Gene 556 (2015) 51–60

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Identification of proteins associated with RNA polymerase III using amodified tandem chromatin affinity purification

Ngoc-Thuy-Trinh Nguyen a,b,c, Cyril Saguez a,b,c,1, Christine Conesa a,b,c, Olivier Lefebvre a,b,c, Joël Acker a,b,c,⁎a CEA, iBiTecS, SBIGeM, FRE 3377, Gif-sur-Yvette F-91191, Franceb CNRS, FRE 3377, Gif-sur-Yvette F-91191, Francec Univ Paris-Sud, FRE 3377, Gif-sur-Yvette F-91191, France

Abbreviations: ChIP, chromatin immunoprecipitatiodine; HRP, horseradish peroxidase; LC–MS/MS, liquid chrotrometry; MS, mass spectrometry; mRNA, messenger RNAII, RNA polymerase II; RNase, ribonuclease; SDS-PAGE, soamide gel electrophoresis; TAP, tandem affinity purificatiofinity purification; TFIIIA, transcription factor IIIA; TFIIIC,transcription factor IIIB; rRNA, ribosomal RNA; tRNA, tran⁎ Corresponding author at: CEA, iBiTec-S, SBIGeM, FR

Yvette F-91191, France.E-mail address: joel.acker@cea.fr (J. Acker).

1 Present address: Institut Pasteur, Unité de GénétiqueUMR3525, University Pierre and Marie Curie UFR927)Paris Cedex 15, France.

http://dx.doi.org/10.1016/j.gene.2014.07.0700378-1119/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 July 2014Received in revised form 25 July 2014Accepted 29 July 2014Available online 30 July 2014

Keywords:RNA polymerase IIITFIIIATAP-MSProtein–protein interaction

To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growingyeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivocross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrastto TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together withPol III bound on its target genes. Transcription factors, elongation factors, RNA‐associated proteins and proteinsinvolved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIBsubunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including his-tones suggest that DNA‐bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Protein–protein interactions are essential for most cellular process-es. The identification of partners in protein complexes and their subse-quent characterization can provide new insights into their molecularfunction and a more complete picture of protein networks. Therefore,a number of methods have been developed not only to identify directinteracting partners but also to reveal all interaction networks presentin macromolecular complexes. Traditional tandem affinity purification(TAP) coupled to high throughput mass spectrometry has been widelyused with success to decipher interactome especially in yeast (Collinset al., 2007; Gavin et al., 2006; Krogan et al., 2006). The developmentof chromatin immunoprecipitation (ChIP) as a major tool for character-izing DNA-associated proteins and complexes was particularly useful toinvestigate interactions between specific proteins and genomic DNA re-gions (Aparicio et al., 2004). However, these procedures have some

n; HBH, histidine–biotin–histi-matography tandemmass spec-; Pol III, RNA polymerase III; Poldium dodecyl sulfate polyacryl-n; TChAP, tandemchromatin af-transcription factor IIIC; TFIIIB,sfer RNA.E3377, Bâtiment 144, Gif-sur-

Moléculaire des Levures (CNRS, Rue du Docteur Roux, 75724

drawbacks.Weak or transient interactions are typically lost using tradi-tional purification methods. Moreover, large protein complexes orDNA–protein macrocomplexes may be insoluble and are often lost dur-ing the preparation of protein extracts as they pellet along with thechromatin (Lambert et al., 2009). On the other hand, ChIP has also lim-itations for its use in proteomic approaches since this method generallyyields poor protein and DNA recovery. ChIP uses in vivo formaldehydecross-link that modifies proteins (Metz et al., 2004) and can reducetheir binding to affinity resins. It also causes high background bindingthat necessitates washing under high stringency conditions. To over-come some limitations of these techniques, a histidine–biotin–histidine(HBH) tag compatible with denaturing purification conditions has beendeveloped to isolate protein complexes after in vivo cross-link(Tagwerker et al., 2006b). The multisubunit proteasome complex orRNA polymerase II (Pol II) as well as their co-purifying proteins havebeen successfully purified using this procedure (Tagwerker et al.,2006a; Tardiff et al., 2007).

Like Pol II which synthesizes protein-encoding mRNAs, Pol III is amacromolecular machine responsible for the transcription of class IIIgenes encoding ribosomal 5S rRNA, tRNAs and other small untranslatedRNAs. The reconstitution of aminimal in vitro transcription systemwithall components necessary for yeast basal Pol III transcription (Pol III andthree transcription factors, TFIIIA, TFIIIB and TFIIIC) revealed that addi-tional auxiliary factors are necessary for optimal transcription (Ducrotet al., 2006). Traditional TAP-MS strategy has been used without muchsuccess to characterize the proteins associated with Pol III transcriptionmachinery. Purification of Pol III or TFIIIC resulted mainly in the recov-ery of the core complex whereas no purification of the complete TFIIIB

52 N.-T.-T. Nguyen et al. / Gene 556 (2015) 51–60

factor could be achieved (Gavin et al., 2006; Krogan et al., 2006). To ob-tain an exhaustive characterization of the proteins associated in vivo toclass III genes, a strategy could consist to take advantage of the highlevels of enrichment obtained when Pol III is immunoprecipitatedfrom cross-linked chromatin (Soragni and Kassavetis, 2008) to identifythe proteins that co-purify with Pol III.

In this work, we first report a HBH-based modified tandem chroma-tin affinity protocol (TChAP) where chromatin preparation was set upaccording to ChIP experiments but adapted to larger amounts of cells.We present here a standard procedure including in vivo cross-link,DNA shearing by sonication and affinity purification providing a repro-ducible and good recovery of HBH-tagged proteins. A complete proce-dure accomplished in less than one week allows the capture ofprotein–DNA complexes in quantities sufficient for analysis by massspectrometry. We used this strategy to determine the co-purifying pro-tein networks of both TFIIIA and Pol III. We showed that TFIIIA is not agood bait for this process since the protein we isolated is neither associ-ated with the Pol III transcriptional machinery nor with the 5S rDNAloci. On the other hand, illustrating the potential worth of the TChAPprocedure, the purification of Pol III allowed the identification of moreco-purifying proteins than traditional TAP-MS protocol (Collins et al.,2007; Gavin et al., 2006; Krogan et al., 2006). In addition to proteinsknown to directly interact with free RNA polymerases, we detectedalso all the subunits of TFIIIB whereas TFIIIC or chromatin factors wereclearly absent. These results suggest that the Pol III purified aftercross-link in exponential phase contains free Pol III and Pol III engagedin transcription reinitiation.

