The Rtt106 Histone Chaperone Is Functionally Linked to Transcription Elongation and Is Involved in the Regulation of Spurious Transcription from Cryptic Promoters in Yeast*♦

Rtt106 is a histone chaperone that has been suggested to play a role in heterochromatin-mediated silencing in Saccharomyces cerevisiae. It interacts physically and functionally with the chromatin assembly factor-1 (CAF-1), which is associated with replication-coupled nucleosomal deposition. In this work, we have taken several approaches to study Rtt106 in greater detail and have identified a previously unknown function of Rtt106. We found genetic interactions between rtt106Δ and mutations in genes encoding transcription elongation factors, including Spt6, TFIIS, and members of the PAF and yeast DSIF complexes. In addition, chromatin immunoprecipitation analysis indicates that Rtt106 is associated with transcribed regions of active genes. Furthermore, our results show that Rtt106 is required for the repression of transcription from a cryptic promoter within a coding region. This observation strongly suggests that Rtt106 is involved in the regulation of chromatin structure of transcribed regions. Finally, we provide evidence that Rtt106 plays a role in regulating the levels of histone H3 transcription-coupled deposition over transcribed regions. Taken together, our results indicate a direct link for Rtt106 with transcription elongation and the chromatin dynamics associated with RNA polymerase II passage.

Rtt106 is a histone chaperone that has been suggested to play a role in heterochromatin-mediated silencing in Saccharomyces cerevisiae. It interacts physically and functionally with the chromatin assembly factor-1 (CAF-1), which is associated with replication-coupled nucleosomal deposition. In this work, we have taken several approaches to study Rtt106 in greater detail and have identified a previously unknown function of Rtt106. We found genetic interactions between rtt106⌬ and mutations in genes encoding transcription elongation factors, including Spt6, TFIIS, and members of the PAF and yeast DSIF complexes. In addition, chromatin immunoprecipitation analysis indicates that Rtt106 is associated with transcribed regions of active genes. Furthermore, our results show that Rtt106 is required for the repression of transcription from a cryptic promoter within a coding region. This observation strongly suggests that Rtt106 is involved in the regulation of chromatin structure of transcribed regions. Finally, we provide evidence that Rtt106 plays a role in regulating the levels of histone H3 transcription-coupled deposition over transcribed regions. Taken together, our results indicate a direct link for Rtt106 with transcription elongation and the chromatin dynamics associated with RNA polymerase II passage.
In eukaryotic organisms, the chromatin structure has a major impact on important nuclear processes such as DNA replication or transcription. Most transcriptional regulation mechanisms involve chromatin modulation by histone-modifying enzymes or ATP-remodeling machines. However, another mechanism emerged from recent advances in chromatin studies that links nucleosomal assembly, disassembly, and histone dynamics to the control of transcription (1). At gene promoters, histone dynamics could modulate histone marks that play a critical role during activation or repression of genes (2).
As is the case for transcription initiation, chromatin structure is also a major obstacle to elongating RNA polymerase II (RNAP II) 3 (3). However, cells have developed mechanisms involving different factors that deal with this challenge and allow RNAP II movement during elongation. Interestingly, these factors also play a role in chromatin refolding in the wake of transcription by promoting different mechanisms such as histone displacement, exchange, and redeposition (4).
The refolding of nucleosomes in the wake of RNAP II is of great importance to the cell because its absence has detrimental consequences. A defect in this process results in transcriptionally permissive chromatin along the transcribed regions (5). This permissive structure allows initiation of spurious transcription from cryptic sites within coding regions (5,6). Importantly, recent observations show that this phenomenon is widespread in yeast. 4 Repression of inappropriate transcription is very important, and key factors involved in this function are highly conserved and essential to cell survival. This includes histone chaperone proteins such as Spt6, Asf1, and the Facilitating Chromatin Transcription (FACT) complex (5)(6)(7).
