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Regulation of RNA Polymerase III Transcription by Maf1 in Mammalian Cells
J. Mol. Biol. (2008) 378, 481–491
doi:10.1016/j.jmb.2008.02.060
Available online at www.sciencedirect.com
Regulation of RNA Polymerase III Transcription by Maf1 in Mammalian Cells Sarah J. Goodfellow 1 †, Emma L. Graham 1 †, Theodoros Kantidakis 1,2 , Lynne Marshall 1,2 , Beverly A. Coppins 1 , Danuta Oficjalska-Pham 3,4 , Matthieu Gérard 5 , Olivier Lefebvre 3 and Robert J. White 1,2 ⁎ 1
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK 2
Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow G61 1BD, UK 3
Laboratoire de Transcription des Gènes, Service de Biochimie et Génétique Moléculaire, CEA, iBiTecS, F-91191 Gif-sur-Yvette Cedex, France
RNA polymerase (pol) III produces essential components of the biosynthetic machinery; therefore, its output is tightly coupled with the rate of cell growth and proliferation. In Saccharomyces cerevisiae, Maf1 is an essential mediator of pol III repression in response to starvation. We demonstrate that a Maf1 ortholog is also used to restrain pol III activity in mouse and human cells. Mammalian Maf1 represses pol III transcription in vitro and in transfected fibroblasts. Furthermore, genetic deletion of Maf1 elevates pol III transcript expression, thus confirming the role of endogenous Maf1 as an inhibitor of mammalian pol III output. Maf1 is detected at chromosomal pol III templates in rodent and human cells. It interacts with pol III as well as its associated initiation factor TFIIIB and is phosphorylated in a serum-sensitive manner in vivo. These aspects of Maf1 function have been conserved between yeast and mammals and are therefore likely to be of fundamental importance in controlling pol III transcriptional activity. © 2008 Elsevier Ltd. All rights reserved.
4
Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland 5
Laboratoire de Transgenèse, Service de Biologie Moléculaire Systémique, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex, France Received 10 September 2007; received in revised form 22 February 2008; accepted 25 February 2008 Available online 4 March 2008 Edited by J. Karn
Keywords: Maf1; RNA polymerase III; TFIIIB; transcription
*Corresponding author. Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow, G61 1BD, UK. E-mail address: [email protected]. † These authors contributed equally to this work. Abbreviations used: pol, RNA polymerase; rRNA, ribosomal RNA; ScMaf1, Saccharomyces cerevisiae Maf1; HsMaf1, Homo sapiens Maf1; ChIP, chromatin immunoprecipitation; RT, reverse transcription; ARPP, acidic ribosomal phosphoprotein; HA, hemagglutinin; ES, embryonic stem.
Introduction In all eukaryotes examined, RNA polymerase (pol) III synthesises several essential components of the biosynthetic machinery, including tRNA and 5S ribosomal RNA (rRNA).1 The rate of pol III transcription is tightly regulated in response to changing conditions.1,2 For example, tRNA synthesis is low when nutrients or mitogens are limiting but is ra-
0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.
482 pidly induced by growth stimuli.3–7 Elevated pol III activity is a recurring feature of mouse and human tumours.2,8 In Saccharomyces cerevisiae, the pol III response to nutrient limitation has been shown to depend on the repressor Maf1.9 Indeed, genetic analyses indicate that Maf1 in budding yeast is an essential mediator of pol III repression under a wide range of adverse conditions, including DNA damage, secretory pathway defects, growth to stationary phase and inhibition of the target of rapamycin signalling pathway.9–12 Such stresses control the phosphorylation state of Maf1, via protein kinase A and protein phosphatase 2A.13–15 Maf1 was initially identified in a yeast screen for mutations that affect tRNA suppressors and was found to interact genetically with pol III.16 Subsequent studies demonstrated a physical interaction between Maf1 and pol III, as well as between Maf1 and the Brf1 subunit of the pol III-specific transcription factor TFIIIB.9,14,15,17,18 S. cerevisiae Maf1 (ScMaf1) is a hydrophilic protein of 395 amino acids that shows no significant similarity to other known proteins.16 However, database searches revealed Maf1 orthologs in other eukaryotes, including humans.17 The overall homology between Homo sapiens Maf1 (HsMaf1) and ScMaf1 is low (∼16% amino acid identity), but these orthologs share three blocks of much higher conservation (28%–47%) arranged in the same order. This study demonstrates that mammalian Maf1 can repress pol III transcription in vitro and in vivo. Consistent with this, genetic loss of Maf1 elevates expression of pol III products. We show that endogenous HsMaf1 associates stably with pol III as well as TFIIIB and is present at tRNA, 5S rRNA, 7SL and 7SK genes in vivo. Scanning chromatin immunoprecipitation (ChIP) analysis detects HsMaf1 around the start region of the 7SL gene but not further downstream, suggesting that it may not associate with actively transcribing pol III. The data are consistent with previous studies of Maf1 in yeast. The conservation of this mechanism for pol III regulation strongly suggests that it is of fundamental importance.
Results Human Maf1 can inhibit pol III transcription in vitro and in vivo An expression vector encoding the mouse protein was transfected into CCL39 rodent fibroblasts to test if Maf1 can regulate pol III transcription in mammals. In parallel, these cells were transfected with empty vector or expression vector encoding the Brf1 subunit of TFIIIB. Reverse transcription (RT) PCR was used to monitor levels of endogenous pol III transcripts. Transfection of Maf1 was found to reduce expression of every pol III product examined, including 5S rRNA, tRNALeu, B2 RNA, 7SK RNA and U6 snRNA (Fig. 1). The genes encoding these transcripts include representatives of each of the
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
Fig. 1. Mouse Maf1 inhibits pol III transcription in vivo. CCL39 fibroblasts were transiently transfected with pcDNA3HA vector (lane 1), pcDNA3HA.Maf1 (lane 2) or pcDNA3HA.Brf1 (lane 3). Extracted RNA was analysed by RT-PCR using primers specific for the indicated transcripts.
promoter types used by pol III, including the type III promoters that are not found in S. cerevisiae.1,19,20 Inhibition is specific, since levels of the mRNA encoding acidic ribosomal phosphoprotein (ARPP) P0 remained unaffected. In contrast, the Brf1 vector selectively induced tRNA and B2 genes, without affecting the other pol III templates, for which it is either not required or not limiting in these cells.19,20 The data indicate that mouse Maf1 can have a repressive effect on the expression of pol III products in fibroblasts. Although specific, the response encompasses all pol III templates examined. This is consistent with whole-genome analyses in S. cerevisiae, which detected Maf1 at all known categories of pol III-transcribed genes.14,15 Purified recombinant HsMaf1 was tested for its ability to regulate transcription in a HeLa cell nuclear extract. As shown in Fig. 2a, 200 ng of recombinant HsMaf1 abolishes transcription of all the pol III templates tested, namely, 5S rRNA, tRNALeu, B2, VA1, 7SL and 7SK genes. The repression is specific, as pol I transcription of a pre-rRNA template shows minimal response. Furthermore, the inhibition of pol III transcription by HsMaf1 is dose dependent, with as little
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
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Fig. 2. HsMaf1 specifically inhibits pol III transcription in human cell extracts. (a) HeLa nuclear extract (20 μg) was used to transcribe the indicated templates in the presence or absence of 200 ng of recombinant HsMaf1, as indicated. (b) HeLa nuclear extract (20 μg) was used to transcribe the indicated templates in the presence of increasing amounts (12.5, 25, 50, 100 or 200 ng) of HsMaf1 or equivalent volumes of HsMaf1 control fraction.
as 12.5 ng of recombinant protein causing a substantial reduction in transcriptional activity (Fig. 2b). HsMaf1 may act at more than one stage in the pol III transcription cycle Order-of-addition experiments can sometimes provide information concerning mechanisms of repression. For example, this approach provided evidence that p53 inhibits pol III transcription complex assembly but has a minimal effect once the complex has formed.21 Subsequent analyses confirmed that p53 can block promoter access of TFIIIB, in vitro and in vivo.22 We therefore carried out order-of-addition experiments to test if HsMaf1 can inhibit a preassembled pol III initiation complex or acts exclusively during complex assembly. Recombinant HsMaf1 was mixed with HeLa extract 15 min prior to addition of VA1 gene template (Fig. 3a, lanes 1 and 2), simultaneously with VA1 addition (lanes 3 and 4) or 15 min after addition of VA1 template (lanes 5 and 6). Nucleotides were then added to allow transcription. Under the conditions of our assays, 15 min is sufficient for stable complex formation on the VA1 promoter.23 Although repression was most efficient when HsMaf1 was present during pre-initiation complex assembly, significant inhibition was still observed from pre-assembled transcription complexes (Fig. 3b). This contrasts with repression by p5321 but resembles the outcome of similar experiments carried out with ScMaf1, which inhibits most effectively if added prior to transcription complex formation, although it is able to significantly repress a pre-assembled complex.18 This finding was interpreted as supporting a model in which ScMaf1 acts at
more than one stage in the transcription cycle, one of which is likely to be recruitment/recycling of the pol.18 This is consistent with the fact that ScMaf1 interacts with two distinct targets, TFIIIB and pol III itself.14,15,17,18 We therefore carried out co-immunoprecipitation assays to test if the same is true for the human proteins. HsMaf1 interacts with the endogenous pol III transcription apparatus Antiserum raised against Maf1 was found to coimmunoprecipitate from HeLa nuclear extract both TFIIIB and pol III, as revealed by Western blotting for Brf1 and for RPC39, respectively (Fig. 4a). In addition, blotting for TFIIIC220 provided evidence for a stable association with TFIIIC. These interactions are specific, as they were not detected using pre-immune serum or an irrelevant antibody against the translation factor 4EBP1. They were also confirmed using alternative antisera (data not shown). DNA does not appear to be required for association of these proteins, as co-immunoprecipitation was undiminished following DNase treatment. The interactions were also retained after 3 days of serum deprivation (Fig. 4b). Maf1 is phosphorylated in fibroblasts in a serum-dependent manner Since phosphorylation of ScMaf1 is linked to its function, we asked whether Maf1 is also phosphorylated in mammalian cells. Vector encoding hemagglutinin (HA)-tagged murine Maf1 was transfected into fibroblasts, which were cultured for 24 h in the
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Mammalian Maf1 Inhibits RNA Polymerase III Transcription
nents TBP and Brf1 as positive controls; in both cases, pol III transcription was inhibited. In contrast, transcription was stimulated by depletion of HsMaf1, using either of two antisera (1166 and 1167). The corresponding pre-immune sera had little or no effect, when compared with extract that was mock depleted in the absence of antibody. These data provide evidence that endogenous HsMaf1 present in HeLa nuclear extracts has a negative effect on transcription by pol III. Pol III transcript expression is suppressed by endogenous Maf1 in mouse embryonic stem cells
Fig. 3. HsMaf1 can inhibit pol III transcription whether it is added before or after transcription complex assembly. (a) Buffer (lanes 1, 3 and 5) or 200 ng of recombinant HsMaf1 (lanes 2, 4 and 6) was added to nuclear extract (20 μg) 15 min before (lanes 1 and 2), simultaneously with (lanes 3 and 4) or 15 min after (lanes 5 and 6) the addition of VA1 template DNA. Transcription was initiated by the addition of nucleotides. (b) Transcriptional outputs for the abovementioned experiment and two repeats were determined by densitometry and are displayed graphically (n = 3; *significantly different from control, p b 0.05; **significantly different from 15 min, p b 0.05).