2. Material and methods

2.1. Yeast strains and culture conditions

Standard yeast media and growth conditions were used (Sherman,1991). All strains used in this study are derivatives of BY4741 strain.HBH tag was integrated at the chromosomal loci of PZF1 and RPO31genes as described previously (Longtine et al., 1998). Epitope integra-tion and expression were confirmed by PCR amplification and Westernblotting using a streptavidin–HRP conjugate (ThermoScientific). For co-purification experiments, the HBH tag was integrated at the chromo-somal locus of the RPO31 gene in the POB3, SPT16 or MAF1 TAP-taggedstrains obtained from the Euroscarf collection.

2.2. Modified tandem chromatin affinity purification (TChAP)

2.2.1. Chromatin preparationThe protocol for chromatin preparation is described for a single 2-

liter culture. For large-scale purifications, multiple 2-liter chromatin ex-tracts were pooled. Cultures were grown to an OD600 of 3 in large erlenscontaining 2 l of YPD rich medium supplemented with adenine.Addition of biotin in the medium was not required since no limitationof biotinylation of the HBH tag was detected. Cells were cross-linked for 20 min with 1% formaldehyde. Cultures were thencentrifugated 5 min at 3500 rpm in a JLA-8.1000. Unreacted formalde-hyde was diluted out by 4 washes of the cell pellets with phosphate-buffered saline (PBS 1×) as described (Déjardin and Kingston,2009). Cell pellets were then washed once with sucrose buffer (PBS1×; 300 mM sucrose; 1% Triton X-100; 2 mM magnesium acetate).Cell pellets were resuspended in 20 ml sucrose buffer containing prote-ase inhibitormixture (Complete™; Roche), frozen and stored at−80 °Cbefore being disrupted by passage through an Eaton-Press. Lysateswerecentrifugated 4 min at 6200 rpm at 4 °C. Pellets were washed 3 timeswith PBST buffer (PBS 1×, 0.5% Triton X-100), once with PBS 1× andonce with LB3JDm buffer adjusted to pH 7.5 (10 mM NaH2PO4/Na2HPO4 pH 7.5, 200 mMNaCl, 0.1% SDS, 0.1% sarkosyl, 10 mM imidaz-ole). Pelletswere resuspended in 15mlfinal of LB3JDm in a 50ml Falcontube before sonication. Chromatin was solubilized and sheared using a

Q700 sonicator with a microtip probe (Qsonica). The sonicator was setto 15 cycles of 10 s ON followed by 50 s OFF with 70% amplitudepower. The probe and the extract were kept cold in ice. The resulting ly-sates were clarified by centrifugation for 15 min at 11,000 g at 4 °C. Analiquot of the supernatant was taken to analyze DNA and proteincontent.

2.2.2. Tandem affinity processChromatin extract prepared from 8-liter cultures (about 40ml sam-

ple) was first subjected to nickel affinity purification using an AKTA pu-rifier system (GE Healthcare). To limit the non-specific binding ofproteins, the supernatant was loaded on a 5ml Sepharose Fast Flow col-umn screwed to another 5 ml nickel Sepharose Fast Flow column,allowing the pre-clearing of the chromatin extract before themetal che-late affinity capture. The samples were injected 3 times at 2.6 ml/minusing the same two column system. After an extensive wash withLB3JDmbuffer, proteinswere eluted with LB3JDm containing 1M imid-azole (pH adjusted to 7.5). Elution fractions were collected, pooled(24 ml) and transferred into three 15 ml tubes containing 140 μl of10% streptavidinMag Sepharose bead slurry (GEHealthcare) equilibrat-ed with the elution buffer. The samples were incubated overnight at10 °C with gentle rotation using an overhead shaker (Intelli-Mixer RM-2, neoLab, Germany). Beads were collected on the side of the tubeusing a magnetic separator (FlexiMag, Spherotech). Supernatant wasdiscarded and the beads were washed with 10 ml of buffer 1 (10 mMNaH2PO4/Na2HPO4 pH 7.5, 200 mM NaCl). One thousandth of thebeads was collected and transferred into a new Eppendorf tube forDNA extraction and further PCR analysis. The rest of the beads werewashed for 5 min with gentle rotation with 10 ml of a more stringentbuffer (10 mM NaH2PO4/Na2HPO4 pH 7.5, 2 M urea). The beads werethen washed again for 5 min with gentle rotation with 10 ml of buffer1 before the final elution. The beads were resuspended in 600 μl ofbuffer 1 together with 300 μl of 3× decrosslink buffer (125 mM Tris–HCl pH 8.8, 5.6% SDS, 0.5 M β-mercaptoethanol) and transferred intotwo 1.5 ml Protein Lobind Eppendorf tubes. Proteins were then elutedand reverse cross-linked by incubation for 5 min at 95 °C at 1000 rpmin a Thermomixer (Eppendorf) followed by 5 min on ice. The elutionstep was repeated 6 times.

To perform TChAP in the absence of RNA, chromatin extractobtained from C160-HBH strain was treated with RNase A (0.6 mg/ml,Sigma-Aldrich R4642) for 30 min at room temperature beforeloading on the two column purification system. Samples wereanalyzed before and after RNase A treatment to confirm the depletionof RNA.

2.2.3. Protein extractionProteins eluted from nickel or streptavidin Sepharose stepwere pre-

cipitated with TCA (20% final), washed with acetone and resuspendedin 10 μl of reversal buffer (250 mM Tris–HCl pH 8.8, 2% SDS, 0.5 M β-mercaptoethanol). The streptavidin sample was diluted with LaneMarker sample buffer (Thermo Scientific) according to the supplier'sprotocol before SDS-PAGE (NuPAGE 4–12%, Life Technologies) andmass spectrometry analysis.

2.2.4. DNA extraction100 μl of the samplewas incubated overnight at 62 °Cwith agitation

with 125 μl of buffer (100mMTris–HCl pH 8, 100mMNaCl, 2% SDS) and12.5 μl of Proteinase K (20 mg/ml, Life Technologies). DNA was thenextracted by phenol/chloroform/isoamyl alcohol followed by ethanolprecipitation. For the samples to be analyzed by PCR amplification,an additional RNase A treatment followed by purification using theQIAquick PCR purification kit (Qiagen) was performed according tothe manufacturer's instructions.

