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Originally published In Press as doi:10.1074/jbc.M600387200 on July 2, 2006

J. Biol. Chem., Vol. 281, Issue 39, 29155-29164, September 29, 2006
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Nucleosome Depletion Activates Poised RNA Polymerase III at Unconventional Transcription Sites in Saccharomyces cerevisiae*

Elisa Guffanti{ddagger}1, Riccardo Percudani{ddagger}, Olivier Harismendy§, Julie Soutourina§, Michel Werner§, Maria Giuseppina Iacovella, Rodolfo Negri, and Giorgio Dieci{ddagger}2

From the {ddagger}Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, 43100 Parma, Italy, the §Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique (CEA)/Saclay, 91191 Gif-sur-Yvette, France, and the Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Biologia Cellulare, Università di Roma La Sapienza, 00185 Roma, Italy

Received for publication, January 13, 2006 , and in revised form, June 9, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RNA polymerase (pol) III, assisted by the transcription factors TFIIIC and TFIIIB, transcribes small untranslated RNAs, such as tRNAs. In addition to known pol III-transcribed genes, the Saccharomyces cerevisiae genome contains loci (ZOD1, ETC1-8) associated to incomplete pol III transcription complexes (Moqtaderi, Z., and Struhl, K. (2004) Mol. Cell. Biol. 24, 4118-4127). We show that a short segment of the ZOD1 locus, containing box A and box B promoter elements and a termination signal between them, directs the pol III-dependent production of a small RNA both in vitro and in vivo. In yeast cells, the levels of both ZOD1- and ETC5-specific transcripts were dramatically enhanced upon nucleosome depletion. Remarkably, transcription factor and pol III occupancy at the corresponding loci did not change significantly upon derepression, thus suggesting that chromatin opening activates poised pol III to transcription. Comparative genomic analysis revealed that the ZOD1 promoter is the only surviving portion of a tDNAIle ancestor, whose transcription capacity has been preserved throughout evolution independently from the encoded RNA product. Similarly, another TFIIIC/TFIIIB-associated locus, close to the YGR033c open reading frame, was found to be the strictly conserved remnant of an ancient tDNAArg. The maintenance, by eukaryotic genomes, of chromatin-repressed, non-coding transcription units has implications for both genome expression and organization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In eukaryotes, RNA polymerase (pol)3 III, assisted by a specific set of basal transcription factors (TFIIIA, TFIIIB, TFIIIC), transcribes at high efficiency the genes for tRNAs, 5 S rRNA, and a few other non-translated RNAs. In Saccharomyces cerevisiae, these include the RNA component of RNase P, the U6 small nuclear RNA, and the cytoplasmic RNA of the signal recognition particle, encoded respectively by the RPR1, SNR6, and SCR1 genes (1-3), as well as a small RNA of unknown function encoded by the RNA170 locus (4). The genomes of higher eukaryotes also contain a variety of extremely abundant, repetitive short interspersed elements (SINEs) that have evolved from tRNA or 7SL RNA genes and maintain a pol III promoter (5). SINE transcription by pol III can influence the expression of other genes, both at the transcriptional and at the post-transcriptional levels (5, 6). Up to now, no tRNA gene-derived transcription unit has been identified in yeast genomes. Genome-wide chromatin immunoprecipitation analyses in S. cerevisiae have recently revealed several new loci that are associated to the pol III transcription machinery (7-9). One of them, SNR52, encodes a C/D box small nucleolar RNA. Other newly identified loci appear to be associated to incomplete transcription machinery. One of these loci has been named ZOD1 (for zone of disparity) because its occupancy by pol III was found to be disproportionately low when compared with TFIIIC occupancy (9). In another study, the same locus (referred to as iYML089c) was found to be associated to appreciable levels of pol III (8). Other loci named ETC (for extra TFIIIC) are occupied by the TFIIIC subunit Tfc4 at levels comparable with those of standard class III genes, whereas TFIIIB and pol III are absent (9). One of the ETC loci, ETC5, corresponds to the previously identified RNA170 gene, whose transcription product was previously detected in vivo (4). In contrast, no transcription products have been identified for the remaining ETC loci and for ZOD1, yet they all contain a box B and additional conserved residues, suggesting a biologically relevant pol III function (9). The lack of ZOD1 expression under standard growth conditions is especially intriguing as pol III seems to be present at this locus.

In this study, we show that nucleosome depletion dramatically enhances the expression of both ZOD1 and ETC5 without affecting their occupancy by the pol III machinery. By comparative genomic analysis, we also found that ZOD1 originates from an ancient tRNAIle gene, whose box B has been strictly conserved throughout evolution of the hemiascomycetes, and we identified another TFIIIC/TFIIIB-associated element in the S. cerevisiae genome as a conserved tDNAArg remnant.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Templates—The S. cerevisiae ZOD1 locus was amplified from yeast genomic DNA (strain S288C) using the high fidelity Pfu DNA polymerase (Promega) and the following oligonucleotide primers: ZOD1_fw, 5'-TTCGGGTGCCAATTTAGCGTGC-3'; ZOD1_rev, 5'-AGTGTCGGCCACCTGATAAAGG-3'. The amplified fragment (420 bp) was inserted into the pCRTOPO vector. The sequence of the insert, containing the ZOD1 transcribed sequence (55 bp) plus 170 bp of 5'-flanking and 195 bp of 3'-flanking sequence, was found to exactly match the sequence retrieved from Saccharomyces Genome Database (SGD). A BamHI-PstI ZOD1-containing restriction fragment was excised from pCRTOPO-ZOD1 and subcloned into pNEB193 and into the YEp352 shuttle vector (for in vivo analysis) (10).