presence or absence of serum and then labelled with [32P]orthophosphate for 3 h prior to harvesting. Immunoprecipitation with anti-HA antibody revealed strong labelling of transfected Maf1 in the cells grown in serum, whereas labelling was much diminished in the serum-starved cells (Fig. 5, top panel). Western blotting confirmed that Maf1 was indeed immunoprecipitated from the starved cells (Fig. 5, bottom panel). It also revealed that Maf1 from the growing cells migrates as a doublet under our gel conditions, whereas the upper band is selectively diminished when extracted from serum-starved cells. Both forms appear to be phosphorylated, as they are labelled with [32P]. We speculate that the slower-migrating form may be most heavily modified. These data provide evidence that Maf1 is phosphorylated in fibroblasts in a serum-sensitive manner. Immunodepletion of HsMaf1 raises pol III transcription Antisera against HsMaf1 were used to immunodeplete the endogenous protein from HeLa cell extract (Fig. 6). We depleted extracts of the TFIIIB compo-
Attempts were made to generate a genetic knockout in mouse embryonic stem (ES) cells. This proved to be unexpectedly difficult, but we did succeed in deleting one allele, to create an ES line that carries a single intact copy of the Maf1 gene. Western blotting indicated that Maf1 protein expression is ∼40% lower in the heterozygous cells, consistent with the loss of one allele (Fig. 7a). RT-PCR showed a specific decrease in Maf1 mRNA to ∼34% of wild-type levels (Fig. 7b). In contrast, expression of B2 and tRNAArg transcripts is elevated approximately two- to threefold. 5S rRNA and tRNALeu, however, only increased by ∼30% in the heterozygous ES cells. The reason for this differential sensitivity is unclear, since 5S rRNA and tRNALeu expression responds strongly to Maf1 in other assays ((Figs. 1, 2 and 6)). Elevated B2 RNA in the heterozygous cells was also observed by Northern blot (Fig. 7c). We conclude that endogenous Maf1 inhibits expression of at least some pol III templates in mouse ES cells. Mammalian Maf1 associates with pol III-transcribed genes in vivo ChIP assays were used to test if Maf1 interacts with pol III-transcribed genes in mammalian cells. Two separate antisera raised against different epitopes within HsMaf1 both detected this factor at chromosomal tRNA and 5S rRNA genes in HeLa cells (Fig. 8a). As might be expected, the HsMaf1 signal was weaker than that obtained using positive control antisera against pol III or TFIIIB but was nevertheless well above the background obtained using beads alone or a negative control antibody against the pol II-specific factor TFIIB. Similar results were obtained with CCL39 and A31 fibroblasts (Fig. 8b and other data not shown). In addition, HsMaf1 was detected at 7SK genes, despite their distinct promoter architecture (Fig. 8c). As expected, only background signal was obtained in this case with the Brf1 antiserum used to detect TFIIIB, because Brf1 is not utilised by such genes as 7SK that have type III promoters.19,20 We conclude that chromosomal pol III-transcribed genes of all promoter types are targeted specifically by Maf1 in mammalian cells. Scanning ChIP analysis has been used previously to investigate the region of a target gene that is occupied by ScMaf1.15 This approach is difficult to
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
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Fig. 4. HsMaf1 associates with TFIIIB, TFIIIC and pol III. (a) Proteins were immunoprecipitated (IP) from HeLa nuclear extract using the indicated antibodies, in the absence (lanes 1–3) or presence (lanes 4–6) of 20 U of DNase 1. Immunoprecipitates were analysed by Western blotting with antibody Ab7 against TFIIIC (220-kD subunit), antibody C39 against pol III (RPC6) and antibody 128 against TFIIIB (Brf1). Lane 7 shows 25% of input material. The asterisk marks an apparently nonspecific band that migrates above TFIIIC220 and is present in all the immunoprecipitations. Note that all three panels are from the same experiment but show samples run on different gels (a gap was left between lanes 3 and 4 in the middle and lower panels). (b) Proteins were immunoprecipitated using the indicated antibodies from HeLa cells cultured in the presence of 10% (lanes 1–3) or 0.5% (lanes 4–6) serum. Immunoprecipitates were analysed by Western blotting with antibody Ab7 against TFIIIC (220kD subunit), antibody C39 against pol III (RPC6) and antibody 128 against TFIIIB (Brf1). Lanes 7 and 8 show 12.5% of input material.
apply to pol III templates, because of their short size. However, it was used successfully for the 522-bp SCR1 gene, the longest pol III transcription unit in the S. cerevisiae genome, which encodes the 7SL RNA component of the signal recognition particle.15 We attempted to do the same with a human 7SL gene, although it is only ∼ 300 bp. Primers were designed to amplify sequences at the start and finish of the 7SL coding region, as well as ∼ 200 and ∼500 bp downstream of the termination site and ∼ 200 bp upstream of the initiation site (Fig. 9a). The size of sonicated chromatin fragments used for ChIP limits resolution and causes overlap between the signals from neighbouring amplicons. Nevertheless, the results obtained for TFIIIB, TFIIIC and pol III conformed sufficiently well to their expected positions to establish a proof of principle. Thus, the Brf1 subunit of TFIIIB is known to locate to the region immediately upstream of the start site and was accordingly detected most strongly using primer set 2, with the neighbouring primer sets 1 and 3 giving weaker but significant signals and the downstream primer sets 4 and 5 close to the background (Fig. 9b and c). In contrast, TFIIIC is positioned over a substantial part of the coding region, extending from the start towards the termination site; consistent with
this, it was detected most strongly using primer sets 2 and 3, while weaker signals were obtained with the flanking primer sets. Pol III was also detected most strongly using primers that amplify the coding region, especially set 3, again with weaker signals from the flanking primer sets. These observations gave us some confidence in our scanning ChIP assay, despite the difficulties in resolving regions of pol III-transcribed genes, because of their small size. The Maf1 signal was weak on the 7SL gene but was nevertheless consistently above the background with primer set 2 (Fig. 9c). Since the Brf1 ChIP is also strongest with set 2, whereas TFIIIC and pol III show different patterns, the data support a model in which Maf1 co-localises with Brf1 in the vicinity of the transcription start site. This is consistent with scanning ChIP analysis of ScMaf1 on the SCR1 gene in yeast.15
Discussion This study demonstrates that the function of Maf1 as a repressor of pol III transcription has been conserved through evolution. Mammalian Maf1 potently inhibits expression of all pol III templates
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Fig. 5. Maf1 displays serum-sensitive phosphorylation in fibroblasts. Anti-HA antibody was used to immunoprecipitate from CCL39 fibroblasts transfected with pcDNA3HA.Maf1 and cultured for 24 h in the absence (lane 1) or presence (lane 2) of serum before radiolabelling for 3 h with [32P]orthophosphate. Immunoprecipitated material was resolved by SDS-PAGE, transferred to nitrocellulose and then visualised by autoradiography (upper panel) and Western blotting with anti-HA antibody (lower panel).
tested in human cell extracts and in transfected fibroblasts. This is not an artefact of overexpression, since pol III transcript levels are elevated by depleting endogenous Maf1 through genetic ablation in mouse ES cells. Furthermore, endogenous Maf1 is detected by ChIP at chromosomal tRNA and 5S rRNA genes in rodent and human cells and co-immunoprecipitates with the endogenous human pol III machinery. We therefore conclude that mammalian Maf1 interacts with the pol III transcription apparatus and restrains its output in vivo. Other studies of human Maf1 have been published recently, reflecting the intense interest in this important factor.24–26 The data from these authors are consistent with ours. For example, they have shown that overexpression of HsMaf1 in human cancer cell lines can repress transfected tRNAArg, VA1 and U6 reporters without affecting a co-transfected pol IIdependent reporter.25,26 We have extended these findings by transfecting a murine Maf1 expression vector and showing that it can selectively repress a range of endogenous pol III-transcribed genes in untransformed rodent fibroblasts (Fig. 1). Reina et al. demonstrated that recombinant HsMaf1 can specifically inhibit pol III transcription in vitro of 5S, VA1, 7SL and U6 genes without affecting pol II transcription from the adenovirus major late promoter.24 We have confirmed this observation for 5S, VA1 and 7SL, while adding tRNALeu , B2 and 7SK genes as res-
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
ponsive targets and demonstrating that pol I transcription is unresponsive (Fig. 2). Two studies showed that RNA interferencemediated depletion of HsMaf1 from human cells can selectively raise expression of pol III transcripts.24,25 We have adopted a different approach to confirm the function of endogenous Maf1. Figure 6 shows that its immunodepletion from HeLa extracts increases pol III transcription in vitro. Furthermore, we have made heterozygous mouse ES cells and shown that genetic deletion of one Maf1 allele raises pol III transcript expression in vivo (Fig. 7). Our work therefore provides independent confirmation that Maf1 is a physiologically relevant repressor of pol III transcription in mammals. This conclusion is supported by ChIP data revealing the presence of endogenous Maf1 at 5S rRNA, tRNALeu, 7SL and 7SK genes in HeLa cells and CCL39 fibroblasts (Figs. 8 and 9). It was also found at tRNALeu genes in human U87 glioblastoma cells.25 The results have been obtained using several different antibodies. Between these studies, Maf1 has been shown to operate in a disparate range of mammalian cell types: human HEK293 embryonic kidney cells and IMR90-TERT fibroblasts,24 two human glioblastoma lines25 and HeLa cells, as well as rodent ES cells and fibroblasts. No exception has been reported. We found that endogenous TFIIIB and pol III coimmunoprecipitate with endogenous HsMaf1 from HeLa cells, as reported by Reina et al.24 Their study also used glutathione S-transferase pull-down assays to show that HsMaf1 targets TFIIIB through its Brf1 subunit and pol III through its RPC1 and RPAC2 subunits.24 The specific targeting of both pol III and Brf1 has therefore been conserved from yeast to man. We were surprised to detect TFIIIC in complexes with Maf1 from human cells as well—an observation that has not been reported previously. Although a direct interaction is certainly possible, we suspect that association is instead mediated through simultaneous binding of Brf1 to both Maf1 and TFIIIC. Scanning ChIP analysis suggests that Maf1 is located
Fig. 6. HeLa nuclear extract was mock depleted (lane 1) or immunodepleted with TBP antibody MTBP-6 (lane 2), Brf1 antibody 128 (lane 3), HsMaf1 antibodies 1166 and 1167 (lanes 5 and 7, respectively) and the corresponding pre-immune sera (lanes 4 and 6, respectively). These extracts were then used to transcribe the indicated templates.