53N.-T.-T. Nguyen et al. / Gene 556 (2015) 51–60

2.3. Real time PCR analysis of DNA purified by TChAP

Quantitative PCR experiments were performed as previously de-scribed (Harismendy et al., 2003) on total genomic DNA purified fromchromatin extracts and onDNA obtained from the final affinity purifica-tion eluates (0.1% of the streptavidin Sepharose beads). Real-time PCRwas performed using an ABI Prism 7300machine (Applied Biosystems).The PCR reactions were carried out in 20 μl containing 0.4 μM eachprimer and 10 μl of Master Mix SYBR green PCR reaction (AppliedBiosystems). All sets of reactions were conducted at least in triplicate.The primers were 5′-GGTTGTTTGGCCGAGCG and 5′-TATTCCCACAGTTAACTGCGGTCA for tDNALeu, 5′-GTTGCGGCCATATCTACCAGA and 5′-TCGCGTATGGTCACCCACTAC for 5S rDNA and 5′-AAAGAAACTTGCACCGGAAA and 5′-GGCCCATATTCGCTTTAACA for GAL1.

2.4. Mass spectrometry and data analysis

Mass spectrometry was performed at the Mass Spectrometry Labo-ratory (IBB, Warsaw, Poland). Proteins were separated by SDS-PAGE,excised from the gel, treated with trypsin and identified by LC–MS/MSusing the Nano-Acquity LC system (Waters) and Orbitrap Velos massspectrometer (Thermo Electron Corp.). Acquired raw data were proc-essed by Mascot distiller followed by database search with the Mascotprogram (Matrix Science) against Saccharomyces Genome Database(http://www.yeastgenome.org/). Search parameters for precursor andproduct ion mass tolerance were 37 ppm and 0.1 Da, respectively. Pep-tideswithMascot score exceeding the threshold value corresponding tob0.5% false positive rate, calculated by Mascot procedure, were consid-ered to be positively identified.

2.5. Co-purification experiments

50ml cells growing exponentially (OD600=1) in YPDmediumwerewashed with lysis buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 0.05%NP40, 0.5mMDTT, 5% glycerol) and resuspended in 0.5ml of lysis buffercontaining 1× protease inhibitor cocktail (Complete™, Roche) and0.5 mM PMSF. Whole-cell extracts prepared using acid-washed glassbeads were adjusted to 300 mM NaCl and incubated for 2 h at 4 °Cwith 30 μl of 10% streptavidin Mag Sepharose bead slurry (GEHealthcare) equilibrated with lysis buffer containing 1 mg/ml insulin.The purified proteins were released from the beads by boiling for10 min. Eluted proteins were resolved by SDS-PAGE and analyzed byWestern blot using a streptavidin–HRP conjugate (Thermo Scientific)or anti-TAP antibodies (Open Biosystems). Quantification was per-formed using Quantity One® software (Bio-Rad).

3. Results and discussion

3.1. A modified tandem chromatin purification procedure (TChAP)

To obtain a global view of the partners of the Pol III transcriptionma-chinery in a chromatin context, we decided to perform a tandem chro-matin affinity purification after in vivo cross-link using the HBH tag thatconsists of a bacterially-derived peptide efficiently biotinylated in vivoby endogenous biotin ligases in yeast and mammalian cells, flanked bytwo hexahistidine tags (Tagwerker et al., 2006b).

This process includes fixing live cells with formaldehyde, bindingsonicated lysates to nickel resin, eluting complexes with imidazoleand binding them to streptavidin Sepharose magnetic beads underhigh stringency conditions to reduce the background. The purified com-plexes are then characterized by MS. Following this strategy, proteinsassociated with purified HBH-tagged Pol II have been successfully iden-tified (Tardiff et al., 2007). The HBH epitope was thus introduced at thechromosomal loci of PZF1 encoding TFIIIA, the 5S rRNA gene-specifictranscription factor and of RPO31 gene encoding C160, the largest sub-unit of Pol III. Preliminary assays were performed using TFIIIA-HBH

protein as a model and published protocols of HBH-based purification.Fig. 1A shows for example a Western blot analysis of TFIIIA-HBH recov-ery after its purification on nickel resin using different protein extrac-tion buffers previously described in proteomic approaches (Déjardinand Kingston, 2009; Tagwerker et al., 2006a; Tardiff et al., 2007).TFIIIA-HBHwas poorly recovered upon extensive denaturing conditions(Urea or GuHCl containing buffers) or with the FA buffer used by Tardiffet al. to purify HBH-tagged Pol II. A higher amount not only of TFIIIA-HBH, but also of Arc1, the major biotinylated protein in yeast (Kimet al., 2004) was obtained using a SDS/sarkosyl containing buffer(LB3JDm, modified from Déjardin and Kingston, 2009).

These preliminary results prompted us to develop our own purifica-tion strategy. We first determined optimal conditions for chromatinpreparation starting from 2-liter cultures of TFIIIA-HBH cells.

3.1.1. Optimization of chromatin preparationSonication is one of the important steps of the process and depends

on various parameters including amount of cells, extent of cross-linking,volume of lysate and specificity of the sonicator used. Preliminary assayswere performedwith the Bioruptor sonicator (Diagenode) we classical-ly used to solubilize and prepare chromatin for ChIP experiments(Tavenet et al., 2009). However, this sonicator appeared to be not ap-propriate for a reproducible treatment of large volumes of lysates. Inthe following experiments, all sonications were thus performed usingthe Q700 sonicator (Qsonica).

Classical fixing of yeast cells is performed using 1% formaldehyde tofreeze interaction networks. On the other hand, extensive cross-linkingin the presence of 3% formaldehyde was found to be required for an ex-haustive characterization of proteins associated with specific genomicloci (Déjardin and Kingston, 2009). We therefore tested the influenceof formaldehyde concentration by adding 0% to 3% formaldehyde in cul-tures of TFIIIA-HBH cells grown to exponential phase. After 20min, cellswere washed, cellular extracts were prepared and lysates were sonicat-ed. DNA fragments obtained after 0 to 25 cycles of sonication were re-solved on agarose gel and visualized by ethidium bromide (Fig. 1B). Asexpected, solubilization was rapidly achieved with a very similar pro-tein and DNA content whatever the formaldehyde concentrationsused (Fig. 1B and data not shown). On the other hand, the extent ofDNA shearing was correlated with formaldehyde concentration andthe number of sonication cycles. DNA fragments with an average sizebelow 2000 bp could not be observed with formaldehyde concentra-tions higher than 1%. In accordance with previous reports (Guerreroet al., 2006), we also observed a reduced recovery of purified proteinsupon increasing concentration of formaldehyde (data not shown). Thefinal conditions for chromatin preparation are indicated in Fig. 1C. Wedecided to use 1% formaldehyde for cross-link and non-extensive soni-cation conditions (15 sonication cycles) that might preserve protein in-tegrity and yielded DNA fragments between 200 and 2000 bp (Fig. 1B).

