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RESEARCH ARTICLE

Reconstitution of human rRNA gene transcription in mouse cellsby a complete SL1 complex

Kensaku Murano1, Mitsuru Okuwaki1, Fumitaka Momose2, Michiko Kumakura1, Shuhei Ueshima1,Robert F. Newbold3 and Kyosuke Nagata1,*

ABSTRACT

An important characteristic of the transcription of a ribosomal RNA

gene (rDNA) mediated by DNA-dependent RNA polymerase (Pol) I

is its stringent species specificity. SL1/TIF-IB is a key complex for

species specificity, but its functional complex has not been

reconstituted. Here, we established a novel and highly sensitive

monitoring system for Pol I transcription to reconstitute the SL1

activity in which a transcript harboring a reporter gene synthesized

by Pol I is amplified and converted into translatable mRNA by

the influenza virus RNA-dependent RNA polymerase. Using this

monitoring system, we reconstituted Pol I transcription from the

human rDNA promoter in mouse cells by expressing four human

TATA-binding protein (TBP)-associated factors (TAFIs) in the SL1

complex. The reconstituted SL1 also re-activated human rDNA

transcription in mouse A9 cells carrying an inactive human

chromosome 21 that contains the rDNA cluster. Chimeric SL1

complexes containing human and mouse TAFIs could be formed,

but these complexes were inactive for human rDNA transcription.

We conclude that four human TAFIs are necessary and sufficient

to overcome the barrier of species specificity for human rDNA

transcription in mouse cells.

KEY WORDS: Ribosomal RNA gene, Transcription, Species

specificity, RNA polymerase I, SL1, TIF-IB

INTRODUCTIONOne of the most important rDNA transcription issues to be

addressed is the molecular mechanism of its stringent species

specificity (Heix and Grummt, 1995). Three decades ago,

Grummt et al. demonstrated that in vitro transcription of human

and mouse rDNAs requires completely homologous extracts

(Grummt, 1981; Grummt et al., 1982), i.e. human rDNA is not

transcribed in mouse cell extracts and vice versa. This distinct

promoter recognition specificity of Pol I is mediated by a multi-

subunit factor called SL1 in humans and TIF-IB in mice. SL1/

TIF-IB recruits Pol I on the rDNA promoter through RRN3/TIF-

IA and stabilizes the binding of the upstream binding factor

(UBF) at the rDNA promoter (Friedrich et al., 2005; Moss and

Stefanovsky, 2002; Russell and Zomerdijk, 2006). Pol I, UBF and

TIF-IA are functionally interchangeable between humans and

mice, whereas the SL1/TIF-IB complex must be derived from the

same species to support rDNA transcription (Bell et al., 1990;

Heix and Grummt, 1995; Learned et al., 1985; Mishima et al.,

1982; Rudloff et al., 1994; Schnapp et al., 1993). The human SL1

complex contains the TATA-binding protein (TBP) and three

TBP-associated factors known as TAFIA (also known as TAFI48

and TAF1A), TAFIB (also known as TAFI63 and TAF1B) and

TAFIC (also known as TAFI110C and TAF1C) (Comai et al.,

1992). Among these SL1 components, TBP is interchangeable

between humans and mice, indicating that TAFIs are responsible

for the promoter selectivity of SL1/TIF-IB (Rudloff et al., 1994).

UV cross-linking experiments have demonstrated that TAFIA and

TAFIB bind to both the homologous and heterologous promoters,

suggesting that a binding of SL1/TIF-IB to the heterologous

promoter precludes a formation of productive initiation

complexes (Rudloff et al., 1994). There are contradictory

reports about reconstituting SL1/TIF-IB activity in vitro. It has

been reported that the three recombinant human TAFIs and the

TBP are necessary and sufficient to reconstitute a

transcriptionally active human SL1 complex (Zomerdijk et al.,

1994). In contrast, three mouse recombinant TAFIs and the TBP

complex did not reconstitute the TIF-IB activity in an in vitro

transcription system (Heix et al., 1997). Later, a further TAFI,

TAFID (also known as TAFI41 and TAF1D), was identified from

the TBP-antibody affinity-purified SL1 fraction (Gorski et al.,

2007). TAFID co-migrates with TBP on SDS-PAGE, so it

remained unidentified for more than 10 years following the initial

identification of the SL1 complex (Comai et al., 1992). TAFID is

involved in rDNA transcription in vivo (Gorski et al., 2007).

Thus, we hypothesize that TAFID is a final component in

reconstituting the human SL1 activity in mouse cells and

overcoming the rDNA transcription species barrier.

To examine this hypothesis, we established a novel and highly

sensitive monitoring system for Pol I transcription. The products

synthesized by Pol I are not translated to proteins. We tried to

overcome this problem using the influenza virus RNA-dependent

RNA polymerases (RdR Pol). Influenza A virus belongs to

the Orthomyxoviridae family, and its genome comprises eight

single-stranded RNAs of negative polarity (Naito et al., 2007).

The viral RNA (vRNA) is associated with the viral RdR Pol,

comprising PB1, PB2 and PA subunits, and nucleoprotein (NP),

forming viral ribonucleoprotein (vRNP) complexes (Nagata et al.,

2008; Portela and Digard, 2002). The transcription promoter and

the replication signal of the viral genome exist at the 39 and 59

untranslated terminal regions (UTRs) of each segment.

Transcription of the influenza virus genome is initiated by the

cap snatching mechanism (Li et al., 2001). The viral RdR Pol

polyadenylates the nascent RNA chain, possibly by a slippage

1Department of Infection Biology, Faculty of Medicine and Graduate School ofComprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai,Tsukuba, Ibaraki 305-8575, Japan. 2Kitasato Institute for Life Sciences, KitasatoUniversity, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. 3Institute of CancerGenetics and Pharmacogenomics, Division of Biosciences, School of HealthSciences and Social Care, Brunel University, Uxbridge, Middlesex UB8 3PH, UK.

*Author for correspondence (knagata@md.tsukuba.ac.jp)

Received 21 November 2013; Accepted 25 May 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3309–3319 doi:10.1242/jcs.146787

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mechanism at the adenylation signal which consists of five toseven uracil residues located near the 59 terminal of vRNA (Poon

et al., 1999). Thus, the synthesized RNA has the authenticeukaryotic mRNA structure. An influenza-virus-like particlegeneration system was established based on the transfection ofplasmid DNAs containing the viral genes under the control of the

Pol I promoter and terminator (Neumann et al., 1999; Zobel et al.,1993). In this system, an influenza virus genome RNA of an exactsize and orientation is synthesized by cellular Pol I. When the

viral coding region is replaced with a reporter gene (see Fig. 1A),the reporter gene is expressed through the viral system describedabove. We examined the mechanism of rDNA transcription. In

particular, we evaluated its species-specific property using thisnovel and highly sensitive assay system, as we considered thepossibility that the species-specificity contradiction might be

caused by reconstituted Pol I with an activity less than thedetectable level of conventional assay systems.

In the present study, we established a viral RdR Pol-basedreporter system to monitor Pol I transcription. Using this

monitoring system, we succeeded in reconstituting Pol Itranscription from human rDNA in mouse cells by exogenouslyexpressing human TAFIA, B, C and D in the SL1 complex, but

not with human TAFIA, TAFIB and TAFIC. Exogenousexpression of four TAFIs not only induced transcription from

the human rDNA promoter in a reporter plasmid but alsoreactivated human rDNA in mouse cells containing the human

chromosome 21 carrying an inactive human rDNA cluster. Theseresults indicate that four TAFIs in human SL1 are necessaryand sufficient to reconstitute a transcriptionally active SL1 andovercome the barrier of species specificity during rDNA

transcription in mouse cells.

