lane 2), and M2-agarose saturated with FLAG-UBF (lane 3) were incubated with extracts from mouse cells. After washing, bead-bound proteins were subjected to western blot analysis using anti-PCAF antibodies. (C) Association of cellular TTF-I with PCAF. Bead-bound FLAG-PCAF (lane 2) or control beads (lane 3) were incubated with extract from mouse cells, and associated TTF-I was identified on immunoblots. In lane 1, the amount of TTF-I present in 10% of the extract is shown. (D) Co-immunoprecipitation of TTF-I and PCAF. Histidine-tagged TTF-I or cyclin A was co-expressed with FLAG-PCAF in Sf9 cells. PCAF was precipitated with anti-FLAG antibodies (M2) and analyzed on western blots for the presence of TTF-I (lanes 1 and 2) or cyclin A (lanes 3 and 4).
Fig. 2. The C-terminal part of TTF-I interacts with PCAF. N-terminal His-tagged deletion mutants TTFΔN185 (lanes 1, 4 and 7), TTFΔN323 (lanes 2, 5 and 8) and TTFΔN445 (lanes 3, 6 and 9) were expressed in Sf9 cells in the presence (lanes 1–3 and 7–9) or absence (lanes 4–6) of FLAG-PCAF. PCAF was bound to M2-agarose by immunoadsorption, and associated TTF-I was identified on immunoblots with anti-TTF-I antibodies. In lanes 1–3, the amount of TTF-I present in 5% of the cell lysates is shown. A schematic representation of TTF-I is shown above. The negative regulatory domain (NRD) and the DNA-binding domain are indicated. The numbers mark amino acid positions.
Fig. 3. PCAF acetylates TAFI68 in vitro. (A) Acetylation of recombinant proteins. A 2 µg aliquot of the proteins indicated was incubated with 500 ng of FLAG-PCAF, 1 µCi of [3H]acetyl-CoA and 0.4 µM TSA in a total volume of 30 µl of buffer AM-100 for 30 min at 30°C. Proteins were separated by 10% SDS–PAGE, and acetylated proteins were visualized by fluorography. (B) TAFI68 interacts with PCAF. GST–PCAF or GST were bound to glutathione–agarose beads and incubated with 35S-labeled TAFI68 (lanes 2 and 3), TTFΔN323 (lanes 5 and 6) or UBF (lanes 8 and 9). Bound proteins were analyzed by 8% SDS–PAGE and autoradiography. Ten percent of the 35S-labeled input proteins are shown in lanes 1, 4 and 7. (C) Acetylation of TAFI68 with CBP, GCN5 and PCAF. A 500 ng aliquot of TAFI68 was incubated for 30 min at 30°C with 1 µCi of [3H]acetyl-CoA, 0.4 µM TSA and comparable units of HAT activity of CBP (lane 2), GCN5 (lane 3) or PCAF (lane 4). After gel electrophoresis, acetylated TAFI68 was visualized by fluorography. A Coomassie Blue stain of 500 ng of TAFI68 is shown on the left.
Fig. 4. TAFI68 is acetylated in vitro and in vivo. (A) PCAF acetylates cellular SL1 in vitro. Immunopurified SL1 (lane 1), recombinant p53 (lane 2) and BSA (lane 3) were incubated with 500 ng of FLAG-PCAF and 1 µCi of [3H]acetyl-CoA, separated by 10% SDS–PAGE and acetylated proteins detected by fluorography. Autoacetylated PCAF is indicated. (B) TAFI68 is acetylated by the cellular PCAF complex. FLAG-tagged PCAF isolated from transfected NIH-3T3 cells (lane 1) or 500 ng of recombinant FLAG-PCAF purified from Sf9 cells (lane 3) was used to acetylate recombinant TAFI68 that was expressed in Sf9 cells (lane 2). Acetylation was monitored on immunoblots with α-acetyl-lysine antibodies. A Coomassie Blue stain of TAFI68 is shown on the left. (C) TAFI68 is acetylated in vivo. TIF-IB/SL1 was immunopurified from PC-1000 fractions (Schnapp and Grummt, 1996) from Ehrlich ascites (lane 1) and HeLa cells (lane 2), and subjected to western blotting using α-acetyl-lysine antibodies (lanes 3 and 4). In lanes 1 and 2, the western blot was reprobed with α-TAFI95 and α-TAFI68 antibodies.