Because of the compaction of the yeast genome and since most PolIII-transcribed genes are only ≈100–150 bp long, we could wonderwhether the larger size of the DNA fragments obtained after chromatinpreparation might affect the protein network co-purifying with Pol IIItranscription machinery. For instance, one could imagine that proteinsbound on neighboring Pol II or Pol I-transcribed genes could be detect-ed. However, it did not appear to be the case. Few peptides from thelargest subunits of Pol I and Pol II were identified by MS analysis butthey were also present in the untagged control strain and were thusconsidered as background proteins (Table 3).

3.1.2. Optimization of tandem affinity purificationWild type untagged control strain (WT) or cells expressing TFIIIA-

HBH or C160-HBH were grown under identical conditions to exponen-tial phase. After cross-link with 1% formaldehyde, chromatin extractswere prepared using the optimized procedure developed previouslywith TFIIIA-HBH cells (Fig. 1C). The profiles of the DNA fragments ob-tained for the 3 strainswere highly similar (Fig. 2A). Chromatin extracts

A

HCOH

Time of sonication/

cycle

Number of sonication

cyclesPower

amplitude

Sonicationconditions 1%

10 sec ON 50 sec OFF 15 70%

B

Number of sonication

cycles

HCOH 0% 1% 2%

kb

1.5

0.5

5

3

0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25

3%

1.5

0.5

5

3

kb

0 5 10 15 20 25

LB

3JD

m

FA Gu

HC

l

Ure

a

TFIIIA-HBH

*

WB: Streptavidin-HRP

Arc1

Buffer

C

Fig. 1. Optimization of chromatin preparation after cross-link of TFIIIA-HBH cells. (A) Determination of the optimal buffer. Chromatin extracts prepared in different buffers used inprevious published protocols were affinity-purified on nickel Sepharose resin. Elution samples were resolved by SDS-PAGE and analyzed by Western blotting using a streptavidin–HRPconjugate. The positions of TFIIIA-HBH and Arc1 are indicated. LB3JDm was modified from the buffer used for PICh (Déjardin and Kingston, 2009) by reducing detergent concentration(0.1% SDS/sarkosyl instead of 0.2%) to allow binding to nickel resin. FAwas used to characterize the proteins associatedwith Pol II (Tardiff et al., 2007). Urea buffer was prepared accordingto the initial HBH-based protocol (Tagwerker et al., 2006a). InGuHCl buffer, guanidine hydrochloride (6M) replaced urea (8M). (B) DNA shearing as a function of formaldehyde cross-linkand sonication process. 6 μg of DNA isolated from chromatin extracts cross-linked with different concentrations of formaldehyde were resolved on a 1% agarose gel and visualized byethidium bromide after the indicated number of sonication cycles. DNA fragments obtained upon the chosen chromatin preparation conditions are surrounded by a rectangle.(C) Summary of optimal conditions determined for chromatin preparation.

54 N.-T.-T. Nguyen et al. / Gene 556 (2015) 51–60

were then subjected to purification on nickel and streptavidinSepharose resins. Two major changes were added as compared topublished HBH-based purifications. First, the use of a SDS/sarkosyl con-taining buffer allowed a better recovery of the HBH-tagged protein(Fig. 1A). Second, we used a chromatographic system (AKTA purifier)that shortened the time required for the nickel affinity step and gave abetter reproducibility and a higher recovery of our tagged proteins. Inaddition, a modified pre-clearing step was performed to reduce thebackground. Chromatin extracts were loaded on a two column systemwith a Sepharose column without nickel coupled to a nickel Sepharosecolumn (see Material and methods for further details).

Table 1List of the proteins identified after TChAP of cross-linked TFIIIA-HBH cells and curation oftheMS data. The number of peptides detected in each replicate, the gene names and com-plex or general functions are indicated.

Usualname

Gene Peptides,exp1

Peptides,exp2

Complex/function

PZF1 YPR186C 51 65 TFIIIAISW1 YBR245C 2 16 Chromatin remodelerRSC8 YFR037C 3 8 RSC complexSPT16 YGL207W 3 29 FACT complexSPT5 YML010W 4 16 Spt4/Spt5 complexNCL1 YBL024W 4 21 tRNA methyltransferaseNSR1 YGR159C 5 15 rRNA processingPAP1 YKR002W 2 4 Poly A polymerasePUS1 YPL212C 2 2 tRNA pseudouridine synthaseTRM82 YDR165W 2 4 tRNA methyltransferaseSUB2 YDL084W 14 19 RNP/splicing/exportDBP3 YGL078C 3 7 rRNA processingNUG1 YER006W 2 4 rRNA processingUTP22 YGR090W 2 10 rRNA processingRRP5 YMR229C 2 27 18S and 5.8S rRNA synthesisUBI4 YLL039C 5 3 UbiquitinETT1 YOR051C 3 4 Unknown functionBLM10 YFL007W 3 35 Proteasome activatorERG10 YPL028W 4 11 Ergosterol biosynthesisLYS21 YDL131W 6 13 Homocytrate synthase

Western blot analysis using a streptavidin–HRP conjugate revealedthat as expected, TFIIIA-HBH and C160-HBH were retained on bothnickel and streptavidin resins (Fig. 2B). The first affinity purificationon nickel resin reduced the co-purification of biotinylated proteins onthe second streptavidin resin, leading to a lower background. However,Arc1, themajor yeast biotinylated protein (Kim et al., 2004)was presentduring all the purification process (Fig. 2B).