RESULTSA novel reporter assay system for transcription by Pol IWe developed a novel and sensitive reporter assay system tomonitor transcription by Pol I in order to precisely analyze the

species barrier for rDNA transcription (Fig. 1A) by modifying theoriginal method (Zobel et al., 1993) for the influenza-virus-likeparticle generation system. In this system, engineered vRNA

containing a firefly luciferase (Luc) gene or an EGFP genesandwiched between 59- and 39-terminal cis-acting regulatoryregions is synthesized by Pol I under the control of the promoterand terminator in transfected cells. PA, PB1, PB2 and NP are

supplied from expression vectors under the control of the PolII promoter (Neumann et al., 1999). mRNA encoding Luc issynthesized by the influenza viral RdR Pol using RNA

transcribed by Pol I as a template and the cellular cap structureas a primer (Li et al., 2001; Poon et al., 1999). The synthesized

Fig. 1. An RdR Pol-based reporter assay system for the transcription by Pol I. (A) A schematic representation of the influenza virus RNA-dependent RNApolymerase (RdR Pol)-mediated Pol I reporter assay system. The influenza virus model RNA genomes are expressed from phPolI-vNS-Luc or phPolI-vNA-EGFP (denoted ‘Luc. or EGFP’) by Pol I. The influenza virus RdR Pol amplifies the synthesized RNA and transcribes mRNA from the model viral RNA ofnegative polarity. (B) Dependency of the Pol I reporter assay system on viral polymerases and NP. The Luc activity was normalized to the Renilla luciferaseactivity expressed under the control of the SV40 promoter, a typical Pol II promoter. AU, arbitrary units. Results are mean6s.d. obtained from threeindependent experiments. (C) Evaluation of Pol I reporter systems. 3 Pol.+NP indicates transfection of plasmids containing RdR Pol components and NP.IRES and PolyA indicate an internal ribosome entry site and a poly(A) signal, respectively. (D) Expression of EGFP in the RdR Pol-based system. HeLa cellswere co-transfected with plasmids expressing PA, PB1, PB2 and NP, and phPolI-vNA-EGFP (left panels). Transfection was also carried out with omission of theplasmid encoding NP (right panels).

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RNA is a typical form of eukaryotic mRNA and is therebysubjected to translation after its transport from the nucleus to the

cytoplasm. First, we examined whether this reporter system wasdependent on viral components (Fig. 1B). phPolI-vNS-Luccontains an Luc gene of reverse orientation sandwichedbetween 59- and 39-terminal UTRs of the influenza virus (A/

WSN/33) segment 8, which encodes a nonstructural (NS) protein.As expected, the Luc activity was detected in HeLa cellsexpressing all components of this system (Fig. 1B, lane 6).

Although phPolI was originally called pHH21 or pPolI in aprevious report (Neumann et al., 1999), we designated thisplasmid as phPolI because it makes it easy to distinguish plasmids

carrying the human or mouse rDNA promoter. Next, weevaluated this system by comparing it with an internalribosome entry site (IRES)-mediated reporter system (Hannan

et al., 1996; Palmer et al., 1993). In the IRES-mediated reportersystem for Pol I used by Palmer et al., activity relative to thebackground was increased ,30-fold. In our experiments, theIRES-mediated reporter system did not produce a significant

level of the Luc activity (Fig. 1C, lanes 3 and 4). In the RdR Pol-mediated system described above, the Luc activity was .104-foldhigher than the background activity. These results show that the

RdR Pol-mediated system is highly sensitive for monitoring Pol Iactivity. We also constructed a reporter system expressing EGFP.The EGFP signal was observed in the presence of four viral

components, whereas it was not observed in the absence of NP(Fig. 1D).

Evaluation of the RdR Pol-based reporter system for Pol INext, we confirmed the reliability of the RdR Pol-based reportersystem for monitoring transcription by Pol I. We constructedthree reporter plasmids carrying the human rDNA promoter

with mutations at residues that abrogate activity (Grummt, 1982;Grummt, 1998; Haltiner et al., 1986; Jones et al., 1988; Kishimotoet al., 1985; Miller et al., 1985; Moss and Stefanovsky, 1995). As

shown in Fig. 2A,B, phPolI-DCore-vNS-Luc and phPolI-DUCE-vNS-Luc lacked the core and upstream control elements of thehuman rDNA promoter, respectively. G residues at 27 and 216

relative to the transcription initiation site are quite important for thetranscription of the mammalian rRNA gene. These G residues arereplaced with A residues in phPolI-G-7,-16A-vNS-Luc. The RdRPol-mediated Luc activity driven by phPolI-DCore-vNS-Luc in

HeLa cells was completely eliminated (Fig. 2C, lanes 4 and 5). Inaddition, the RdR Pol-mediated Luc activity driven by phPolI-DUCE-vNS-Luc and phPolI-G-7,-16A-vNS-Luc in HeLa cells

were ,10% of that driven by phPolI-v-NS-Luc (Fig. 2C, lanes 6–9). These results were consistent with previous reports about theproperties of the rDNA promoter.

We also constructed the pmPolI-vNS-Luc reporter plasmid,which carries the mouse rRNA gene promoter instead ofthe human rRNA gene promoter in phPolI-vNS-Luc. Although

pmPolI was originally called as pHMP1 in a previous report(Turan et al., 2004), we redesignated this plasmid pmPolI becauseit makes it easy to distinguish plasmids carrying the human ormouse rDNA promoter. As shown in the human reporter system,

we constructed three reporter plasmids carrying mutations in themouse rDNA promoter that abrogate activity (Fig. 2D,E). Therewas no RdR Pol-mediated Luc activity driven by pmPolI-DCore-

vNS-Luc in mouse NIH3T3 cells (Fig. 2F, lanes 4 and 5). TheRdR Pol-mediated Luc activity driven by pmPolI-DUCE-vNS-Luc and pmPolI-G-7,-16A-vNS-Luc in mouse NIH3T3 cells was

dramatically lower compared with that by pmPolI-v-NS-Luc

(Fig. 2F, lanes 6–9). These results clearly demonstrate that ourRdR-mediated reporter system detects Pol I activity.

The transcription rate of rDNA by Pol I is closely correlatedwith the cell growth rate and depends on the cell cultureconditions (Grummt, 1999). We examined RdR Pol-mediated Lucactivity in HeLa cells maintained with 0.5%, 2% and 10% serum

(supplementary material Fig. S1A). The Pol I transcriptionlevel, as monitored by the Luc activity, increased in a serum-concentration-dependent manner, as previously reported by

conventional assay systems monitoring the incorporation of[3H]uridine into the 45S rRNA precursor (Stefanovsky et al.,2006). Furthermore, we confirmed that the RdR Pol-based

reporter system responded to exogenous expression of UBF,an essential transcription factor for transcription by Pol I(supplementary material Fig. S1B,C; Grummt, 1999). CX-5461

inhibits Pol I via the disruption of SL1 recruitment on the rDNApromoter (Drygin et al., 2011). Treatment with CX-5461 reducedthe Luc activity in the RdR Pol-based reporter system whennormalized to c-Myc- or b-actin-encoding mRNAs as previously

described (Drygin et al., 2011) (supplementary material Fig.S1D). Taken together, we conclude that this RdR Pol-basedreporter system is highly sensitive and useful for analyzing the

transcription mechanism by Pol I.

