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 Previous Article

Molecular and Cellular Biology, September 2008, p. 5543-5554, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00416-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Remodeling Yeast Gene Transcription by Activating the Ty1 Long Terminal Repeat Retrotransposon under Severe Adenine Deficiency{triangledown}

Géraldine Servant, Carole Pennetier,{dagger} and Pascale Lesage*

Université Paris Diderot-Paris 7, CNRS, UPR 9073, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, F-75005 Paris, France

Received 12 March 2008/ Returned for modification 8 May 2008/ Accepted 20 June 2008


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ABSTRACT
 
The Ty1 long terminal repeat (LTR) retrotransposon of Saccharomyces cerevisiae is a powerful model to understand the activation of transposable elements by stress and their impact on genome expression. We previously discovered that Ty1 transcription is activated under conditions of severe adenine starvation. The mechanism of activation is independent of the Bas1 transcriptional activator of the de novo AMP biosynthesis pathway and probably involves chromatin remodeling at the Ty1 promoter. Here, we show that the 5' LTR has a weak transcriptional activity and is sufficient for the activation by severe adenine starvation. Furthermore, we demonstrate that Ty1 insertions that bring Ty1 promoter sequences into the vicinity of a reporter gene confer adenine starvation regulation on it. We provide evidence that similar coactivation of genes adjacent to Ty1 sequences occurs naturally in the yeast genome, indicating that Ty1 insertions can mediate transcriptional control of yeast gene expression under conditions of severe adenine starvation. Finally, the transcription pattern of genes adjacent to Ty1 insertions suggests that severe adenine starvation facilitates the initiation of transcription at alternative sites, partly located in the 5' LTR. We propose that Ty1-driven transcription of coding and noncoding sequences could regulate yeast gene expression in response to stress.


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INTRODUCTION
 
Long terminal repeat (LTR) retrotransposons are widespread transposable elements in eukaryotes. They resemble retroviruses in both their structure and their mode of replication, but they are not infectious (8). Their structure consists of two direct LTRs almost identical in sequence and two open reading frames (ORFs), analogs of the retroviral gag and pol genes. Retrotransposons propagate via reverse transcription (RT) of their mRNA and integration of the newly synthesized cDNA onto the chromosomes. In addition to full-length elements, genomes are scattered with solo LTRs, which originate from recombination between the two LTRs of an ancestral element. Full-length elements and solo LTRs constitute a significant percentage of genomes: 3% of the yeast Saccharomyces cerevisiae genome, up to 50% of the maize genome, 10% of the mouse genome, and 8% of the human genome (17, 23, 47, 68).

The mobility and the abundance of LTR retrotransposons can be harmful to the organisms. There is also increasing evidence that their recruitment as alternative exons or promoters has been a driving force in genome evolution in many organisms (30, 38, 46, 66). LTR sequences confer tissue-specific transcription of the neuronal apoptosis inhibitory gene (NAIP) family in humans, rats, and mice (55). The synchronous expression of a subset of genes expressed in oocytes and preimplantation embryos during mouse development depends on MalR LTR retrotransposons, which provide alternative promoters and 5' exons to these genes (51). In the yeast Schizosaccharomyces pombe, the Tf2 LTR functions as an oxygen-dependent promoter and can also direct transcription of adjacent coding and noncoding sequences when oxygen is limiting (59). Although LTR retrotransposons are generally repressed at both transcriptional and posttranscriptional levels by a wide variety of mechanisms of gene silencing or by host factors that restrict different steps of their life cycle (32, 44, 60), they can be activated by stress and environmental challenges in several organisms (24, 40, 62).

The yeast Ty1 element is an interesting model to understand the activation of LTR retrotransposons by stress and the impact of such activation on gene expression. Ty1 is the most abundant LTR retrotransposon in S. cerevisiae, with 32 full-length elements and 185 solo LTRs in the sequenced genome of the S288C strain (34). These insertions are mainly located in a 1-kb window upstream of genes transcribed by RNA polymerase III (Pol III), where they have no or minor effects on tRNA gene expression (10, 34). Rare insertions in the promoter or 5' noncoding regions of Pol II genes have also been recovered by genetic screens (for review, see references 7 and 40). These insertions were selected by their ability to inactivate or activate gene expression, indicating that Ty1 insertions are not neutral to Pol II gene expression. Solo LTR insertions inactivate gene expression probably via promoter competition (69). Full-length insertions that inactivate gene expression are mainly in the same transcriptional orientation as the target gene and separate the structural gene from its promoter, while activating insertions are in the divergent transcriptional orientation and bring Ty1 promoter sequences in proximity to the 5' end of the target gene. The consequence is that the transcription of the adjacent gene depends on Ty1 promoter sequences. A well-documented example of this effect are Ty1 insertions upstream of the his3{Delta}4 promoterless allele that restore histidine prototrophy to yeast cells (57).

Different kinds of environmental stress, such as ionizing radiation, DNA damage, nitrogen starvation, and severe adenine starvation, activate Ty1 transcription and mobility (11, 45, 49, 54, 56, 61, 64). Studies to understand the impact of Ty1 insertions on adjacent gene transcription and to decipher the mechanisms of Ty1 activation by stress have provided several clues on Ty1 promoter structure and on the transcription factors involved in Ty1 transcription. The full Ty1 promoter extends over 1 kb both upstream and downstream of two TATA boxes and includes the 5' LTR and part of the TYA (gag) ORF. Several transcription factors bind to the Ty1 promoter and regulate its transcription (19, 20, 26, 27, 36, 42, 48, 49, 65) (Fig. 1). The Swi/Snf, SAGA, and ISWI complexes that act on chromatin structures also regulate Ty1 transcription (14, 25, 33, 53).


Figure 1
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  		FIG. 1. (A) Ty1 structure and promoter. Ty1 transcription regulatory sequences are located within the first kilobase of the retrotransposon. Ty1 transcription starts in the 5' LTR (symbolized by an arrow pointing to the right) and terminates in the 3' LTR (symbolized by a circle on top of a vertical line). A, B, C, D, and E indicate Gcn4 binding sites. FRE and MIR binding sites are also shown. The ATG initiation codon is located in the LTR, upstream of Gcn4E binding site. Gcn4, Gcr1, Ste12, Tec1, and Tea1 are activators of Ty1 transcription, while Mot3 is a repressor. Although the MIR region contributes positively to Ty1 transcription, the exact roles of Mcm1 and Rap1 in Ty1 transcription have not been clearly defined. The presence of the five Gcn4 binding sites is a signature of highly expressed Ty1 elements. (B) Multiple alignment of the 5' LTR sequences of Ty1-PR1, Ty-DR3, and Ty1-PR1. Ty1-PR1 is a highly expressed element (H) and contains five Gcn4 binding sites in its LTR (in boldface). Ty1-DR3 and Ty1-ML1 are weakly expressed Ty1 elements (W). Ty1-DR3 lacks the Gcn4E binding site, while Ty1-ML1 lacks the full set of Gcn4 binding sites and TATA box 1.

Recently, we reported that Ty1 transcription is activated under conditions that strongly affect cellular adenine nucleotide synthesis: i.e., severe adenine starvation. The activation is followed by a proportional increase in retrotransposition events (64). The activation was observed in adenine-deprived cells, lacking the Bas1 transcriptional activator responsible for the induction of the ADE genes of the de novo AMP biosynthesis when adenine is limiting. This observation suggested a new mode of regulation of Ty1 transcription by adenine nucleotide levels. Since adenylic nucleotide availability probably reflects the energy status of the cell, severe adenine starvation could mimic conditions that impair the energetic metabolism in the yeast's natural environment.

Ty1 elements can be divided into two basic classes according to their level of expression: the weakly transcribed elements and the highly transcribed elements. Based on genetic data, it was proposed that Ty1 transcription is repressed by chromatin structure that is alleviated at the highly expressed elements by chromatin remodeling complexes (48). The activation by severe adenine starvation occurs mainly on weakly transcribed Ty1 elements. The activation is abolished in an hta1-htb1{Delta} mutant that opens chromatin structure at these elements by depleting histones H2A and H2B or in an snf2{Delta} mutant that inactivates Swi/Snf, suggesting that the activation involves chromatin remodeling at the Ty1 promoter (64). To obtain more information about the mechanism of activation of Ty1 by severe adenine starvation, we have characterized the regions of the Ty1 promoter important for the activation. Although previous work suggested that important Ty1 regulatory sequences reside mostly in Ty1 internal sequences (21, 72), we demonstrate here that the 5' LTR possesses a weak transcriptional activity that is stimulated by severe adenine starvation. By investigating the impact of several Ty1 insertions on the transcription of different reporter genes, we also discovered that severe adenine starvation can modify the transcription pattern of genes located up to 300 bp from Ty1 insertions.