3.2. Large scale purification of TFIIIA-HBH and C160-HBH protein networksusing TChAP

A five-day process of TChAP purification is presented in Fig. 3A. Atotal of 9 independent purifications were sent for LC–MS/MS analysesto identify the proteins co-purifying with TFIIIA (2 replicates), Pol III(3 replicates including 1 with RNase A treatment) and the proteins de-tected in the control untagged strain (4 replicates). Cells were grown toexponential phase in 8-liter cultures. Four chromatin extracts preparedfrom 2-liter cultures were pooled and subjected to TChAP purification.After one stringent wash (urea 2 M, for details see Section 2.2.2) re-quired to lower non-specific binding, proteins bound on streptavidinmagnetic beads were released using denaturing conditions.

3.2.1. Protein analysis of co-purifying networksThe purified proteins were resolved on a 4–12% NuPAGE gel and

stained using Imperial blue, a sensitive Coomassie blue dye reagentcompatible with mass spectrometry analysis. The protein profiles ob-served for TFIIIA-HBH and C160-HBH were clearly distinct (Fig. 3B). Aspecific protein pattern could also be detected when the complete pro-cedure was applied to the wild type strain suggesting the presence ofcontaminant proteins. The gels were sliced into 4 sections and sent formass spectrometry analysis.

3.2.2. Analysis of the DNA purified after the TChAP processTo investigate towhich extent class III genes loci were captured dur-

ing the TChAP purification process, DNAwas purified from an aliquot of

Table 2List of the proteins identified after TChAP of cross-linked C160-HBH cells and curation of theMS data. Five genes of interest (italics) that were identified in only one replicate are added tothe list. Thenumber of peptides detected in each replicate, the genenames and complex or general functions are indicated. One replicate (exp3)wasperformed on chromatin extracts afterRNase treatment.

Usual name Gene Peptides, exp1 Peptides, exp2 Peptides, exp3 Complex/function

RPO31 YOR116C 273 264 317 RNA Pol IIIRET1 YOR207C 179 145 224 RNA Pol IIIRPC82 YPR190C 102 95 111 RNA Pol IIIRPC53 YDL150W 43 41 71 RNA Pol IIIRPC40 YPR110C 26 23 27 RNA Pol IIIRPC37 YKR025W 37 19 36 RNA Pol IIIRPC34 YNR003C 20 18 29 RNA Pol IIIRPC31 YNL151C 10 5 20 RNA Pol IIIRPC25 YKL144C 12 4 16 RNA Pol IIIRPC19 YNL113W 4 3 7 RNA Pol IIIRPC17 YJL011C 15 14 17 RNA Pol IIIRPC11 YDR045C 8 4 10 RNA Pol IIIRPC10 YHR143W-A 2 3 RNA Pol IIIRPB5 YBR154C 15 11 22 RNA Pol IIIRPO26 YPR187W 5 8 RNA Pol IIIRPB8 YOR224C 4 2 3 RNA Pol IIIBDP1 YNL039W 10 12 30 TFIIIBBRF1 YGR246C 13 12 33 TFIIIBSPT15 (TBP) YER148W 2 4 6 TFIIIBBUD27 YFL023W 48 28 66 RNA Pol III assemblyGPN3 YLR243W 2 4 RNA Pol III assemblyNPA3 YJR072C 5 7 11 RNA Pol III transportMAF1 YDR005C 5 3 11 RNA Pol III represseurSPT16 YGL207W 5 12 8 FACT complexMTR4 YJL050W 3 4 RNA transport surveillanceNAB3 YPL190C 2 2 Nrd1 complexNCL1 YBL024W 2 2 tRNA methyl transferaseNSR1 YGR159C 7 3 17 rRNA processingSGN1 YIR001C 2 4 RNA metabolismSMT3 YDR510W 5 4 12 SUMO ubiquitinETT1 YOR051C 4 4 4 Unknown functionAML1 YGR001C 2 4 Unknown functionPOL30 YBR088C 3 2 2 PCNAERG10 YPL028W 4 2 13 Ergosterol biosynthesisERG13 YML126C 9 6 12 Ergosterol biosynthesisTAL1 YLR354C 7 13 TransaldolaseLYS20 YDL182W 9 10 6 Lysine biosynthesis pathwayRRP5 YMR229C 5 2 6 18S and 5.8S rRNA synthesisDBP3 YGL078C 3 7 rRNA processingRPB10 YOR210W 2 RNA Pol IIITFC4 YGR047C 5 TFIIICDST1 (TFIIS) YGL043W 3 TFIISSUB1 YMR039C 2 Transcriptional coactivatorPOB3 YML069W 8 FACT complex

55N.-T.-T. Nguyen et al. / Gene 556 (2015) 51–60

the final streptavidin elutions of TFIIIA-HBH and C160-HBH. The pres-ence of selected Pol III-transcribed genes was analyzed by real-timePCR. As shown in Fig. 3C, a clear enrichment of the 5S rRNA and tRNALeu

genes was detected after the purification of Pol III. In contrast, no signif-icant specific DNAenrichment could be detected after TFIIIA purificationsuggesting that TFIIIA was not a good bait to capture 5S rDNA upon ourexperimental conditions.

3.3. Analysis of MS data

3.3.1. Curation of MS dataWe noted that the background of the TChAP performed on cells in

exponential phase is quite high. 232 proteins, a large majority of thepolypeptides detected in our MS analysis were also present in the un-tagged control strain and thus considered as background proteins(Table 3). On the other hand, improvements inmass spectrometry tech-nology have greatly increased the sensitivity of the analyses yieldingtherefore large lists of proteins. Thus, the next challenge relies now inthe discrimination of true interactors from nonspecific binders. Efficientanalysis of theMS results is critical to discern bona fide interactors. Thus,the set of proteins identified by MS was refined according to differentcriteria. First, proteins should be identified by at least two independent

peptides (Criterion n°1). Second, proteins should not be present inthe list of common contaminants shown in Table 3 generated afterpurifications from the control untagged strain (Criterion n°2). Third,proteins should be detected in at least two independent purifications,considering reproducibility as one of the major criteria (Criterion n°3).Last, ribosomal proteins that are common contaminants in manyproteomic approaches (Lambert et al., 2010) were removed from thefinal lists.

Curation of our data generated restrictive lists of co-purifying pro-teins. Only 20 proteins were identified in the purification of TFIIIA(Table 1) whereas 39 were found in the purification of Pol III (Table 2).

