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• W1992238166 abstract "Article15 March 2001free access Acetylation of TAFI68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription Viola Muth Viola Muth Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Present address: INSERM U525, Faculté de Médecine, 91 Boulevard de l'Hôpital, 75013 Paris, France Search for more papers by this author Sophie Nadaud Sophie Nadaud Present address: INSERM U525, Faculté de Médecine, 91 Boulevard de l'Hôpital, 75013 Paris, France Search for more papers by this author Ingrid Grummt Ingrid Grummt Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Renate Voit Corresponding Author Renate Voit Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Viola Muth Viola Muth Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Present address: INSERM U525, Faculté de Médecine, 91 Boulevard de l'Hôpital, 75013 Paris, France Search for more papers by this author Sophie Nadaud Sophie Nadaud Present address: INSERM U525, Faculté de Médecine, 91 Boulevard de l'Hôpital, 75013 Paris, France Search for more papers by this author Ingrid Grummt Ingrid Grummt Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Renate Voit Corresponding Author Renate Voit Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Author Information Viola Muth1,2, Sophie Nadaud2, Ingrid Grummt1 and Renate Voit 1 1Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 2Present address: INSERM U525, Faculté de Médecine, 91 Boulevard de l'Hôpital, 75013 Paris, France ‡V.Muth and S.Nadaud contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1353-1362https://doi.org/10.1093/emboj/20.6.1353 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 TAFI68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAFI68 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 deacetyl ates TAFI68 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. Introduction Packaging of DNA into chromatin inhibits transcription at the initiation and elongation step. Therefore, for transcription to proceed the cell must recruit proteins to alleviate nucleosome-mediated transcriptional repression. Several laboratories have pursued the mechanisms by which nucleosome and chromatin structure are altered in order to facilitate binding of sequence-specific transcription factors to DNA and transcription initiation. These studies have revealed the participation of multiprotein complexes in the remodeling of chromatin structures. One group of such complexes are ATP-dependent chromatin remodeling machines, such as SWI/SNF, NURF, CHRAC, ACF and RSC, that are thought to facilitate transcription factor binding or function through alteration of nucleosome structure or position (for reviews see Wu, 1997; Kadonaga, 1998). A second group includes transcriptional coactivators with histone acetyltransferase (HAT) activity that, through acetylation of N-terminal lysines, alter histone–DNA interactions and nucleosome stability functions (for reviews see Wade and Wolffe, 1997; Struhl, 1998). Increased levels of histone acetylation are often associated with enhanced transcriptional activity (Hebbes et al., 1988), whereas hypoacetylation of histones correlates with transcriptional silencing (reviewed by Pazin and Kadonaga, 1997). In previous studies, we have demonstrated that alleviation of nucleosome-mediated transcriptional repression by DNA-bound proteins is also a key event in class I gene transcription on nucleosomal templates. Transcription of rDNA on chromatin requires binding of the transcription termination factor TTF-I to the promoter-proximal terminator element, known as T0, which is positioned 160 bp upstream of the transcription start site (Längst et al., 1997). Binding of TTF-I to T0 results in alterations of the chromatin structure, including the establishment of DNase I-hypersensitive sites adjacent to the bound factor and relocation of a nucleosome. Chromatin remodeling induced by TTF-I is a prerequisite for transcriptional activation on otherwise repressed nucleosomal rDNA templates. Apparently, TTF-I-mediated chromatin remodeling facilitates the access of basal RNA polymerase I (Pol I) transcription factors or recruits chromatin-modifying activities to the rDNA promoter (Längst et al., 1998). Consistent with this, sequence-specific transcription factors and coactivators have been demonstrated to target HATs and deacetylases to promoters. The finding that many coactivators (e.g. GCN5, CBP/p300, PCAF, ACTR, SRC-1, BRCA2 