Yeast Strains—The thermosensitive, slow growth brf1-II.6 strain harbors a double aspartate to alanine substitution in Brf1, one of the components of TFIIIB (11, 12). The 13 Myc-tagged strains were constructed following a previously described strategy (13). All strains were derived from UKY403 (14, 15). The epitope coding sequence was inserted between the last codon and the stop codon of BDP1, RET1, and TFC4 ORFs at the corresponding genomic loci. Expression of Myc-tagged proteins was verified by Western blot analysis using the anti-Myc monoclonal antibody. No growth defect was detected in the tagged strains. A {Delta}zod1 S. cerevisiae strain was constructed by one-step gene replacement integrating kanMX in the ZOD1 locus of YPH500 strain, that removed all the ZOD1 transcribed region plus 146 bp of 5'-flanking and 87 bp of 3'-flanking sequence. The disruption cassette was obtained by PCR amplification of the kanMX coding sequence from the {Delta}gcn4 strain of the yeast gene knock-out collection (Open Biosystems, Huntsville, AL) using the following oligonucleotide primers: ZOD1DEL_fw, 5'-CGATCTAACTATTCTGTAAATTCTGTAGTGACTCTTTAGGGCGTACGCTGCAGGTCGAC-3'; ZOD1DEL_rev, 5'-AAATGGAAAGGGAAAAATTAAGGCTGCGAAAATTAATAATAATCGATGAATTCGAGCTCG-3'.

For phenotype analysis, ZOD1 deletion and overexpression (YPH500 transformed with YEp352-ZOD1) strains were grown in liquid YPD cultures to the same density and then spotted at increased dilutions on either rich or minimal media containing either glucose, glycerol, or galactose as a carbon source and various drugs (hydroxyurea, rapamycin, cycloheximide), at 15, 30, or 37 °C.

In Vitro Transcription and DNA Binding Assays—ZOD1 in vitro transcription and primer extension analysis were carried out using a reconstituted transcription system from S. cerevisiae, as described previously (16). For gel retardation assays, a 420-bp ZOD1-containing fragment, 5'-end-labeled on the sense strand, was generated by PCR using 5'-labeled ZOD1_fw and unlabeled ZOD1_rev as primers. 3 fmol of DNA were then incubated with 0.6 µg of TFIIIC purified up to the DEAE-Sephadex A-25 step (16, 17). Binding conditions and native gel electrophoresis were as described previously (3). For DNase I footprinting analysis, 4 fmol of the same DNA fragment were incubated with 0.6 µg of TFIIIC in an 18-µl binding mixture containing 166 mM KCl, 10 µg/ml pBlueScript-KS plasmid DNA, 0.1 mg/ml bovine serum albumin, 5% glycerol, 20 mM HEPES, pH 8, 4 mM MgCl2, 0.5 mM dithiothreitol. Complexes were treated with 0.5 ng (0.05 units) of pancreatic DNaseI for 1 min at 25 °C. The digestion was stopped with 20 mM EDTA. Digested DNA was phenol-extracted, ethanol-precipitated, and fractionated as described (3).

In Vivo Transcription Analyses—Total RNA was purified as described (18) from yeast cells grown to A600 = 0.8-1 in the appropriate selective medium and temperature. For Northern blot analysis, 15 µg of total RNA were loaded on a 6% polyacrilamide-7M urea minigel and then transferred to nylon membrane (Gene Screen PlusTM, PerkinElmer Life Sciences) and hybridized as described (3). For ZOD1 RNA detection, the probe was the radiolabeled oligonucleotide 5'-GCTAAGTGAACTACTTATCCAAAGC-3'. For detection of ETC5/RNA170 transcript, a double-stranded DNA probe was produced by random priming labeling using an ETC5 fragment amplified from yeast genomic DNA with the oligonucleotides ETC5_fw, 5'-TAGCGTTACGTTCGATACCTTCAC-3', and ETC5_rev (5'-GGGCTAGTCTTAGTTTGATTGAGC-3'. The probe used for detection of tRNAAla(AGC) was an oligonucleotide complementary to the mature tRNA sequence. The U3-specific probe was 5'-CTTTGCCGTTGCATTTGTAGTTTTTTCCTTTGGAAGT-3'. Strains UKY403 and MHY308 (14, 15) were employed to analyze the effects of nucleosome depletion on ZOD1 and ETC5 transcription. The glucose shift experiments were carried out as described previously (19).

Chromatin Analysis by Micrococcal Nuclease—Strains MHY308 and UKY403 (14, 15) were grown overnight at 30 °C in galactose-containing medium to an A600 between 0.6 and 1.2. Each culture was then halved, spun down, and resuspended: half in galactose-containing medium and half in glucose-containing medium. These cultures were incubated for different time periods at 30 °C and then subjected to micrococcal nuclease (MNase) treatment, as follows. Each culture was incubated with 1% formaldehyde for 10 min at room temperature for cross-linking, and then the reaction was blocked by adding glycine to 330 mM for 10 min. Cells were collected by centrifugation and resuspended in sorbitol buffer (0.9 M sorbitol, 10 mM Tris-HCl, pH 8.0, 40 mM beta-mercaptoethanol) plus zymolyase 100T (0.1 mg/A600). After 15 min of incubation at 25 °C, the spheroplasts were washed twice with 20 ml of sorbitol buffer and resuspended in 0.4 ml/10 A600 units of Nonidet P-40 buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM CaCl2, 0.5 mM spermidin, 0.075% Nonidet P-40). The spheroplasts were then split into 0.4-ml aliquots and digested with increasing amounts of MNase (Roche Applied Science) for 20 min. The digestion was stopped with 5 mM EDTA, 1% SDS. Proteinase K (80 µg) was added, and the samples were incubated for at least 6 h at 56 °C to revert the cross-linking. Genomic DNA was phenolextracted and ethanol-precipitated. For the analyses reported in Fig. 5, MNase-digested bulk chromatin was fractionated on 1% agarose gels. The probe for the Southern hybridization shown in Fig. 5B was a radiolabeled double-stranded DNA fragment PCR-amplified from genomic DNA with the following oligonucleotide primers: fw_A1, 5'-AGGGCGAAGTACGTATTTC-3'; rev_B2, 5'-GCATGATAAAGCCTCACCC-3'. For the analysis in Fig. 4, genomic DNA was restricted with XbaI (30 units/sample) for 12 h at 37 °C. After phenol extraction and ethanol precipitation, the restricted DNA was used for a low resolution analysis by indirect labeling. DNA was fractionated on a 1.4% agarose gel and blotted onto BioBondTM-Plus nylon membrane following Southern's method. The probe for Southern blotting was a radiolabeled DNA fragment PCR-amplified from genomic DNA with the following oligonucleotide primers: fw, 5'-GTCTTTGCTTATTGTATGCC-3'; rev, 5'-CAGAGTCGGTAGTAGATACC-3'.