In contrast to these factors, there is no evidence of the involvement of the replication-associated histone chaperone chromatin assembly factor-1 (CAF-1) in the repression of spurious transcription. This suggests the existence of a specific role for some histone chaperones associated with transcription. Interestingly, the newly discovered factor Rtt106 was exclusively linked to the CAF-1 function (8). Rtt106 possess a histone chaperone activity and interacts both functionally and physically with CAF-1 and histones H3/H4 (8). Moreover, Rtt106 and CAF-1 play an important role in heterochromatin silencing by controlling the spreading of the Sir proteins in yeast (9). Therefore, the new histone chaperone Rtt106 was proposed to connect the S-phase to epigenetic inheritance in yeast (8).
In this report, we describe a previously uncharacterized function of Rtt106 that is independent from its functional interaction with CAF-1. Our data demonstrate that Rtt106 is a histone chaperone involved in transcription elongation. Indeed, we found that Rtt106 interacts functionally and genetically with various elongation factors. Importantly, in the absence of Rtt106, we observe a derepression of spurious transcription from the cryptic promoter of the model gene FLO8. This phenotype is generally associated with a defect in the adequate refolding of chromatin structure after RNAP II passage (5,10). Therefore, both molecular evidence and genetic evidence link Rtt106 to the modulation of chromatin associated with elongation. In addition, ChIP assays clearly show that Rtt106 associ-ates with transcribed regions of chromatin. Finally, using an in vivo histone deposition assay, we found that Rtt106 is important for the deposition of new histones in the transcribed regions of active genes. Altogether, our data show a new role for the histone chaperone Rtt106 in the chromatin modulations within transcribed regions.

MATERIALS AND METHODS
Saccharomyces cerevisiae Strains, and Genetics Methods-All genotypes of the S. cerevisiae strains used in this study are listed in supplemental Table S1. The synthetic genetic array screens were conducted as described in Ref. 11. Deletions or promoter replacement of genes were performed by homologous recombination and standard yeast genetics methods. The GAL1-FLO8-HIS3 was described previously (12). Efficient G 1 arrest (at least 95%) of cells was achieved by adding 500 ng/ml ␣-factor for 2-3 h. All oligonucleotide sequences are listed in supplemental Table S2.
Chromatin Immunoprecipitation-ChIP analyses were performed as described previously (12,13). Northern blot analyses were performed as described in Ref. 12.

RESULTS
We showed that the high mobility group-like factor Spt2/ Sin1 plays an important role in chromatin structure modifications during transcription elongation (12). We conducted a synthetic genetic array (SGA) using the spt2⌬ strain as the query strain (12). This method allowed us to construct and analyze double mutants in which spt2⌬ was combined with deletions in most of the non-essential genes of S. cerevisiae. We found several candidates, among which was the gene encoding the histone chaperone Rtt106 (supplemental Fig. S1). It has specific affinity toward histone H3 and H4 and was suggested to function in the S-phase (8). In vivo, Rtt106 interacts physically with the CAF-1 that is involved in chromatin assembly coupled to replication and also in heterochromatin silencing in yeast (8,9). Since Spt2 function is tightly associated with transcription elongation, our finding of synthetic interaction between spt2⌬ and the mutation in RTT106 could suggest a specific replication-independent role of this new histone chaperone.
To get more insights into the biological function of Rtt106, we conducted another SGA screen using, this time, rtt106⌬ as the query strain. In Fig. 1A, we show all the potential interactions of Rtt106 identified in our SGA screen. We found 58 candidates representing different cellular functions. Interestingly, our analysis of gene ontology terms revealed an enrichment of genes involved in DNA replication and CAF-1 function (p value of 0.00015). This is consistent with previous reports showing a functional link between Rtt106 and CAF-1 (8,14). Importantly, we also found an enrichment of genes involved in transcription by RNAP II (Fig. 1A, genes in green). It is interesting to note that this group is significantly enriched in factors that are mainly involved in the elongation step (p value of 0.003). As shown in Fig. 1A (in green), Rtt106 interacts with Spt4, which is a subunit of the elongation factor DSIF, the PAF elongation complex component Leo1, elongation factor 1 (Elf1), and the Ctk1 kinase, which controls the transition from transcription initiation to elongation through the phosphorylation of the largest RNAP II subunit Rpb1 C-terminal domain repeats on the serine 2 residue. These data suggest a potential role of Rtt106 in transcription that is independent from its function with CAF-1 in both chromatin assembly coupled to DNA replication and heterochromatin silencing.