and TAFII250) are HATs that are contained in large multiprotein complexes has led to an appealing model that transcription activators recruit these enzymes to certain gene promoters to destabilize nucleosomes. These structural transitions enhance the accessibility of DNA to various factors, thereby reducing the repressive effect of nucleosomes. Most studies concerning the effect of acetylation on the structure and function of chromatin have focused on the reversible acetylation of core histones. In addition to histones, other chromatin-associated proteins are also modified by HATs, such as the non-histone proteins HMG-17 and HMGI(Y) (Munshi et al., 1998; Herrera et al., 1999) as well as several transcription factors including p53 (Gu and Roeder, 1997; Sakaguchi et al., 1998; Liu et al., 1999), GATA-1 (Boyes et al., 1998), EKLF (Zhang and Bieker, 1998), E2F1 (Martínez-Balbás et al., 2000), TCF (Waltzer and Bienz, 1998), HNF-4 (Soutoglou et al., 2000) and the basal factors TFIIE and TFIIF (Imhof et al., 1997). The list of acetylated proteins is increasing rapidly. Acetylation can have either stimulatory or inhibitory effects on transcription. In the case of p53, E2F1, GATA-1 and EKLF, acetylation occurs directly adjacent to the DNA-binding domain and acetylation enhances DNA binding in vitro and in vivo (Gu and Roeder, 1997; Sakaguchi et al., 1998; Liu et al., 1999; Martínez-Balbás et al., 2000). Specific acetylation of HMGI(Y) by CBP, on the other hand, leads to enhanceosome disassembly and turn-off of interferon-β gene expression (Munshi et al., 1998). In addition, the stability of proteins and protein–protein interactions are affected by acetylation, indicating that acetylation regulates a variety of cellular functions. To elucidate the role of TTF-I binding to the upstream terminator, we have examined whether TTF-I can interact with the p300/CBP-associated factor PCAF, thereby recruiting HAT activity to the rDNA promoter. Indeed, we found that PCAF interacts with TTF-I and acetylates TAFI68, the second largest subunit of the TATA box-binding protein (TBP)-containing promoter selectivity factor TIF-IB/SL1. Acetylation by PCAF increases the DNA binding activity of TAFI68 and enhances Pol I transcription. mSir2a, a murine NAD+-dependent histone deacetylase that, by analogy to yeast Sir2, may play a role in silencing of the rDNA locus, can deacetylate TAFI68 and repress Pol I transcription in vitro. The results indicate that HATs modify component(s) of the Pol I transcriptional machinery and suggest acetylation as a novel regulatory modification that activates rDNA transcription. Results TTF-I interacts with PCAF To examine whether TTF-I may recruit HAT(s) to the rDNA promoter, recombinant TTF-I was immobilized on agarose beads and incubated with extracts from mouse cells. TTF-I-associated HAT activity was determined using core histones as substrates. As shown in Figure 1A, significant levels of HAT activity were associated with bead-bound TTF-I but not with control beads saturated with the FLAG peptide (lanes 3 and 4). Notably, whereas the endogenous HAT activity of the extract preferentially acetylated histone H4 (lane 2), the HAT that was retained on TTF-I beads preferentially acetylated histone H3. No labeling of histones H2A and H2B was observed. The preferential acetylation of histone H3 suggests that PCAF has been pulled down by TTF-I. Indeed, western blot analysis of bead-bound proteins revealed that significant levels of PCAF protein were retained on the TTF-I beads (Figure 1B, lane 1). No PCAF was detected on control beads saturated with cyclin A (lane 2) or upstream binding factor (UBF) (lane 3). In a reciprocal experiment, immobilized PCAF was incubated with mouse cell extract and specific retention of cellular TTF-I was demonstrated on immunoblots using anti-TTF-I antibodies (Figure 1C, lane 2). Figure 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). Download figure Download PowerPoint To demonstrate interaction of TTF-I with PCAF in vivo, histidine-tagged TTF-I was expressed in Sf9 cells in the absence or presence of FLAG-tagged PCAF, precipitated with anti-FLAG (M2) antibodies, and co-immunoprecipitated TTF-I was visualized on western blots (Figure 1D). Consistent with TTF-I interacting with PCAF in vivo, TTF-I was found to co-precipitate with PCAF (lane 1). When extracts from Sf9 cells were used that overexpress cyclin A in the absence or presence of PCAF, no