In vivo reconstitution of human rDNA transcription in mousecellsWe used the RdR Pol-based reporter system to uncover themolecular mechanism underlying the species-specific transcription

by Pol I. phPolI and pmPolI, used in Fig. 3, contain the human andmouse Pol I promoters, respectively (Neumann et al., 1999; Turanet al., 2004). In human HeLa cells, the Luc activity was observed inthe presence of viral RdR Pol components and NP (Fig. 3, 3Pol+NP)

when the human Pol I vector (phPolI-vNS-Luc) was transfected. Incontrast, the Luc activity was not detected when the mouse Pol Ivector (pmPolI-vNS-Luc) was transfected (Fig. 3A, upper panel).

Furthermore, the mouse reporter, but not the human reporter,showed Luc activity in mouse NIH3T3 cells (Fig. 3A, lower panel).These results indicate that species-specific transcription by Pol I is

reproduced in the RdR Pol-based reporter system.To reconstitute the human SL1 activity in mouse cells,

we cloned four human TAFIs (hTAFIs) and performedimmunoblotting using extracts from 293T cells transfected with

vectors encoding hTAFIs (Fig. 3B). The effect of hTAFIs ontranscription from the human rDNA promoter was examined inNIH3T3 cells using the RdR Pol-based reporter system for Pol I.

The human rDNA promoter was not transcribed in NIH3T3 cellswithout the human SL1 complex or even in the presence of thethree factors: hTAFIA, hTAFIB, and hTAFIC (Fig. 3C, lanes 3

and 5). In sharp contrast, with hTAFIA, hTAFIB, hTAFIC andhTAFID (Fig. 3C, lanes 4 and 6) Luc activities were detectedfrom the human reporter (phPolI-vNS-Luc). Next, NIH3T3

cells were co-transfected with the human reporter plasmid inthe presence of all combinations of hTAFIs (Fig. 3D). Nocombination of the three hTAFIs reconstituted the efficienttranscription activity driven by the human rDNA promoter

(Fig. 3D, lanes 3–6), although a low transcription levelwas observed when either hTAFIA or hTAFIB was omitted.Importantly, human rDNA transcription was not at all stimulated

in NIH3T3 cells when a single factor or any combination of twofactors were expressed (Fig. 3D, lanes 7–16). These resultsindicate that the four hTAFIs are required for maximum

transcription from the human Pol I promoter in mouse cells. In

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addition, hTAFIC and hTAFID played an essential role in the SL1

activity and species specificity (Fig. 3D, lanes 3 and 4), andmouse TAFIA (mTAFIA) and mTAFIB could partially replacehuman cognate proteins for human rDNA transcription in mouse

cells (Fig. 3D, lanes 5 and 6). This finding is consistent withthat from UV crosslinking experiments stating that TAFIA andTAFIB bind to both homologous and heterologous promoters

(Rudloff et al., 1994). As a control, we examined the effect of allcombinations of hTAFIs on the mouse reporter system in NIH3T3cells (supplementary material Fig. S2). Exogenous expression of

the four hTAFIs did not increase the level of the mouse Pol Iactivity but rather interfered with mouse Pol I activity. Takentogether, the four hTAFIs in human SL1 are necessary andsufficient to reconstitute a transcriptionally active SL1 and

overcome the barrier of species specificity in human rDNAtranscription in mouse cells.

Transcriptional reactivation of inactive human rDNA in A9cellsAs shown above, the RdR Pol-based reporter system wassensitive for detecting Pol I activity. However, we could not

completely exclude the possibility that human rDNA wastranscribed in mouse cells by an unexpected effect betweenRdR Pol and the four hTAFIs. To exclude this possibility and

directly show the function of the human SL1 in mouse cells, weused mouse A9 cells carrying a single intact human chromosome21 (A9ch21 cells), which contains human rDNAs, but not

any human TAFI genes (Cuthbert et al., 1995). Human rDNAtranscription could not be detected in A9ch21 cells (Fig. 4A, leftpanel). Because the inactive human rDNA promoter region wasreported to be highly methylated (Guetg et al., 2012; Santoro and

Grummt, 2001), we examined the DNA methylation pattern onhuman rDNAs in A9ch21 cells using methylation-sensitive

Fig. 2. Pol I promoter element-dependent expression in the RdR Pol-based reporter system. (A) The schematic representation of deletion (break in thegray bar) and substitution (**) mutants in the human rDNA promoter. (B) The sequence of the human rDNA promoter in phPolI-vNS-Luc. Nucleotideposition is indicated relative to the transcription start site (+1). Blue letters indicate the 59 UTR of influenza virus segment 8, encoding a nonstructural protein.phPolI-Dcore-vNS-Luc and phPolI-DUCE-vNS-Luc lack regions indicated by the red and orange letters, respectively. The underlined G residues at 27 and216 are replaced with A residues in phPolI-G-7,-16A-vNS-Luc. (C) The activity of the human rDNA promoters containing mutations. HeLa cells were co-transfected with plasmids encoding viral components (3 Pol+NP) and reporter plasmids as indicated below each lane. (D) The schematic representation ofdeletion and substitution mutants in the mouse rDNA promoter. (E) The sequence of the mouse rDNA promoter in pmPolI-vNS-Luc. pmPolI-Dcore-vNS-Luc andpmPolI-DUCE-vNS-Luc lack the regions indicated by red and orange letters, respectively. The underlined G residues at 27 and 216 are replaced with Aresidues in pmPolI-G-7,-16A-vNS-Luc. (F) The activity of mouse rDNA promoters containing mutations. NIH3T3 cells were co-transfected with plasmidsencoding viral components (3 Pol+NP) and reporter plasmids as indicated below each lane. Results in C and F are mean6s.d. obtained from three independentexperiments. + and ++ indicate 30 ng and 90 ng of plasmid DNA, respectively. AU, arbitrary units.

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(HpaII) and -insensitive (MspI) restriction enzymes. Although

human rDNAs were transcriptionally inactive in A9ch21 cells,.40% of human rDNA promoters were unmethylated, and themethylation status of the human rDNA promoter was not

significantly different from that of the mouse rDNA promotersin A9ch21 cells (Fig. 4B). Moreover, the epigenetic profile on thehuman rDNA promoter in A9ch21 cells was examined by

chromatin immunoprecipitation (ChIP) assays. Both acetylationand trimethylation at histone H3 lysine 9 (H3K9) on humanrDNAs were slightly higher than those on mouse rDNAs in

A9ch21 cells (Fig. 4C). In addition, the amount of UBF boundto the human rDNA promoter in A9ch21 cells was quite low.However, micrococcal nuclease (MNase) digestion assays

demonstrated that the human rDNA promoter region was moreresistant to MNase than the mouse rDNA promoter in A9ch21cells comprising both active and inactive rDNAs (Fig. 4D). These

results suggest that inactive human rDNAs are not assembled intoa typical heterochromatin structure in mouse cells, but they ratherjust form compact chromatin structure resistant to nuclease.

Next, we examined the effect of exogenous hTAFIs expression

on transcription from the human rDNA promoter in A9ch21 cells.First, we confirmed that the SL1 activity was reconstituted byfour exogenously expressed hTAFIs in A9ch21 cells (Fig. 4E).