Genetic Evidence That Rtt106 Interacts with Transcription Elongation Factors-To examine this possibility further, we tested the interaction between rtt106⌬ and the rpb1⌬-104 mutant, which is deleted in several C-terminal domain repeats. As shown in Fig. 1B, the double mutant rtt106⌬ rpb1⌬-104 grew more slowly than either single mutant. This synthetic phenotype confirms a link between Rtt106 and the transcription process. In addition, we found that rtt106⌬ strains containing the suppressor of Ty insertion (SPT) phenotype reporter lys2-128␦ are also able to grow on medium lacking lysine, indicating an SPT phenotype of these mutants (Fig. 1C). An SPT phenotype is presumably associated with genes that have a clear connection to transcription and chromatin structure regulation. It is interesting to note that in contrast to rtt106⌬ and in parallel to the wild type strain, the CAF-1 subunit mutant cac1⌬ is not able to grow on medium lacking lysine (Fig. 1C). This further indicates that Rtt106 possesses a specific function in transcription that is independent of the CAF-1 factor. B, Rtt106 interacts genetically with the RNA polymerase II largest subunit Rpb1. Serial dilutions of cell cultures from wild type (WT), rtt106⌬, rpb1⌬-104, and rtt106⌬ rpb1⌬-104 strains were grown on the indicated media for 3 days at the indicated temperature. C, the mutant rtt106⌬ has an SPT phenotype. Serial dilutions of cell cultures from wild type, rtt106⌬, cac1⌬, and cac1⌬ rtt106⌬ containing the SPT reporter allele lys2-128␦ were grown on rich (YPD) or minimal media lacking lysine (SC-Lysine) at the indicated temperature for 3-4 days. D, rtt106⌬ interacts genetically with members of the PAF elongation factor complex. Serial dilutions of cell cultures from wild type, rtt106⌬, paf1⌬, rtt106⌬ paf1⌬, ctr9⌬, and rtt106⌬ ctr9⌬ were grown on rich media (YPD) for 3 days at the indicated temperatures.
To confirm a potential Rtt106 function in elongation, we studied the possible interaction with the PAF complex members. The PAF complex plays an important role in chromatin modulation associated with transcription elongation. It coordinates the recruitment of different epigenetic marks that are of great importance for the regulation of chromatin structure dur-ing elongation (reviewed in Ref. 4). We constructed pairwise double mutants of rtt106⌬ and each of the PAF genes (Fig. 1D). In addition to Leo1 that was identified in our SGA screen, we found that the rtt106⌬ paf1⌬ and rtt106⌬ ctr9⌬ grew more slowly than each single mutant. Moreover, the double mutants did not grow at 33°C, indicating a strong synthetic phenotype.
The genetic interactions between Rtt106, Spt2, Spt4, PAF, and Elf1 factors indicate a strong link between Rtt106 and the transcription elongation process. This is further supported by our observation that rtt106⌬ genetically interacts with mutations in genes encoding several other elongation factors, including TFIIS, the DSIF subunit Spt5, and the histone H3 Lys-36 methyltransferase gene Set2 (supplemental Fig. S2).