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MATERIALS AND METHODS
 
Yeast strains and plasmids. All strains used in this study are S288C derivatives. FYBL1-23D (MAT{alpha} flo8-1 ura3{Delta}851 trp1{Delta}63 his3{Delta}200), LV426 (FYBL1-23D bas1{Delta}::TRP1) (64), LV69 (FYBL1-23D his3{Delta}4), and LV150 (FYBL1-23D Ty1-his3{Delta}4) (49) have already been described. Null alleles of BAS1 and STE12 were obtained in LV150 by one-step gene replacement using a PCR fragment of the KanMX and TRP1 cassettes flanked with 5' and 3' sequences of BAS1 and STE12, respectively. The resulting strains are LV922 (LV150 bas1{Delta}::KanMX), LV993 (LV150 ste12{Delta}::TRP1), and LV926 (LV150 bas1{Delta}::KanMX, ste12{Delta}::TRP1). The strain LV1107 (FYBL1-23D LTR-his3{Delta}4 bas1{Delta}::KanMX) was obtained in two steps. We first introduced in LV922 the URA3 gene at position 5560 of the Ty1 element, which is adjacent to his3{Delta}4. We then selected uracil auxotrophic cells, on 5-fluorouracil plates (6). We confirmed by PCR (Eppendorf PCR extender system) that these cells lost URA3 and Ty1 internal regions, by homologous recombination between the two LTRs of the Ty1 element, leaving a solo LTR.

Strains containing a TYA-lacZ fusion at the chromosomal locus of a native Ty1 element, with lacZ expressed from all of the Ty1 transcription control sequences, have already been described (48). Briefly, lacZ was fused in frame to TYA at an MfeI restriction site (at coordinate 1569 of Ty1-H3) (5), and followed by the URA3 gene and TYB sequences (coordinates 2171 to 3726 of Ty1-H3). The Munich Information Center for Protein Sequences (MIPS) annotation is used to name Ty1 elements (http://mips.gsf.de/genre/proj/yeast/). Strains containing an LTR-lacZ fusion at Ty1-DR3, Ty1-ML1, and Ty1-PR1 loci were obtained upon transformation of FYBL1-23D with a Ty1up-LTR-lacZ-URA3-"ty1b" DNA fragment. For each Ty1 element, The Ty1up-LTR portion was obtained by high-fidelity PCR amplification (Eppendorf PCR extender system) of a region covering approximately 100 bp upstream of the element and the 5' LTR. The Ty1up sequence allows targeting of the integration to the right Ty1 locus. The "ty1b" sequences come from Ty1-H3 (coordinates 2171 to 3726). LTR sequences were checked for errors introduced during the amplification process. Null alleles of BAS1 and SNF2 were obtained in these strains by one-step gene replacement, using a PCR fragment of the KanMX and TRP1 cassettes flanked with 5' and 3' sequences of BAS1 and SNF2, respectively (Eppendorf PCR extender system). All constructs and gene replacements were checked by PCR analysis. Yeast transformations were performed by the lithium acetate procedure.

To construct pPL143, pPL144, and pPL147 plasmids, which express lacZ from the complete promoters of Ty1-DR3, Ty1-ML1, and Ty1-PR1, respectively (TYA-lacZ fusions), sequences extending from the first nucleotide and the MfeI restriction site of each element (at coordinate 1569 of Ty1-H3) (5) were amplified by high-fidelity PCR (Roche) and cloned into Yep356R (2µm and URA3) (50). A similar strategy was used to obtain pGS7, pGS8, pGS15, which are Yep356 (2µm and URA3) derivatives carrying LTR-lacZ fusions, with 5' LTR sequences of Ty1-DR3, Ty1-PR1, and Ty1-ML1, respectively.

Plasmids pIL10, pIL11, and pIL12 carry Ty1 portions upstream of the UAS-less TDH3-lacZ reporter gene, and pIL5 carries a UAS-less TDH3-lacZ fusion. These plasmids were kindly provided by E. Dubois (37). A PCR-amplified DNA fragment corresponding to Ty1-DR3 5' LTR (Eppendorf PCR extender system) was introduced at the HindIII site of pIL5 to create pGS17.

For all of the constructs, we checked that the amplification process did not introduce detrimental errors into the sequences. Details can be obtained upon request.

Growth conditions. Yeast strains were grown in rich yeast extract-peptone-dextrose, Hartwell's synthetic complete (HC), and synthetic minimum (SDc, which is SD minimal medium containing arginine, isoleucine, tryptophan, leucine and valine) media, all supplemented with 2% glucose (1). Adenine was added to a final concentration of 0.3 mM.

For β-galactosidase assays, Northern blotting, and primer extension analyses, precultures were grown to saturation in liquid HC or SDc medium supplemented with adenine at 30°C and diluted 100-fold in the same medium, in the presence or absence of adenine. Ten-milliliter cultures were grown to mid-log phase between three and five generations at 22°C, which is the permissive temperature for Ty1 retrotransposition, or at 30°C. The media used in each experiment are given in the corresponding figure legends.

Northern blotting and primer extension analysis. Total RNA was extracted by the hot acid phenol procedure as previously described (58). For Northern blotting, 5 to 15 µg of each RNA sample was loaded onto a 1% agarose-1x Tris-borate-EDTA gel. The size-fractionated RNA was transferred to a Hybond-N membrane (Amersham). Transfer and hybridization procedures were performed as recommended by the supplier. ACT1, ESF1, and Ty1 probes were generated by random priming (Roche). The Ty1 probe was derived from a region of Ty1 with no homology to Ty2 (coordinates 3137 to 3682 in Ty1-H3). An anti-HIS3 RNA probe corresponding to HIS3 618 to 213 with a GG sequence at the 5' end was synthesized by T7 RNA polymerase (Promega), yielding uniformly labeled RNA with [{alpha}-32P]UTP as a tracer, from a DNA fragment obtained by PCR amplification of genomic DNA, using the T7 RNA polymerase promoter containing oligonucleotide 5'-TAATACGACTCACTATAGGGCGAGGTGGCTTCTCTTATGG as a forward primer and 5'-GCATTCCGGCTGGTCGCTAATC as a reverse primer.

Primer extension experiments were performed with 0.1 pmol of 5'-labeled (32P) primer and 100 µg of RNA, denatured at 65°C for 15 min in 10 µl (final volume) reverse transcriptase buffer (50 mM Tris-HCl [pH 8.3], 10 mM MgCl2, 80 mM KCl). The denatured mixture was cooled slowly to room temperature for 10 min. A 10.4-µl mix of 2 mM each deoxynucleoside triphosphate (dNTP), 8 mM dithiothreitol, and 8 U avian myeloblastosis virus (Finnzymes) reverse transcriptase in reverse transcriptase buffer was then added. Reaction mixtures were incubated for 60 min at 42°C. RNA templates were degraded with 20 µmol of NaOH. Samples were ethanol precipitated, and the pellets were resuspended in 8 µl 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol. Samples were loaded on 9% sequencing gels. The primers used in this study were O-GS71 (ESF1, coordinates –190 to –211 relative to the initiation codon) and O-GS73 (HIS3, coordinates +102 to +81 relative to the initiation codon). Sequences were performed in parallel, using the same primers (Thermosequenase; USB).

Northern blotting and primer extension analyses were reproduced at least three times. Results were quantified on a Molecular Dynamics PhosphorImager with ImageQuant software.

β-Galactosidase assays. β-Galactosidase assays were performed as already described (49). β-Galactosidase units are expressed in nanomoles of 2-nitrophenyl-β-D-galactopyranoside hydrolyzed per minute per milligram of protein. Values are averages of at least three independent measurements.


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RESULTS
 
Severe adenine starvation activates LTR-lacZ fusions. The promoter of Ty1 contains three regions important for its transcription: the 5' LTR and two regions called FRE (filamentous response element) and MIR (Mcm1, Tea1 [previously Ibf1], and Rap1), which are located in the TYA ORF (Fig. 1). We previously showed that severe adenine starvation activates the transcription of lacZ fusions, carrying all Ty1 promoter sequences. The activation was more efficient on weakly expressed elements than on highly expressed elements (64). These results are exemplified in the right panel of Fig. 2A, with TYA-lacZ fusions introduced at three Ty1 elements located on different chromosomes, either Ty1-DR3 and Ty1-ML1, which are weakly expressed, or Ty1-PR1, which is highly expressed. The increase in expression was independent of the Ste12 transcriptional activator, which binds to the FRE site (64). Furthermore, the activation was conserved with lacZ fusions containing the 5' portion of Ty1 promoter, including the 5' LTR and the FRE site (64). These observations suggested that the 5' LTR might be sufficient to promote the activation. To test this idea, we assayed the expression of LTR-lacZ fusions driven from Ty1-DR3, Ty1-ML1, and Ty1-PR1. The 5' LTRs of Ty1-PR1 and Ty1-DR3 are very similar in their sequences, except that Ty1-DR3 lacks the Gcn4E binding site, which is a signature of highly expressed Ty1 elements (Fig. 1) (48). The sequence of the Ty1-ML1 5' LTR differs at several positions, mostly in the five potential Gcn4 binding sites and TATA box 1, which are far from consensus. In the three fusions, lacZ is translated from the Ty1 initiation codon, which is located in the 5' LTR. Conditions of severe adenine starvation were obtained in bas1{Delta} cells grown in the absence of adenine, as described previously (64). For each LTR-lacZ fusion, there was no significant difference in the β-galactosidase activities of wild-type cells grown with or without adenine, and their activity was similar to that observed in bas1{Delta} cells grown in the presence of adenine (data not shown) (Fig. 2A, left panel). When the bas1{Delta} cells were grown in the absence of adenine, we observed 10-, 3.5-, and 1.6-fold increases in β-galactosidase activity with LTR-lacZ fusions at Ty1-DR3, Ty1-ML1, and TY1-PR1, respectively. Except for Ty1-ML1, these activation factors were almost identical to those observed with TYA-lacZ fusions. In addition, the LTR-lacZ fusions were expressed at lower levels at Ty1-DR3 and Ty1-ML1 than at Ty1-PR1, indicating that the hierarchy in the level of expression observed with TYA-lacZ fusions was conserved. Taken together, these observations suggest that the 5' LTR contains regulatory elements which are determinant for both the expression level of Ty1 elements and for their level of activation by severe adenine starvation.