We then examined transcription from human rDNA in A9ch21cells in the presence of the four hTAFIs. Total RNA was preparedfrom A9ch21 cells transiently expressing hTAFIs, followed by

quantitative RT-PCR using primer sets for human 45S pre-rRNA.The inactive human rDNA in mouse cells was reactivated by fourexogenously expressed hTAFIs (Fig. 4F). Human pre-rRNAcould not be observed in A9ch21 cells in the absence of TAFID

(Fig. 4G, lane 2). These results indicate that human rDNAtranscription was reconstituted by four hTAFIs in mouse A9ch21cells in addition to that monitored by the RdR Pol-based reporter

system.

Formation of a chimeric SL1 complexHuman rDNA transcription in mouse cells required four hTAFIs,whereas three hTAFIA, hTAFIB and hTAFIC did not confertranscription, as shown in Figs 3 and 4. It is possible that

hTAFIA, hTAFIB and hTAFIC cannot interact with mouseTAFID (mTAFID). To examine this hypothesis, Flag-taggedmTAFID was co-expressed with HA-tagged hTAFIA, hTAFIBand hTAFIC in 293T cells, followed by immunoprecipitation with

anti-Flag antibody. HA-tagged hTAFIA, hTAFIB and hTAFIC co-immunoprecipitated with Flag-tagged mTAFID, as they werewith Flag-tagged hTAFID (Fig. 5A). Endogenous TBP was also

co-immunoprecipitated with Flag-tagged hTAFID and mTAFID(Fig. 5B). These results indicate that mTAFID is able to form achimeric SL1 complex, including TBP and hTAFIA, hTAFIB and

hTAFIC, in agreement with a previous report (Heix et al., 1997).Furthermore, Flag-tagged mTAFID formed a chimeric SL1complex with HA-tagged hTAFIA, hTAFIB and hTAFIC, andendogenous TBP did not confer transcription of human rDNA

(Fig. 5C, lane 4). In addition, no combination of three hTAFIsand a single mTAFI conferred maximum transcription of human

Fig. 3. Reconstitution of human rDNA transcription in mouse cells.(A) The species-specific transcription by Pol I in vivo. phPolI and pmPolIcontain human and mouse Pol I promoters, respectively. Black and whitebars indicate the Luc activity derived from the human rDNA (phPolI-vNS-Luc)and the mouse rDNA promoters (pmPolI-vNS-Luc), respectively (mean6s.d.obtained from three independent experiments). (B) Exogenous expressionof human TAFIs (hTAFIs). 293T cells were transfected with plasmidsencoding hTAFIs. The expression levels of each exogenous hTAFI andhuman b-actin (Act b) were detected by immunoblotting using anti-Flag andanti-b-actin antibodies, respectively. (C) Transcription of the human rDNA inmouse cells. NIH3T3 cells were transfected with a plasmid set for the RdRPol-based reporter system (3Pol+NP and phPolI-vNS-Luc) and expressionplasmids for hTAFIs as indicated below each lane. pmPolI-vNS-Luc wasintroduced to NIH3T3 cells for positive control of the Luc assay (lane 8).Black bar and white bars indicate the Luc activity from human and mouserDNA promoters, respectively (mean6s.d. obtained from three independentexperiments). *P,0.0002. (D) The effect of all possible combinations ofhTAFIs on Luc activity was examined by the RdR Pol-based reporter systemfor the human rDNA transcription in NIH3T3 cells (mean6s.d. obtained fromthree independent experiments). *P,0.0002; **P,0.01 versus lane 1. AU,arbitrary units.

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rDNA, which was driven by four hTAFIs (supplementary materialFig. S3A). These data suggest that any chimeric SL1 complexcomprising three hTAFIs and a single mTAFI cannot formfunctional SL1 complexes.

Chimeric SL1 complexes did not bind to the human rDNApromoterUV crosslinking experiments have demonstrated that TAFIA andTAFIB bind to both homologous and heterologous promoters(Rudloff et al., 1994). This report encouraged us to examine

whether the mouse SL1/TIF-IB complex and chimeric complexare associated with the human rDNA promoter in mouse A9 cellscarrying the human chromosome 15 (A9ch15 cells). The four

exogenously expressed hTAFIs activated transcription of humanrDNA in A9ch15 and A9ch21 cells (supplementary material Fig.S3B). The reactivation level of human rDNA transcription inA9ch15 cells by the four hTAFIs was 9.1-fold higher than that of

A9ch21 cells (supplementary material Fig. S3C). Therefore, weused A9ch15 cells instead of A9ch21 cells for additional ChIPassays. We performed ChIP assays using anti-TBP antibody. TBP

bound to the mouse rDNA promoter, but not to the human rDNApromoter, in mouse A9ch15 cells (Fig. 6A, left panel). However,TBP bound to the human rDNA promoter in HeLa cells (Fig. 6A,

right panel). These results clearly indicate that the mouse SL1/TIF-IB complexes cannot associate with the human rDNApromoter. As shown in Fig. 6B, the four exogenously expressedhuman TAFIs recruited TBP on the human rDNA promoter in

A9ch15 cells. In contrast, chimeric SL1 complexes and fourmTAFIs could not recruit TBP on the human rDNA promoter inA9ch15 cells compared with the four hTAFIs. Furthermore, theoccupancy of UBF at the human rDNA promoter increased

slightly in A9ch15 cells in the presence of the four exogenoushTAFIs (Fig. 6C), which is in good agreement with a reportstating that human SL1 stabilizes the binding of UBF at the

human rDNA promoter (Friedrich et al., 2005). A large amount ofRPA194, the largest subunit of Pol I, was also recruited on humanrDNA in mouse A9ch15 cells by the four exogenously expressed

human TAFIs. We conclude that SL1 complexes comprising fourhuman TAFIs are required for the recognition of the human rDNApromoter.

DISCUSSIONIn this report, we describe an RdR Pol-based reporter system fortranscription by Pol I. The RdR Pol-based reporter system

showed high sensitivity, because RNA synthesized by Pol I isamplified and converted into translatable mRNA by RdR Pol.Transcription of human rDNA was reconstituted in mouse cells

by exogenously expressing four TAFIs from the human SL1complex. In addition, we demonstrated that silent human rDNAtranscription in mouse A9ch21 cells was reactivated by the four

exogenously expressed hTAFIs. These results suggest that thefour human TAFIs are necessary and sufficient to overcome thespecies-specific barrier of human rDNA transcription in mousecells.

Fig. 4. Transcription reactivation of inactive human rDNA in mouse A9 cells. (A) Human rDNA is transcription-inactive in A9ch21 cells. RT-PCR wascarried out using primer sets for human pre-rRNA (left panel) and mouse pre-rRNA (right panel) in the presence or absence of reverse transcriptase (RT) asindicated below each lane. (B) Methylation pattern on mouse and human rDNA promoters in A9ch21 cells. The methylation pattern was determined withMspI (which is methylation insensitive) or HpaII (which is methylation sensitive). Resistance to restriction enzyme (RE) was determined by quantitative PCR(qPCR) using primer sets for the human (black bar) and mouse rDNA (rDNA) promoters (white bar). (C) Chromatin property on human and mouse rDNAs inA9ch21 cells. A ChIP assay was performed using indicated antibodies. The amount (percentage of input) of rDNAs obtained by immunoprecipitation wasdetermined by qPCR using the indicated primer sets for the mouse and human rDNA promoters. (D) MNase resistance of rDNAs in A9ch21 cells. Resistance toMNase was determined by qPCR using the indicated primer sets for mouse and human rDNA promoters. (E) Human rDNA promoter activity detected bythe RdR Pol-based reporter system in A9ch21 cells in the presence of four human TAFIs (hTAFIs). (F,G) Reactivation of human rDNA transcription in A9ch21cells. Total RNA was prepared from A9ch21 cells transfected with pCHA as a control or pCHA-based plasmid set expressing the four hTAFIs at the indicated timepost transcription. The level of human pre-rRNA was measured by quantitative RT-PCR and normalized by level of mouse b-actin. Results are mean6s.d.obtained from three independent experiments. AU, arbitrary units.