Rtt106 Is Recruited to Transcribed Chromatin Regions-Taken together, the results described above strongly indicate that Rtt106 is involved in transcription elongation. To test whether this new Rtt106 function is direct, we asked whether this factor is physically associated with actively transcribed regions. To this end, we analyzed by ChIP assays the association of Rtt106 across the constitutively active gene PMA1 and to a non-transcribed locus (intergenic region of chromosome V, hereafter called NoORF). As shown in Fig. 2A, after immunoprecipitation with an antibody directed against Rtt106-Myc, we observed a significant enrichment at both the coding region (probe C) and the 3Ј-untranslated region (probe D) versus the non-transcribed region (NoORF:probe A). In contrast, we did not observe a significant enrichment at the non-transcribed upstream activation sequence of PMA1 (probe B). In Fig. 2B, in addition to different PMA1 locations, we analyzed by ChIP-QPCR the association of Rtt106 to several transcribed regions of active genes. Our data confirm that Rtt106 is significantly associated with transcribed chromatin. Since Rtt106 has been suggested to play a role during the S-phase, we wanted to exclude the possibility that its association with chromatin is linked to DNA replication. To this end, FIGURE 2. Rtt106 is functionally and physically associated with transcription elongation. A, Rtt106 is associated with the transcribed regions of PMA1. Chromatin immunoprecipitations were performed using anti-Myc antibody to immunoprecipitate Rtt106-13Myc. The horizontal bars in the diagram represent the regions analyzed by quantitative PCR; each reaction analyzing PMA1 and intergenic V regions (NoORF) was separated on a polyacrylamide gel. A representative of duplicate experiments is shown. UAS, upstream activation sequence. B, Rtt106 is associated with the transcribed regions of several genes. ChIP assays were performed as described in A except that the DNA was quantified using real-time PCR. The chromatin used as control was extracted from an untagged strain, and the values shown represent the average and standard error of three independent experiments. C, Rtt106 is essential to the inhibition of transcription initiation from the cryptic promoter of the pGAL1::FLO8::HIS3 reporter gene. The reporter construct is diagrammed in the top part of the figure. Shown below are serial dilutions of wild type (WT), spt2⌬, and rtt106⌬ strains containing the pGAL1::FLO8::HIS3 reporter construct that were grown on synthetic complete medium (SC) or medium lacking histidine and containing galactose as the carbon source (SC-His-Gal). D, rtt106⌬ interacts genetically with Spt6. Cells from wild type, rtt106⌬, spt6-1004, and rtt106⌬ spt6-1004 were grown on rich medium (YPD) for 3 days at the indicated temperature. E, Rtt106 inhibits transcription initiation from the FLO8-HIS3 cryptic promoter. Total RNAs from wild type and rtt106⌬ strains were analyzed by a Northern blot with a probe for HIS3. SCR1 served as a loading control. F, Rtt106 and Spt6 collaborate to inhibit transcription initiation from the FLO8 cryptic promoter. Total RNAs from wild type, rtt106⌬, spt6-1004, rtt106⌬, and spt6-1004 strains grown at 25°C were analyzed by a Northern blot with a probe for FLO8. SCR1 served as a loading control.
we analyzed by ChIP assay the association of Rtt106 with the coding regions of several genes in G 1 -arrested cells but did not find it to be significantly different from that seen in exponentially growing cells (supplemental Fig. S3). Therefore, our data show association of Rtt106 with the coding region of active genes and exclude the possibility that this recruitment is linked to the role of Rtt106 during S-phase.
Rtt106 Is Critical for the Repression of Spurious Transcription from Cryptic Promoter-To test further the role of Rtt106 in elongation, we used a reporter system sensitive to transcription elongation defects in vivo. This reporter is based on previous studies showing that cryptic promoters exist within the coding regions of certain genes and can become active in particular transcription elongation mutants (5). This phenotype has been most extensively characterized for the FLO8 gene (5,10,12,15). To test whether an rtt106⌬ mutation allows cryptic initiation, we used a reporter for FLO8 cryptic initiation in which the 3Ј-coding region of FLO8 has been replaced with the HIS3 coding region such that HIS3 is only expressed when the FLO8 cryptic promoter is active (12). Using this reporter, we tested the expression of FLO8-HIS3 in wild type, rtt106⌬, and spt2⌬ mutant strains by assaying growth on medium lacking histidine. Our results (Fig. 2C) show that, similar to spt2⌬, rtt106⌬ allows growth on medium lacking histidine, indicating HIS3 expression from the FLO8 cryptic promoter. After that, we tested cryptic initiation at FLO8-HIS3 more directly and performed Northern hybridization analysis using a HIS3 probe. In addition to the full-length FLO8-HIS3 transcript, we observed a short HIS3 transcript only in the rtt106⌬ mutant (Fig. 2E). This observation confirms that in the absence of Rtt106, the repression of cryptic transcription is impaired and suggests that Rtt106 plays a direct role in regulation of chromatin structure during elongation.