Figure 2
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  		FIG. 2. β-Galactosidase activity of LTR-lacZ and TYA-lacZ fusions. (A) β-Galactosidase activity of LTR-lacZ and TYA-lacZ fusions at Ty1-DR3, Ty1-ML1, and Ty1-PR1 in bas1{Delta} cells. Cultures were grown at 22°C in SDc minimum medium supplemented (+) or not (–) with 0.3 mM adenine. AF, activation factor (versus adenine). Data represent the average with standard error from three independent cultures. Exact average β-galactosidase specific activities are given above the bars and are expressed in nanomoles of 2-nitrophenyl-β-D-galactopyranoside hydrolyzed per minute per milligram of protein. (B) β-Galactosidase activity of LTR-lacZ and TYA-lacZ fusions carried on high-copy-number plasmids in bas1{Delta} cells. Growth conditions and data representations are described in the legend to panel A. (C) β-Galactosidase activity of LTR-lacZ fusions at Ty1-DR3 and Ty1-PR1 in bas1{Delta}, snf2{Delta}, and bas1{Delta} snf2{Delta} cells. Growth conditions and data representations are described in the legend to panel A.

The less-efficient activation by severe adenine starvation of LTR-lacZ fusions than of TYA-lacZ fusions observed at Ty1-ML1 might suggest that promoter sequences located in TYA are necessary for complete activation in the context of degenerated TATA boxes. Likewise, in bas1{Delta} cells grown in the presence of adenine, the β-galactosidase activity of the LTR-lacZ fusion at Ty1-PR1 was markedly lower than the activity of the TYA-lacZ fusion. Together, these differences confirm that TYA sequences are also important for the full expression and possibly the activation of these elements.

Since the β-galactosidase activities produced by the LTR-lacZ fusions were low, especially at Ty1-DR3 and Ty1-ML1, we wished to confirm the activation by severe adenine starvation with equivalent LTR-lacZ fusions carried on high-copy-number plasmids. In bas1{Delta} cells transformed with these plasmids and grown in the presence of adenine, the β-galactosidase activities were between 70- and 300-fold higher than the activities obtained with chromosomal fusions (compare Fig. 2A, left panel, and B). Interestingly, LTR-lacZ and TYA-lacZ fusions remained activated by severe adenine starvation when carried on plasmids, although the level of activation varied less between weakly and highly expressed elements (Fig. 2B). The hierarchy of expression between Ty1 elements was also maintained on plasmids, although not as clearly. From this experiment, we conclude that the chromosomal environment is not essential for the activation of Ty1 by severe adenine starvation or the hierarchy of expression between elements.

The activation of LTR-lacZ fusions by severe adenine starvation is compromised in an snf2 mutant. We previously reported that the activation by severe adenine starvation of several TYA-lacZ fusions is abolished when the Swi/Snf chromatin remodeling complex is inactivated, suggesting that Swi/Snf could be involved in the mechanism of activation (64). Having established that the 5' LTR is sufficient to promote the activation by severe adenine starvation, we asked whether Swi/Snf contributes to the activation through the 5' LTR. Since SNF2 encodes an ATPase essential for Swi/Snf remodeling activity (39), the levels of expression of LTR-lacZ fusions at Ty1-DR3 and Ty1-PR1 were compared in bas1{Delta} and bas1{Delta} snf2{Delta} cells grown with and without adenine. At the weakly expressed Ty1-DR3 element, the SNF2 deletion led to a significant decrease in the activation of LTR-lacZ by severe adenine starvation, indicating that Swi/Snf is necessary for the activation to occur (Fig. 2C, compare the 10-fold activation in bas1{Delta} to the 2.2-fold activation in bas1{Delta} snf2{Delta} cells). At the highly expressed Ty1-PR1 element, the activation of LTR-lacZ, which was already low in bas1{Delta} cells, was completely abolished in bas1{Delta} snf2{Delta} cells (Fig. 2C, compare the 1.6-fold activation in bas1{Delta} cells to the 0.9-fold activation in bas1{Delta} snf2{Delta} cells). The loss of activation by severe adenine starvation of LTR-lacZ fusions at Ty1-DR3 and Ty1-PR1, in the absence of SNF2, is consistent with a role of the Swi/Snf chromatin remodeling complex in the mechanism of activation.

It is noteworthy that the SNF2 single deletion led to an increase, although less than twofold, in the expression of LTR-lacZ at Ty1-PR1, independently of the adenine availability. This observation is surprising since previous data clearly established that Ty1 mRNA levels synthesized from all Ty1 elements strongly decrease in snf2{Delta} cells (14, 28). In addition, the expression of a TYA-lacZ fusion at Ty1-PR1 is abolished in snf2{Delta} cells, indicating that Swi/Snf plays a major role in Ty1 transcription (48). One explanation is that Swi/Snf plays antagonistic roles at Ty1-PR1: a minor repressive effect at the 5' LTR and a major positive effect at downstream Ty1 promoter sequences.

The expression of a promoterless his3{Delta}4 allele adjacent to a full-length Ty1 element is activated by severe adenine starvation. Ty1 insertions can activate the expression of adjacent genes (reviewed in references 7 and 40). In all cases, Ty1 elements are inserted within a window of 175 bp upstream of the target gene, in such a way that Ty1 and the adjacent gene are divergently transcribed. These insertions have been extremely useful in the identification of Ty1 transcription factors, such as Ste12, which binds to the FRE site in TYA and is important for basal Ty1 transcription in haploid cells (3, 14, 36, 49). Thus, we speculated that such Ty1 insertions might also be helpful to understand the regulation of Ty1 transcription by severe adenine starvation. To investigate whether conditions of severe adenine starvation could activate the expression of a gene adjacent to a Ty1 element, we used a reporter Ty1-his3{Delta}4 allele, which consists of a Ty1 insertion 56 bp upstream of the promoterless his3{Delta}4 allele (Fig. 3). Cells containing the Ty1-his3{Delta}4 allele were able to grow in the absence of histidine, while cells containing the his3{Delta}4 allele alone could not, indicating that his3{Delta}4 expression depends on the presence of the Ty1 insertion (Fig. 3, rows 1 and 2). Furthermore, the ability of Ty1-his3{Delta}4 cells to grow in the absence of histidine required the Ste12 transcriptional activator (Fig. 3, row 5). We showed previously that STE12 is not required for the activation of TYA-lacZ fusions by severe adenine starvation (64). Therefore, we analyzed whether the activation of Ty1 by severe adenine starvation could rescue the histidine auxotrophy of ste12{Delta} cells, by stimulating his3{Delta}4 transcription from the adjacent Ty1 sequences. The growth of ste12{Delta} and ste12{Delta} bas1{Delta} cells was compared on plates lacking either histidine only or both histidine and adenine. Although ste12{Delta} cells grew poorly on both plates, the growth of ste12{Delta} bas1{Delta} cells was greatly improved in the absence of adenine (Fig. 3, compare rows 4 and 5). The suppression of the growth defect of an ste12{Delta} mutant on plates lacking histidine by severe adenine starvation was strictly dependent on the presence of Ty1 sequences upstream of his3{Delta}4, since severe adenine starvation did not rescue the histidine auxotrophy of bas1{Delta} cells containing the his3{Delta}4 allele alone (data not shown). These results indicate that Ty1 can activate the transcription of an adjacent his3{Delta}4 allele under conditions of severe adenine starvation. The impact of severe adenine starvation on Ty1-his3{Delta}4 could not be detected in a bas1{Delta} single mutant (Fig. 3, row 3), probably because in these cells, STE12 activates Ty1-his3{Delta}4 transcription at sufficient levels to allow the growth in the absence of histidine, independently of adenine availability (see below).


Figure 3
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  		FIG. 3. Severe adenine starvation activates Ty1-his3{Delta}4 expression. (A) BAS1 STE12 (wild type [WT]), bas1{Delta}, ste12{Delta}, and bas1{Delta} ste12{Delta} cells containing either his3{Delta}4, Ty1-his3{Delta}4, or LTR-his3{Delta}4 alleles were spotted onto plates in a series of 10-fold dilutions. All plates were incubated at 30°C for 4 days.

Since severe adenine starvation can activate Ty1-his3{Delta}4 and LTR-lacZ expression, we asked whether the 5' LTR would also be sufficient to promote activation of his3{Delta}4 under these conditions of starvation. We obtained an LTR-his3{Delta}4 allele in bas1{Delta} cells, by eviction of the Ty1 internal region in Ty1-his3{Delta}4, through homologous recombination between the two LTRs of the element. In the resulting LTR-his3{Delta}4 allele, the LTR is at the same distance from his3{Delta}4 and in the same orientation as it is in Ty1-his3{Delta}4. Unlike the Ty1-his3{Delta}4 allele, the LTR-his3{Delta}4 allele did not confer histidine prototrophy to bas1{Delta} cells, and severe adenine starvation did not suppress the histidine deficiency of these cells (Fig. 3, row 6). Together, these results indicate that severe adenine starvation can activate the expression of a gene adjacent to a Ty1 element and that the activation requires Ty1 internal sequences.