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It has been reported that the human SL1 activity can bereconstituted in vitro by the three recombinant hTAFIs, hTAFIA,hTAFIB and hTAFIC, in addition to recombinant TBP, with a

relatively higher background (Zomerdijk et al., 1994). Thoseauthors used DNA-affinity-purified UBF and recombinanthTAFIA, hTAFIB, hTAFIC and TBP for reconstituting in vitro

transcription by Pol I. Furthermore, it has been shown that UBFinteracts with hTAFID in vivo and in vitro (Gorski et al., 2007).Based on those results, it is quite likely that a low level ofhTAFID, which was associated with either DNA-affinity-purified

UBF or the purified Pol I fraction, was present in their in vitro

reaction. This assumption is supported by a report that mouseTIF-IB activity is not reconstituted by recombinant protein of

three mTAFIs, mTAFIA, mTAFB and mTAFC, in addition torecombinant TBP and UBF (Heix et al., 1997). Taken together,we conclude that hTAFID is the putative final component in

completing the SL1 activity.The four mouse TAFIA, TAFIB, TAFIC and TAFID proteins

show 90%, 87%, 75% and 68% similarity, respectively, to their

human counterparts, according to BLAST2 (NCBI) analyses.Their similarity is in good agreement with the observation thatTAFIA and TAFIB are partially replaceable for human cognate

proteins, whereas TAFIC and TAFID have crucial roles in speciesspecificity, as shown in Fig. 3D. We found that mouse Flag-tagged TAFID forms a complex with human HA-tagged TAFIA,

TAFIB, TAFIC and TBP (Fig. 5). This result is partiallysupported by a report that mouse and human TAFIs (hTAFIA,

Fig. 5. Formation of the chimeric SL1 complex.(A,B) Immunoprecipitation (IP) with anti-Flag antibody. 293T cells weretransfected with combinations of pCHA or pCFlag-based plasmids encodinghuman TAFIs (HA-hA to HA-hD, Flag-hD) and mouse TAFID (Flag-mD).Immunoprecipitation was carried out with anti-Flag antibody, followed byimmunoblotting (WB) using anti-HA, anti-Flag and anti-TBP antibodies,respectively. (C) Human rDNA promoter activity detected by the RdR Pol-based reporter system. A9ch21 cells were transfected with combinations ofpCHA-based plasmids encoding human TAFIs (HA-hA to HA-hD) and/orpCFlag-based plasmids encoding mouse TAFIs (Flag-mA to Flag-mD).Results are mean6s.d. obtained from three independent experiments. AU,arbitrary units.

Fig. 6. Recruitment of transcription machineries to the human rDNApromoter by exogenous four human TAFIs in A9ch15 cells. (A) Binding ofTBP to the rDNA promoter in A9ch15 cells and HeLa cells. ChIP assays wereperformed using anti-TBP antibody. The DNAs obtained byimmunoprecipitation were determined by quantitative PCR using the primersets for the mouse and human rDNA promoters. (B) Binding of TBP to thehuman rDNA promoter in A9ch15 cells. A9ch15 cells were transfected withcombinations of pCHA-based plasmids encoding human TAFIs (HA-hA toHA-hD) and/or pCFlag-based plasmids encoding mouse TAFIs (Flag-mA toFlag-mD). Asterisks indicate P,161026 versus lane 2. (C) The occupancy ofUBF and Pol I at the human rDNA promoter in A9ch15 cells. ChIP assayswere performed using anti-UBF and RPA194 (largest subunit of Pol I)antibodies. The DNAs obtained by immunoprecipitation were determined byquantitative PCR using the primer sets for the human rDNA promoter.Results are mean6s.d. obtained from three independent experiments.*P,0.05.

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hTAFIB and hTAFIC) can form chimeric TBP–TAFI complexes(Heix et al., 1997). It appears that subtle differences between

individual TAFIs might affect the overall conformation of thechimeric complex between TBP and the TAFIs, which could betranscriptionally inactive, as mentioned by Heix and Grummt(Heix and Grummt, 1995). Our results clearly indicate that the

human SL1 activity requires four hTAFIs, at least in mouse cells.For further analysis of the molecular mechanism of the promoterselectivity by Pol I, SL1/TIF-IB activity should be reconstituted

in vitro by four TAFIs and TBP recombinant proteins.Although the human SL1 activity was reconstituted in mouse

cells, the four mouse TAFIs did not confer transcription of mouse

rDNA in HeLa cells (supplementary material Fig. S4). Theseresults are consistent with a report that the human SL1 canreprogram mouse nuclear extracts during in vitro transcription

from the human rDNA promoter (Learned et al., 1985), but TIF-IB cannot reprogram human nuclear extracts during in vitro

transcription from the mouse rDNA promoter (Schnapp et al.,1991). TIF-IB requires mouse Pol I to reprogram human nuclear

extracts, whereas mouse SL1/TIF-IB, human UBF and human PolI can reconstitute transcription from the mouse rDNA promoter in

vitro (Bell et al., 1990; Schnapp et al., 1991). It is assumed that

there is an unknown factor(s) preventing TIF-IB activity in HeLacells, suggesting that the species barrier(s) to rDNA transcriptionmight be different between humans and mice.

The level of SL1/TIF-IB in cells is too low to study themolecular mechanism of promoter recognition and species-specific transcription by Pol I (Heix et al., 1997). Thus, to

confirm the exogenous expression of hTAFIs, we performedimmunoblotting using extracts from 293T cells transfectedwith vectors encoding hTAFIs (Fig. 3B). Despite considerableefforts, exogenously expressed TAFIs were not detected by

immunoblotting using NIH3T3, A9ch15, A9ch21 or HeLa cellextracts. In these cells, the abundance of SL1 might be under thecontrol of an unknown mechanism, whereas 293T cells lack the

possible mechanism, which keeps the SL1 level low. Similarly,the p53 tumor suppressor protein level and activity are controlledby a ubiquitin-proteasome pathway involving MDM2 (Hock and

Vousden, 2014). The possible mechanism to maintain a low levelof SL1 is currently an open question.

We used two mouse A9 cell lines (A9ch21 and A9ch15)carrying a human chromosome to show the SL1 activity in the

presence of four human TAFIs. During the course of this study,we found that the level of human rDNA transcription in A9ch15cells was 9.1-fold higher than that in A9ch21 cells in the presence

of the four human TAFIs (supplementary material Fig. S3C). Thehuman rDNA copy number in an A9ch15 cell was smaller thanthat in an A9ch21 cell (supplementary material Fig. S3D).