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
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Fig. 7. Endogenous Maf1 inhibits pol III transcription in mouse cells. (a) Western blot of whole cell extract (10 μg) from matched Maf1+/+ and Maf1+/− ES cells using antibodies to Maf1 and actin, as indicated. (b) RNA from matched Maf1+/+ or Maf1+/− ES cells was analysed by RT-PCR using primers specific for the indicated transcripts. The tRNA primers recognise unprocessed primary transcripts, whereas all other primers recognise mature products. (c) Northern blots with RNA (10 μg) from matched Maf1+/+ or Maf1+/− ES cells were probed for B2 and ARPP P0 RNAs.
near the start of the 7SL gene, where Brf1 is expected to be found, as well as pol III prior to transcript initiation and the upstream end of TFIIIC. The Maf1 signal is close to the background further downstream, despite strong occupancy by pol III and TFIIIC within the transcribed region. This is consistent with the hypothesis that TFIIIC and actively transcribing pol III do not bind Maf1. However, interpretation has to be tentative, since epitope masking or inefficient cross-linking may also be involved. It would have been preferable to use a gene that gave a stronger Maf1 signal than 7SL, but our choice was severely constrained because most pol III templates are too short for scanning ChIP analysis. The low Maf1 signal on 7SL is consistent with its high rate of transcription. The data indicate that Maf1 performs a significant inhibitory role in apparently unstressed mammalian cells. Although it also functions under normal growth conditions in S. cerevisiae, the repressive activity of ScMaf1 is substantially enhanced when these yeasts are subjected to stresses.14,15 Indeed, ScMaf1 appears to mediate the pol III response to all forms of stress that have been tested, including DNA damage and growth into stationary phase.9,10 Mammals possess a substantial number of regulatory factors that impact on pol III output and are not present in yeast. Examples include the tumour suppressors RB and p53, both of which are potent repressors of pol III transcription in mammalian cells.27 RB and its relatives p107 and p130 perform dominant roles in restraining pol III output in growth-arrested murine fibroblasts.28,29 Furthermore, a wide range of stresses are known to induce p53, including DNA damage.30 It will be important to determine how mammalian Maf1 is integrated with other pathways that influence
pol III activity. One might guess that regulators such as RB and p53 have evolved to deal with conditions that are specific for Metazoa, whereas mammalian Maf1 may mediate responses to stress conditions that are also encountered by unicellular organisms. However, this hypothesis is too simplistic. Strong p53 responses are triggered by depletion of ribonucleotides31 or ribosomal protein imbalances,32,33 stresses that must also affect lower eukaryotes. Especially surprising is the fact that these responses do not appear to be redundant in mammalian cells and are lost in the absence of p53.31–33 This suggests that rather than simply providing additional levels of control, metazoan-specific response pathways may have actually replaced some of the more ancient mechanisms for coping with stress. Although Maf1 is essential for pol III regulation under all forms of stress that have been examined in budding yeast,9,10 we predict that it will have a more specialised role in mammals. Characterisation of this role and its interaction with other regulatory pathways will require extensive analysis. Johnson et al. provided striking evidence that the role of Maf1 has evolved through evolution.25 They demonstrated that as well as repressing pol III, HsMaf1 can bind and directly regulate a select group of pol II-transcribed genes, including the gene encoding TBP.25 By influencing TBP expression, HsMaf1 can also indirectly affect transcription of additional templates, including the pol I-dependent rRNA genes.25 This contrasts strongly with budding yeast, where ScMaf1 does not affect TBP levels9 and where genome-wide ChIP analyses did not implicate targets other than pol III templates.14,15 Evidently, some aspects of Maf1 function have changed dramatically
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citing to determine if Maf1 function is compromised in tumours. With its capacity to restrict a cell's biosynthetic capacity, it is conceivable that HsMaf1 may turn out to be a physiological tumour suppressor that is targeted in cancers.
Materials and Methods Cell culture Cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U/ ml of penicillin and 100 μg/ml of streptomycin. ES cells were cultured on feeder cells (mouse embryonic fibroblasts inactivated by mitomycin C), exactly as previously described.34 Transient transfection CCL39 cells were seeded onto six-well plates at a density of 1 × 105 cells/well. After 24 h, cells were transfected with 2 μg/well of plasmid DNA using Lipofectamine (Life Technologies, Inc.). pcDNA3HA.Brf1 is a human Brf1 cDNA in the pcDNA3HA expression vector.35 pcDNA3HA.Maf1 was created by subcloning a cDNA encoding Mus musculus Maf1 into BamHI/XbaI-digested pcDNA3HA. RNA analysis RNA extraction and RT were performed as previously described.36 Primers and cycling parameters have been described for 5S rRNA, ARPP P0 mRNA,37 7SK RNA, pretRNALeu38 and U6 snRNA.39 Other primers were as follows: Fig. 8. Endogenous Maf1 associates with class III genes in mammalian cells. ChIPs were performed using HeLa cells (a and c) or CCL39 fibroblasts (b) with beads alone or antibodies against Brf1, pol III, TFIIB or alternative Maf1 epitopes, as indicated. Association of each factor with the indicated genes was analysed by PCR with gene-specific primers. Input genomic DNA (10%, 2% and 0.4% of that used in ChIPs) was analysed in parallel.
during evolution. The absence of pol I repression by recombinant Maf1 in vitro does not conflict with the results of Johnson et al.,25 who found no interaction of HsMaf1 with ribosomal DNA promoters in ChIP assays; although pre-rRNA levels are responsive to HsMaf1 in transfected cells, this appears to be an indirect effect mediated through changes in TBP expression.25 Depletion of HsMaf1 by RNA interference was found to cause morphological changes.25 Furthermore, overexpression of HsMaf1 inhibits anchorageindependent growth of a glioblastoma cell line.25 On this basis, Johnson et al. suggested that HsMaf1 might influence oncogenic transformation.25 It is not yet established whether such effects require the ability of HsMaf1 to repress pol III transcription. However, it is noteworthy that elevated pol III output is strongly associated with cell transformation.2,8 It will be ex-
B2: 5′-GGG GCT GGA GAG ATG GCT-3′ and 5′-CCA TGT GGT TGC TGG GAT-3′ as well as mouse Maf1: 5′-GCA GTT CTG CCA GGA GGG CCA3′ and 5′-CTC CAT GGT GCT GGT CTC CTC-3′. PCR was performed using the following cycling parameters: B2: 95 °C for 3 min; 18 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s; and 72 °C for 5 min as well as mouse Maf1: 95 °C for 2 min; 25 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min; and 72 °C for 5 min. Northern blotting was performed as previously described.21 B2 and ARPP P0 probes have been described.40,41 Recombinant Maf1 and transcription assays Recombinant Maf1 was produced by cloning the HsMaf1 gene into the pFastBac vector from the Bac-to-Bac Baculovirus Expression System (Invitrogen) using the BamHI and SalI restriction sites. The recombinant protein was expressed in insect cells infected with baculovirus and has a 6-histidine tag. His-tagged HsMaf1 was then purified to ∼ 95% homogeneity using a Poros MC20 (Boeringer Mannheim) cobalt-chelate affinity column and was eluted using 50 mM imidazole. A fraction obtained from control purification of HsMaf1 with no His tag was used as a control in transcription reactions. In vitro transcription assays used 20 μg of HeLa nuclear extract and were carried out as previously described.42
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
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Fig. 9. HsMaf1 co-localises with Brf1 on a 7SL gene. (a) Schematic illustration of the positions of primer sets 1–5 relative to the coding region of a 7SL gene. (b) ChIPs were performed using HeLa cells and preimmune serum or antiserum against Maf1 (1166), Brf1 (128), pol III (1900) and TFIIIC (Ab7), as indicated. PCR analyses are shown using each of the five 7SL primer sets carried out for the same set of ChIP DNA. (c) Schematic representation of the ChIP signals obtained for Maf1 (blue), Brf1 (red), TFIIIC (green) and pol III (purple) with the five 7SL primer sets (as indicated along the x-axis). The y-axis values show the mean relative strengths of ChIP signal from three independent experiments, expressed in arbitrary units after normalisation against input and the signal with pre-immune serum. Template plasmids were pHu5S3.1 for 5S rRNA,43 pLeu for tRNA,44 pTB14 for B2,40 pVA1 for VA1,45 p7L30.1 for 7SL,46 7SK-pAT/153 for 7SK47 and EcoRI-linearized pHrP2 for pre-rRNA.48 For immunodepletion, extract was pre-incubated with protein A–Sepharose beads carrying pre-bound antibodies for 2 h on ice, with gentle agitation every 5 min.