Figure 1
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  		FIGURE 1.
In vitro transcription properties of the ZOD1 transcription unit. A, nucleotide sequence of the ZOD1 locus. The sequence of the transcribed region is in bold characters. The box A and box B sequences are underlined. The arrow marks the transcription start site. B, a 420-bp radiolabeled DNA fragment containing the ZOD1 promoter was incubated with partially purified TFIIIC, and then bound and free DNA molecules were fractionated by electrophoresis on a native polyacrylamide gel. C, the same ZOD1-containing DNA fragment, radiolabeled on the sense strand, was incubated in the presence (lane 6) or in the absence (lane 5) of TFIIIC and then digested with DNase I. The digestion products were run on a sequencing gel together with sequencing reactions conducted with the same radiolabeled oligonucleotide used to amplify the ZOD1 fragment (lanes 1-4). The positions of the promoter elements are reported on the right. The dotted lines to the left of boxes A and B indicate the actual extension of the TFIIIC-protected regions. D, ZOD1 in vitro transcription was carried out either in the presence (lane 1) or in the absence (lane 2) of TFIIIC. The migration position and approximate size (established on the basis of radiolabeled RNAs of known size run on the same gel) of the ZOD1 transcript are indicated on the left. E, in vitro-produced ZOD1 transcripts were subjected to primer extension analysis (lane 5). The migration position of the fully extended ZOD1-specific primer, as well as the sequence of the non-transcribed DNA strand around the start site (+1), are indicated on the right.

 
Chromatin Immunoprecipitation—Cross-linked chromatin was prepared essentially as described (20, 21). Cultures (200 ml) were grown exponentially in galactose-containing medium to A600 = 0.6. 100 ml of culture were then crosslinked with 1% formaldehyde for 10 min, whereas the rest of the culture was collected, washed, diluted to A600 = 0.3, incubated in a glucose-containing medium for 6 h, and then fixed. Immunoprecipitation was performed using the anti-Myc 9E10 mouse monoclonal antibody (a gift from the DRIP, CEA/Saclay). Immunoprecipitated DNA was purified as described (20) and analyzed by quantitative PCR on an ABI Prism 7000 instrument (Applied Biosystems). The PCR reactions were carried out in 25-µl mixtures containing 0.4 µM each primer and 12.5 µl of Platinum SYBR Green qPCR SuperMix UDG (Invitrogen). Data were collected on the ABI Prism 7000. The associated software was used to calculate cycle threshold values with hand-refining. Relative quantification using a standard curve method was performed, and the occupancy level for a specific fragment was defined as the ratio of immunoprecipitated DNA over total DNA. Three independents experiments were averaged. The following PCR primers were used: fw, 5'-CTTTTGGCGCTTTGGATAAG-3', rev, 5'-CACATGCGAGAAAATGGAAA-3' for ZOD1; fw, 5'-CGTATAAGCCGCAAGGAAAA-3', rev, 5'-TCTGAACCCAGTGTCAAACG-3' for ETC5; fw, 5'-ACATTTCCACACCCTGGAAC-3', rev, 5'-TTCTTCGCGAGAACAATTCA-3' for GAL1 ORF; fw, 5'-GCTTCAGTAGCTCAGTAGGAA-3', rev, 5'-TGCTCCAGGGGAGGTTC-3' for tM(CAU)D; fw, 5'-CGCTGAGTGAGACGCTAACA-3', rev, 5'-ATCCAAAGCCGTAGCAAGTG-3' for ETC2; fw, 5'-TCGAGAAAGCCTGGATGAGT-3', rev, 5'-TATTACTGCCATCGCCACAA-3' for ETC6; fw, 5'-CCTGGTGCCGTTAGCTATTC-3', rev, 5'-GAAACGAACCCAACGAGTGT-3' for ETC7; fw, 5'-CTTCTTGTCCCCGTTAACCA-3', rev, 5'-AGCCGGACGGAATTTATAGC-3' for iYMR103c; fw, 5'-TTCCGAAACTTCCCAAGAAA-3', rev, 5'-TCGAACCTGGACATGAAAAA-3' for iYGR033c. Control chromatin immunoprecipitation experiments were performed with the untagged UKY403 strain.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The ZOD1 Promoter Supports Efficient pol III-dependent Transcription—Sequence analysis of the ZOD1 locus revealed the presence of putative box A and box B promoter elements separated by ~30 bp (Fig. 1A) (8). A stretch of nine Ts is positioned between box A and box B, a situation reminiscent of both SNR6 and SNR52 genes, where the box B is located downstream of a poly(dT) termination signal (22, 23). A gel retardation assay (Fig. 1B) confirmed that TFIIIC binds stably to ZOD1, as observed in vivo. DNase I protection analysis further showed that TFIIIC specifically contacts the box B region, whereas box A protection was less evident (Fig. 1C). Protection in the box B region extended well downstream of the conserved box B sequence motif and ended with a strongly hypersensitive site 20 bp downstream of the box B. In vitro transcription analysis using a pol III-specific reconstituted system showed the TFIIIC-dependent production of an ~55-nucleotide transcript (Fig. 1D). As revealed by primer extension analysis (Fig. 1E), such a transcript starts at a G located 17 bp upstream of the box A. The transcript size is perfectly explained by termination at the run of T residues between box A and box B.

Detection and pol III Dependence of ZOD1 RNA in Vivo—The above results suggest that the previously reported failure to detect ZOD1 RNA in yeast cells (9) is not due to intrinsic promoter defects of ZOD1. As shown in Fig. 2A, we also failed to detect a ZOD1 RNA in vivo in the YPH500 strain by Northern blot analysis (lane 1). The DNA oligonucleotide probe efficiently detected the in vitro-produced ZOD1 transcript (lane 4), but a ZOD1-specific RNA could only be detected in vivo after transformation of YPH500 with a YEp352-ZOD1 multicopy plasmid (cf. lanes 2 and 3). This RNA had the same size of the one produced in vitro. As shown in Fig. 2B, in vivo accumulation of the ZOD1 RNA was dependent on the pol III transcription machinery as its levels were 7-fold reduced in the pol III transcription-defective brf1-II.6 mutant strain (11) (cf. lanes 3 and 4 with lanes 1 and 2).