Notably, the level of DNA methylation on the human rDNApromoter in A9ch15 cells was significantly lower than that inA9ch21 cells (supplementary material Fig. S3E). The level of

DNA methylation on the promoter may determine the level ofresponse to exogenous expression of the four human TAFIs. Inaddition, the maintenance of the DNA methylation level on thehuman rDNA promoter was independent of human rDNA

transcription in mouse A9 cells. Noncoding promoter RNA(pRNA) synthesized by RNA polymerase I recruits PARP1 andNoRC complexes to rDNA, so pRNA is involved in the formation

and maintenance of silent rDNA chromatin (Guetg et al., 2012;Mayer et al., 2006; Santoro et al., 2010). Nucleolar localization ofNoRC in NIH3T3 cells is mediated by pRNAs synthesized from

mouse and human rDNA proximal Pol I promoters (Mayer et al.,

2008). The results shown in supplementary material Fig. S3 raisefundamental questions whether mouse pRNA is involved in DNA

methylation on human rDNA promoter, and whether humanpRNA is transcribed by mouse Pol I machinery. In conclusion,the A9 cell line carrying human chromosomes 15 and 21 mayprovide a good model system for studying rDNA epigenetics.

Studies on the mechanism of rDNA transcription mediatedby Pol I have become a focus again. It is well known thatthe regulation of rDNA transcription is related to cancer cell

proliferation (White, 2005). c-Myc, a representative transcriptionfactor involved in tumorigenesis, binds to rDNA and increasesrDNA transcription (Arabi et al., 2005; Grandori et al., 2005).

Several protein kinases, including casein kinase II and ERK,have the ability to upregulate rDNA transcription through thephosphorylation of UBF and TIF-IA in cancer cells (Bierhoff

et al., 2008; Lin et al., 2006; Panova et al., 2006; Stefanovskyet al., 2001). Thus, the Pol I transcription machinery is being re-evaluated as an emerging target for treating cancer (Drygin et al.,2010; Hein et al., 2013). It is possible that the RdR Pol-based

reporter system for Pol I activity would be a convenient andhighly sensitive tool for monitoring Pol I activity and screeninginhibitors for Pol I transcription.

MATERIALS AND METHODSCell cultures and luciferase assayHeLa cells, NIH3T3 cells, A9ch15 cells and A9ch21 cells were used for

transfection experiments. They were maintained at 37 C in Dulbecco’s

modified Eagle’s medium (Nissui) supplemented with 10% fetal bovine

serum. In addition, A9ch15 cells and A9ch21 cells were cultured in the

presence of 50 mg/ml hygromycin B (InvivoGen) to keep the human

chromosome in the mouse cells. The Luc and the Renilla Luc activities

were determined using Luciferase assay reagent (Promega) and

the Renilla Luciferase Assay System (Promega) according to the

manufacturer’s protocol. The relative luminescence intensity was

measured for 10 seconds with a MiniLumat (Berthold).

DNA transfectionCells were transfected with DNA using Lipofectamine 2000 (Invitrogen)

or GeneJuice (Novagen) transfection reagent kit according to the

manufacturers’ instructions. For Fig. 1B, HeLa cells were co-

transfected with 25 ng each of pCAGGS-PA, pCAGGS-PB1,

pCAGGS-PB2, pCAGGS-NP (Kawaguchi et al., 2005; Naito et al.,

2007), which leads to synthesis of PA, PB1, PB2 and NP mRNAs,

respectively, by Pol II, 90 ng of phPolI-vNS-Luc and 10 ng of pRL-SV40

(lane 6). Assays were also carried out under omission of either one of the

plasmids encoding viral components, pCAGGS-PA (lane 2), pCAGGS-

PB1 (lane 3), pCAGGS-PB2 (lane 4), or pCAGGS-NP (lane 5) or in the

absence of all viral components (lane 1). For Fig. 1C, HeLa cells were

co-transfected with phPolI (40 ng, lane 1) or phPolI-vNS-Luc (40 ng,

lane 2) together with plasmids (38 ng for each) encoding the four viral

components and pRL-SV40 (10 ng). phPolI-IRES-Luc (190 ng, lane 3),

phPolI-IRES-Luc-PolyA (190 ng, lane 4) and pSV40-Luc (190 ng, lane

5) were introduced to HeLa cells together with pRL-SV40 (10 ng). For

Fig. 1D, HeLa cells were co-transfected with 80 ng of each plasmid

expressing PA, PB1, PB2 and NP and 80 ng of phPolI-vNA-EGFP

(Fig. 1D, left panels). Transfection was also carried out with the omission

of the plasmid encoding NP (Fig. 1D, right panels). For Fig. 2C, HeLa

cells were transfected with one of phPolI-based plasmids (90 ng), pRL-

SV40 (10 ng) for expression of Renilla Luc, and plasmids (each 25 ng)

expressing PA, PB1 and PB2 subunits, and NP. For Fig. 2F, NIH3T3

cells were transfected with one of pmPolI-based plasmids (90 ng), pRL-

SV40 (10 ng) for expression of Renilla Luc, and plasmids (each 25 ng)

expressing PA, PB1 and PB2 subunits, and NP. For Fig. 3C, NIH3T3

cells were transfected with pCHA-TAFIA, pCHA-TAFIB, pCHA-TAFIC

and pCHA-TAFID (25 ng for each) as indicated below each lane in the

presence of the plasmid set (3Pol+NP) for the RdR Pol-based reporter

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system: phPolI-vNS-Luc (45 ng), pCAGGS-PA (11.3 ng), pCAGGS-

PB1 (11.3 ng), pCAGGS-PB2 (11.3 ng), pCAGGS-NP (11.3 ng) and

pRL-SV40 (10 ng). NIH3T3 cells were transfected with pCFlag-TAFIA,

pCFlag-TAFIB, pCFlag-TAFIC and pCFlag-TAFID (25 ng for each) as

indicated below each lane in the presence of plasmid set (100 ng) for the

RdR Pol-based reporter system. The total amount of plasmid DNA was

adjusted to 200 ng with the empty plasmid pCHA or pCFlag. For

Fig. 3D, NIH3T3 cells were transfected with the plasmid set (100 ng) for

the RdR Pol-based reporter system as mentioned in Fig. 3C, and together

with pCHA-based plasmids encoding TAFIs, as indicated below each

lane (25 ng each). The total amount of plasmid DNA was adjusted to

200 ng with empty plasmid, pCHA. The Luc assay was carried out at

48 hours post transfection. For Fig. 4E, A9ch21 cells were transfected

with pCHA-TAFIA, pCHA-TAFIB, pCHA-TAFIC and pCHA-TAFID

(50 ng for each) as indicated below each lane in the presence of plasmid

set for the RdR Pol-based reporter system: phPolI-vNS-Luc (90 ng),

pCAGGS-PA (22.6 ng), pCAGGS-PB1 (22.6 ng), pCAGGS-PB2

(22.6 ng), pCAGGS-NP (22.6 ng) and pRL-SV40 (20 ng). For

Fig. 4F,G, A9ch21 cells were transfected with pCHA-TAFIA, pCHA-

TAFIB, pCHA-TAFIC and pCHA-TAFID (100 ng for each) as indicated

below each lane. Total amount of plasmid DNA was adjusted to 400 ng

with empty plasmid, pCHA.