Rtt106 Cooperates with the Essential Elongation Factor Spt6 to Inhibit Spurious Transcription-Recent studies show that Spt6 plays a central role in the refolding of normal chromatin in the wake of transcription. It controls the level of histone H4 over transcribed regions and also modulates the histone H3 and H4 acetylation through the control of H3 Lys-36 methylation (5,10). Our results indicate that Rtt106 may play an important role in the refolding of repressing chromatin structure at the transcribed region of active genes and suggest a potential functional link between Rtt106 and the essential elongation factor Spt6. To address this possibility, we constructed the double mutant rtt106⌬ spt6-1004 and assayed growth in different conditions. We observed synthetic growth defects in the double mutant at 33°C (Fig. 2D). This phenotype could suggest that both factors are required to regulate chromatin structure at the transcribed regions and thereby inhibit spurious transcription. We addressed this possibility by asking whether the double mutant rtt106⌬ spt6-1004 produces higher levels of transcripts from cryptic promoters. For that, we performed Northern blot analyses using a FLO8 probe to study the endogenous FLO8 gene transcripts in different strains (Fig. 2F). We found that both rtt106⌬ and spt6-1004 produce short transcript indicative of spurious transcription. However, the level of FLO8 short transcript is significantly increased in the double mutant rtt106⌬ spt6-1004, suggesting that the two factors cooperate to inhibit cryptic promoters and spurious transcription.
Rtt106 Plays an Important Role in Chromatin Assembly Coupled to Transcription-These genetic and molecular data suggest a role for Rtt106 in the regulation of chromatin structure during transcription elongation. To gain additional insights into the role of Rtt106 in this process, we wanted to know whether this histone chaperone is involved in the histone dynamics associated with transcription elongation. To address this, we asked whether the deletion of RTT106 would affect the levels of histone H3 exchange in the coding region of the inducible GAL1 or the constitutive PMA1 genes. We used a histone exchange assay previously described by us and others (13,16,17). In this system, there are two different sources of histone H3 in the cell, the endogenous histone tagged with the Myc epitope and a galactose-inducible form fused to the FLAG tag coexpressed with histone H4. To eliminate the contribution of DNA replication-dependent histone deposition, exponentially growing cells containing the double histone H3 tag system are blocked in G 1 with ␣-factor. After incubation with ␣-factor, cells are either fixed or induced to express FLAG-H3 prior to formaldehyde treatment to cross-link chromatin. Next, the levels of total H3, FLAG-H3, and RNAP II are assayed by standard ChIP-QPCR (Fig. 3A). As shown in Fig. 3B, after induction of the new histone H3 in galactose medium, we observe a high FIGURE 3. Rtt106 contributes to the transcription-dependent histone H3 deposition in transcribed regions of active genes. A, a schematic explaining the experimental procedure. B, Rtt106 is required for the incorporation of new histones H3 at transcribed regions of active genes. Yeast cells from the wild type (WT) or rtt106⌬ strains containing the histone double tag system described in Ref. 13 were arrested in G 1 by ␣-factor. After that, the cells were formaldehyde-fixed or shifted to galactose medium for 60 min prior to formaldehyde treatment. ChIP assays were then performed using anti-FLAG, antihistone H3, and anti-Rpb1 antibodies. The values reported for the incorporation of new H3 represent the percentage of IP relative to histone H3 occupancy. The value of the wild type after galactose induction was set to 1. All values shown represent the average and standard error of three independent experiments.