Ty1 internal sequences are required to activate the expression of an adjacent TDH3-lacZ fusion from the Ty1 promoter under conditions of severe adenine starvation. To further analyze the requirement for Ty1 promoter sequences in the activation of adjacent gene expression under conditions of severe adenine starvation, we used centromeric plasmids carrying different portions of the Ty1 promoter, inserted 140 bp upstream of a TDH3-lacZ reporter construct, with the same divergent organization as for the Ty1-his3{Delta}4 allele (Fig. 4A). These plasmids have been previously used to demonstrate the involvement of the Ty1 FRE site in the activation of Ty1-adjacent genes (37). TDH3 encodes a glyceraldehyde-3-phosphate dehydrogenase involved in glycolysis and neoglucogenesis. In these plasmids, TDH3-lacZ expression is dependent upon Ty1 promoter sequences. The β-galactosidase activity of bas1{Delta} cells grown in the presence of adenine was weak with all of the constructs (Fig. 4B). When TDH3-lacZ expression was driven from the complete Ty1 promoter, a 10-fold increase in β-galactosidase activity was observed in adenine-starved cells (Fig. 4B, plasmid pIL11). The activation decreased to 4.9- and 6.2-fold, in the absence of the 3' portion of Ty1 promoter including MIR, suggesting that MIR contributes to achieving complete activation of TDH3-lacZ under conditions of severe adenine starvation (Fig. 4B, plasmids pIL10 and pIL12). This observation is consistent with our previous report that severe adenine starvation does not fully activate TYA-lacZ fusions lacking MIR (64) and with the observation that two and three tandem copies of MIR inserted upstream of a CYC1-lacZ reporter gene confer the activation by severe adenine starvation to the construct (data not shown). Furthermore, the activation by severe adenine starvation was abolished when TDH3-lacZ was expressed from a solo LTR (Fig. 4B, plasmid pGS17). These results are consistent with those obtained with Ty1-his3{Delta}4 and LTR-his3{Delta}4 alleles. Together, they indicate that Ty1 internal sequences are important for the activation of adjacent gene expression under conditions of severe adenine starvation.


Figure 4
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  		FIG. 4. β-Galactosidase activity of TDH3-lacZ fusions expressed from Ty1 sequences. (A) Schematic of the TDH3-lacZ reporter gene. (B) β-Galactosidase activity in bas1{Delta} cells of TDH3-lacZ fusions carried on centromeric plasmids. The structures of the different TDH3-lacZ fusions and the names of the plasmids are indicated. Gray and dotted boxes indicate the MIR and the FRE regions. The arrow indicates the LTR sequences. Cultures were grown at 22°C in SDc minimum medium supplemented (+ade) or not (–ade) with 0.3 mM adenine. β-Galactosidase specific activities are expressed in nanomoles of 2-nitrophenyl-β-D-galactopyranoside hydrolyzed per minute per milligram of protein. Data represent the average with standard error from three independent cultures.

Ty1-his3{Delta}4 and LTR-his3{Delta}4 transcription profiles are altered upon severe adenine starvation. To understand the impact of severe adenine starvation on the transcription of genes adjacent to Ty1, Northern blotting analysis was undertaken with cells carrying Ty1-his3{Delta}4 and LTR-his3{Delta}4 alleles. As a control, Ty1 mRNA levels increased severalfold in bas1{Delta} cells grown in the absence of adenine and were extremely low in ste12{Delta} cells (Fig. 5A, lanes 7, 8, and 9). In bas1{Delta} ste12{Delta} cells grown in the absence of adenine, the amounts of Ty1 mRNA increased to reach a level similar to that observed in wild-type cells (Fig. 5A, compare lane 13 with lanes 4 and 5). This increase is consistent with the activation of TYA-lacZ by severe adenine starvation in the absence of STE12 (64). It is worth noting that the decrease in Ty1 transcript observed in ste12{Delta} cells was accompanied by the appearance of a transcript of smaller size (Fig. 5A, lanes 8 and 9). A similar short species has been described in spt3 mutants that affect Ty1 transcription and corresponds to an initiation of transcription in the TYA ORF (70).


Figure 5
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  		FIG. 5. Pattern of transcription of Ty1-his3{Delta}4 and LTR-his3{Delta}4 alleles. RNA extracted from BAS1 STE12 (wild type [WT]), bas1{Delta}, ste12{Delta}, and bas1{Delta} ste12{Delta} cells containing either his3{Delta}4, Ty1-his3{Delta}4, or LTR-his3{Delta}4 alleles and from BAS1 STE12 HIS3 (wild type [HIS3]) cells was subjected to Northern blotting (A) and primer extension analysis (B). Cultures were grown in HC medium supplemented with (+) or without (–) 0.3 mM adenine at 22°C. (A) For each sample, approximately 15 µg of RNA was loaded onto the gel. The blot was sequentially hybridized to HIS3, ACT1, and Ty1 probes. The sizes of the mRNA molecules are 5.6 kb (Ty1), 0.7 kb (HIS3), and 1.3 kb (ACT1). Lanes are numbered for clarity as cited in the text. The HIS3 signal in the right panel has been overexposed to permit detection of weak bands. (B) For each sample, 100 µg of RNA was analyzed by primer extension using 32P-end-labeled O-GS73 primer. A sequencing ladder obtained with O-GS73 is shown (TGCA). The 5' ends of the different products are indicated with respect to the +1 nucleotide of the HIS3 ATG codon and the +1 nucleotide of the 5' LTR. Black circles indicate specific RT stops. Open circles indicate nonspecific arrests. Lanes are numbered for clarity as cited in the text. (C) Sequences encompassing the Ty1 LTR and his3{Delta}4 with the position of the different RT stops observed by primer extension with the O-GS73 primer. HIS3 and Ty1 LTR sequences are indicated in boldface. Sequences upstream of the HIS3 ORF, which are conserved in his3{Delta}4, are in capital letters. Two ORFs present in the sequences between the Ty1 LTR and HIS3 are underlined. The HIS3 ATG initiation codon is indicated by an arrow pointing to the right, and the +1 coordinate of the Ty1 LTR is indicated by an arrow pointing to the left. TATA-rich sequences upstream of LTR coordinates +15 and +45 are indicated by shaded boxes.

Two mRNA species were detected in Ty1-his3{Delta}4 wild-type cells, using a HIS3 riboprobe specific for HIS3 mRNA (Fig. 5A, lanes 4 and 5). Using an RNA ladder, we estimated an approximately 180-nucleotide difference in lengths between these two mRNA species (data not shown). Their synthesis was dependent on the presence of the full-length Ty1 element upstream of his3{Delta}4 since they were not detected in wild-type cells carrying the Ty1-less his3{Delta}4 or LTR-his3{Delta}4 alleles (Fig. 5A, lanes 2, 3, 14, and 15). These species were only detected in cells able to grow in the absence of histidine: i.e., in wild-type and bas1{Delta} cells and in adenine-deprived ste12{Delta} bas1{Delta} cells (compare Fig. 3 and 5A, lanes 4 to 7 and 10 to 13). The mRNA species of lower molecular weight comigrated with functional HIS3 mRNA (Fig. 5A, lane 1). Furthermore, its amount increased under severe adenine starvation: i.e., in bas1{Delta} and ste12{Delta} bas1{Delta} cells grown in the absence of adenine (Fig. 5A, compare lanes 6 and 7 and lanes 12 and 13). Altogether, these observations indicate that the mRNA species of lower molecular weight allows the synthesis of a functional His3 protein and that its expression is stimulated by severe adenine starvation. Although the amount of the mRNA species of higher molecular weight showed no obvious correlation with the adenine availability in wild-type and bas1{Delta} cells, its expression was stimulated by the absence of adenine in ste12{Delta} bas1{Delta} cells (Fig. 5A, compare lanes 12 and 13). We conclude that severe adenine starvation activates the synthesis of both HIS3 mRNA species.

Primer extension experiments were also performed to map the 5' end of the different mRNA species produced by HIS3, Ty1-his3{Delta}4, and LTR-his3{Delta}4, respectively (Fig. 5B and 5C). In HIS3 wild-type cells, two major products were identified, which correspond well to the two already characterized HIS3 transcripts (Fig. 5B, lane 1: RT stops at positions –11 and –24 relative to HIS3 initiation ATG codon [63]). These products were also detected in Ty1-his3{Delta}4 cells that were His+: i.e., in wild-type and bas1{Delta} cells and in adenine-deprived ste12{Delta} bas1{Delta} cells (Fig. 5B, lanes 4, 5, 6, 7, and 9). Another major product, which was specific for Ty1-his3{Delta}4, was identified in these cells. This product corresponds to a 5' end located in the LTR sequence, approximately at position –71 relative to the HIS3 ATG codon (Fig. 5B, position +15 relative to the +1 nucleotide of the LTR). In addition to these products, minor products were also revealed, which may correspond to other 5' ends or may be the consequence of secondary structures that block RT (Fig. 5B: RT stops at positions –5 and –27 relative to the HIS3 ATG and positions +5, +45, and +71 relative to the +1 nucleotide of the LTR). Interestingly, all of these products were absent in Ty1-less his3{Delta}4 cells, LTR-his3{Delta}4 cells, Ty1-his3{Delta}4 ste12{Delta} cells and Ty1-his3{Delta}4 ste12{Delta} bas1{Delta} cells grown in the presence of adenine (Fig. 5B, lanes 2, 3, 8, and 10 to 13). Together, these observations suggest that the corresponding mRNA species are under the control of Ty1 internal sequences and respond to severe adenine starvation. Among these species, only the HIS3 –11 and –24 species should be functional, since species with 5' ends in the LTR contain two short ORFs that might impair HIS3 translation (Fig. 5C).