3.3.2. TFIIIA is not a good bait for TChAPOur proteomic and qPCR data strongly suggested that the TFIIIA pu-

rified using TChAP is neither associated with the Pol III transcriptionalmachinery (Table 1) nor with the 5S rDNA loci (Fig. 3C). Using conven-tional TAP-MS approaches, TFIIIA was found to interact with only twoproteins (Krogan et al., 2006; Ziv et al., 2011) including Ubi4 thatwas also detected in our analysis suggesting that TFIIIA could beubiquitinylated. The 75-aa HBH epitope could thus not be accessiblewhen TFIIIA is bound to 5S rDNA within the Pol III transcriptional ma-chinery. Conversely, we did not detect any peptide of TFIIIA in our

Table 3List of contaminant proteins identified after purification of crosslinked BY4741 wt controlstrain.

Usual name Gene Yeast biotinylated proteins

ABP1 YCR088WACC1 YNR016C Al-Feel et al. (1992)ACS2 YLR153CACT1 YFL039CADE4 YMR300CADH1 YOL086CADH2 YMR303CALA1 YOR335CALD6 YPL061WAPA1 YCL050CARB1 YER036CARC1 YGL105W Kim et al. (2004)ARF1 YDL192WARO2 YGL148WASC1 YMR116CASN1 YPR145WASN2 YGR124WBAT2 YJR148WBEM2 YER155CBFR1 YOR198CBMH1 YER177WBRE5 YNR051CCAR2 YLR438WCCT3 YJL014WCCT8 YJL008CCDC19 YAL038WCDC39 YCR093WCDC48 YDL126CCDC60 YPL160WCHC1 YGL206CCKA2 YOR061WCOP1 YDL145CCPR6 YLR216CCRM1 YGR218WCYS4 YGR155WDAK1 YML070WDBP2 YNL112WDBP5 YOR046CDED1 YOR204WDED81 YHR019CDHH1 YDL160CDPS1 YLL018CDUF1 YOL087CDUR1,2 YBR208C Pirner and Stolz (2006)EFT2 YDR385WELP3 YPL086CEMW1 YNL313CENO1 YGR254WENO2 YHR174WERG20 YJL167WFAS1 YKL182WFAS2 YPL231WFBA1 YKL060CFKS1 YLR342WFUN12 YAL035WGAL83 YER027CGCD11 YER025WGCN1 YGL195WGCN20 YFR009WGCS1 YDL226CGET3 YDL100CGFA1 YKL104CGLK1 YCL040WGLN1 YPR035WGLN4 YOR168WGLT1 YDL171CGLY1 YEL046CGND1 YHR183WGPM1 YKL152CGPP1 YIL053WGRS1 YBR121CGSH1 YJL101CGSP1 YLR293CGUA1 YMR217WGUK1 YDR454C

Table 3 (continued)

Usual name Gene Yeast biotinylated proteins

GUS1 YGL245WHEF3 YNL014WHEK2 YBL032WHIS4 YCL030CHOG1 YLR113WHRK1 YOR267CHSC82 YMR186WHSP104 YLL026WHSP82 YPL240CHXK2 YGL253WHYP2 YEL034WIKI3 YLR384CILS1 YBL076CIMD3 YLR432WIPP1 YBR011CIST2 YBR086CKRS1 YDR037WLEU1 YGL009CLIA1 YJR070CLSG1 YGL099WLSP1 YPL004CMAP1 YLR244CMCM4 YPR019WMES1 YGR264CMHP1 YJL042WMMT1 YMR177WMMT2 YPL224CMRN1 YPL184CMSC2 YDR205WMSN5 YDR335WMYO2 YOR326WNAM7 YMR080CNAT1 YDL040CNEW1 YPL226WNMA1 YLR328WNMA2 YGR010WNMT1 YLR195CNST1 YNL091WNTH1 YDR001CNUM1 YDR150WOLA1 YBR025CPAB1 YER165WPBP1 YGR178CPDC1 YLR044CPDR5 YOR153WPFK1 YGR240CPFK2 YMR205CPGI1 YBR196CPGK1 YCR012WPHO8 YDR481CPIL1 YGR086CPMA1 YGL008CPUB1 YNL016WPYC1 YGL062W Pirner and Stolz (2006)PYC2 YBR218C Pirner and Stolz (2006)RNA1 YMR235CRNQ1 YCL028WRNR1 YER070WROM2 YLR371WRPA135 YPR010CRPA190 YOR341WRPA43 YOR340CRPB2 YOR151CRPC53 YDL150WRPG1 YBR079CRPL19A YBR084C-ARPL27B YDR471WRPL3 YOR063WRPL40A YIL148WRPO21 YDL140CRPO31 YOR116CRPS0A YGR214WRPS1B YML063WRPS3 YNL178WRPS31 YLR167WRPS6A YPL090CRPS8A YBL072C

56 N.-T.-T. Nguyen et al. / Gene 556 (2015) 51–60

Table 3 (continued)

Usual name Gene Yeast biotinylated proteins

RSP5 YER125WSAC1 YKL212WSAC6 YDR129CSAH1 YER043CSAM1 YLR180WSAM2 YDR502CSAR1 YPL218WSBP1 YHL034CSEC14 YMR079WSEC18 YBR080CSEC21 YNL287WSEC23 YPR181CSEC24 YIL109CSEC27 YGL137WSEC28 YIL076WSEC53 YFL045CSFB2 YNL049CSHM2 YLR058CSIP1 YDR422CSNF1 YDR477WSNF4 YGL115WSRO9 YCL037CSSA1 YAL005CSSA2 YLL024CSSA3 YBL075CSSB1 YDL229WSSB2 YNL209WSSC1 YJR045CSSE1 YPL106CSST2 YLR452CSSZ1 YHR064CSTI1 YOR027WSTM1 YLR150WSUI3 YPL237WSUP35 YDR172WSUP45 YBR143CSXM1 YDR395WTDH1 YJL052WTDH2 YJR009CTDH3 YGR192CTEF2 YBR118WTEF4 YKL081WTHR4 YCR053WTHS1 YIL078WTIF2 YJL138CTIF3 YPR163CTIF4631 YGR162WTIF4632 YGL049CTIF5 YPR041WTKL1 YPR074CTMA19 YKL056CTOM70 YNL121CTRL1 YJL087CTRP2 YER090WTRP5 YGL026CTUB2 YFL037WURA2 YJL130CURA7 YBL039CVAC8 YEL013WVAS1 YGR094WVMA1 YDL185WVMA2 YBR127CVTC2 YFL004WVTC3 YPL019CVTC4 YJL012CXRN1 YGL173CYAR010C YAR010CYBR238C YBR238CYDJ1 YNL064CYEF3 YLR249WZPR1 YGR211WZRC1 YMR243CZRT3 YKL175WZUO1 YGR285C