Figure 2
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  		FIGURE 2.
pol III-dependent production of ZOD1 RNA in vivo. A, total RNA extracted from YPH500 strain, either untransformed (lane 1) or transformed with the empty YEp352 vector (lane 2) or the YEp352-ZOD1 plasmid (lane 3), was gel-fractionated and probed with a radiolabeled oligonucleotide complementary to the ZOD1 transcript. The migration position of ZOD1 RNA is indicated on the left. The same blot was hybridized with a probe specific for tRNAAla(AGC), used as an internal standard. ZOD1 RNA produced in vitro with the pol III reconstituted system was loaded in lane 4 as a control. B, total RNA extracted from either wild type or brf1-II.6 strains transformed with the YEp352-ZOD1 plasmid, prior to (lanes 1 and 3) or after (lanes 2 and 4) a temperature shift (6 h at 37 °C), was gel-fractionated and probed with a radiolabeled oligonucleotide complementary to the ZOD1 transcript. The same blot was hybridized with a probe specific for U3 RNA as an internal standard. The migration position of ZOD1 RNA is indicated on the left. The asterisk indicates a longer ZOD1-related transcript likely deriving from pol III readthrough of the internal run of T6 and termination at a run of T5 ~35 bp downstream (see Fig. 1A).

 
Nucleosome Depletion Strongly Activates ZOD1 and ETC5 Expression—Class III genes are generally characterized by extremely high transcription rates (24, 25). We considered chromatin-mediated repression as a possible reason for the unusually low levels of in vivo ZOD1 expression. To test this hypothesis, we used a yeast strain (UKY403) that survives with a single histone H4 gene under the control of the GAL1 promoter. Shifting the UKY403 strain to a glucose-containing medium blocks histone H4 gene expression, thus causing a global nucleosome loss and consequent growth arrest. The same shift is without effect on the isogenic MHY308 control strain, in which the histone H4 gene is under the control of its own promoter (14, 15). As shown in Fig. 3A, upon histone H4 depletion, the 55-nucleotide ZOD1 RNA became detectable in UKY403 even in the absence of ZOD1 extra copies (lane 2). The low levels of ZOD1 RNA observed in the presence of extra gene copies (lane 6) were increased 10-fold upon histone H4 depletion in UKY403 (lane 7), whereas they were unchanged in the MHY308 control strain (lane 9). Note that no activation was observed for the tRNAAla(AGC) gene family upon nucleosome depletion (Fig. 3A).4 The levels of other pol III-synthesized RNAs, such as SNR52 and SCR1 transcripts, were unaffected by nucleosome depletion (data not shown and Ref. 3), in agreement with an early study showing that there is no global change in pol III transcription upon nucleosome depletion (15). We also analyzed the expression profile, before and after nucleosome loss, of another locus associated to incomplete pol III machinery, ETC5 (9). This locus has previously been called RNA170 because it encodes a pol III transcript of ~170 nucleotides (4). As shown in Fig. 3B, nucleosome depletion produced a 15-fold increase in the levels of RNA170 transcript (cf. lanes 2 and 3).

Chromatin Structure of the ZOD1 Locus—Nucleosome organization of the ZOD1 locus under standard conditions or after histone H4 depletion was analyzed by MNase digestion using the UKY403 and MHY308 strains. As shown in Fig. 4, low resolution chromatin analysis of both strains grown in galactose-containing medium (lanes 2 and 5) showed an organized nucleosomal ladder starting immediately downstream of the ZOD1 transcription unit that appears extensively protected from MNase digestion, possibly due to the presence of a nucleosome or of bound transcription factors. This MNase digestion pattern did not change upon the shift of MHY308 to glucose-containing medium (lanes 3 and 4). In contrast, shifting to glucose, the UKY403 strain led, after 6 h, to a dramatic increase in MNase sensitivity in the entire region, suggesting a diffuse loss of chromatin organization (Fig. 4, cf. lanes 6 and 7 with lane 5).


Figure 3
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  		FIGURE 3.
Activation of ZOD1 and ETC5 expression in vivo upon nucleosome depletion. A, total RNA extracted before (lanes 1, 3, 6, and 8) and after (lanes 2, 4, 7, and 9) glucose shift either from untransformed UKY403 (lanes 1 and 2) and MHY308 (lanes 3 and 4) or from the same strains transformed with the YEp352-ZOD1 plasmid (lanes 6-9) was gel-fractionated and probed with a radiolabeled oligonucleotide complementary to the ZOD1 transcript. The same blot was hybridized with a probe specific for tRNAAla(AGC), used as an internal standard. Total RNA extracted from the YPH500 strain grown in glucose-containing medium was loaded on lane 5. B, total RNA extracted before (lanes 2 and 4) or after (lanes 3 and 5) glucose shift from either UKY403 (lanes 2 and 3) or MHY308 (lanes 4 and 5) strains was gel-fractionated and probed with an ETC5-specific probe. The migration position of the ETC5 transcript and of U3 RNA, used as an internal standard, are indicated on the right. Total RNA extracted from the YPH500 strain grown in glucose-containing medium was loaded on lane 1.

 


Figure 4
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  		FIGURE 4.
Micrococcal nuclease mapping of chromatin structure at the ZOD1 locus. Chromatin from MHY308 (lanes 1-4) or UKY403 (lanes 5-7) cells, grown in galactose (lanes 2 and 5) or shifted to glucose-containing medium for 6 h (lanes 3, 4, 6, and 7), was digested with MNase (for lanes 4 and 7, a 2-fold higher concentration was used with respect to the other lanes). Lane 8, digestion of deproteinized DNA. Lane 1, no digestion. The migration of DNA size markers run in parallel is shown on the left (M). Nucleosome positioning, under conditions of unperturbed chromatin structure (observed in lanes 2-5), is schematically illustrated on the left (positioned nucleosomes are indicated as orange ovals). Crescents indicate the putative position of components of the transcription machinery. In the scheme on the right, disorganized nucleosomes are indicated by dotted ovals. Black arrows indicate the positions of primers used to prepare the Southern hybridization probe. The position of the ZOD1 transcription unit is also indicated on the right.