III have been described previously.21,49 Antisera 1166 and 1167 against HsMaf1 were raised by immunizing rabbits with synthetic peptides YNPDLDSDPFGEDGSL and CRSISGSTYTPSEAGN, corresponding to HsMaf1 residues 168–183 and 203–218, respectively. Antiserum 1767 was raised by immunizing rabbits with recombinant mouse Maf1 protein; this antiserum was used for Western blotting.
Immunoprecipitation and Western blotting
Phosphate labelling in vivo
HeLa nuclear extract (250 μg) was incubated at 4 °C for 3 h on a rotating wheel with 25 μl of protein G–Sepharose beads that had been pre-incubated with affinity-purified antiserum 1166 against Maf1, the corresponding pre-immune serum or an anti-4EBP1 antibody (Santa Cruz Biotechnology). Bound material was resolved by SDS-PAGE on 7.8% polyacrylamide gels, and specific proteins were detected by Western blotting as previously described.42 Peptide antiserum 128 against the TFIIIB subunit Brf1 and monoclonal antiserum C39 against the RPC6 subunit of pol
CCL39 cells were transfected with pcDNA3HA.Maf1 and then, after 48 h, were labelled for 3 h with 0.3 mCi/ml of [32P]orthophosphate in phosphate-free medium, before harvesting for immunoprecipitation, as previously described.50 ChIP assays ChIP assays were performed as previously described51 using antibody 128 against Brf1,21 antiserum Ab7 against
490 TFIIIC,52 antiserum 1900 against pol III,53 antibody FL109 against TFIIA as well as antibody C18 against TFIIB (both from Santa Cruz Biotechnology), antisera 1166 and 1167 against Maf1 (see above) and antiserum 2966 against Maf1. Antiserum 2966 against Maf1 was raised by immunizing rabbits with synthetic peptides YDFSTARSHEFSREPS (corresponding to residues 108–123 of human and mouse Maf1) and GGEGRAEETSTMEEDR (which matches residues 240–250 of HsMaf1 and residues 237–252 of mouse Maf1). Immunoprecipitated DNA was analysed by PCR using the indicated primers. Apart from the 7SL scanning ChIP assays, primers and amplification conditions have been previously described.37,51,54 The 7SL primers were as follows: 7SL-1: 5′-CCGTGGCCTCCTCTACTTG-3′ and 5′TTTACCTCGTTGCACTGCTG-3′, 7SL-2: 5′-CGTCACCATACCACAGCTTC-3′ and 5′CGGGAGGTCACCATATTGAT-3′, 7SL-3: 5′-GTTGCCTAAGGAGGGGTGA-3′ and 5′TCTCTTGAGAGTCCAAAATTAA-3′, 7SL-4: 5′-TTTTTGACACACTCCTCCAAGA-3′ and 5′ATCTGGTCAAAGCAACATACACTG-3′ and 7SL-5: 5′-TGCCTCCAGATAAAACTGCTC-3′ and 5′ACCCCACTAGAACCCTGACA-3′. PCR was performed using the following cycling parameters: 95 °C for 2 min; 25 cycles of 95 °C for 1 min, 58 °C for 30 s as well as 72 °C for 1 min; and 72 °C for 3 min. Maf1 gene deletion Genomic fragments flanking exons 2 and 5 of the mouse Maf1 gene were PCR amplified and subcloned with LacZ, neomycin and diphtheria toxin cassettes. The construct was transfected into ES cells by electroporation, and Southern blotting was used to identify cells carrying the mutant Maf1 allele.
Acknowledgements This work was funded by grants 068710 and 081977 from the Wellcome Trust, grant BBS/B/0711X from the Biotechnology and Biological Sciences Research Council, grants C1288/A3143 and C1288/A4411 from Cancer Research UK (to R. J. W.) and grant 3982 from the Association pour la Recherche contre le Cancer (to O. L.). We are grateful to Arnie Berk and Yuhong Shen for providing antiserum Ab7 and VA1 plasmid, to Peter Cook for providing monoclonal antibody C39 and to Shona Murphy for providing 7SL and 7SK plasmids. We also thank Peter Geiduschek for encouraging us to use the scanning ChIP assay.
References 1. White, R. J. (2002). RNA polymerase III transcription, 3rd Edition Landes Bioscience, Austin, TX, http://www. eurekah.com 2. White, R. J. (2005). RNA polymerases I and III, growth control and cancer. Nat. Rev. Mol. Cell Biol. 6, 69–78.
Mammalian Maf1 Inhibits RNA Polymerase III Transcription 3. Johnson, L. F., Abelson, H. T., Green, H. & Penman, S. (1974). Changes in RNA in relation to growth of the fibroblast: I. Amounts of mRNA, rRNA, and tRNA in resting and growing cells. Cell, 1, 95–100. 4. Mauck, J. C. & Green, H. (1974). Regulation of pretransfer RNA synthesis during transition from resting to growing state. Cell, 3, 171–177. 5. Garber, M., Panchanathan, S., Fan, R. S. & Johnson, D. L. (1991). The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, induces specific transcription by RNA polymerase III in Drosophila Schneider cells. J. Biol. Chem. 266, 20598–20601. 6. Sethy, I., Moir, R. D., Librizzi, M. & Willis, I. M. (1995). In vitro evidence for growth regulation of tRNA gene transcription in yeast. J. Biol. Chem. 270, 28463–28470. 7. Clarke, E. M., Peterson, C. L., Brainard, A. V. & Riggs, D. L. (1996). Regulation of the RNA polymerase I and III transcription systems in response to growth conditions. J. Biol. Chem. 271, 22189–22195. 8. White, R. J. (2004). RNA polymerase III transcription— a battleground for tumour suppressors and oncogenes. Eur. J. Cancer, 40, 21–27. 9. Upadhya, R., Lee, J. & Willis, I. M. (2002). Maf1 is an essential mediator of diverse signals that repress RNA polymerase III transcription. Mol. Cell, 10, 1489–1494. 10. Willis, I. M., Desai, N. & Upadhya, R. (2004). Signaling repression of transcription by RNA polymerase III in yeast. Prog. Nucleic Acid Res. 77, 323–353. 11. Geiduschek, E. P. & Kassavetis, G. A. (2006). Transcription: adjusting to adversity by regulating RNA polymerase. Curr. Biol. 16, R849–R851. 12. Willis, I. M. & Moir, R. D. (2007). Integration of nutritional and stress signaling pathways by Maf1. Trends Biochem. Sci. 32, 51–53. 13. Moir, R. D., Lee, J., Haeusler, R. A., Desai, N., Engelke, D. R. & Willis, I. M. (2006). Protein kinase A regulates RNA polymerase III transcription through the nuclear localization of Maf1. Proc. Natl Acad. Sci. USA, 103, 15044–15049. 14. Oficjalska-Pham, D., Harismendy, O., Smagowicz, W. J., Gonzalez de Peredo, A., Boguta, M., Sentenac, A. & Lefebvre, O. (2006). General repression of RNA polymerase III transcription is triggered by protein phosphatase type 2A-mediated dephosphorylation of Maf1. Mol. Cell, 22, 623–632. 15. Roberts, D. N., Wilson, B., Huff, J. T., Stewart, A. J. & Cairns, B. R. (2006). Dephosphorylation and genomewide association of Maf1 with pol III-transcribed genes during repression. Mol. Cell, 22, 633–644. 16. Boguta, M., Czerska, K. & Zoladek, T. (1997). Mutation in a new gene MAF1 affects tRNA suppressor efficiency in Saccharomyces cerevisiae. Gene, 185, 291–296. 17. Pluta, K., Lefebvre, O., Martin, N. C., Smagowicz, W. J., Stanford, D. R., Ellis, S. R. et al. (2001). Maf1p, a negative effector of RNA polymerase III in Saccharomyces cerevisiae. Mol. Cell. Biol. 21, 5031–5040. 18. Desai, N., Lee, J., Upadhya, R., Chu, Y., Moir, R. D. & Willis, I. M. (2005). Two steps in Maf1-dependent repression of transcription by RNA polymerase III. J. Biol. Chem. 280, 6455–6462. 19. Geiduschek, E. P. & Kassavetis, G. A. (2001). The RNA polymerase III transcription apparatus. J. Mol. Biol. 310, 1–26. 20. Schramm, L. & Hernandez, N. (2002). Recruitment of RNA polymerase III to its target promoters. Genes Dev. 16, 2593–2620. 21. Cairns, C. A. & White, R. J. (1998). p53 is a general repressor of RNA polymerase III transcription. EMBO J. 17, 3112–3123.