YGR038C-BYGR161C-DYGR234W

Table 3 (continued)

Usual name Gene Yeast biotinylated proteins

YHR020WYJR029WYNL010W

57N.-T.-T. Nguyen et al. / Gene 556 (2015) 51–60

purifications of Pol III (Table 2) whereas qPCR results clearly showed anenrichment of 5S rDNA genes (Fig. 3C). In fact, to our knowledge, thepresence of TFIIIA on 5S rDNA has never been evidenced in vivo. NoChIP experiments using TFIIIA as a bait have ever been reported andTFIIIA has not been identified by MS analysis after the purification of5S rDNA domains in their native chromatin context (Hamperl et al.,2013). On the other hand, TFIIIA also binds with high affinity and spec-ificity to 5S rRNA (WangandWeil, 1989).Most of the proteins identifiedafter TFIIIA-HBH purification correspond to chromatin or RNA associat-ed factors suggesting that they might not directly interact with TFIIIA.Further work will be required to determine whether they are trueinteractors or non-specific binders.

3.3.3. Analysis of the proteins associated with C160-HBHC160 appeared to be a good bait to capture the proteins associated

with Pol III bound on class III genes as suggested by qPCR data(Fig. 3C). Previous TAP-MS experiments using C160 as a bait have iden-tified 24 co-purifying proteins (Collins et al., 2007; Gavin et al., 2006;Krogan et al., 2006). When compared to the 39 proteins detected

A S P S P S P

kb

1.5

0.5

5

WT TFIIIA-HBH

C160-HBH

B

WB

: S

trep

tavi

din

-HR

P

Nickel elution Streptavidin elution

TFIIIA-HBH

C160-HBH

* Arc1** * *

WT TFIIIA-HBH

C160-HBH

WT TFIIIA-HBH

C160-HBH

Fig. 2. Characterization of tandem chromatin affinity purification steps. (A)DNA (6 μg) ex-tracted from the pellet (P) and the supernatant (S) after the sonication of wild type (WT),TFIIIA-HBH or C160-HBH cross-linked cells was resolved on a 1% agarose gel and visual-ized by ethidium bromide. (B) Western blot analysis using a streptavidin–HRP conjugateof elution samples after nickel (left panel) or nickel/streptavidin fractionations (rightpanel). The positions of specific HBH-tagged proteins are indicated. The band detectedin all samples (asterisk) corresponds to Arc1, the major biotinylated contaminant of thisprocedure.

Fo

lden

rich

men

t (%

)C

C160-HBH

TFIIIA-HBH

GAL1 RDN5tDNALeu

10

30

BA

Streptavidin-Sepharose affinityusing magnetic beads

In vivo cross-link with1% formaldehyde

4-12% SDS-PAGEImperial blue staining

LC-MS/MS

Day1

Chromatin preparation:mechanical lysis, sonication

and centrifugation

Pre-clearing (Sepharose without Ni2+)and Ni2+-Sepharose affinity on AKTA

purifier

Day3

Day4

Day5

8-liter cultures

Day2

220

120160

100

50

20

70

40

30

Imperial blue stainingkDa

WT TFIIIA-HBH

C160-HBH

0

Fig. 3. Large scale purification of TFIIIA-HBH and C160-HBH protein networks using TChAP. (A) Schematic of TChAP procedure. Eight liters of YPD were inoculated to reach exponentialphase the following day. Cellswere cross-linked, lysed, sonicated and affinity-purifiedover consecutive nickel and streptavidin resins before protein identification byMS. (B) The completeTChAPprocedurewas performed fromwild type (WT), TFIIIA-HBH or C160-HBHcells. 50% of thefinal elution fractionswere analyzedby SDS-PAGE and Imperial blue staining before beingsent tomass spectrometry analysis. (C) Real-time PCR analysis of DNA enrichment after TChAP. The amounts of tDNALeu or 5S rDNA in final elution fractions affinity-purified from TFIIIA-HBH or C160-HBH cells are expressed as a value relative to that of the whole cell extracts and are shown as histograms. The GAL1 gene was used as a control. Error bars represent thestandard deviation between three independent replicates.

58 N.-T.-T. Nguyen et al. / Gene 556 (2015) 51–60

using ourmethod, an overlap of 18 proteins (Fig. 4A) was observed thatcomprised 16 subunits of Pol III, Bdp1 and Maf1.

Interestingly, all the three subunits of TFIIIB were identified in ourstudy whereas neither Brf1 nor TBP has ever been found associatedwith Pol III using conventional TAP-MS approaches (Gavin et al., 2006;Krogan et al., 2006). On the other hand, only one subunit of TFIIIC wasidentified in one of our purifications of Pol III (Table 2). Such resultscould be correlated with previous observations suggesting that TFIIICis an assembly factor whose main role in Pol III transcription is to pro-mote the assembly of TFIIIB. First, in vitro data showed that TFIIIC wasnot required for transcriptional reinitiation on short class III genes(Ferrari et al., 2004) and that TFIIIC might be displaced by Pol III duringtranscriptional elongation (Bardeleben et al., 1994) leaving a Pol III–TFIIIB reinitiation complex (Dieci and Sentenac, 1996). On the otherhand, the low levels of TFIIIC occupancy observed at all transcriptionallyactive genes relatively to TFIIIB and Pol III (Harismendy et al., 2003;Moqtaderi and Struhl, 2004; Roberts et al., 2003; Soragni andKassavetis, 2008) may indicate that, once bound to DNA, TFIIIB is suffi-cient for Pol III recruitment in vivo. The presence of both TFIIIB and PolIII in our purifications suggested that TFIIIB–Pol III reinitiation com-plexes were purified, with Pol III engaged in transcription cycles as ex-pected in exponential phase where transcriptional levels are high. Ingood agreement with this result, we did not identify any histone pro-teins in our analysis. Although active tRNA genes are largely devoid ofnucleosomes, Pol III was easily detected in previous TAP-MS worksusing histone proteins as baits (Gilmore et al., 2011). Moreover, recent

studies clearly showed that nucleosome and Pol III occupancy levels(hence transcription) on tDNAs were inversely correlated (Kumar andBhargava, 2013).