 
The correlation between transcriptional derepression and nucleosome loss at the ZOD1 locus was investigated in more detail by doing time course analyses of both phenomena. Fig. 5A, lanes 1-17, shows an ethidium bromide staining of MNase-treated bulk chromatin purified from UKY403 cells grown in galactose (lanes 1-5) or incubated in glucose for 60 (lanes 6-9), 120 (lanes 10-13), or 240 min (lanes 14-17). A nucleosomal ladder is evident in the samples from cells grown in galactose. Chromatin from cells shifted 60 or 120 min in glucose maintains a general nucleosomal organization, although the ladder appears slightly smeared, suggesting a partial loss of nucleosomes in several defined genomic loci due to histone H4 depletion (14). After 240 min in glucose, chromatin does not show any sign of nucleosomal organization (lanes 14-17). Since under these conditions, UKY403 cells appear more resistant to MNase treatment, we repeated the treatment on both UKY403 and MHY308 cells, after a 240-min glucose shift, in the presence of increased MNase concentrations (Fig. 5A, lanes 18-29). The MNase digestion profile of bulk chromatin from MHY308 control cells produced a well detectable nucleosomal ladder (lanes 21-23), whereas no nucleosomal ladder was observed in the case of UKY403 cells (cf. lanes 28-29 with lanes 22-23). We conclude that chromatin from UKY403 cells shifted to glucose starts to show a partial disorganization after 1 h and appears to be seriously nucleosome-depleted only 240 min after the glucose shift. This result is coherent with the previously observed cell cycle block at 4 h from shift (15). We next analyzed global changes in chromatin organization at the ZOD1 locus (Fig. 5B). The same gels shown in panel A (lanes 1-17) were blotted and Southern-hybridized with a ZOD1-specific probe. Although cells grown in galactose (Fig. 5B, lanes 1-5) show a perfectly normal nucleosomal ladder, chromatin from cells grown 60, 120, or 240 min in glucose (lanes 6-9, 10-13, and 14-17, respectively) produced only a smeared profile, which is diagnostic of complete chromatin disorganization. From this evidence, we can conclude that the ZOD1 locus chromatin is especially sensitive to histone H4 depletion. To see whether there is a correlation between chromatin disorganization and transcriptional derepression at the ZOD1 locus, we analyzed by Northern blot the time course of ZOD1 transcript accumulation following glucose shift of UKY403 cells. As shown in Fig. 5C, the levels of ZOD1 RNA were already increased significantly 1 h after glucose shift, and a 30-fold activation was observed after 4 h (Fig. 5C, cf. lanes 1 and 6; see also the plot on the right). Note that, at variance with the experiment in Fig. 3A (lane 1), very low levels of ZOD1 RNA could be detected in this experiment even in the absence of nucleosome depletion. Altogether, the data in Fig. 5 show that pol III transcription derepression at the ZOD1 locus parallels, without any appreciable lag, chromatin disorganization deriving from reduced nucleosome density.


Figure 5
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  		FIGURE 5.
Time course analysis of nucleosome loss and transcriptional derepression at the ZOD1 locus. A, ethidium bromide staining of MNase-treated bulk chromatin purified from UKY403 or MHY308 cells grown as indicated and treated with the indicated units of MNase (MN). M, molecular weight markers. B, Southern blots of the gels shown in panel A (lanes 1-17) using a radiolabeled double-stranded DNA probe encompassing the entire ZOD1 locus (see "Experimental Procedures"). C, total RNA extracted before (lane 1) and at different times after (lanes 2-7) glucose shift from untransformed UKY403 cells was gel-fractionated and probed with a radiolabeled oligonucleotide complementary to the ZOD1 transcript. The same blot was hybridized with a probe specific for the U3 snRNA, used as an internal standard. The plot on the right, derived from phosphorimaging device quantification of the same experiment, reports the ratios between ZOD1 transcripts observed at the different times after glucose shift and ZOD1 transcripts observed in galactose-grown cells (lane 1). Note that very low levels of ZOD1 RNA could be detected in this experiment, at variance with the experiment in Fig. 3A (lane 1).

 
Occupancy of Unconventional pol III Loci before and after Nucleosome Depletion—To gain insight into the mechanism of ZOD1 and ETC5 derepression observed upon nucleosome depletion, the BDP1, TFC4, and RET1 ORFs, coding for subunits of TFIIIB, TFIIIC, and pol III, respectively, were C-terminally fused with the 13-Myc epitope coding sequence in the UKY403 genetic background, and the association of the transcription machinery to the ZOD1 and ETC5 loci before and after nucleosome loss was analyzed by chromatin immunoprecipitation. As shown in Fig. 6A (upper plot), under unperturbed chromatin conditions, the relative TFIIIB and pol III occupancies at ZOD1 were lower than TFIIIC occupancy, in agreement with previous analysis (9). In contrast, relative TFIIIC and pol III occupancies at the ETC5 locus were found to be similar. Fig. 6A further shows that pol III association to ZOD1 and ETC5, relative to pol III-tDNAMet association, is about the same. This contrasts with the observation that the steady state levels of ZOD1 and ETC5 RNAs are very different (ETC5 RNA is much more abundant; Fig. 3). Such a discrepancy might be explained by a much lower stability of ZOD1 RNA with respect to ETC5 RNA. Upon nucleosome depletion (Fig. 6A, lower plot), the only appreciable changes were a 1.9-fold increase of relative pol III occupancy at ZOD1 and a 1.5-2-fold increase of relative TFIIIC and Bdp1 occupancies (not accompanied by a proportional increase of pol III occupancy) at the ETC5 locus. Note that absolute occupancies at the reference tM(CAU)D locus, as well as at several other tested tDNA loci, did not change significantly upon glucose shift (data not shown). The dramatic increase in ZOD1 and ETC5 expression upon nucleosome depletion is thus not paralleled by a proportional increase in occupancy by the transcription machinery. The observation that pol III is already present at the ZOD1 locus before nucleosome depletion is in agreement with the results in Fig. 5, showing that transcriptional derepression takes place without any significant lag with respect to chromatin disorganization.