co-immunoprecipitation with PCAF was observed (lanes 3 and 4). This specific interaction between PCAF and TTF-I suggests that PCAF may be recruited to the rDNA promoter by TTF-I. The C-terminal part of TTF-I interacts with PCAF In order to delimit the part of TTF-I that interacts with PCAF, a series of N-terminal deletions of TTF-I were expressed in insect cells (Evers et al., 1995; Sander et al., 1996) and tested for their ability to interact with PCAF. Two of these mutants, TTFΔN185 and TTFΔN323, are functionally active, i.e. mediate Pol I transcription termination and transcriptional activation on chromatin templates. Mutant TTFΔN445, however, although able to bind to the 'sal box’ target sequence on naked DNA, fails to mediate transcription termination and activate transcription on chromatin templates (Evers et al., 1995; Längst et al., 1997). To assay the ability of the three N-terminally truncated versions of TTF-I to interact with PCAF, the respective proteins were overexpressed in Sf9 cells either alone or together with FLAG-PCAF (Figure 2). The cell lysates were incubated with anti-FLAG antibodies and the immunoprecipitates were assayed for TTF-I on western blots. Under stringent conditions, no TTF-I was retained on the anti-FLAG resin if cells were used that express TTF-I in the absence of FLAG-PCAF (lanes 4–6). In contrast, a significant amount (5%) of TTF-I present in the lysate co-precipitated with PCAF. Significantly, all three mutants exhibited similar PCAF binding capacities, indicating that the C-terminal part of TTF-I, which harbors the DNA-binding domain, mediates the interaction with PCAF. Figure 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. Download figure Download PowerPoint PCAF acetylates TAFI68 Having established that TTF-I is capable of interacting with PCAF, we sought to investigate whether components of the transcription initiation complex are targeted by PCAF. For this, we carried out in vitro protein acetyltransferase assays using several Pol I-specific transcription factors as substrates. Subunits of the promoter selectivity factor TIF-IB/SL1 (TAFI110, TAFI68, TAFI48 and TBP), UBF and the polymerase and transcript release factor (PTRF; Jansa et al., 1998) were expressed in Sf9 cells and purified to near homogeneity. p53, which is known to be acetylated by PCAF, was used as a positive control. Equal amounts of protein were incubated with PCAF and [3H]acetyl-CoA, and acetylated proteins were visualized by fluorography. In agreement with previous data demonstrating acetylation of p53 by PCAF (Sakaguchi et al., 1998; Liu et al., 1999), p53 was labeled efficiently in this in vitro assay (Figure 3A, lane 1). Significantly, two subunits of the promoter selectivity factor TIF-IB/SL1, i.e. TAFI68 and TAFI48, were acetylated, the acetylation of TAFI68 being ∼3-fold stronger than that of TAFI48 (lanes 4 and 5). Acetylation of TAFI68 was even more pronounced than that of p53 (lane 1), suggesting that TAFI68 is acetylated at multiple sites. None of the other proteins tested, i.e. TAFI110, TBP, UBF or PTRF, was acetylated by PCAF in vitro, demonstrating the specificity of the acetylation reaction. Figure 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. Download figure Download PowerPoint As acetylated proteins are usually associated with their respective HATs, we examined whether PCAF interacts with TAFI68. Immobilized glutathione S-transferase (GST)–PCAF was incubated with 35S-labeled TAFI68, TTF-I and UBF, respectively, and bead-bound labeled proteins were visualized by electrophoresis and autoradiography. As shown in Figure 3B, ∼10% of TAFI68 was found to be associated with GST–PCAF but not with GST control beads (lanes 1–3). The efficiency of TAFI68 binding was comparable to that of TTF-I (lanes 4–6). In contrast, almost no UBF (<1% of input) was retained on the PCAF affinity resin (lane 8). This result demonstrates that TAFI68 and TTF-I, but not UBF, interact with PCAF. Next, we tested whether TAFI68 is specifically targeted by PCAF or whether other HATs also acetylate this subunit of TIF-IB/SL1. We therefore compared the ability of similar amounts (in terms of HAT activity) of CBP, GCN5 and PCAF to acetylate TAFI68 (Figure 3C). All three HATs modified p53 with comparable efficiency (data not shown). TAFI68, on the other hand, was acetylated efficiently by PCAF (lane 4), to a lesser extent by GCN5 (lane 3), but not by CBP (lane 2). Thus, acetylation of TAFI68 appears to be mediated