The difference in sizes between the two HIS3 mRNA species detected by Northern blot analysis (around 180 nucleotides) suggests that the 5' end of the mRNA species of higher molecular weight must be in the LTR sequence. However, this difference was bigger than the difference in sizes of the products obtained by primer extension analysis (65 to 120 nucleotides). We made several unsuccessful attempts to detect 5'-end products that could correspond to this mRNA species by using other primers or longer migration of electrophoresis gels. In conclusion, severe adenine starvation activates the synthesis of several mRNA species in Ty1-his3{Delta}4 cells. Their 5' ends are located in the LTR and at normal HIS3 start sites. Their synthesis requires Ty1 internal sequences, since these are absent in LTR-his3{Delta}4 cells. Among these transcripts, the mature HIS3 mRNA might be the only one to produce a functional His3 protein.

Severe adenine starvation alters the transcription pattern of ESF1, a gene adjacent to Ty1-DR6 in the yeast genome. Our findings that severe adenine starvation can activate the transcription of his3{Delta}4 and TDH3-lacZ, when they are 56 and 140 bp downstream of Ty1 promoter sequences, respectively, prompted us to analyze whether this activation could also happen with yeast genes naturally located in proximity to Ty1 insertions. On chromosome IV, the full-length Ty1-DR6 (YDRWTy1-5) element is located 320 bp upstream of the ESF1 gene that encodes an essential nucleolar protein involved in pre-rRNA processing (52). Two species of ESF1 mRNA were identified by Northern blot analysis in wild-type and bas1{Delta} cells (Fig. 6A, lanes 1 to 4). Interestingly, the levels of the higher-molecular-weight species increased in bas1{Delta} cells grown in the absence of adenine (Fig. 6A, lane 4). This species was also detected in bas1{Delta} ste12{Delta} cells grown in the absence of adenine, whereas it could not be detected in ste12{Delta} cells, independently of the adenine availability or in ste12{Delta} bas1{Delta} cells grown in the presence of adenine (Fig. 6A, compare lane 8 with lanes 5, 6, and 7). This profile of expression is similar to that of Ty1 and suggests that the transcription of this ESF1 mRNA species is under the control of Ty1 transcription regulatory signals and is stimulated by severe adenine starvation. The situation was different for the ESF1 mRNA of lower molecular weight, as its amounts remained relatively constant in the wild-type and bas1{Delta} cells grown with and without adenine (Fig. 6A, lanes 1 to 4) and increased in ste12{Delta} cells and in ste12{Delta} bas1{Delta} cells grown in the presence of adenine (Fig. 6A, lanes 5, 6, and 7). Moreover, in ste12{Delta} bas1{Delta} cells, the amounts of this species decreased concomitantly with the increase of the higher-molecular-weight species, in the absence of adenine. (Fig. 6A, compare lanes 7 and 8). This balance between the two species suggests a competition in their synthesis.


Figure 6
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  		FIG. 6. The ESF1 gene is under the control of the Ty1-DR6 endogenous element. RNA extracted from cultures of BAS1 STE12 (wild type [WT]), bas1{Delta}, ste12{Delta}, and bas1{Delta} ste12{Delta} cells was subjected to Northern blotting (A) and primer extension analysis (B). Cultures were grown at 22°C in SC medium supplemented with (+) or without (–) 0.3 mM adenine. (A) For each sample, approximately 15 µg of RNA was loaded onto the gel. The blot was sequentially hybridized to ESF1, ACT1, and Ty1 probes. The sizes of the mRNA molecules are 5.6 kb (Ty1), 1.9 kb (ESF1), and 1.3 kb (ACT1). Lanes are numbered for clarity as cited in the text. (B) One hundred micrograms of RNA was analyzed by primer extension using a 32P-end-labeled O-GS71 primer. A sequencing ladder obtained with the same primer is shown (TGCA). The 5' ends of the different products are indicated with respect to ESF1 +1 and LTR +1. Black circles indicate specific 5' ends. Lanes are numbered for clarity as cited in the text. (C) Sequences encompassing the Ty1 LTR and ESF1, with the positions of the different RT stops observed by primer extension with the O-GS71 primer. ESF1 and Ty1 LTR sequences are indicated in boldface. Three short ORFs present in the sequences between Ty1 LTR and ESF1 are underlined. The ESF1 ATG initiation codon is indicated by an arrow pointing to the right, and the +1 coordinate of Ty1 LTR is indicated by an arrow pointing to the left. TATA-rich sequences upstream of LTR coordinates +15 and +45 are indicated by shaded boxes.

Primer extension experiments were performed in parallel (Fig. 6B and C). Using a primer complementary to sequences in the ESF1 gene (coordinates +49 and +28 relative to ESF1 +1), we characterized four products corresponding to 5' ends upstream of ESF1 at coordinates –306, –63, –70, and –88 relative to the ESF1 initiation codon (data not shown). The ESF1 –306 product was also identified with primer O-GS71, which is complementary to sequences upstream of ESF1 (Fig. 6B and C). Among these products, the ESF1 –70 and –88 products probably correspond to transcription start sites previously observed in a global mapping of yeast transcription start sites (73). The level of these products did not change in response to severe adenine starvation.

The O-GS71 primer also revealed four products corresponding to 5' ends at positions –326, –336, –366, and –392 upstream of ESF1, in the Ty1 LTR (Fig. 6B and C, LTR +5, +15, +45, and +71). Remarkably, these 5' ends were identical to those observed in the LTR sequence of Ty1-his3{Delta}4 mRNA (Fig. 5B and C). Moreover, the presence of these products in the different samples fit perfectly with the presence of the higher-molecular-weight RNA species detected by Northern blotting in the different cell cultures, as they were not detected in ste12{Delta} cells and in ste12{Delta} bas1{Delta} cells grown in the presence of adenine (Fig. 6B, lanes 5, 7, and 8). These results demonstrate for the first time that a full-length Ty1 element controls the transcription of a yeast gene naturally located in proximity to a Ty1 promoter. It is noteworthy that Ty1-DR6 promoter sequences are located more than 300 bp upstream of ESF1, as this suggests a relatively long-distance effect of Ty1 on adjacent gene transcription.


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DISCUSSION
 
The Ty1 5' LTR possesses a weak transcription activity and is sufficient for the activation by severe adenine starvation. The first striking observation is that the 5' LTR can activate the transcription of lacZ reporter fusions in the absence of other activating sequences. Previous work has shown that while Ty1 transcription starts in the 5' LTR, there is no upstream activating sequence (UAS) in the LTR and important regulatory sequences reside mostly in the TYA ORF (21, 72). Yet, an upstream activating activity (UAS) has been described with the his4-912{delta} composite promoter, consisting of the juxtaposition of HIS4 UAS and a Ty1 LTR (18). In this case, Gcn4 acting at the HIS4 UAS and Gcr1 acting at the Ty1 LTR are the primary activators required for his4-912{delta} expression. Here, we show that the LTRs of two weakly expressed elements, Ty1-DR3 and Ty1-ML1, and one highly expressed element, Ty1-PR1, activate the transcription of lacZ fusions. The activation is effective with LTR-lacZ fusions, either introduced at endogenous Ty1 locations or carried on multicopy plasmids. Interestingly, the hierarchy of expression previously observed with endogenous TYA-lacZ fusions carrying the full Ty1 promoter sequences is conserved with LTR-lacZ fusions, although the expression of LTR-lacZ fusions is weak compared to that of TYA-lacZ. This confirms the important role of Ty1 internal sequences for full transcription. Collectively, these data demonstrate that Ty1 LTRs possess a weak transcription activity and contribute to the variability in expression between Ty1 elements. A binding site for the Gcr1 transcriptional activator of genes encoding glycolytic enzymes, ribosomal proteins, and cyclins has been found in the Ty1 LTR, and Gcr1 activates Ty1 transcription (2, 41, 65). Likewise, five potential Gcn4 binding sites are located in the 5' LTRs of several Ty1 elements (mostly those expressed at high levels) and Gcn4 overproduction stimulates the transcription of these elements (48). Thus, Gcr1 and Gcn4 might contribute to Ty1 LTR transcription activity. Since the yeast genome contains over 200 LTR sequences, our results raise the possibility that the intrinsic transcription activity of the LTRs contributes to intergenic transcription in yeast.

Our previous work has shown that severe adenine starvation activates TYA-lacZ fusions containing the full Ty1 promoter sequences (64). We now show that the activation also occurs on LTR-lacZ fusions. Interestingly, the results obtained with LTR-lacZ fusions recapitulate several previous observations made with TYA-lacZ fusions, such as the more efficient activation of weakly expressed elements and the loss of activation in a snf2{Delta} mutant that affects Swi/Snf chromatin remodeling activity. These results emphasize the important role of the 5' LTR in the regulation of Ty1 transcription. Since Ty1 transcription starts in the 5' LTR, our results also suggest that severe adenine starvation could facilitate the initiation of Ty1 transcription by Swi/Snf-dependent chromatin remodeling at Ty1 promoters. Alternatively, chromatin remodeling at the Ty1 promoter could be a prerequisite to the activation.