Mammalian Maf1 Inhibits RNA Polymerase III Transcription 22. Crighton, D., Woiwode, A., Zhang, C., Mandavia, N., Morton, J. P., Warnock, L. J. et al. (2003). p53 represses RNA polymerase III transcription by targeting TBP and inhibiting promoter occupancy by TFIIIB. EMBO J. 22, 2810–2820. 23. Larminie, C. G. C., Cairns, C. A., Mital, R., Martin, K., Kouzarides, T., Jackson, S. P. & White, R. J. (1997). Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein. EMBO J. 16, 2061–2071. 24. Reina, J. H., Azzouz, T. N. & Hernandez, N. (2006). Maf1, a new player in the regulation of human RNA polymerase III transcription. PLoS ONE, 1, e134. 25. Johnson, S. S., Zhang, C., Fromm, J., Willis, I. M. & Johnson, D. L. (2007). Mammalian Maf1 is a negative regulator of transcription by all three nuclear RNA polymerases. Mol. Cell, 26, 367–379. 26. Rollins, J., Veras, I., Cabarcas, S., Willis, I. M. & Schramm, L. (2007). Human Maf1 negatively regulates RNA polymerase III transcription via the TFIIB family members Brf1 and Brf2. Int. J. Biol. Sci. 3, 292–302. 27. White, R. J. (2004). RNA polymerase III transcription and cancer. Oncogene, 23, 3208–3216. 28. Sutcliffe, J. E., Cairns, C. A., McLees, A., Allison, S. J., Tosh, K. & White, R. J. (1999). RNA polymerase III transcription factor IIIB is a target for repression by pocket proteins p107 and p130. Mol. Cell. Biol. 19, 4255–4261. 29. Scott, P. H., Cairns, C. A., Sutcliffe, J. E., Alzuherri, H. M., Mclees, A., Winter, A. G. & White, R. J. (2001). Regulation of RNA polymerase III transcription during cell cycle entry. J. Biol. Chem. 276, 1005–1014. 30. Vousden, K. H. & Lu, X. (2002). Live or let die: the cell's response to p53. Nat. Rev. Cancer, 2, 594–604. 31. Linke, S. P., Clarkin, K. C., Di Leonardo, A., Tsou, A. & Wahl, G. M. (1996). A reversible, p53-dependent G0/ G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage. Genes Dev. 10, 934–947. 32. Lohrum, M. A. E., Ludwig, R. L., Kubbutat, M. H. G., Hanlon, M. & Vousden, K. H. (2003). Regulation of HDM2 activity by the ribosomal protein L11. Cancer Cell, 3, 577–587. 33. Sulic, S., Panic, L., Barkic, M., Mercip, M., Uzelac, M. & Volarevic, S. (2005). Inactivation of S6 ribosomal protein gene in T lymphocytes activates a p53-dependent checkpoint response. Genes Dev. 19, 3070–3082. 34. Tessarollo, L. (2001). Manipulating mouse embryonic stem cells. Methods Mol. Biol. 158, 47–63. 35. Sutcliffe, J. E., Brown, T. R. P., Allison, S. J., Scott, P. H. & White, R. J. (2000). Retinoblastoma protein disrupts interactions required for RNA polymerase III transcription. Mol. Cell. Biol. 20, 9192–9202. 36. Larminie, C. G. C., Sutcliffe, J. E., Tosh, K., Winter, A. G., Felton-Edkins, Z. A. & White, R. J. (1999). Activation of RNA polymerase III transcription in cells transformed by Simian virus 40. Mol. Cell. Biol. 19, 4927–4934. 37. Winter, A. G., Sourvinos, G., Allison, S. J., Tosh, K., Scott, P. H., Spandidos, D. A. & White, R. J. (2000). RNA polymerase III transcription factor TFIIIC2 is overexpressed in ovarian tumours. Proc. Natl Acad. Sci. USA, 97, 12619–12624. 38. Daly, N. L., Arvanitis, D. A., Fairley, J. A., GomezRoman, N., Morton, J. P., Graham, S. V. et al. (2005).
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Deregulation of RNA polymerase III transcription in cervical epithelium in response to high-risk human papillomavirus. Oncogene, 24, 880–888. Goodfellow, S. J., Innes, F., Derblay, L. E., MacLellan, W. R., Scott, P. H. & White, R. J. (2006). Regulation of RNA polymerase III transcription during hypertrophic growth. EMBO J. 25, 1522–1533. White, R. J., Stott, D. & Rigby, P. W. J. (1989). Regulation of RNA polymerase III transcription in response to F9 embryonal carcinoma stem cell differentiation. Cell, 59, 1081–1092. Hurford, R. K., Cobrinik, D., Lee, M.-H. & Dyson, N. (1997). pRB and p107/p130 are required for the regulated expression of different sets of E2F responsive genes. Genes Dev. 11, 1447–1463. White, R. J., Gottlieb, T. M., Downes, C. S. & Jackson, S. P. (1995). Mitotic regulation of a TATA-binding-proteincontaining complex. Mol. Cell. Biol. 15, 1983–1992. Sorensen, P. D. & Frederiksen, S. (1991). Characterization of human 5S rRNA genes. Nucleic Acids Res. 19, 4147–4151. McLaren, A. & Goddard, J. P. (1986). The nucleotide sequence of a human tRNALeu gene. Nucleic Acids Res. 14, 5938. Dean, N. & Berk, A. J. (1988). Ordering promoter binding of class III transcription factors TFIIIC1 and TFIIIC2. Mol. Cell. Biol. 8, 3017–3025. Ullu, E. & Weiner, A. M. (1985). Upstream sequences modulate the internal promoter of the human 7SL RNA gene. Nature, 318, 371–374. Kleinert, H., Bredow, S. & Benecke, B. J. (1990). Expression of a human 7S K RNA gene in vivo requires a novel pol III upstream element. EMBO J. 9, 711–718. Lescure, A., Lutz, Y., Eberhard, D., Jacq, X., Krol, A., Grummt, I. et al. (1994). The N-terminal domain of the human TATA-binding protein plays a role in transcription from TATA-containing RNA polymerase II and III promoters. EMBO J. 13, 1166–1175. Jones, E., Kimura, H., Vigneron, M., Wang, Z., Roeder, R. G. & Cook, P. R. (2000). Isolation and characterization of monoclonal antibodies directed against subunits of human RNA polymerases I, II and III. Exp. Cell Res. 254, 163–172. Johnston, I. M., Allison, S. J., Morton, J. P., Schramm, L., Scott, P. H. & White, R. J. (2002). CK2 forms a stable complex with TFIIIB and activates RNA polymerase III transcription in human cells. Mol. Cell. Biol. 22, 3757–3768. Gomez-Roman, N., Grandori, C., Eisenman, R. N. & White, R. J. (2003). Direct activation of RNA polymerase III transcription by c-Myc. Nature, 421, 290–294. Shen, Y., Igo, M., Yalamanchili, P., Berk, A. J. & Dasgupta, A. (1996). DNA binding domain and subunit interactions of transcription factor IIIC revealed by dissection with poliovirus 3C protease. Mol. Cell. Biol. 16, 4163–4171. Fairley, J. A., Scott, P. H. & White, R. J. (2003). TFIIIB is phosphorylated, disrupted and selectively released from tRNA promoters during mitosis in vivo. EMBO J. 22, 5841–5850. Fairley, J. A., Kantidakis, T., Kenneth, N. S., Intine, R. V., Maraia, R. & White, R. J. (2005). Human La is found at RNA polymerase III-transcribed genes in vivo. Proc. Natl Acad. Sci. USA, 102, 18350–18355.
doi:10.1016/j.jmb.2008.02.060
Available online at www.sciencedirect.com
Regulation of RNA Polymerase III Transcription by Maf1 in Mammalian Cells Sarah J. Goodfellow 1 †, Emma L. Graham 1 †, Theodoros Kantidakis 1,2 , Lynne Marshall 1,2 , Beverly A. Coppins 1 , Danuta Oficjalska-Pham 3,4 , Matthieu Gérard 5 , Olivier Lefebvre 3 and Robert J. White 1,2 ⁎ 1
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK 2
Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow G61 1BD, UK 3
Laboratoire de Transcription des Gènes, Service de Biochimie et Génétique Moléculaire, CEA, iBiTecS, F-91191 Gif-sur-Yvette Cedex, France
RNA polymerase (pol) III produces essential components of the biosynthetic machinery; therefore, its output is tightly coupled with the rate of cell growth and proliferation. In Saccharomyces cerevisiae, Maf1 is an essential mediator of pol III repression in response to starvation. We demonstrate that a Maf1 ortholog is also used to restrain pol III activity in mouse and human cells. Mammalian Maf1 represses pol III transcription in vitro and in transfected fibroblasts. Furthermore, genetic deletion of Maf1 elevates pol III transcript expression, thus confirming the role of endogenous Maf1 as an inhibitor of mammalian pol III output. Maf1 is detected at chromosomal pol III templates in rodent and human cells. It interacts with pol III as well as its associated initiation factor TFIIIB and is phosphorylated in a serum-sensitive manner in vivo. These aspects of Maf1 function have been conserved between yeast and mammals and are therefore likely to be of fundamental importance in controlling pol III transcriptional activity. © 2008 Elsevier Ltd. All rights reserved.
4
Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland 5
Laboratoire de Transgenèse, Service de Biologie Moléculaire Systémique, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex, France Received 10 September 2007; received in revised form 22 February 2008; accepted 25 February 2008 Available online 4 March 2008 Edited by J. Karn
Keywords: Maf1; RNA polymerase III; TFIIIB; transcription
*Corresponding author. Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow, G61 1BD, UK. E-mail address: [email protected]. † These authors contributed equally to this work. Abbreviations used: pol, RNA polymerase; rRNA, ribosomal RNA; ScMaf1, Saccharomyces cerevisiae Maf1; HsMaf1, Homo sapiens Maf1; ChIP, chromatin immunoprecipitation; RT, reverse transcription; ARPP, acidic ribosomal phosphoprotein; HA, hemagglutinin; ES, embryonic stem.
Introduction In all eukaryotes examined, RNA polymerase (pol) III synthesises several essential components of the biosynthetic machinery, including tRNA and 5S ribosomal RNA (rRNA).1 The rate of pol III transcription is tightly regulated in response to changing conditions.1,2 For example, tRNA synthesis is low when nutrients or mitogens are limiting but is ra-
0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.
482 pidly induced by growth stimuli.3–7 Elevated pol III activity is a recurring feature of mouse and human tumours.2,8 In Saccharomyces cerevisiae, the pol III response to nutrient limitation has been shown to depend on the repressor Maf1.9 Indeed, genetic analyses indicate that Maf1 in budding yeast is an essential mediator of pol III repression under a wide range of adverse conditions, including DNA damage, secretory pathway defects, growth to stationary phase and inhibition of the target of rapamycin signalling pathway.9–12 Such stresses control the phosphorylation state of Maf1, via protein kinase A and protein phosphatase 2A.13–15 Maf1 was initially identified in a yeast screen for mutations that affect tRNA suppressors and was found to interact genetically with pol III.16 Subsequent studies demonstrated a physical interaction between Maf1 and pol III, as well as between Maf1 and the Brf1 subunit of the pol III-specific transcription factor TFIIIB.9,14,15,17,18 S. cerevisiae Maf1 (ScMaf1) is a hydrophilic protein of 395 amino acids that shows no significant similarity to other known proteins.16 However, database searches revealed Maf1 orthologs in other eukaryotes, including humans.17 The overall homology between Homo sapiens Maf1 (HsMaf1) and ScMaf1 is low (∼16% amino acid identity), but these orthologs share three blocks of much higher conservation (28%–47%) arranged in the same order. This study demonstrates that mammalian Maf1 can repress pol III transcription in vitro and in vivo. Consistent with this, genetic loss of Maf1 elevates expression of pol III products. We show that endogenous HsMaf1 associates stably with pol III as well as TFIIIB and is present at tRNA, 5S rRNA, 7SL and 7SK genes in vivo. Scanning chromatin immunoprecipitation (ChIP) analysis detects HsMaf1 around the start region of the 7SL gene but not further downstream, suggesting that it may not associate with actively transcribing pol III. The data are consistent with previous studies of Maf1 in yeast. The conservation of this mechanism for pol III regulation strongly suggests that it is of fundamental importance.