by PCAF. Cellular TIF-IB/SL1 contains acetylated TAFI68 As recombinant TAFI68 and TAFI48 can be acetylated by PCAF, we examined whether these subunits are accessible for acetylation within the cellular TBP–TAF complex. For this, the human factor SL1 was affinity purified, incubated with PCAF and [3H]acetyl-CoA, and [3H]acetate incorporation was monitored by fluorography (Figure 4A). As a positive control, acetylation of p53 was assayed. Consistent with TAFI68 being a bona fide substrate for PCAF, TAFI68 was labeled in the TBP–TAF complex. TAFI48, on the other hand, was not acetylated under these conditions, suggesting that the acetylation site(s) may be masked by interaction with other TAFIs or TBP. Figure 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. Download figure Download PowerPoint Cellular PCAF interacts with its substrates in the context of a large multiprotein complex. To examine whether cellular PCAF would acetylate TAFI68 as well, the PCAF complex was immunopurified from NIH-3T3 cells overexpressing FLAG-tagged PCAF and used to acetylate recombinant TAFI68. As shown in Figure 4B, both the cellular PCAF complex (lane 1) and recombinant PCAF isolated from Sf9 cells (lane 3) are capable of acetylating TAFI68 (lane 2), the latter being more active than the cellular complex. In vitro acetylation of proteins does not establish that they are substrates for modification in vivo. To determine whether acetylation of TAFI68 occurs in vivo, we used an antibody, anti-AK, which reacts with acetyl-lysine residues but not with unacetylated lysine residues (Hebbes et al., 1988). The human factor SL1 and the homologous murine factor TIF-IB were immunopurified from HeLa and Ehrlich ascites cells, respectively, and subjected to western blotting using anti-AK and anti-TAFI antibodies. As shown in Figure 4C, in both the human and mouse complex TAFI68 was recognized by the anti-AK antibodies, demonstrating that this subunit of TIF-IB/SL1 is acetylated in vivo. Treatment with trichostatin A does not affect rRNA synthesis in cycling cells Given that TIF-IB/SL1 is acetylated in vivo, one would expect inhibition of deacetylases by butyrate or trichostatin A (TSA) to have an effect on cellular rRNA synthesis. To address this issue, pre-rRNA synthesis was monitored in FT210 cells, a murine mammary tumor cell line carrying a temperature-sensitive mutant of cdc2 (Yasuda et al., 1991). Cells were cultured in the absence or presence of TSA, and pre-rRNA synthesis was compared on northern blots using a labeled antisense RNA that hybridizes to 5′-terminal nucleotides (from +1 to +155) of mouse 45S pre-rRNA. To exclude the possibility that inhibition of deacetylases affects rDNA transcription only in distinct phases of the cell cycle, the experiment was performed with synchronized cells. Cells were synchronized at the G2–M boundary by culturing at the non-permissive temperature (39°C), and then shifted to the permissive temperature (33°C) to allow the cells to re-enter the cell cycle. In agreement with previous data (Klein and Grummt, 1999), there are drastic changes in the overall rRNA synthetic activity during cell cycle progression, being maximal at G2, shut down at mitosis and slowly recovering during progression through G1 phase (Figure 5B). However, there was no difference in pre-rRNA levels, regardless of whether the cells were cultured in the absence or presence of TSA. Thus, inhibition of TSA-sensitive deacetylases does not affect cellular pre-rRNA synthesis. Figure 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. Download figure Download PowerPoint Acetylation augments the DNA-binding activity of TAFI68 Acetylation changes the size and charge of lysine residues, and thus alters the functional properties of proteins. TAFI68 contains three zinc finger motifs, and cross-linking studies have demonstrated that it binds to the rDNA promoter (Rudloff et al., 1994; Beckmann et al., 1995; Heix et al., 1997). Given the stimulation of DNA binding activity of p53 (Gu and Roeder, 1997), MyoD (Sartorelli et al., 1999), E2F-1 (Martínez-Balbás et al., 2000), GATA-1 (Boyes et al., 1998) and HNF-4 (Soutoglou et al., 2000), we investigated whether the DNA binding properties of TAFI68 were affected by acetylation. FLAG-tagged TAFI68 was incubated with a labeled DNA fragment covering murine rDNA sequences from −54 to +7, and protein–DNA complexes were separated from free DNA by electrophoresis. As shown in Figure 6A, TAFI68 