Figure 6
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  		FIGURE 6.
Occupancy at unconventional pol III loci before and after nucleosome depletion. A, cross-linked chromatin from UKY403-derived strains expressing the indicated tagged proteins was immunoprecipitated with anti-Myc antibody and analyzed by quantitative real-time PCR. Upper and lower plots report, on the y axis, the occupancy values at the four loci indicated on the x axis, measured before and 6 h after the glucose shift, respectively. All values are shown relative to the occupancies at tM (CAU)D, which were set to 1. The GAL1 locus was used as a negative control. The error bars indicate the standard deviation of three independent experiments. Gray, white, and checked bars refer to Bdp1p, Ret1p, and Tfc4p occupancy, respectively. B, the association of components of the pol III machinery to other unconventional pol III loci (reported on the x axis) was analyzed as described for panel A.

 
Genome-wide studies of pol III machinery localization have identified several other loci, in addition to ZOD1 and ETC5, that are associated to one or more components of the pol III machinery. In particular, seven B box-containing ETC loci were identified by Moqtaderi and Struhl (9) in addition to ETC5, whereas Roberts et al. (8) reported a locus, referred to as iYGR033c, that is occupied by the whole pol III machinery. Both iYGR033c and another intergenic region, iYMR103c, were also identified as pol III machinery-associated in the genome-wide study by Harismendy et al. (7).5 Our attempts to detect in vitro and in vivo transcription products from these loci were unsuccessful (data not shown). We thus decided to look in more detail at their occupancy by components of the pol III machinery in the presence or absence of chromatin perturbation. As shown in Fig. 6B, ETC7 and iYMR103c were not found associated to any of the pol III machinery components. ETC2 and ETC6, as expected, were found to be appreciably associated to TFIIIC but not to TFIIIB and pol III, whereas iYGR033c was occupied by relatively low levels of pol III with respect to TFIIIC and TFIIIB. Upon nucleosome depletion, no significant changes were observed on any one of the tested loci in terms of TFIIIC, TFIIIB, and pol III occupancy (Fig. 6B), and no in vivo transcriptional derepression of ETC2, ETC6, and iYGR033c could be observed by Northern analysis (data not shown).


Figure 7
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  		FIGURE 7.
Comparison and evolution of the ZOD1 and iYGR033c loci in hemiascomycetes. A, sequence alignment of the ZOD1 locus with syntenic regions of related yeast genomes. Numbers in parentheses indicate the position with respect to the start codon of UFO1 (S. cerevisiae) or UFO1 homologous (other yeasts). Identical nucleotide residues of the multiple alignment are shaded in black. Conserved nucleotide residues are shaded in gray. The sequence corresponding to a tRNAIle(AAT) in K. lactis and A. gossypii is framed with a dotted box. The relative orientation of UFO1 ORF and tDNAIle-derived element is represented on the top; the indicated distance is the one found in S. cerevisiae. B, sequence alignment of the iYGR033c locus with syntenic regions of related yeast genomes. Numbers in parentheses indicate the position with respect to the start codon of TIM21(YGR033c)(S. cerevisiae) or TIM21 homologous (other yeasts). The relative orientation of TIM21 ORF and tDNAArg-derived element is represented on the top; the indicated distance is the one found in S. cerevisiae. C, approximate phylogenetic relationships among hemiascomycetes, showing the origin of ZOD1 and iYGR033c loci. The tree (not drawn to scale) is according to a multigene phylogeny (39). The supposed inactivation event of the tDNA ancestor is indicated by a filled arrowhead for ZOD1 and by an open arrowhead for iYGR033c; species branching after that point do not encode for a functional tRNA, whereas preserving a box B element.