Severe adenine starvation activates the transcription of genes adjacent to Ty1 insertions by allowing divergent transcription initiation from cryptic sites in the LTR. The discovery that severe adenine starvation can activate the transcription of genes adjacent to Ty1 insertions is also of interest. This conclusion is exemplified by the Ty1-his3{Delta}4 allele, the TDH3-lacZ reporter construct expressed from different Ty1 sequences, and the ESF1 gene, which is naturally located in the vicinity of Ty1-DR6 in the yeast genome. In all of these cases, Ty1 is transcribed divergently from its associated gene. Previous studies on the impact of Ty1 insertions on adjacent gene expression suggested that the activation strongly depends on the Ste12 transcriptional activator (reviewed in references 7 and 40). Thus, it is noteworthy that severe adenine starvation can activate the expression of these genes in the absence of STE12. Yet, it is consistent with our previous observation that the activation of Ty1 transcription by severe adenine starvation occurs in ste12{Delta} cells (64). Furthermore, although the 5' LTR is sufficient to promote activation by severe adenine starvation when fused to lacZ in the same orientation, it is unable to activate the transcription of his3{Delta}4 or TDH3-lacZ reporter genes when it is inserted in the opposite orientation. An explanation for the different impact of the LTR on lacZ, his3{Delta}4, and TDH3-lacZ transcription is that Ty1 internal sequences could be essential for adjacent gene transcription. This hypothesis is supported by the activation of TDH3-lacZ, observed with constructs that contain some Ty1 internal sequences, and by the characterization of several HIS3 mRNA species in Ty1-his3{Delta}4 cells that are absent in LTR-his3{Delta}4 cells. Since chromatin structures play an important role in Ty1 transcription, one idea is that Ty1 internal sequences help to organize a chromatin structure permissive for transcription over the Ty1 LTR and adjacent regions, allowing activation by severe adenine starvation (48, 69). Also of note is the identification of ESF1 and his3{Delta}4 mRNA, with common 5'ends in the 5' LTR of Ty1-DR6 in the case of ESF1 and in the 5' LTR of the Ty1-his3{Delta}4 allele. Although we cannot rule out that some of these mRNA species correspond to the maturation of a precursor RNA by RNases, their increase under severe adenine starvation suggests that severe adenine starvation could stimulate the initiation of transcription at cryptic sites, especially in the LTR. It is noteworthy that AT-rich sequences, which might function as TATA boxes, are present in the LTR at a canonical distance from the +15 and +45 cryptic initiation sites (Fig. 5C and 6C).

Yeast genes are under the control of endogenous Ty1 elements. There are two yeast genes transcribed by RNA polymerase II, ESF1 and NDJ1, which are naturally located in proximity to full-length endogenous Ty1 elements, in a configuration similar to that of the Ty1-his3{Delta}4 allele and the Ty1-TDH3-lacZ construct (22). With ESF1, this study provides the first example of a yeast gene naturally under the control of an endogenous Ty1 insertion. Here, we identify two classes of ESF1 mRNA species. The first class contains several mRNA species with 5' ends in the LTR of Ty1-DR6. All of these transcripts are likely not functional since they contain several short ORFs upstream of the ESF1 initiation codon. The second class corresponds to mRNA species with 5' ends in the region comprised between the 5' LTR and ESF1 and includes functional ESF1 mRNA. Ty1-DR6 affects the expression of these two classes by different means. Transcripts with 5' ends in the LTR of Ty1-DR6 are under the control of Ty1-DR6 regulatory sequences and follow the regulation of Ty1 transcription, as they are absent in ste12{Delta} cells and their level increases in adenine-deprived cells. In contrast, conditions that inhibit Ty1 transcription (i.e., ste12{Delta}) increase the amounts of the second class of ESF1 mRNA and conversely conditions that stimulate Ty1 transcription (i.e., severe adenine starvation in ste12{Delta} cells) decrease their amounts. This regulation could be explained by a competition for the recruitment of transcription factors at the ESF1 promoter region and the 5' LTR or by transcription interference with transcription from the Ty1-LTR masking the ESF1 promoter region.

Remarkably, NDJ1, which encodes a meiosis-specific telomere protein, is another gene potentially under the control of an adjacent endogenous Ty1 insertion since it is located 250 bp downstream of Ty1-OL promoter sequences (22). Although we were unable to detect NDJ1 mRNA by Northern blotting, microarray studies performed to characterize yeast genes upregulated under conditions of severe adenine starvation identified NDJ1 (Servant et al. unpublished data). Strikingly, the distance between ESF1 and Ty1-DR6 or between NDJ1 and Ty1-OL (320 and 250 bp, respectively) is wider than the classical distance of Ty1 insertions reported to activate his3{Delta}4 expression (within a 175-bp window [9]). Hence, Ty1 can influence the expression of genes located at a distance larger than predicted from previous studies. However, the distance of these genes from Ty1 sequences probably influences the way Ty1 will affect their expression. Effectively, in the case of Ty1-his3{Delta}4, the activation of Ty1 transcription allows the transcription of functional HIS3 mRNA, whereas with ESF1, conditions that inactivate Ty1 transcription favor the transcription of functional ESF1 mRNA. It has already been reported that an endogenous Ty2 insertion inhibits the transcription of the high-affinity copper transporter gene CTR3 (35). With ESF1 and NDJ1, we provide the first evidence that endogenous Ty1 insertions can remodel yeast gene transcription under certain growth conditions.

Genome-wide studies in cells deficient in RNA degradation reveal that the yeast transcriptome includes numerous antisense transcripts from coding regions and cryptic unstable transcripts from intergenic regions (15, 16, 71). In addition, important regulatory functions have been assigned to antisense transcripts at IME4, PHO84, and Ty1; to noncoding interfering transcripts at SER3; and to cryptic transcripts in the ribosomal DNA arrays (4, 13, 29, 31, 43, 67). Our study indicates that full-length Ty1 insertions can drive the transcription of adjacent regions. We also provide evidence with the LTR-lacZ fusions that transcription can be initiated from solo LTRs. Since 32 full-length Ty1 elements and 185 solo LTRs are scattered in the yeast genome (34), an attractive hypothesis is that Ty1-driven transcription could contribute to the production of noncoding RNAs. Since Ty1 transcription is activated by different kinds of environmental stress conditions, Ty1-driven transcription of noncoding sequences could perhaps influence the reprogramming of yeast gene expression in response to stress. This hypothesis makes particular sense with the nonrandom clustering of stress-related genes, tRNA genes, and Ty1 sequences described in the S. cerevisiae genome (12).


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ACKNOWLEDGMENTS
 
We are grateful L. Bénard and M. Springer for stimulating discussions and comments on the manuscript. Thanks also go to M. Bétermier and C. Condon for helpful comments on the manuscript. We are grateful to E. Dubois for providing plasmids and to J. Lederout for technical help with riboprobe synthesis.

This work was supported by grants from the CNRS (UPR 9073) and Université Paris-Diderot Paris 7. G.S. was a recipient of a fellowship from the CNRS and of a fellowship from the Association pour la Recherche contre le Cancer.


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FOOTNOTES
 
* Corresponding author. Present address: CNRS UMR 7151, Hôpital Saint-Louis, 1 Avenue C, Vellefaux F-75475, Paris Cedex 10, France. Phone: 33 1 58 41 51 25. Fax: 33 1 58 41 50 20. E-mail: pascale.lesage{at}univ-paris-diderot.fr Back

{triangledown} Published ahead of print on 30 June 2008. Back

{dagger} Present address: UMR 144 CNRS, Department of Cell Biology, Institut Curie, 26 Rue d'Ulm, F-75248 Paris Cedex 05, France. Back