Immunoblotting and ChIPAntibodies specific for Flag epitope tag (Sigma; M2), actin b (Sigma;

AC-74), UBF (Santa Cruz Biotechnology; H-300), acetylated histone H3

(Upstate/Millipore), tri-methylated histone H3K9 (Abcam; ab8898),

RPA194 (Santa Cruz Biotechnology; C-1) and TBP (Abcam; 51841),

were used for immunoblotting and the ChIP assay. Immunoblotting was

carried out essentially as described previously (Murano et al., 2008).

ChIP assays were performed according to the manual for the ChIP assay

kit (Upstate). Quantitative PCR was carried out using FastStart SYBR

Green Master (Roche) in the presence of primer sets, 59-AAGCCC-

TCTCTGTCCCTGTCAC-39 and 59-GGAGAACTGATAAGACCGAC-

AGGT-39 for the mouse rRNA gene (rDNA) promoter, 59-TTGA-

CCAGAGGGACCCCGG-39 and 59-AATAACCCGGCGGCCCAAA-

ATG-39 for the human rDNA promoter.

Restriction enzyme and MNase sensitivity assaysCpG methylation was assayed by digestion with HpaII (a methylation-

sensitive restriction enzyme) and MspI (a methylation-insensitive

restriction enzyme) as described previously (Guetg et al., 2012;

Santoro et al., 2002). Genomic DNA (500 ng) prepared from A9ch21

cells was digested with 20 units of HpaII or MspI for 3 hours at 37 C.

Resistance to restriction enzymes was determined by quantitative PCR

using FastStart SYBR Green Master (Roch) in the presence of a primer

set, 59-AAGCCCTCTCTGTCCCTGTCAC-39 and 59-GGAGAACTGA-

TAAGACCGACAGGT-39 for the mouse rDNA promoter, and 59-

TTGACCAGAGGGACCCCGG-39 and 59-AATAACCCGGCGGCCCA-

AAATG-39 for the human rDNA promoter. Nucleotide positions of

2262 bp (where ‘2’ indicates the position upstream of the transcription

site) for the mouse promoter, and 2167 bp, 290 bp, 262 bp and

256 bp for the human promoter relative to the transcription start site

were found to be methylated, and the methylation sites were located

between the forward and reverse primer positions.

For the MNase-sensitivity assay, A9ch21 cells (56105) were fixed in

1% formaldehyde, followed by permeabilization with MNase buffer

(15 mM Tris-HCl pH 7.4, 15 mM NaCl, 60 mM KCl, 2 mM CaCl2,

0.5 mM DTT, 0.1% NP-40). Permeabilized cells were treated with

MNase (Worthington Biochemical Corporation) at 37 C for 10 minutes,

and then the reaction was stopped by same volume of 26Protein K buffer

(20 mM Tris-HCl pH 7.9, 10 mM EDTA, 1% SDS). The reaction was

incubated at 95 C for 15 minutes for reversal of crosslinking, followed by

treatment with RNase A and Proteinase K for purification of DNA. The

resistance to MNase was determined by quantitative PCR using FastStart

SYBR Green Master (Roch) in the presence of a primer sets, 59-

ACCTCCCCAGGTATGACTTCCAG-39 and 59-AGTACCTATCTCCA-

GGTCCAATAGGAAC-39 for the mouse rDNA promoter (nucleotide

positions of 2110 to +3 relative to transcription start site), and 59-

TTGACCAGAGGGACCCCGG-39 and 59-AATAACCCGGCGGCCCA-

AAATG-39 for the human rDNA promoter (nucleotide positions of 2182

to 21 relative to transcription start site).

Quantitative RT-PCRQuantitative determination of pre-rRNA levels by RT-qPCR was

carried out essentially as described previously (Murano et al., 2008).

Total RNA was prepared with MagExtractor-RNA (TOYOBO) and

treated with RNase-free DNase I (Invitrogen). For analysis of human

45S pre-rRNA, total RNA was subjected to reverse transcription with a

primer, 59-ACACACCACCGTTCGGCCTC-39, which corresponds to

the 59 external transcribed spacer (ETS) of the human rDNA. The

synthesized cDNA was quantitated by real-time PCR using FastStart

SYBR Green Master (Roch) in the presence of a primer set, 59-

CGTGCGTTCAGGCGTTCTCGTC-39 and 59-CGGCCGGCCAGC-

GAGCCGATCG-39, corresponding to the 59 ETS of the human

rDNA. For analysis of mouse 45S pre-rRNA, reverse transcription was

performed in the presence of a primer, 59-GTATGCAACGCCAC-

CGGCCA-39, followed by real-time PCR using a primer set, 59-

GATGTGTGAGGCGCCCGGTT-39 and 59-GTATGCAACGCCACC-

GGCCA-39. For analysis of mouse b-actin-encoding mRNA, reverse

transcription was performed in the presence of oligo(dT) as a primer,

followed by real-time PCR using a primer set, 59-TGTTACCA-

ACTGGGACGACA-39 and 59- GGGGTGTTGAAGGTCTCAAA-39.

For analysis of human b-actin- and c-Myc-encoding mRNA, reverse

transcription was performed in the presence of oligo(dT) as a primer,

followed by real-time PCR using a primer set, 59-ATGGGTCAGAAG-

GATTCCTATGT-39 and 59-GGTCATCTTCTCGCGGTT-39 for b-

actin, and 59-AGCGACTCTGAGGAGGAACA-39 and 59-CCCTC-

TTGGCAGCAGGATAG-39 for c-Myc.

Construction of plasmid vectorsTo construct phPolI-vNS-Luc, a DNA fragment of the Luc gene was

amplified by PCR with primers, 59- CGTCTCGGGGAGCAAAAG-

CAGGGTGACAAAGACATAATGGAAGACGCCAAAAACATAAAG-

39 and 59-CGTCTCCTATTAGTAGAAACAAGGGTGTTTTTTATTAT-

TACAATTTGGACTTTCCGCCC-39, using PGV-C (TOYO Ink) as a

template. The amplified DNA fragment was digested with BsmBI, and then

cloned into pHH21 (Neumann et al., 1999) digested with BsmBI. To

generate phPolI-vNA-EGFP, phPolI-WSN-NA (Neumann et al., 1999)

backbone containing both the 39 and 59 UTR regions of the influenza viral

genome was amplified by PCR with primers, 59-TCCGTAGATTGGT-

CTTGGCCAGACGG-39 and 59-TCCCATATGTTAAACTCCTGCTTT-

CGCTCCCCC-39 using phPolI-WSN-NA as a template. A DNA fragment

of EGFP gene was amplified by PCR with primers 59-GGGGAT-

CCCATATGGTGAGCAAGGGCGAGGAG-39 and 59-CCGGATCCT-

TACTTGTACAGCTCGTCCATGCCG-39 using pEGFP-C1 (Clontech)

as a template. The amplified DNA fragment was digested with NdeI and

BamHI, and then cloned into the same site of phPolI-WSN-NA backbone.