 
The TFIIIC Binding Regions of ZOD1 and iYGR033c Originated from tRNA Genes—Having precisely defined the ZOD1 transcription unit, we looked in more detail at its conservation in the genomes of the hemiascomycetes fungi. As demonstrated by the alignment in Fig. 7A, the ZOD1 box B has been strictly conserved, upstream of the UFO1 ORF, from the ancient hemiascomycete Ashbia gossypii up to S. cerevisiae, whereas the regions immediately surrounding the box B element, including box A, have generally diverged among the different yeast species. We noticed, however, an extended region of high sequence homology between Kluyveromyces lactis and A. gossypii. Strikingly, this region corresponds to the transcribed sequence of a tDNAIle(AAT). This tRNAIle gene copy has been lost in the more recent yeast species but, significantly, its box B has been rigorously preserved in both sequence and genomic location. Phylogenetic analysis revealed a similar origin for the B box-containing region of iYGR033c that was found to originate from a tRNAArg(CCG) gene (Fig. 7B). This tDNA was lost more recently than the ZOD1-associated tDNA as it is still present in Candida glabrata (Fig. 7C). Again, the box B has been strictly conserved in both sequence and genomic location. None of the ETC loci was found to originate from tDNAs. In particular, we observed that the ETC5 locus originated much more recently than ZOD1 and iYGR033c as it could not be detected in yeasts more ancient than Saccharomyces bayanus.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that the S. cerevisiae ZOD1 locus encodes a novel pol III-transcribed, non coding RNA whose expression is subject to chromatin repression. Chromatin-mediated repression was similarly found to operate at the ETC5/RNA170 locus. Our data suggest that derepression of ZOD1 and ETC5 transcription upon nucleosome depletion involves activation of poised RNA polymerase III, rather than enhanced recruitment of transcription components, thus providing the first example of post-recruitment mechanism of pol III activation. By comparative genomic analysis, we have traced the evolutionary history of ZOD1 back to a tDNAIle(AAT) ancestor that is still present and syntenic in K. lactis and A. gossypii. We have further shown that iYGR033c, another locus associated in vivo with components of the pol III machinery, also derives from a tDNAArg ancestor. ZOD1 and iYGR033c thus represent the first examples of tDNA-derived genetic elements in yeast. Such an origin is reminiscent of that of higher eukaryote SINEs, although ZOD1 and iYGR033c evolution from their tDNA ancestors is unlikely to have occurred through retrotransposition such as in the case of SINEs (see Ref. 5). The fact that the tDNA box B has been selectively maintained, whereas RNA coding features have diverged, excludes that ZOD1 and iYGR033c are tDNA-derived pseudogenes and rather suggests a physiologically relevant function of their transcription factorinteracting regions. Such a function is likely to require the very act of transcription (at least in the case of ZOD1) or transcription complex assembly, rather than RNA products. The ZOD1 function, however, remains unknown, as the conditions under which ZOD1 transcription might be induced in yeast cells are unknown. Yeast strains that either overexpress or are deleted for ZOD1 do not show obvious phenotypes under a variety of conditions (data not shown). Both the ZOD1 and the ETC5 transcription units lie close to the 5'-end of pol II-transcribed genes (UFO1 and ERG2, respectively). The process of ZOD1 and ETC5 transcription by pol III might positionally influence the expression of neighboring genes under some conditions, as recently reported for SER3 regulation by an upstream, non-coding transcription unit (26) and as it is known to occur in some cases of tDNA-mediated transcriptional regulation (27-30). In this respect, it is worth noting that UFO1 mRNA levels are ~2-fold increased, when compared with wild type, in two different TFIIIC mutants (12). On the other hand, UFO1 expression was not found to be appreciably changed in a strain in which the ZOD1 locus had been replaced by a kanMX cassette.6 Moreover, no UFO1 expression changes were observed in a genome-wide analysis of the yeast transcriptome in histone-depleted UKY403 strain (31).7 It is possible that ZOD1-dependent modulation of neighboring genes only occurs under particular conditions. More generally, chromatin-sensitive, pol III-associated sites such as ZOD1 and ETC5 might be important for genome organization and dynamics, as exemplified by the involvement of class III genes in Ty retrotransposon integration (32, 33) and by the global chromatin marking activity recently attributed to tDNAs (34). This hypothesis receives strong support by a very recently published study showing that, in Schizosaccharomyces pombe, TFIIIC localizes without pol III at many B box-containing sites that act as boundary elements in genome organization (35). Many of these sites in fission yeast genome are located between divergent promoters, as is the case for ZOD1, iYGR033c, ETC5, and several other ETC loci in the S. cerevisiae genome (9). How the presence of active pol III at some of these loci might contribute to boundary activity is not known. A previous study has shown that the heterochromatin barrier activity of the HMR tDNA mainly requires efficient TFIIIC and TFIIIB assembly, but an involvement of ongoing pol III transcription in the barrier effect has not been excluded (28). Whatever the actual stimulus for and physiological significance of ZOD1 transcription might be, chromatin reorganization is likely to play a role in some step of the process. Chromatin-mediated repression of ZOD1 has unusual features when compared with known cases of class III gene repression by chromatin. TFIIIC has previously been shown to compete with repressive chromatin for binding to class III gene loci, thus leading to the notion of a reciprocal interference between chromatin components and transcription complexes at the level of recruitment (3, 19, 36). In contrast, since there are no major changes in ZOD1 and ETC5 occupancy before and after nucleosome depletion, we suggest that derepression of transcription at these loci is related to activation of pol III already present at the units rather than to enhancement of recruitment of the components needed for transcription to occur. Chromatin thus interferes with ZOD1 and ETC5 transcription by affecting a post-recruitment step in the pol III transcription cycle. Such a step might be the transition of the recruited polymerase to an elongation-competent state or its ability to reinitiate efficiently on the same template through the facilitated recycling pathway (24, 25). The maintenance of poised RNA polymerase molecules at repressed genes is classically exemplified by the case of heat-shock promoter (37), and it has been recently observed in a genome-wide analysis of pol II location during stationary phase in S. cerevisiae (38). Our observation that pol III can be maintained in a poised state at its promoters by a chromatin-mediated mechanism further suggests that post-recruitment mechanisms of transcriptional regulation might be more widespread that commonly thought.

By exploiting regulated nucleosome depletion (14), we have been able to unmask an otherwise silent transcription unit. We speculate that S. cerevisiae and other eukaryotic genomes might contain, within the so-called intergenic regions, many of such chromatin-repressed, cryptic transcription sites that could be revealed using the same strategy.


   FOOTNOTES
 
* This work was supported by the Human Frontier Science Program Grant RGY0011/2002-C (to G. D.) and by a grant from the Italian Ministry of Education, University and Research (PRIN and FIRB programs). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of an EMBO Short-term Fellowship. Back

2 To whom correspondence should be addressed: Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Parco Area delle Scienze 23/A, 43100 Parma, Italy. Tel.: 39-0521-905649; Fax: 39-0521-905151; E-mail: giorgio.dieci{at}unipr.it.

3 The abbreviations used are: pol, RNA polymerase; TF, transcription factor; MNase, micrococcal nuclease; ORF, open reading frame; SINE, short interspersed elements; fw, forward; rev, reverse. Back

4 Although the tRNAAla(AGC) detected in Fig. 3A is the mature tRNA form and not the primary transcript, transcriptional derepression of the corresponding tDNAs would have resulted in increased levels of the mature tRNA. Back

5 O. Harismendy, unpublished data. Back

6 E. Guffanti and G. Dieci, unpublished observations. Back

7 We note, however, that the expression of ERG2, an ORF adjacent to ETC5, was found to be 2-fold down-regulated upon histone depletion in the same genome-wide study. Back