ACCELERATED PUBLICATION: Rtt106 Role in Transcription OCTOBER 10, 2008 • VOLUME 283 • NUMBER 41 level of incorporation at both GAL1 and PMA1 coding regions in wild type strain. These levels are consistent with those previously reported (13). Importantly, although we do not observe a significant change in Rpb1 association (Fig. 3B), deletion of RTT106 results in a significantly reduced incorporation of histone H3 at the coding regions of GAL1 and PMA1. It is interesting to note that in the absence of Rtt106, histone deposition at the non-transcribed intergenic V region (NoORF) is not decreased. These observations clearly indicate that in the absence of Rtt106, new histone deposition in the wake of the RNAP II is reduced. Therefore, our data strongly suggest that the Rtt106 histone chaperone regulates transcription-dependent histone H3 deposition during elongation to ensure that transcribed regions regain normal chromatin structure in the wake of transcription.

DISCUSSION
Previous studies have suggested that Rtt106 is a histone chaperone protein that plays a role in heterochromatin silencing through a link to the S-phase and DNA replication-dependent chromatin assembly (8,9). The present study shows several new results that strongly indicate an important role of Rtt106 during transcription elongation. First, our synthetic lethal screen using rtt106⌬ and the yeast deletion library uncovered several genes involved in transcription and, more specifically, in transcription elongation. Second, a direct test of genetic interactions between rtt106⌬ and mutants of genes encoding elongation factors confirmed a strong genetic link between this histone chaperone and transcription elongation. Third, our ChIP data clearly show that Rtt106 is physically associated with transcribed regions. Fourth, in the absence of Rtt106, we observe a derepression of spurious transcription from the FLO8 cryptic promoter. This provides strong evidence that Rtt106 is required for chromatin structure refolding in the wake of transcription elongation. Finally, we show that Rtt106 is important for normal transcription-dependent histone H3 deposition at active genes. This result indicates that Rtt106 is involved in the histone dynamics that are tightly associated with an elongating RNA polymerase II.
We have previously shown that in highly expressed genes, newly synthesized histone H3 is incorporated in the transcribed regions of those genes (13). This observation suggested that nucleosomes are disassembled in front of RNAP II and reassembled in its wake using new histones at these locations. Our new data clearly indicate that Rtt106 is important for the deposition of new histones in the highly transcribed genes GAL1 and PMA1. This effect is similar to the one we observed in asf1⌬ in a previous study (13). Therefore, it is possible that Rtt106 provides new histones H3/H4 to the machinery that reassemble nucleosomes in the wake of transcription at highly transcribed genes. Alternatively, Rtt106 could deposit histones directly after the RNAP II passage. Regardless of the exact mechanism, redeposition of nucleosomes in the wake of RNAP II is impaired in the absence of Rtt106, and this provides strong evidence that this factor plays an important function in transcription-coupled chromatin assembly.
The Spt6 histone chaperone is essential for the maintenance of histone H4 over transcribed regions and thereby for normal nucleosomal occupancy (5). Moreover, Spt6 is required for methylation of histone H3 on lysine 36, which mediates subsequent deacetylation of histone H3 and H4 by Rpd3 at coding regions (10). This deacetylation plays an essential role in stabilizing the chromatin structure after RNAP II passage, resulting in inhibition of cryptic promoters (4). Our genetic and molecular data show that Rtt106 cooperates with Spt6 to repress the FLO8 cryptic promoter. It is possible that Rtt106 plays a significant role in the Spt6 pathway described above. It may cooperate with Spt6 to maintain a normal level of histones and, therefore, adequate nucleosomal occupancy. Alternatively, similarly to other histone chaperones that were shown to be required for some histone modifications, Rtt106 may stimulate the histone methyltransferase activity of Set2 and therefore regulate indirectly the acetylation status of histones H3 and H4. A future study distinguishing between these possible mechanisms will be undoubtedly interesting.