For construction of the IRES-mediated reporter system for Pol I, a DNA

fragment of IRES derived from ECMV was generated by PCR

amplification from pMX-IRES-GFP with specific primers, 59-CGG-

TCTAGATAACGTTACTGGCCGAAGC-39 and 59-CGGTCTAGAAGC-

GGCCGCATTGGAATTCCCGGGATCCGGTTGTGGCAAGCTTATCAT-

39. The IRES fragment was phosphorylated with T4 polynucleotide kinase

and cloned into pHH21, which was digested with KpnI and blunted with

T4 DNA polymerase, resulting in construction of phPolI-IRES. The DNA

fragment for Luc gene was amplified by PCR using primers 59-

CGCGGATCCATGGAAGACGCCAAAAACATAAAGAAAGGC-39 and

59-ATAGTTTAGCGGCCGCTTACAATTTGGACTTTCCGCCCTTCTTG-

39 and PGV-C as a template. The amplified PCR product was digested with

BamHI and NotI, and cloned into phPolI-IRES digested with BamH I and

NotI, resulting in phPolI-IRES-Luc. To construct phPolI-IRES-Luc-PolyA,

a DNA fragment of the poly(A) signal (PolyA) was generated by PCR

amplification with the primer set 59-GTAAAGCGGCCGCGACTCTA-

GATCATAATC-39 and 59-GTAAAGCGGCCGCTTACGCCTTAAGA-

TACATTGATGAG-39 using pEGFP-C1 as a template. The amplified

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fragment was treated with NotI and cloned into the NotI site in phPolI-

IRES-Luc, resulting in phPolI-IRES-Luc-PolyA. For expression of Flag-

tagged UBF1 and UBF2, cDNAs of UBF1 and UBF2 were amplified by

PCR with primers, 59-AAAGGATCCATGAACGGAGAAGCCGA-

CTG-39 and 59-AAAGAATTCTCAGTTGGAGTCAGAGTCTG-39,

using cDNA derived from HeLa cells as a template. The amplified DNA

fragments of UBF1 and UBF2 were digested with EcoRI and BamHI and

cloned into the EcoRI and BamHI sites of plasmid pcDNA3.1(+)-Flag. To

construct expression vectors for human TAFIA, TAFIB, TAFIC and

TAFID, DNA fragments were amplified by PCR using cDNA prepared

from HeLa cells. The primers used here were as follows: 59-

CTAGCTAGCATGAGTGATTTCAGTGAAGAATTAAAAGGGC-39 and

59-CGCGGATCCTCAGAGTCTTGGATTTACAATACTGTATTTT-39 for

TAFIA cDNA, 59-CTAGCTAGCATGGACCTCGAGGAGGCGGAA-39

and 59-CGCGGATCCTCAATGTCGTCTCACTTTCTTGGATCTTG-39

for TAFIB cDNA, 59-CTAGCTAGCATGGACTTCCCCAGCTCCCTC-

CG-39 and 59-GGAAGATCTTCAGAAGCCCATTCGAGGCTTCTTCC-

39 for TAFIC cDNA, and 59-CTAGCTAGCATGGATAAATCAGGA-

ATAGATTCTCTTGACC-39 and 59-GGAAGATCTTCACATTTTCA-

GGCCTCTCTGTCCAGTA-39 for TAFID cDNA. cDNA fragments of

TAFIA and B were digested with NheI and BamHI, and cDNA

fragments of TAFIC and TAFID were digested with NheI and BglII.

These cDNA fragments were cloned into the restriction site of NheI and

BglII in pCHA vector containing an HA epitope tag or pCFlag vector

containing the Flag epitope tag. pCFlag was constructed by cloning Flag

DNA fragment into EcoRI site of pCAGGS. Flag DNA fragment

(91 bp) was generated by PCR and digested with EcoRI. The

oligonucleotides used in the PCR were as follows, 59-CCGGAATTC-

GCCGCCATGGACTACAAGGATGACGACGACAAGGGCGCTAGC-

GGGG-39 and 59-CCGGAATTCAGTCACTTAAGATATCACGCGTG-

GTGACCCCGCTAGCGCCCTTGTC-39.

To construct expression vector for mouse TAFIs, cDNA fragments

were amplified by PCR using cDNA prepared from A9ch21 cells. The

primers used here were as follows: 59-CTAGCTAGCATGATGAGT-

GATTTCGGTGAAGAGTTGACA-39 and 59-CGCACGCGTCTAGGT-

GTGAACACCTGGATTTACAATAGT-39 for TAFIA cDNA, 59-

CTAGCTAGCATGGATGTGGAGGAGGTGAAAGCGTTCA-39 and

59-CGCACGCGTTCAGTGTCTTCTTCCTTTCTTGGATCCTGA-39 for

TAFIB cDNA, 59-CGCACGCGTATGGACTTCCCTGGCACCCTGCG-

39 and 59-GGAAGATCTTCAGAAGCCCATTCGTGGCTTCTTCTGG-

39 for TAFIC cDNA, and 59-CTAGCTAGCATGGCTCAATCAGAAG-

TGAATTCTGTCTGT-39 and 59-GGAAGATCTTTATCTATGTTCTT-

TACCTTCTTCAATAGTC-39 for TAFID cDNA. These cDNA fragments

of TAFIA and B were digested with NheI and MluI, and cloned into the

restriction site of NheI and MluI in pCFlag vector containing the Flag

epitope tag. The cDNA fragment of TAFIC was digested with MluI and

BglII, and cloned into the MluI and BglII site in pCFlag. The cDNA

fragments of TAFID was digested with NheI and BglII, and cloned into

NheI and BglII site in pCFlag.

To generate phPolI-Dcore-vNS-Luc, phPolI-DUCE-vNS-Luc and

phPolI-G-7,-16A-vNS-Luc, phPolI-vNS-Luc used as template for

amplification by PCR with primers, 59-CGAAAGATATACCTCC-

CCCGG-39 and 59-GTTATTAGTAGAAACAAGGGTGTTTTTTATT-

ATTA-39, 59-AGCCACACACGGAGCGCCCG-39 and 59-GTCCGTGT-

CGCGCGTCGCCT-39, and 59-AGTTATTAGTAGAAACAAGGGT-

GTTTTTTATTATTA-39 and 59-CGGCGGCCTAAAATGCCGACTC-

GGA-39, respectively. These DNA fragments were phosphorylated by T4

polynucleotide kinase, followed by ligation. To generate pmPolI-Dcore-

vNS-Luc, pmPolI-DUCE-vNS-Luc, and pmPolI-G-7,-16A-vNS-Luc,

pmPolI-vNS-Luc were used as template for amplification by PCR with

primers 59-ATAGGTAGTAGAAACAAGGGTGTTTTTTA-39 and 59-

AACAGATAGAAAAGATCACAAGCATAAAAG-39, 59-ACCTCCA-

CAGGTATGACTTCCAGGT-39 and 59-CGGACCTCAAAGGAAC-

AACTGGT-39, and 59-AATAGGTAGTAGAAACAAGGGTGTTTTT-

TA-39 and 59-TCCAGGTCTAATAGGAACAGATAG-39, respectively.

These DNA fragments were phosphorylated by T4 polynucleotide kinase,

followed by ligation. The structure of human and mouse rDNA promoter

regions in these plasmids is represented on Fig. 2.

AcknowledgementsWe thank Takeshi Sekiya and Kenji Sugiyama (University of Tsukuba, Japan) forvaluable discussion.

Competing interestsThe authors declare no competing interests.

Author contributionsK.M. and K.N. designed research; K.M. and M.O. performed experiments; F.M.,M.K. and S.U. provided the plasmid vectors; R.F.N. provided A9 cell lines; andK.M. and K.N. wrote the paper.

FundingThis work was supported in part by grants-in-aid from the Ministry of Education,Culture, Sports, Science, and Technology of Japan [grant numbers 25118504 toK.M., and 24115001 and 24115002 to K.N.].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.146787/-/DC1

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