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REFERENCES
 
    1
  1. Adams, A., D. E. Gottschling, C. A. Kaiser, and T. Stearns. 1997. Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    2. 2
  2. Barbara, K. E., T. M. Haley, K. A. Willis, and G. M. Santangelo. 2007. The transcription factor Gcr1 stimulates cell growth by participating in nutrient-responsive gene expression on a global level. Mol. Genet. Genomics 277:171-188.[CrossRef][Medline]
    3. 3
  3. Baur, M., R. K. Esch, and B. Errede. 1997. Cooperative binding interactions required for function of the Ty1 sterile responsive element. Mol. Cell. Biol. 17:4330-4337.[Abstract]
    4. 4
  4. Berretta, J., M. Pinskaya, and A. Morillon. 2008. A cryptic unstable transcript mediates transcriptional trans-silencing of the Ty1 retrotransposon in S. cerevisiae. Genes Dev. 22:615-626.[Abstract/Free Full Text]
    5. 5
  5. Boeke, J. D., D. Eichinger, D. Castrillon, and G. R. Fink. 1988. The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1. Mol. Cell. Biol. 8:1432-1442.[Abstract/Free Full Text]
    6. 6
  6. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197:345-346.[CrossRef][Medline]
    7. 7
  7. Boeke, J. D., and S. B. Sandmeyer. 1991. Yeast transposable elements, p. 193-261. In J. R. Broach, J. R. Pringle, and E. W. Jones (ed.), The molecular and cellular biology of the yeast Saccharomyces, vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    8. 8
  8. Boeke, J. D., and J. P. Stoye. 1997. Retrotransposons, endogenous retroviruses, and the evolution of retroelements, p. 343-436. In J. M. Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses, vol. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    9. 9
  9. Boeke, J. D., C. A. Styles, and G. R. Fink. 1986. Saccharomyces cerevisiae SPT3 gene is required for transposition and transpositional recombination of chromosomal Ty elements. Mol. Cell. Biol. 6:3575-3581.[Abstract/Free Full Text]
    10. 10
  10. Bolton, E. C., and J. D. Boeke. 2003. Transcriptional interactions between yeast tRNA genes, flanking genes and Ty elements: a genomic point of view. Genome Res. 13:254-263.[Abstract/Free Full Text]
    11. 11
  11. Bradshaw, V. A., and K. McEntee. 1989. DNA damage activates transcription and transposition of yeast Ty retrotransposons. Mol. Gen. Genet. 218:465-474.[CrossRef][Medline]
    12. 12
  12. Burhans, D. T., L. Ramachandran, J. Wang, P. Liang, H. G. Patterton, M. Breitenbach, and W. C. Burhans. 2006. Non-random clustering of stress-related genes during evolution of the S. cerevisiae genome. BMC Evol. Biol. 6:58.[CrossRef][Medline]
    13. 13
  13. Camblong, J., N. Iglesias, C. Fickentscher, G. Dieppois, and F. Stutz. 2007. Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S. cerevisiae. Cell 131:706-717.[CrossRef][Medline]
    14. 14
  14. Ciriacy, M., K. Freidel, and C. Lohning. 1991. Characterization of trans-acting mutations affecting Ty and Ty-mediated transcription in Saccharomyces cerevisiae. Curr. Genet. 20:441-448.[CrossRef][Medline]
    15. 15
  15. David, L., W. Huber, M. Granovskaia, J. Toedling, C. J. Palm, L. Bofkin, T. Jones, R. W. Davis, and L. M. Steinmetz. 2006. A high-resolution map of transcription in the yeast genome. Proc. Natl. Acad. Sci. USA 103:5320-5325.[Abstract/Free Full Text]
    16. 16
  16. Davis, C. A., and M. Ares, Jr. 2006. Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 103:3262-3267.[Abstract/Free Full Text]
    17. 17
  17. Deininger, P. L., and M. A. Batzer. 2002. Mammalian retroelements. Genome Res. 12:1455-1465.[Abstract/Free Full Text]
    18. 18
  18. Dudley, A. M., L. J. Gansheroff, and F. Winston. 1999. Specific components of the SAGA complex are required for Gcn4- and Gcr1- mediated activation of the his4-912delta promoter in Saccharomyces cerevisiae. Genetics 151:1365-1378.[Abstract/Free Full Text]
    19. 19
  19. Errede, B. 1993. MCM1 binds to a transcriptional control element in Ty1. Mol. Cell. Biol. 13:57-62.[Abstract/Free Full Text]
    20. 20
  20. Errede, B., M. Company, and C. A. Hutchison III. 1987. Ty1 sequence with enhancer and mating-type-dependent regulatory activities. Mol. Cell. Biol. 7:258-265.[Abstract/Free Full Text]
    21. 21
  21. Fulton, A. M., P. D. Rathjen, S. M. Kingsman, and A. J. Kingsman. 1988. Upstream and downstream transcriptional control signals in the yeast retrotransposon, TY. Nucleic Acids Res. 16:5439-5458.[Abstract/Free Full Text]
    22. 22
  22. Garfinkel, D. J. 2005. Genome evolution mediated by Ty elements in Saccharomyces. Cytogenet. Genome Res. 110:63-69.[CrossRef][Medline]
    23. 23
  23. Goffeau, A., B. G. Barrell, H. Bussey, R. W. Davis, B. Dujon, H. Feldmann, F. Galibert, J. D. Hoheisel, C. Jacq, M. Johnston, E. J. Louis, H. W. Mewes, Y. Murakami, P. Philipsen, H. Tettelin, and S. G. Oliver. 1996. Life with 6000 genes. Science 274:546-567.[Abstract/Free Full Text]
    24. 24
  24. Grandbastien, M. A., C. Audeon, E. Bonnivard, J. M. Casacuberta, B. Chalhoub, A. P. Costa, Q. H. Le, D. Melayah, M. Petit, C. Poncet, S. M. Tam, M. A. Van Sluys, and C. Mhiri. 2005. Stress activation and genomic impact of Tnt1 retrotransposons in Solanaceae. Cytogenet. Genome Res. 110:229-241.[CrossRef][Medline]
    25. 25
  25. Grant, P. A., L. Duggan, J. Cote, S. M. Roberts, J. E. Brownell, R. Candau, R. Ohba, T. Owen-Hughes, C. D. Allis, F. Winston, S. L. Berger, and J. L. Workman. 1997. Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev. 11:1640-1650.[Abstract/Free Full Text]
    26. 26
  26. Gray, W. M., and J. S. Fassler. 1996. Isolation and analysis of the yeast TEA1 gene, which encodes a zinc cluster Ty enhancer-binding protein. Mol. Cell. Biol. 16:347-358.[Abstract]
    27. 27
  27. Gray, W. M., and J. S. Fassler. 1993. Role of Saccharomyces cerevisiae Rap1 protein in Ty1 and Ty1-mediated transcription. Gene Expr. 3:237-251.[Medline]
    28. 28
  28. Happel, A. M., M. S. Swanson, and F. Winston. 1991. The SNF2, SNF5 and SNF6 genes are required for Ty transcription in Saccharomyces cerevisiae. Genetics 128:69-77.[Abstract]
    29. 29
  29. Hongay, C. F., P. L. Grisafi, T. Galitski, and G. R. Fink. 2006. Antisense transcription controls cell fate in Saccharomyces cerevisiae. Cell 127:735-745.[CrossRef][Medline]
    30. 30
  30. Horie, K., E. S. Saito, V. W. Keng, R. Ikeda, H. Ishihara, and J. Takeda. 2007. Retrotransposons influence the mouse transcriptome: implication for the divergence of genetic traits. Genetics 176:815-827.[Abstract/Free Full Text]
    31. 31
  31. Houseley, J., and D. Tollervey. 2008. The nuclear RNA surveillance machinery: the link between ncRNAs and genome structure in budding yeast? Biochim. Biophys. Acta 1779:239-246.[Medline]
    32. 32
  32. Irwin, B., M. Aye, P. Baldi, N. Beliakova-Bethell, H. Cheng, Y. Dou, W. Liou, and S. Sandmeyer. 2005. Retroviruses and yeast retrotransposons use overlapping sets of host genes. Genome Res. 15:641-654.[Abstract/Free Full Text]
    33. 33
  33. Kent, N. A., N. Karabetsou, P. K. Politis, and J. Mellor. 2001. In vivo chromatin remodeling by yeast ISWI homologs Isw1p and Isw2p. Genes Dev. 15:619-626.[Abstract/Free Full Text]
    34. 34
  34. Kim, J. M., S. Vanguri, J. D. Boeke, A. Gabriel, and D. F. Voytas. 1998. Transposable elements and genome organization: a comprehensive survey of retrotransposons revealed by the complete Saccharomyces cerevisiae genome sequence. Genome Res. 8:464-478.[Abstract/Free Full Text]
    35. 35
  35. Knight, S. A. B., S. Labbe, L. F. Kwon, D. J. Kosman, and D. J. Thiele. 1996. A widespread transposable element masks expression of a yeast copper transport gene. Genes Dev. 10:1917-1929.[Abstract/Free Full Text]
    36. 36
  36. Laloux, I., E. Dubois, M. Dewerchin, and E. Jacobs. 1990. TEC1, a gene involved in the activation of Ty1 and Ty1-mediated gene expression in Saccharomyces cerevisiae: cloning and molecular analysis. Mol. Cell. Biol. 10:3541-3550.[Abstract/Free Full Text]
    37. 37
  37. Laloux, I., E. Jacobs, and E. Dubois. 1994. Involvement of SRE element of Ty1 transposon in TEC1-dependent transcriptional activation. Nucleic Acids Res. 22:999-1005.[Abstract/Free Full Text]
    38. 38
  38. Landry, J. R., A. Rouhi, P. Medstrand, and D. L. Mager. 2002. The Opitz syndrome gene Mid1 is transcribed from a human endogenous retroviral promoter. Mol. Biol. Evol. 19:1934-1942.[Abstract/Free Full Text]
    39. 39
  39. Laurent, B. C., I. Treich, and M. Carlson. 1993. The yeast SNF2/SWI2 protein has DNA-stimulated ATPase activity required for transcriptional activation. Genes Dev. 7:583-591.[Abstract/Free Full Text]