interacted with the rDNA promoter probe and formed a defined DNA–protein complex (CI). If the amounts of TAFI68 were increased, a second, more slowly migrating complex (CII) was generated (data not shown), indicating that two TAFI68 molecules have bound to DNA. The DNA–protein complex could be competed by a DNA fragment encompassing the core element of the rDNA promoter (lanes 3–5), but not by a 3′-terminal rDNA fragment containing the terminator element T1 (lanes 6–8), underscoring the sequence selectivity of TAFI68 binding. Figure 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. Download figure Download PowerPoint To examine the effect of acetylation on the DNA-binding properties of TAFI68, recombinant TAFI68 was pre-incubated either with acetyl-CoA (mock) or with several HATs and acetyl-CoA before the DNA binding activity was assayed (Figure 6B). Consistent with the data in Figure 6A, in the absence of de novo acetylation, binding of TAFI68 to its cognate site yielded complex CI (lanes 1–3). Acetylation by PCAF increased the overall DNA binding efficiency of TAFI68, leading to enhanced formation of complex CI and generation of complex CII (lanes 4–6). Like PCAF, GCN5 augmented DNA binding and formation of complex CII (lanes 7–9), whereas CBP had no effect (lanes 10–12). Acetylation of TAFI68 by PCAF enhances rDNA transcription in vitro The correlation between acetylation of TAFI68 and the increase of DNA binding suggests that acetylation-induced enhancement of TIF-IB/SL1 binding could be a means that the cell may use to modulate initiation complex formation and regulate rDNA transcription. To examine whether acetylation by PCAF would stimulate Pol I transcription, we assayed the effect of GST–PCAF in a reconstituted transcription system. The system used contained low amounts of TIF-IB and, therefore, transcriptional activity was low (Figure 7A, lane 1). Addition of increasing amounts of GST–PCAF enhanced transcription 7- and 13-fold, respectively (lanes 2 and 3). In the presence of exogenous acetyl-CoA, transcription was elevated further, resulting in 15- and 17-fold activation (lanes 4 and 5). Figure 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. Download figure Download PowerPoint The fact that significant levels of PCAF-mediated transcriptional activation were observed in the absence of exogenous acetyl-CoA suggests that either PCAF or a component(s) of the transcription system contains endogenous acetyl-CoA. To demonstrate unambiguously that transcriptional enhancement by PCAF was caused by acetylation, we compared the effect of GST–PCAF with that of GST–PCAF/ΔHAT, a mutant that lacks HAT activity, on transcriptional activity. Clearly, the mutant protein failed to activate Pol I transcription to a significant extent (Figure 7B, lanes 4 and 5). Thus, the HAT activity of PCAF is required for activation of the basal Pol I transcription apparatus. Deacetylation by Sir2p impairs rDNA transcription in vitro Recent studies indicated that Sir2, a highly conserved member of the family of 'silent information regulators’ which localizes to the nucleolus, exhibits NAD+-dependent histone deacetylase activity (Imai et al., 2000; Smith et al., 2000). To examine whether mSir2a would be a candidate enzyme that deacetylates TAFI68, recombinant murine mSir2a was incubated with acetylated TAFI68 in the absence and presence of NAD+, and deacetylation was monitored on western blots using anti-AK antibodies that recognize acetylated lysines (Figure 8A). In the absence of NAD+, mSir2a did not affect the acetylation level of TAFI68 (lanes 1–4). In contrast, in the presence of NAD+, a time-dependent deacetylation of TAFI68 was observed (lanes 5–8), indicating that acetylated TAFI68 is targeted by mSir2a. In support of this, incubation of purified TIF-IB with mSir2a showed an NAD+-dependent reduction of TAFI68 acetylation, demonstrating the accessibility of acetylated lysine residues within the TBP–TAF complex (Figure 8B). Autoacetylated PCAF, on the other hand, was no" @default.
• W1992238166 created "2016-06-24" @default.
• W1992238166 creator A5005683105 @default.
• W1992238166 creator A5017883670 @default.
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• W1992238166 date "2001-03-15" @default.
• W1992238166 modified "2023-10-16" @default.
• W1992238166 title "Acetylation of TAFI68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription" @default.
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