Results Human Maf1 can inhibit pol III transcription in vitro and in vivo An expression vector encoding the mouse protein was transfected into CCL39 rodent fibroblasts to test if Maf1 can regulate pol III transcription in mammals. In parallel, these cells were transfected with empty vector or expression vector encoding the Brf1 subunit of TFIIIB. Reverse transcription (RT) PCR was used to monitor levels of endogenous pol III transcripts. Transfection of Maf1 was found to reduce expression of every pol III product examined, including 5S rRNA, tRNALeu, B2 RNA, 7SK RNA and U6 snRNA (Fig. 1). The genes encoding these transcripts include representatives of each of the
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
Fig. 1. Mouse Maf1 inhibits pol III transcription in vivo. CCL39 fibroblasts were transiently transfected with pcDNA3HA vector (lane 1), pcDNA3HA.Maf1 (lane 2) or pcDNA3HA.Brf1 (lane 3). Extracted RNA was analysed by RT-PCR using primers specific for the indicated transcripts.
promoter types used by pol III, including the type III promoters that are not found in S. cerevisiae.1,19,20 Inhibition is specific, since levels of the mRNA encoding acidic ribosomal phosphoprotein (ARPP) P0 remained unaffected. In contrast, the Brf1 vector selectively induced tRNA and B2 genes, without affecting the other pol III templates, for which it is either not required or not limiting in these cells.19,20 The data indicate that mouse Maf1 can have a repressive effect on the expression of pol III products in fibroblasts. Although specific, the response encompasses all pol III templates examined. This is consistent with whole-genome analyses in S. cerevisiae, which detected Maf1 at all known categories of pol III-transcribed genes.14,15 Purified recombinant HsMaf1 was tested for its ability to regulate transcription in a HeLa cell nuclear extract. As shown in Fig. 2a, 200 ng of recombinant HsMaf1 abolishes transcription of all the pol III templates tested, namely, 5S rRNA, tRNALeu, B2, VA1, 7SL and 7SK genes. The repression is specific, as pol I transcription of a pre-rRNA template shows minimal response. Furthermore, the inhibition of pol III transcription by HsMaf1 is dose dependent, with as little
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
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Fig. 2. HsMaf1 specifically inhibits pol III transcription in human cell extracts. (a) HeLa nuclear extract (20 μg) was used to transcribe the indicated templates in the presence or absence of 200 ng of recombinant HsMaf1, as indicated. (b) HeLa nuclear extract (20 μg) was used to transcribe the indicated templates in the presence of increasing amounts (12.5, 25, 50, 100 or 200 ng) of HsMaf1 or equivalent volumes of HsMaf1 control fraction.
as 12.5 ng of recombinant protein causing a substantial reduction in transcriptional activity (Fig. 2b). HsMaf1 may act at more than one stage in the pol III transcription cycle Order-of-addition experiments can sometimes provide information concerning mechanisms of repression. For example, this approach provided evidence that p53 inhibits pol III transcription complex assembly but has a minimal effect once the complex has formed.21 Subsequent analyses confirmed that p53 can block promoter access of TFIIIB, in vitro and in vivo.22 We therefore carried out order-of-addition experiments to test if HsMaf1 can inhibit a preassembled pol III initiation complex or acts exclusively during complex assembly. Recombinant HsMaf1 was mixed with HeLa extract 15 min prior to addition of VA1 gene template (Fig. 3a, lanes 1 and 2), simultaneously with VA1 addition (lanes 3 and 4) or 15 min after addition of VA1 template (lanes 5 and 6). Nucleotides were then added to allow transcription. Under the conditions of our assays, 15 min is sufficient for stable complex formation on the VA1 promoter.23 Although repression was most efficient when HsMaf1 was present during pre-initiation complex assembly, significant inhibition was still observed from pre-assembled transcription complexes (Fig. 3b). This contrasts with repression by p5321 but resembles the outcome of similar experiments carried out with ScMaf1, which inhibits most effectively if added prior to transcription complex formation, although it is able to significantly repress a pre-assembled complex.18 This finding was interpreted as supporting a model in which ScMaf1 acts at
more than one stage in the transcription cycle, one of which is likely to be recruitment/recycling of the pol.18 This is consistent with the fact that ScMaf1 interacts with two distinct targets, TFIIIB and pol III itself.14,15,17,18 We therefore carried out co-immunoprecipitation assays to test if the same is true for the human proteins. HsMaf1 interacts with the endogenous pol III transcription apparatus Antiserum raised against Maf1 was found to coimmunoprecipitate from HeLa nuclear extract both TFIIIB and pol III, as revealed by Western blotting for Brf1 and for RPC39, respectively (Fig. 4a). In addition, blotting for TFIIIC220 provided evidence for a stable association with TFIIIC. These interactions are specific, as they were not detected using pre-immune serum or an irrelevant antibody against the translation factor 4EBP1. They were also confirmed using alternative antisera (data not shown). DNA does not appear to be required for association of these proteins, as co-immunoprecipitation was undiminished following DNase treatment. The interactions were also retained after 3 days of serum deprivation (Fig. 4b). Maf1 is phosphorylated in fibroblasts in a serum-dependent manner Since phosphorylation of ScMaf1 is linked to its function, we asked whether Maf1 is also phosphorylated in mammalian cells. Vector encoding hemagglutinin (HA)-tagged murine Maf1 was transfected into fibroblasts, which were cultured for 24 h in the
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Mammalian Maf1 Inhibits RNA Polymerase III Transcription
nents TBP and Brf1 as positive controls; in both cases, pol III transcription was inhibited. In contrast, transcription was stimulated by depletion of HsMaf1, using either of two antisera (1166 and 1167). The corresponding pre-immune sera had little or no effect, when compared with extract that was mock depleted in the absence of antibody. These data provide evidence that endogenous HsMaf1 present in HeLa nuclear extracts has a negative effect on transcription by pol III. Pol III transcript expression is suppressed by endogenous Maf1 in mouse embryonic stem cells
Fig. 3. HsMaf1 can inhibit pol III transcription whether it is added before or after transcription complex assembly. (a) Buffer (lanes 1, 3 and 5) or 200 ng of recombinant HsMaf1 (lanes 2, 4 and 6) was added to nuclear extract (20 μg) 15 min before (lanes 1 and 2), simultaneously with (lanes 3 and 4) or 15 min after (lanes 5 and 6) the addition of VA1 template DNA. Transcription was initiated by the addition of nucleotides. (b) Transcriptional outputs for the abovementioned experiment and two repeats were determined by densitometry and are displayed graphically (n = 3; *significantly different from control, p b 0.05; **significantly different from 15 min, p b 0.05).
presence or absence of serum and then labelled with [32P]orthophosphate for 3 h prior to harvesting. Immunoprecipitation with anti-HA antibody revealed strong labelling of transfected Maf1 in the cells grown in serum, whereas labelling was much diminished in the serum-starved cells (Fig. 5, top panel). Western blotting confirmed that Maf1 was indeed immunoprecipitated from the starved cells (Fig. 5, bottom panel). It also revealed that Maf1 from the growing cells migrates as a doublet under our gel conditions, whereas the upper band is selectively diminished when extracted from serum-starved cells. Both forms appear to be phosphorylated, as they are labelled with [32P]. We speculate that the slower-migrating form may be most heavily modified. These data provide evidence that Maf1 is phosphorylated in fibroblasts in a serum-sensitive manner. Immunodepletion of HsMaf1 raises pol III transcription Antisera against HsMaf1 were used to immunodeplete the endogenous protein from HeLa cell extract (Fig. 6). We depleted extracts of the TFIIIB compo-
Attempts were made to generate a genetic knockout in mouse embryonic stem (ES) cells. This proved to be unexpectedly difficult, but we did succeed in deleting one allele, to create an ES line that carries a single intact copy of the Maf1 gene. Western blotting indicated that Maf1 protein expression is ∼40% lower in the heterozygous cells, consistent with the loss of one allele (Fig. 7a). RT-PCR showed a specific decrease in Maf1 mRNA to ∼34% of wild-type levels (Fig. 7b). In contrast, expression of B2 and tRNAArg transcripts is elevated approximately two- to threefold. 5S rRNA and tRNALeu, however, only increased by ∼30% in the heterozygous ES cells. The reason for this differential sensitivity is unclear, since 5S rRNA and tRNALeu expression responds strongly to Maf1 in other assays ((Figs. 1, 2 and 6)). Elevated B2 RNA in the heterozygous cells was also observed by Northern blot (Fig. 7c). We conclude that endogenous Maf1 inhibits expression of at least some pol III templates in mouse ES cells. Mammalian Maf1 associates with pol III-transcribed genes in vivo ChIP assays were used to test if Maf1 interacts with pol III-transcribed genes in mammalian cells. Two separate antisera raised against different epitopes within HsMaf1 both detected this factor at chromosomal tRNA and 5S rRNA genes in HeLa cells (Fig. 8a). As might be expected, the HsMaf1 signal was weaker than that obtained using positive control antisera against pol III or TFIIIB but was nevertheless well above the background obtained using beads alone or a negative control antibody against the pol II-specific factor TFIIB. Similar results were obtained with CCL39 and A31 fibroblasts (Fig. 8b and other data not shown). In addition, HsMaf1 was detected at 7SK genes, despite their distinct promoter architecture (Fig. 8c). As expected, only background signal was obtained in this case with the Brf1 antiserum used to detect TFIIIB, because Brf1 is not utilised by such genes as 7SK that have type III promoters.19,20 We conclude that chromosomal pol III-transcribed genes of all promoter types are targeted specifically by Maf1 in mammalian cells. Scanning ChIP analysis has been used previously to investigate the region of a target gene that is occupied by ScMaf1.15 This approach is difficult to
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
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Fig. 4. HsMaf1 associates with TFIIIB, TFIIIC and pol III. (a) Proteins were immunoprecipitated (IP) from HeLa nuclear extract using the indicated antibodies, in the absence (lanes 1–3) or presence (lanes 4–6) of 20 U of DNase 1. Immunoprecipitates were analysed by Western blotting with antibody Ab7 against TFIIIC (220-kD subunit), antibody C39 against pol III (RPC6) and antibody 128 against TFIIIB (Brf1). Lane 7 shows 25% of input material. The asterisk marks an apparently nonspecific band that migrates above TFIIIC220 and is present in all the immunoprecipitations. Note that all three panels are from the same experiment but show samples run on different gels (a gap was left between lanes 3 and 4 in the middle and lower panels). (b) Proteins were immunoprecipitated using the indicated antibodies from HeLa cells cultured in the presence of 10% (lanes 1–3) or 0.5% (lanes 4–6) serum. Immunoprecipitates were analysed by Western blotting with antibody Ab7 against TFIIIC (220kD subunit), antibody C39 against pol III (RPC6) and antibody 128 against TFIIIB (Brf1). Lanes 7 and 8 show 12.5% of input material.