    40. 40
  40. Lesage, P., and A. L. Todeschini. 2005. Happy together: the life and times of Ty retrotransposons and their hosts. Cytogenet. Genome Res. 110:70-90.[CrossRef][Medline]
    41. 41
  41. López, M. C., and H. V. Baker. 2000. Understanding the growth phenotype of the yeast gcr1 mutant in terms of global genomic expression patterns. J. Bacteriol. 182:4970-4978.[Abstract/Free Full Text]
    42. 42
  42. Madison, J. M., A. M. Dudley, and F. Winston. 1998. Identification and analysis of Mot3, a zinc finger protein that binds to the retrotransposon Ty long terminal repeat ({delta}) in Saccharomyces cerevisiae. Mol. Cell. Biol. 18:1879-1890.[Abstract/Free Full Text]
    43. 43
  43. Martens, J. A., P. Y. Wu, and F. Winston. 2005. Regulation of an intergenic transcript controls adjacent gene transcription in Saccharomyces cerevisiae. Genes Dev. 19:2695-2704.[Abstract/Free Full Text]
    44. 44
  44. Maxwell, P. H., and M. J. Curcio. 2007. Host factors that control long terminal repeat retrotransposons in Saccharomyces cerevisiae: implications for regulation of mammalian retroviruses. Eukaryot. Cell 6:1069-1080.[Free Full Text]
    45. 45
  45. McClanahan, T., and K. McEntee. 1984. Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage. Mol. Cell. Biol. 4:2356-2363.[Abstract/Free Full Text]
    46. 46
  46. Medstrand, P., J. R. Landry, and D. L. Mager. 2001. Long terminal repeats are used as alternative promoters for the endothelin B receptor and apolipoprotein C-I genes in humans. J. Biol. Chem. 276:1896-1903.[Abstract/Free Full Text]
    47. 47
  47. Meyers, B. C., S. V. Tingey, and M. Morgante. 2001. Abundance, distribution, and transcriptional activity of repetitive elements in the maize genome. Genome Res. 11:1660-1676.[Abstract/Free Full Text]
    48. 48
  48. Morillon, A., L. Bénard, M. Springer, and P. Lesage. 2002. Differential effects of chromatin and Gcn4 on the 50-fold range of expression among individual yeast Ty1 retrotransposons. Mol. Cell. Biol. 22:2078-2088.[Abstract/Free Full Text]
    49. 49
  49. Morillon, A., M. Springer, and P. Lesage. 2000. Activation of the Kss1 invasive-filamentous growth pathway induces Ty1 transcription and retrotransposition in Saccharomyces cerevisiae. Mol. Cell. Biol. 20:5766-5776.[Abstract/Free Full Text]
    50. 50
  50. Myers, A. M., A. Tzagoloff, D. M. Kinney, and C. J. Lusty. 1986. Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene 45:299-310.[CrossRef][Medline]
    51. 51
  51. Peaston, A. E., A. V. Evsikov, J. H. Graber, W. N. de Vries, A. E. Holbrook, D. Solter, and B. B. Knowles. 2004. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Dev. Cell 7:597-606.[CrossRef][Medline]
    52. 52
  52. Peng, W. T., N. J. Krogan, D. P. Richards, J. F. Greenblatt, and T. R. Hughes. 2004. ESF1 is required for 18S rRNA synthesis in Saccharomyces cerevisiae. Nucleic Acids Res. 32:1993-1999.[Abstract/Free Full Text]
    53. 53
  53. Pollard, K. J., and C. L. Peterson. 1997. Role for ADA/GCN5 products in antagonizing chromatin-mediated transcriptional repression. Mol. Cell. Biol. 17:6212-6222.[Abstract]
    54. 54
  54. Rolfe, M., A. Spanos, and G. Banks. 1986. Induction of yeast Ty element transcription by ultraviolet. Nature 319:339-340.[CrossRef]
    55. 55
  55. Romanish, M. T., W. M. Lock, L. N. van de Lagemaat, C. A. Dunn, and D. L. Mager. 2007. Repeated recruitment of LTR retrotransposons as promoters by the anti-apoptotic locus NAIP during mammalian evolution. PLoS Genet. 3:e10.[CrossRef][Medline]
    56. 56
  56. Sacerdot, C., G. Mercier, A. L. Todeschini, M. Dutreix, M. Springer, and P. Lesage. 2005. Impact of ionizing radiation on the life cycle of Saccharomyces cerevisiae Ty1 retrotransposon. Yeast 22:441-455.[CrossRef][Medline]
    57. 57
  57. Scherer, S., C. Mann, and R. W. Davis. 1982. Reversion of a promoter deletion in yeast. Nature 298:815-819.[CrossRef][Medline]
    58. 58
  58. Schmitt, M. E., T. A. Brown, and B. L. Trumpower. 1990. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 18:3091-3092.[Free Full Text]
    59. 59
  59. Sehgal, A., C. Y. Lee, and P. J. Espenshade. 2007. SREBP controls oxygen-dependent mobilization of retrotransposons in fission yeast. PLoS Genet. 3:e131.[CrossRef][Medline]
    60. 60
  60. Slotkin, R. K., and R. Martienssen. 2007. Transposable elements and the epigenetic regulation of the genome. Nat. Rev. Genet. 8:272-285.[Medline]
    61. 61
  61. Staleva Staleva, L., and P. Venkov. 2001. Activation of Ty transposition by mutagens. Mutat. Res. 474:93-103.[Medline]
    62. 62
  62. Strand, D. J., and J. F. McDonald. 1985. Copia is transcriptionally responsive to environmental stress. Nucleic Acids Res. 13:4401-4410.[Abstract/Free Full Text]
    63. 63
  63. Struhl, K. 1986. Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms. Mol. Cell. Biol. 6:3847-3853.[Abstract/Free Full Text]
    64. 64
  64. Todeschini, A.-L., A. Morillon, M. Springer, and P. Lesage. 2005. Severe adenine starvation activates Ty1 transcription and retrotransposition in Saccharomyces cerevisiae. Mol. Cell. Biol. 25:7459-7472.[Abstract/Free Full Text]
    65. 65
  65. Turkel, S., X. B. Liao, and P. J. Farabaugh. 1997. Gcr1-dependent transcriptional activation of yeast retrotransposon Ty2-917. Yeast 13:917-930.[CrossRef][Medline]
    66. 66
  66. van de Lagemaat, L. N., J. R. Landry, D. L. Mager, and P. Medstrand. 2003. Transposable elements in mammals promote regulatory variation and diversification of genes with specialized functions. Trends Genet. 19:530-536.[CrossRef][Medline]
    67. 67
  67. Vasiljeva, L., M. Kim, N. Terzi, L. M. Soares, and S. Buratowski. 2008. Transcription termination and RNA degradation contribute to silencing of RNA polymerase II transcription within heterochromatin. Mol. Cell 29:313-323.[CrossRef][Medline]
    68. 68
  68. Waterston, R. H., K. Lindblad-Toh, E. Birney, J. Rogers, J. F. Abril, P. Agarwal, R. Agarwala, R. Ainscough, M. Alexandersson, P. An, S. E. Antonarakis, J. Attwood, R. Baertsch, J. Bailey, K. Barlow, S. Beck, E. Berry, B. Birren, T. Bloom, P. Bork, M. Botcherby, N. Bray, M. R. Brent, D. G. Brown, S. D. Brown, C. Bult, J. Burton, J. Butler, R. D. Campbell, P. Carninci, S. Cawley, F. Chiaromonte, A. T. Chinwalla, D. M. Church, M. Clamp, C. Clee, F. S. Collins, L. L. Cook, R. R. Copley, A. Coulson, O. Couronne, J. Cuff, V. Curwen, T. Cutts, M. Daly, R. David, J. Davies, K. D. Delehaunty, J. Deri, E. T. Dermitzakis, C. Dewey, N. J. Dickens, M. Diekhans, S. Dodge, I. Dubchak, D. M. Dunn, S. R. Eddy, L. Elnitski, R. D. Emes, P. Eswara, E. Eyras, A. Felsenfeld, G. A. Fewell, P. Flicek, K. Foley, W. N. Frankel, L. A. Fulton, R. S. Fulton, T. S. Furey, D. Gage, R. A. Gibbs, G. Glusman, S. Gnerre, N. Goldman, L. Goodstadt, D. Grafham, T. A. Graves, E. D. Green, S. Gregory, R. Guigo, M. Guyer, R. C. Hardison, D. Haussler, Y. Hayashizaki, L. W. Hillier, A. Hinrichs, W. Hlavina, T. Holzer, F. Hsu, A. Hua, T. Hubbard, A. Hunt, I. Jackson, D. B. Jaffe, L. S. Johnson, M. Jones, T. A. Jones, A. Joy, M. Kamal, E. K. Karlsson, et al. 2002. Initial sequencing and comparative analysis of the mouse genome. Nature 420:520-562.[CrossRef][Medline]
    69. 69
  69. Winston, F. 1992. Analysis of SPT genes: a genetic approach toward analysis of TFIID, histones, and other transcription factors of yeast, p. 1271-1293. In S. L. McKnight and K. R. Yamamoto (ed.), Transcriptional regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    70. 70
  70. Winston, F., K. J. Durbin, and G. R. Fink. 1984. The SPT3 gene is required for normal transcription of Ty elements in S. cerevisiae. Cell 39:675-682.[CrossRef][Medline]
    71. 71
  71. Wyers, F., M. Rougemaille, G. Badis, J. C. Rousselle, M. E. Dufour, J. Boulay, B. Regnault, F. Devaux, A. Namane, B. Seraphin, D. Libri, and A. Jacquier. 2005. Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121:725-737.[CrossRef][Medline]
    72. 72
  72. Yu, K., and R. T. Elder. 1989. A region internal to the coding sequences is essential for transcription of the yeast Ty1-D15 element. Mol. Cell. Biol. 9:3667-3678.[Abstract/Free Full Text]
    73. 73
  73. Zhang, Z., and F. S. Dietrich. 2005. Mapping of transcription start sites in Saccharomyces cerevisiae using 5' SAGE. Nucleic Acids Res. 33:2838-2851.[Abstract/Free Full Text]

Molecular and Cellular Biology, September 2008, p. 5543-5554, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00416-08
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