Several proteins known to be associated with Pol III or with Pol III-transcribed genes (Acker et al., 2013) were present in our purifications.This was the case of Maf1, the repressor of Pol III transcription that wasfound to interact directly with Pol III (Oficjalska-Pham et al., 2006).Bud27, a protein required for the correct assembly of all three RNApoly-merases (Sung et al., 2013) was one of the top hits of each experiment(Table 2). A direct interaction between Bud27 and Pol III was detectedrecently by co-IP (Miron-Garcia et al., 2013). We also identified Npa3and Gpn3, two paralogous small GTPases that are involved in the bio-genesis of Pol III (Minaker et al., 2013). Npa3 was shown to co-purifywith Pol III using a TAP strategy after in vivo cross-linking (Alonsoet al., 2013). The presence of Smt3was consistent with the fact that sev-eral subunits of Pol III are sumoylated (Panse et al., 2004; Sung et al.,2013) and suggested that such modification was not altered duringour purification process.

Interestingly, Spt16 and Pob3, two components of the yeast FACTcomplex, were also found to be associated with Pol III in our study.FACT activity required the presence of Nhp6, a high mobility group pro-tein that plays a role in Pol III transcription (Braglia et al., 2007). We didnot detect Nhp6 in our study. However, Nhp6 is a small size protein(11 kDa) that did not stably associate with Spt16–Pob3 heterodimer.Since human FACT complex was shown by co-IP to interact with Pol III(Birch et al., 2009), strains expressing both C160-HBH and Spt16-TAP

18 621

C160-HBHTChAP

BioGRIDAffinity-MS

Purification of C160A

7 1332

C160-HBH TFIIIA-HBH

TChAP Purification B

Fig. 4. Analysis of the composition of TFIIIA and C160 protein networks identified usingTChAP procedure. (A) Venn diagrams of unique and shared associated proteins detectedby TChAP or conventional TAP-MS (data obtained from BioGRID) using C160 as a baitare shown. (B) Venn diagrams are used to compare the TFIIIA and C160 protein networksidentified after TChAP process.

59N.-T.-T. Nguyen et al. / Gene 556 (2015) 51–60

or Pob3-TAP were generated. No specific co-purification of Spt16 withC160-HBH could be detected after the purification of whole cell extractson streptavidin magnetic beads (data not shown). In contrast, Pob3 wasco-purifiedwith C160-HBHwithout cross-link but to a lesser extent thanMaf1 (Fig. 5). A role of FACT complex in the regulation of the SUP4 tRNAgene transcription has previously been described (Mahapatra et al.,2011). Since FACT complexwas the only chromatin remodeling complexwe identified in our study, we can wonder whether its presence on classIII genesmay be attributed to another of itsmultiple functions such as itsrole in elongation described for Pol II (Orphanides et al., 1998).

Pob3-TAP Maf1-TAP

Input

Co-purification

PurificationC160-HBH

Pob3-TAP

Maf1-TAP

C160-HBH

Pob3-TAP

Maf1-TAP

19.5!Co-purif/Input

16.6Co-purif/Input

Fig. 5. Pob3 co-purifies with C160-HBH. Whole cell extracts from Pob3-TAP or Maf1-TAPstrains expressing or not C160-HBH were purified on streptavidin magnetic beads.Proteins in inputs (upper panel) or eluted fractions (lower panel) were resolved by SDS-PAGE and analyzed byWestern blot using a streptavidin–HRP conjugate or anti-TAP anti-bodies. The co-purification/input quantified ratios are indicated for each strain withcontrol lanes set to 1.

Some of the other proteins identified in this work are related to RNAmaturation and we could wonder whether their association was due tothe presence of RNA. This was not the case since they were also co-purified in a control experiment where chromatin extracts from C160-HBH were treated with RNase A (Table 2, exp3). We also noticed that7 proteins were present in both purifications of TFIIIA and Pol III(Fig. 4B). Further work will be required to determine whether theyare or not bona fide interactors.

4. Conclusions

Although our study gave more co-purifying targets than previoustraditional TAP-MS approaches, several Pol III transcription known ef-fectors (Acker et al., 2013) were not identified using our approach. Forinstance, we hardly detected Sub1 and Dst1 (identified only in one ofour purifications, see Table 2) and did not identify any peptides fromcomplexes involved in chromatin remodeling (except FACT complex)or modifications. This was also the case for Pol II transcription factorslike Yox1, Fkh1, Reb1 and Yap6 that are enriched at class III genes(Horak and Snyder, 2002; Venters et al., 2011). We could wonderwhether this discrepancy comes from the fact that some effectors occu-py only a small subset of tRNA genes as suggested by genomewide anal-ysis of RSC chromatin remodeling complexes or Pol II transcriptionfactors (Horak and Snyder, 2002; Ng et al., 2002; Venters et al., 2011).Another possibility could be that as reported previously, formaldehydewas inefficient to cross-link some proteins like Rpd3 (Kurdistani et al.,2002). In this case, it could be interesting to test whether the use ofother cross-linkers that allow the detection of weak or even transientprotein–protein or protein–DNA interactions, alone or in combinationwith formaldehyde, could provide new associated partners. On theother hand, the application of TChAP approach to other Pol III transcrip-tion componentsmight allow the generation of amore exhaustive list ofproteins associated with the Pol III transcription machinery. To addressthese questions, we are currently performing TChAP experiments usingTFIIIB and TFIIIC subunits as baits.

Another interesting question concerns the regulation of Pol III tran-scription in response to various cellular and environmental changes.The characterization of the set of proteins detected by TChAP usingcells treated with distinct drugs might allow the identification ofstress-specific response pathways involved in Pol III transcription regu-lation. Such approaches under investigation in our lab should give newopportunities to study Pol III transcription regulation.

Acknowledgments

We would like to thank M. Vandamme for efficient technicalassistance, A. Veillet and J. Déjardin for helpful discussions and A.Malinowska (Mass Spectrometry Laboratory, Institute of Biochemistryand Biophysics, Polish Academy of Sciences, Warsaw, Poland) for pro-tein identification. We also thank Pr P. Kaiser (University of California,Irvine, USA) for the generous gift of pFA6a-HBH-kanMX6 plasmid.This work was supported by grants from the French National ResearchAgency (2010-BLAN-1605-01 and 2013-13-BSV3-0012-02). N-T-TNguyen is a recipient of the International PhD program of the CEA.

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