apply to pol III templates, because of their short size. However, it was used successfully for the 522-bp SCR1 gene, the longest pol III transcription unit in the S. cerevisiae genome, which encodes the 7SL RNA component of the signal recognition particle.15 We attempted to do the same with a human 7SL gene, although it is only ∼ 300 bp. Primers were designed to amplify sequences at the start and finish of the 7SL coding region, as well as ∼ 200 and ∼500 bp downstream of the termination site and ∼ 200 bp upstream of the initiation site (Fig. 9a). The size of sonicated chromatin fragments used for ChIP limits resolution and causes overlap between the signals from neighbouring amplicons. Nevertheless, the results obtained for TFIIIB, TFIIIC and pol III conformed sufficiently well to their expected positions to establish a proof of principle. Thus, the Brf1 subunit of TFIIIB is known to locate to the region immediately upstream of the start site and was accordingly detected most strongly using primer set 2, with the neighbouring primer sets 1 and 3 giving weaker but significant signals and the downstream primer sets 4 and 5 close to the background (Fig. 9b and c). In contrast, TFIIIC is positioned over a substantial part of the coding region, extending from the start towards the termination site; consistent with
this, it was detected most strongly using primer sets 2 and 3, while weaker signals were obtained with the flanking primer sets. Pol III was also detected most strongly using primers that amplify the coding region, especially set 3, again with weaker signals from the flanking primer sets. These observations gave us some confidence in our scanning ChIP assay, despite the difficulties in resolving regions of pol III-transcribed genes, because of their small size. The Maf1 signal was weak on the 7SL gene but was nevertheless consistently above the background with primer set 2 (Fig. 9c). Since the Brf1 ChIP is also strongest with set 2, whereas TFIIIC and pol III show different patterns, the data support a model in which Maf1 co-localises with Brf1 in the vicinity of the transcription start site. This is consistent with scanning ChIP analysis of ScMaf1 on the SCR1 gene in yeast.15
Discussion This study demonstrates that the function of Maf1 as a repressor of pol III transcription has been conserved through evolution. Mammalian Maf1 potently inhibits expression of all pol III templates
486
Fig. 5. Maf1 displays serum-sensitive phosphorylation in fibroblasts. Anti-HA antibody was used to immunoprecipitate from CCL39 fibroblasts transfected with pcDNA3HA.Maf1 and cultured for 24 h in the absence (lane 1) or presence (lane 2) of serum before radiolabelling for 3 h with [32P]orthophosphate. Immunoprecipitated material was resolved by SDS-PAGE, transferred to nitrocellulose and then visualised by autoradiography (upper panel) and Western blotting with anti-HA antibody (lower panel).
tested in human cell extracts and in transfected fibroblasts. This is not an artefact of overexpression, since pol III transcript levels are elevated by depleting endogenous Maf1 through genetic ablation in mouse ES cells. Furthermore, endogenous Maf1 is detected by ChIP at chromosomal tRNA and 5S rRNA genes in rodent and human cells and co-immunoprecipitates with the endogenous human pol III machinery. We therefore conclude that mammalian Maf1 interacts with the pol III transcription apparatus and restrains its output in vivo. Other studies of human Maf1 have been published recently, reflecting the intense interest in this important factor.24–26 The data from these authors are consistent with ours. For example, they have shown that overexpression of HsMaf1 in human cancer cell lines can repress transfected tRNAArg, VA1 and U6 reporters without affecting a co-transfected pol IIdependent reporter.25,26 We have extended these findings by transfecting a murine Maf1 expression vector and showing that it can selectively repress a range of endogenous pol III-transcribed genes in untransformed rodent fibroblasts (Fig. 1). Reina et al. demonstrated that recombinant HsMaf1 can specifically inhibit pol III transcription in vitro of 5S, VA1, 7SL and U6 genes without affecting pol II transcription from the adenovirus major late promoter.24 We have confirmed this observation for 5S, VA1 and 7SL, while adding tRNALeu , B2 and 7SK genes as res-
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
ponsive targets and demonstrating that pol I transcription is unresponsive (Fig. 2). Two studies showed that RNA interferencemediated depletion of HsMaf1 from human cells can selectively raise expression of pol III transcripts.24,25 We have adopted a different approach to confirm the function of endogenous Maf1. Figure 6 shows that its immunodepletion from HeLa extracts increases pol III transcription in vitro. Furthermore, we have made heterozygous mouse ES cells and shown that genetic deletion of one Maf1 allele raises pol III transcript expression in vivo (Fig. 7). Our work therefore provides independent confirmation that Maf1 is a physiologically relevant repressor of pol III transcription in mammals. This conclusion is supported by ChIP data revealing the presence of endogenous Maf1 at 5S rRNA, tRNALeu, 7SL and 7SK genes in HeLa cells and CCL39 fibroblasts (Figs. 8 and 9). It was also found at tRNALeu genes in human U87 glioblastoma cells.25 The results have been obtained using several different antibodies. Between these studies, Maf1 has been shown to operate in a disparate range of mammalian cell types: human HEK293 embryonic kidney cells and IMR90-TERT fibroblasts,24 two human glioblastoma lines25 and HeLa cells, as well as rodent ES cells and fibroblasts. No exception has been reported. We found that endogenous TFIIIB and pol III coimmunoprecipitate with endogenous HsMaf1 from HeLa cells, as reported by Reina et al.24 Their study also used glutathione S-transferase pull-down assays to show that HsMaf1 targets TFIIIB through its Brf1 subunit and pol III through its RPC1 and RPAC2 subunits.24 The specific targeting of both pol III and Brf1 has therefore been conserved from yeast to man. We were surprised to detect TFIIIC in complexes with Maf1 from human cells as well—an observation that has not been reported previously. Although a direct interaction is certainly possible, we suspect that association is instead mediated through simultaneous binding of Brf1 to both Maf1 and TFIIIC. Scanning ChIP analysis suggests that Maf1 is located
Fig. 6. HeLa nuclear extract was mock depleted (lane 1) or immunodepleted with TBP antibody MTBP-6 (lane 2), Brf1 antibody 128 (lane 3), HsMaf1 antibodies 1166 and 1167 (lanes 5 and 7, respectively) and the corresponding pre-immune sera (lanes 4 and 6, respectively). These extracts were then used to transcribe the indicated templates.
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
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Fig. 7. Endogenous Maf1 inhibits pol III transcription in mouse cells. (a) Western blot of whole cell extract (10 μg) from matched Maf1+/+ and Maf1+/− ES cells using antibodies to Maf1 and actin, as indicated. (b) RNA from matched Maf1+/+ or Maf1+/− ES cells was analysed by RT-PCR using primers specific for the indicated transcripts. The tRNA primers recognise unprocessed primary transcripts, whereas all other primers recognise mature products. (c) Northern blots with RNA (10 μg) from matched Maf1+/+ or Maf1+/− ES cells were probed for B2 and ARPP P0 RNAs.
near the start of the 7SL gene, where Brf1 is expected to be found, as well as pol III prior to transcript initiation and the upstream end of TFIIIC. The Maf1 signal is close to the background further downstream, despite strong occupancy by pol III and TFIIIC within the transcribed region. This is consistent with the hypothesis that TFIIIC and actively transcribing pol III do not bind Maf1. However, interpretation has to be tentative, since epitope masking or inefficient cross-linking may also be involved. It would have been preferable to use a gene that gave a stronger Maf1 signal than 7SL, but our choice was severely constrained because most pol III templates are too short for scanning ChIP analysis. The low Maf1 signal on 7SL is consistent with its high rate of transcription. The data indicate that Maf1 performs a significant inhibitory role in apparently unstressed mammalian cells. Although it also functions under normal growth conditions in S. cerevisiae, the repressive activity of ScMaf1 is substantially enhanced when these yeasts are subjected to stresses.14,15 Indeed, ScMaf1 appears to mediate the pol III response to all forms of stress that have been tested, including DNA damage and growth into stationary phase.9,10 Mammals possess a substantial number of regulatory factors that impact on pol III output and are not present in yeast. Examples include the tumour suppressors RB and p53, both of which are potent repressors of pol III transcription in mammalian cells.27 RB and its relatives p107 and p130 perform dominant roles in restraining pol III output in growth-arrested murine fibroblasts.28,29 Furthermore, a wide range of stresses are known to induce p53, including DNA damage.30 It will be important to determine how mammalian Maf1 is integrated with other pathways that influence
pol III activity. One might guess that regulators such as RB and p53 have evolved to deal with conditions that are specific for Metazoa, whereas mammalian Maf1 may mediate responses to stress conditions that are also encountered by unicellular organisms. However, this hypothesis is too simplistic. Strong p53 responses are triggered by depletion of ribonucleotides31 or ribosomal protein imbalances,32,33 stresses that must also affect lower eukaryotes. Especially surprising is the fact that these responses do not appear to be redundant in mammalian cells and are lost in the absence of p53.31–33 This suggests that rather than simply providing additional levels of control, metazoan-specific response pathways may have actually replaced some of the more ancient mechanisms for coping with stress. Although Maf1 is essential for pol III regulation under all forms of stress that have been examined in budding yeast,9,10 we predict that it will have a more specialised role in mammals. Characterisation of this role and its interaction with other regulatory pathways will require extensive analysis. Johnson et al. provided striking evidence that the role of Maf1 has evolved through evolution.25 They demonstrated that as well as repressing pol III, HsMaf1 can bind and directly regulate a select group of pol II-transcribed genes, including the gene encoding TBP.25 By influencing TBP expression, HsMaf1 can also indirectly affect transcription of additional templates, including the pol I-dependent rRNA genes.25 This contrasts strongly with budding yeast, where ScMaf1 does not affect TBP levels9 and where genome-wide ChIP analyses did not implicate targets other than pol III templates.14,15 Evidently, some aspects of Maf1 function have changed dramatically
488
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
citing to determine if Maf1 function is compromised in tumours. With its capacity to restrict a cell's biosynthetic capacity, it is conceivable that HsMaf1 may turn out to be a physiological tumour suppressor that is targeted in cancers.