Fig. 5. TSA treatment does not affect cellular pre-rRNA synthesis. (A) FACS analysis. FT210 cells were synchronized at the G2–M boundary by shifting to 39°C for 18 h and released from the G2–M block by shifting to the permissive temperature (33°C). Aliquots of cells were subjected to FACS analysis at the times indicated. (B) Measurement of pre-rRNA synthesis. RNA was prepared from synchronized cells grown in the absence or presence of the indicated amounts of TSA, and 10 µg were used for northern blots to determine 45S pre-rRNA levels.
Fig. 6. Acetylation augments binding of TAFI68 to the rDNA promoter. (A) TAFI68 binds specifically to the rDNA promoter. A 300 ng aliquot of recombinant FLAG-TAFI68 was incubated with 5 fmol of radiolabeled rDNA probe, and DNA–protein complexes were analyzed by electrophoresis on 5% native polyacrylamide gels. For competition, reactions were supplemented with 250, 500 and 1000 fmol, respectively, of an unlabeled fragment harboring sequences of either the rDNA promoter (lanes 3–5) or the rDNA terminator (lanes 6–8). (B) Binding of TAFI68 to the rDNA promoter is enhanced by acetylation. Increasing amounts (50–150 ng) of FLAG-TAFI68 were acetylated with PCAF, hGCN5 and CBP, respectively, and assayed for DNA binding by EMSA. In lanes 1–3, the reactions contained FLAG-TAFI68 alone.
Fig. 7. Acetylation by PCAF stimulates rDNA transcription in vitro. (A) PCAF-mediated transcriptional activation. Transcription factors and Pol I were pre-incubated in the absence (lane 1) or presence of 60 (lanes 2 and 4) and 160 ng (lanes 3 and 5) of purified GST–PCAF. Reactions 4 and 5 were supplemented with 10 µM acetyl-CoA. After pre-incubation for 30 min at 30°C, transcriptions were started by adding 20 ng of template DNA and ribonucleotides. (B) Transcriptional stimulation by PCAF requires a functional HAT domain. Transcription reactions were performed in the absence (lane 1) or presence of GST–PCAF (lanes 2 and 3) or GST–PCAF/ΔHAT (lanes 4 and 5) and 10 µM acetyl CoA as above.
Fig. 8. mSir2a deacetylates TAFI68 and decreases rDNA transcription. (A) mSir2a deacetylates TAFI68. FLAG-TAFI68 and FLAG-PCAF were co-expressed in Sf9 cells and immunopurified with α-FLAG antibodies. A 500 ng aliquot of purified protein FLAG-TAFI68 was incubated with 200 ng of recombinant mSir2a in the absence (lanes 1–4) or presence (lanes 5–8) of 1 mM NAD+ at 30°C for the times indicated. Reactions were subjected to 8% SDS–PAGE, and acetylated proteins were detected on immunoblots using α-acetyl-lysine antibodies. (B) Deacetylation of TAFI68 in the TBP–TAF complex. Recombinant TIF-IB was purified from Sf9 cells that co-expressed the three TAFIs, TBP and PCAF. A 40 ng aliquot of immunopurified complexes was incubated with 200 ng of mSir2 in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of 1 mM NAD+, subjected to 11% SDS–PAGE, and acetylated proteins were visualized on immunoblots using α-acetyl-lysine antibodies. (C) Deacetylation of TAFI68 is reversed by PCAF. Acetylated TAFI68 was incubated with mSir2a immobilized on Ni+-NTA–agarose for 60 min at 30°C in the absence (lane 1) or presence (lane 2) of 1 mM NAD+. Bead-bound Sir2a was removed by centrifugation, and an aliquot of the NAD+-treated reaction was incubated with 500 ng of GST–PCAF and 10 µM acetyl-CoA (lane 3). Acetylated proteins were detected on immunoblots with α-acetyl-lysine antibodies. (D) Deacetylation by Sir2p decreases rDNA transcription in vitro. Transcription factors and RNA polymerase I were pre-incubated for 60 min at 30°C with 200 ng of purified mSir2 immobilized on Ni+-NTA–agarose in the presence (lane 2) or absence (lane 3) of 1 mM NAD+. Bead-bound mSir2a was removed, and the transcriptional activity of the supernatant was measured in the presence of ribonucleotides and template DNA. In lane 1, NAD+ was added to the transcription reaction after the pre-incubation step.
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