   ACKNOWLEDGMENTS
 
We are grateful to Michael Grunstein (UCLA) for yeast strains, to Olivier Lefebvre (CEA-Saclay), Roberto Ferrari (University of Parma), and Elisabetta Soragni (Scripps Research Institute) for helpful suggestions, and to Alessandra Parente (University of Rome La Sapienza) for help with chromatin analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lee, J. Y., Evans, C. F., and Engelke, D. R. (1991) Proc. Natl. Acad. Sci. U. S. A., 88, 6986-6990[Abstract/Free Full Text]
  2. Brow, D. A., and Guthrie, C. (1988) Nature 334, 213-218[CrossRef][Medline] [Order article via Infotrieve]
  3. Dieci, G., Giuliodori, S., Catellani, M., Percudani, R., and Ottonello, S. (2002) J. Biol. Chem. 277, 6903-6914[Abstract/Free Full Text]
  4. Olivas, W. M., Muhlrad, D., and Parker, R. (1997) Nucleic Acids Res. 25, 4619-4625[Abstract/Free Full Text]
  5. White, R. J. (2002) RNA Polymerase III Transcription, Third Ed., Landes Bioscience, Austin, TX
  6. Allen, T. A., Von Kaenel, S., Goodrich, J. A., and Kugel, J. F. (2004) Nat. Struct. Mol. Biol. 11, 816-821[CrossRef][Medline] [Order article via Infotrieve]
  7. Harismendy, O., Gendrel, C. G., Soularue, P., Gidrol, X., Sentenac, A., Werner, M., and Lefebvre, O. (2003) EMBO J., 22, 4738-4747[CrossRef][Medline] [Order article via Infotrieve]
  8. Roberts, D. N., Stewart, A. J., Huff, J. T., and Cairns, B. R. (2003) Proc. Natl. Acad. Sci. U. S. A., 100, 14695-14700[Abstract/Free Full Text]
  9. Moqtaderi, Z., and Struhl, K. (2004) Mol. Cell. Biol. 24, 4118-4127[Abstract/Free Full Text]
  10. Hill, J. E., Myers, A. M., Koerner, T. J., and Tzagoloff, A. (1986) Yeast 2, 163-167[CrossRef][Medline] [Order article via Infotrieve]
  11. Andrau, J. C., Sentenac, A., and Werner, M. (1999) J. Mol. Biol. 288, 511-520[CrossRef][Medline] [Order article via Infotrieve]
  12. Conesa, C., Ruotolo, R., Soularue, P., Simms, T. A., Donze, D., Sentenac, A., and Dieci, G. (2005) Mol. Cell. Biol. 25, 8631-8642[Abstract/Free Full Text]
  13. Longtine, M. S., McKenzie, A., III, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998) Yeast 14, 953-961[CrossRef][Medline] [Order article via Infotrieve]
  14. Han, M., and Grunstein, M. (1988) Cell 55, 1137-1145[CrossRef][Medline] [Order article via Infotrieve]
  15. Kim, U. J., Han, M., Kayne, P., and Grunstein, M. (1988) EMBO J. 7, 2211-2219[Medline] [Order article via Infotrieve]
  16. Dieci, G., Percudani, R., Giuliodori, S., Bottarelli, L., and Ottonello, S. (2000) J. Mol. Biol. 299, 601-613[CrossRef][Medline] [Order article via Infotrieve]
  17. Huet, J., Manaud, N., Dieci, G., Peyroche, G., Conesa, C., Lefebvre, O., Ruet, A., Riva, M., and Sentenac, A. (1996) Methods Enzymol. 273, 249-267[Medline] [Order article via Infotrieve]
  18. Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucleic Acids Res. 18, 3091-3092[Free Full Text]
  19. Marsolier, M. C., Tanaka, S., Livingstone-Zatchej, M., Grunstein, M., Thoma, F., and Sentenac, A. (1995) Genes Dev. 9, 410-422[Abstract/Free Full Text]
  20. Kuras, L., and Struhl, K. (1999) Nature 399, 609-613[CrossRef][Medline] [Order article via Infotrieve]
  21. Kuras, L. (2004) Methods Mol. Biol. 284, 147-162[Medline] [Order article via Infotrieve]
  22. Brow, D. A., and Guthrie, C. (1990) Genes Dev. 4, 1345-1356[Abstract/Free Full Text]
  23. Braglia, P., Percudani, R., and Dieci, G. (2005) J. Biol. Chem. 280, 19551-19562[Abstract/Free Full Text]
  24. Dieci, G., and Sentenac, A. (2003) Trends. Biochem. Sci. 28, 202-209[CrossRef][Medline] [Order article via Infotrieve]
  25. Ferrari, R., Rivetti, C., Acker, J., and Dieci, G. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13442-13447[Abstract/Free Full Text]
  26. Martens, J. A., Laprade, L., and Winston, F. (2004) Nature 429, 571-574[CrossRef][Medline] [Order article via Infotrieve]
  27. Hull, M. W., Erickson, J., Johnston, M., and Engelke, D. R. (1994) Mol. Cell. Biol. 14, 1266-1277[Abstract/Free Full Text]
  28. Donze, D., and Kamakaka, R. T. (2001) EMBO J. 20, 520-531[CrossRef][Medline] [Order article via Infotrieve]
  29. Bolton, E. C., and Boeke, J. D. (2003) Genome Res. 13, 254-263[Abstract/Free Full Text]
  30. Simms, T. A., Miller, E. C., Buisson, N. P., Jambunathan, N., and Donze, D. (2004) Nucleic Acids Res. 32, 5206-5213[Abstract/Free Full Text]
  31. Wyrick, J. J., Holstege, F. C., Jennings, E. G., Causton, H. C., Shore, D., Grunstein, M., Lander, E. S., and Young, R. A. (1999) Nature 402, 418-421[CrossRef][Medline] [Order article via Infotrieve]
  32. Chalker, D. L., and Sandmeyer, S. B. (1992) Genes Dev. 6, 117-128[Abstract/Free Full Text]
  33. Devine, S. E., and Boeke, J. D. (1996) Genes Dev. 10, 620-633[Abstract/Free Full Text]
  34. Bachman, N., Gelbart, M. E., Tsukiyama, T., and Boeke, J. D. (2005) Genes Dev. 19, 955-964[Abstract/Free Full Text]
  35. Noma, K., Cam, H. P., Maraia, R. J., and Grewal, S. I. (2006) Cell 125, 859-872[CrossRef][Medline] [Order article via Infotrieve]
  36. Burnol, A. F., Margottin, F., Huet, J., Almouzni, G., Prioleau, M. N., Mechali, M., and Sentenac, A. (1993) Nature 362, 475-477[CrossRef][Medline] [Order article via Infotrieve]
  37. Lis, J., and Wu, C. (1993) Cell 74, 1-4[CrossRef][Medline] [Order article via Infotrieve]
  38. Radonjic, M., Andrau, J. C., Lijnzaad, P., Kemmeren, P., Kockelkorn, T. T., van Leenen, D., van Berkum, N. L., and Holstege, F. C. (2005) Mol. Cell 18, 171-183[CrossRef][Medline] [Order article via Infotrieve]
  39. Kurtzman, C. P., and Robnett, C. J. (2003) FEMS Yeast Res. 3, 417-432[CrossRef][Medline] [Order article via Infotrieve]

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