Materials and Methods Cell culture Cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U/ ml of penicillin and 100 μg/ml of streptomycin. ES cells were cultured on feeder cells (mouse embryonic fibroblasts inactivated by mitomycin C), exactly as previously described.34 Transient transfection CCL39 cells were seeded onto six-well plates at a density of 1 × 105 cells/well. After 24 h, cells were transfected with 2 μg/well of plasmid DNA using Lipofectamine (Life Technologies, Inc.). pcDNA3HA.Brf1 is a human Brf1 cDNA in the pcDNA3HA expression vector.35 pcDNA3HA.Maf1 was created by subcloning a cDNA encoding Mus musculus Maf1 into BamHI/XbaI-digested pcDNA3HA. RNA analysis RNA extraction and RT were performed as previously described.36 Primers and cycling parameters have been described for 5S rRNA, ARPP P0 mRNA,37 7SK RNA, pretRNALeu38 and U6 snRNA.39 Other primers were as follows: Fig. 8. Endogenous Maf1 associates with class III genes in mammalian cells. ChIPs were performed using HeLa cells (a and c) or CCL39 fibroblasts (b) with beads alone or antibodies against Brf1, pol III, TFIIB or alternative Maf1 epitopes, as indicated. Association of each factor with the indicated genes was analysed by PCR with gene-specific primers. Input genomic DNA (10%, 2% and 0.4% of that used in ChIPs) was analysed in parallel.
during evolution. The absence of pol I repression by recombinant Maf1 in vitro does not conflict with the results of Johnson et al.,25 who found no interaction of HsMaf1 with ribosomal DNA promoters in ChIP assays; although pre-rRNA levels are responsive to HsMaf1 in transfected cells, this appears to be an indirect effect mediated through changes in TBP expression.25 Depletion of HsMaf1 by RNA interference was found to cause morphological changes.25 Furthermore, overexpression of HsMaf1 inhibits anchorageindependent growth of a glioblastoma cell line.25 On this basis, Johnson et al. suggested that HsMaf1 might influence oncogenic transformation.25 It is not yet established whether such effects require the ability of HsMaf1 to repress pol III transcription. However, it is noteworthy that elevated pol III output is strongly associated with cell transformation.2,8 It will be ex-
B2: 5′-GGG GCT GGA GAG ATG GCT-3′ and 5′-CCA TGT GGT TGC TGG GAT-3′ as well as mouse Maf1: 5′-GCA GTT CTG CCA GGA GGG CCA3′ and 5′-CTC CAT GGT GCT GGT CTC CTC-3′. PCR was performed using the following cycling parameters: B2: 95 °C for 3 min; 18 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s; and 72 °C for 5 min as well as mouse Maf1: 95 °C for 2 min; 25 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min; and 72 °C for 5 min. Northern blotting was performed as previously described.21 B2 and ARPP P0 probes have been described.40,41 Recombinant Maf1 and transcription assays Recombinant Maf1 was produced by cloning the HsMaf1 gene into the pFastBac vector from the Bac-to-Bac Baculovirus Expression System (Invitrogen) using the BamHI and SalI restriction sites. The recombinant protein was expressed in insect cells infected with baculovirus and has a 6-histidine tag. His-tagged HsMaf1 was then purified to ∼ 95% homogeneity using a Poros MC20 (Boeringer Mannheim) cobalt-chelate affinity column and was eluted using 50 mM imidazole. A fraction obtained from control purification of HsMaf1 with no His tag was used as a control in transcription reactions. In vitro transcription assays used 20 μg of HeLa nuclear extract and were carried out as previously described.42
Mammalian Maf1 Inhibits RNA Polymerase III Transcription
489
Fig. 9. HsMaf1 co-localises with Brf1 on a 7SL gene. (a) Schematic illustration of the positions of primer sets 1–5 relative to the coding region of a 7SL gene. (b) ChIPs were performed using HeLa cells and preimmune serum or antiserum against Maf1 (1166), Brf1 (128), pol III (1900) and TFIIIC (Ab7), as indicated. PCR analyses are shown using each of the five 7SL primer sets carried out for the same set of ChIP DNA. (c) Schematic representation of the ChIP signals obtained for Maf1 (blue), Brf1 (red), TFIIIC (green) and pol III (purple) with the five 7SL primer sets (as indicated along the x-axis). The y-axis values show the mean relative strengths of ChIP signal from three independent experiments, expressed in arbitrary units after normalisation against input and the signal with pre-immune serum. Template plasmids were pHu5S3.1 for 5S rRNA,43 pLeu for tRNA,44 pTB14 for B2,40 pVA1 for VA1,45 p7L30.1 for 7SL,46 7SK-pAT/153 for 7SK47 and EcoRI-linearized pHrP2 for pre-rRNA.48 For immunodepletion, extract was pre-incubated with protein A–Sepharose beads carrying pre-bound antibodies for 2 h on ice, with gentle agitation every 5 min.
III have been described previously.21,49 Antisera 1166 and 1167 against HsMaf1 were raised by immunizing rabbits with synthetic peptides YNPDLDSDPFGEDGSL and CRSISGSTYTPSEAGN, corresponding to HsMaf1 residues 168–183 and 203–218, respectively. Antiserum 1767 was raised by immunizing rabbits with recombinant mouse Maf1 protein; this antiserum was used for Western blotting.
Immunoprecipitation and Western blotting
Phosphate labelling in vivo
HeLa nuclear extract (250 μg) was incubated at 4 °C for 3 h on a rotating wheel with 25 μl of protein G–Sepharose beads that had been pre-incubated with affinity-purified antiserum 1166 against Maf1, the corresponding pre-immune serum or an anti-4EBP1 antibody (Santa Cruz Biotechnology). Bound material was resolved by SDS-PAGE on 7.8% polyacrylamide gels, and specific proteins were detected by Western blotting as previously described.42 Peptide antiserum 128 against the TFIIIB subunit Brf1 and monoclonal antiserum C39 against the RPC6 subunit of pol
CCL39 cells were transfected with pcDNA3HA.Maf1 and then, after 48 h, were labelled for 3 h with 0.3 mCi/ml of [32P]orthophosphate in phosphate-free medium, before harvesting for immunoprecipitation, as previously described.50 ChIP assays ChIP assays were performed as previously described51 using antibody 128 against Brf1,21 antiserum Ab7 against
490 TFIIIC,52 antiserum 1900 against pol III,53 antibody FL109 against TFIIA as well as antibody C18 against TFIIB (both from Santa Cruz Biotechnology), antisera 1166 and 1167 against Maf1 (see above) and antiserum 2966 against Maf1. Antiserum 2966 against Maf1 was raised by immunizing rabbits with synthetic peptides YDFSTARSHEFSREPS (corresponding to residues 108–123 of human and mouse Maf1) and GGEGRAEETSTMEEDR (which matches residues 240–250 of HsMaf1 and residues 237–252 of mouse Maf1). Immunoprecipitated DNA was analysed by PCR using the indicated primers. Apart from the 7SL scanning ChIP assays, primers and amplification conditions have been previously described.37,51,54 The 7SL primers were as follows: 7SL-1: 5′-CCGTGGCCTCCTCTACTTG-3′ and 5′TTTACCTCGTTGCACTGCTG-3′, 7SL-2: 5′-CGTCACCATACCACAGCTTC-3′ and 5′CGGGAGGTCACCATATTGAT-3′, 7SL-3: 5′-GTTGCCTAAGGAGGGGTGA-3′ and 5′TCTCTTGAGAGTCCAAAATTAA-3′, 7SL-4: 5′-TTTTTGACACACTCCTCCAAGA-3′ and 5′ATCTGGTCAAAGCAACATACACTG-3′ and 7SL-5: 5′-TGCCTCCAGATAAAACTGCTC-3′ and 5′ACCCCACTAGAACCCTGACA-3′. PCR was performed using the following cycling parameters: 95 °C for 2 min; 25 cycles of 95 °C for 1 min, 58 °C for 30 s as well as 72 °C for 1 min; and 72 °C for 3 min. Maf1 gene deletion Genomic fragments flanking exons 2 and 5 of the mouse Maf1 gene were PCR amplified and subcloned with LacZ, neomycin and diphtheria toxin cassettes. The construct was transfected into ES cells by electroporation, and Southern blotting was used to identify cells carrying the mutant Maf1 allele.
Acknowledgements This work was funded by grants 068710 and 081977 from the Wellcome Trust, grant BBS/B/0711X from the Biotechnology and Biological Sciences Research Council, grants C1288/A3143 and C1288/A4411 from Cancer Research UK (to R. J. W.) and grant 3982 from the Association pour la Recherche contre le Cancer (to O. L.). We are grateful to Arnie Berk and Yuhong Shen for providing antiserum Ab7 and VA1 plasmid, to Peter Cook for providing monoclonal antibody C39 and to Shona Murphy for providing 7SL and 7SK plasmids. We also thank Peter Geiduschek for encouraging us to use the scanning ChIP assay.
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