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,
Joseph T. Wade, and
Kevin Struhl*
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Received 14 October 2005/ Returned for modification 23 December 2005/ Accepted 16 February 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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25 kDa, and they contain HMG boxes that partially intercalate the DNA in the minor groove and cause a sharp bend. HMGB proteins have a preference for binding to distorted DNA, and they are believed to function as architectural proteins that stabilize multiprotein complexes on DNA. HMGB proteins are found in many eukaryotic species, and they are involved in a number of cellular processes, including transcription, replication, and DNA repair. However, little is known about the genomic association and biological functions of these proteins. The yeast Saccharomyces cerevisiae contains 10 HMG proteins, including the HMGB protein, Hmo1. Hmo1 has two HMG boxes, A and B. Box A has low affinity for DNA with some structural specificity, whereas box B has higher affinity for DNA with lower structural specificity (16). Strains lacking Hmo1 show a decreased growth rate, higher rates of plasmid loss, and increased sensitivity of chromatin to micrococcal nuclease treatment (23). Hmo1 interacts genetically and physically with FKBP12, a conserved prolyl isomerase (8), and it also plays a role in mutagenesis control (2). Hmo1 also plays a role in transcription of the rRNA (9).
In S. cerevisiae, the ribosome consists of the 5S, 5.8S, 18S, and 25S rRNAs as well as 137 ribosomal proteins (RPs). The 5S RNA is transcribed by RNA polymerase (Pol) III, the 5.8S, 18S, and 25S RNAs are transcribed as a single unit by Pol I, and the RP genes are transcribed by Pol II. Together this represents more than 70% of total cellular RNA and approximately 50% of all mRNA (43). Pol I-directed transcription of rRNA requires the factors TATA binding protein (TBP), core factor (CF), and upstream activation factor (UAF), whereas an additional factor, upstream binding factor (UBF), is required for mammalian cells. UBF contains six HMG boxes and binds throughout the repeated rRNA gene locus (30), and it shows remote sequence similarity to Hmo1 (9).
Transcription of all these genes is regulated positively in response to growth stimuli and negatively in response to environmental stress, and coordinated regulation of these processes is critical for ribosome synthesis and cell growth (43). During rapid growth, Fhl1 and Rap1 bound at RP promoters recruit Ifh1, which in turn activates transcription to maximal levels (25, 34, 35, 42). Following stress, Ifh1 dissociates from RP promoters and Fhl1 recruits the inhibitory factor Crf1, resulting in decreased transcription (25). An additional factor, Sfp1, is also required for maximal transcription from RP promoters. Sfp1 associates with RP promoters specifically under conditions of rapid growth and is exported from the nucleus following stress (15, 24), but its precise role in RP transcription is unclear.
Here we use chromatin immunoprecipitation coupled with microarray analysis to identify the physiological targets of Hmo1 on a genome-wide level. Although Hmo1 exhibits minimal DNA binding specificity in vitro, we show that Hmo1 associates strongly and specifically with most RP promoters at an IFHL sequence motif. Our results strongly suggest that Hmo1 binds cooperatively with Rap1 and Fhl1 to RP promoters, whereas Hmo1 binding at the rRNA gene locus occurs independently of Rap1 and Fhl1. Surprisingly, loss of Hmo1 abolishes binding of Fhl1 and Ifh1 to RP promoters but does not significantly affect the level of transcriptional activity, suggesting that proteins other than Fhl1 and Ifh1 play an important role in RP transcription. Lastly, we show that Hmo1 is also required for both transcription and processing of the rRNA and, like mammalian UBF, associates throughout the rRNA gene locus. We propose that Hmo1 is involved in the coordination of rRNA and RP gene transcription.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hmo1 (yDH544) strain was generated from the wild-type strain (BY4741), which was obtained from Research Genetics. This was done because the hmo1 deletion strain in the Research Genetics collection contains a copy of the wild-type HMO1 locus and does not show the expected slow-growth phenotype. Strain yDH419, which was used for the experiments depicted in Fig. 1 and 3, is derived from BY4742 (Research Genetics) with Hmo1 C-terminally tagged with three myc epitopes using a previously described method (36). For strains containing derivatives of the RPS11B promoter (see Fig. 4A), yDH419 was integrated at the HIS3 locus with HPIpV4 plasmid containing RPS11B promoter derivatives (26). For the experiment depicted in Fig. 4B, strain yDH419 was transformed with plasmid YCplac111, containing sequence from the PGK1 promoter from position –480 to position –420 relative to the ATG with the binding sites for Gcr1 mutated and with or without an optimized IFHL motif (CCAGGCGGAA). Strain yDH548 (see Fig. 7A) was created by sporulating a fhl1 derivative of the diploid BY4743 (Research Genetics) to yield MATa his31 leu20 ura30 met150 lys20 fhl1, which was tagged at the C terminus of Hmo1 with three myc epitopes as described for yDH419. Strain yJTW5 (see Fig. 7B) was created by crossing yDH544 with yJTW4 (TAP-tagged Ifh1, myc-tagged Fhl1 [42]). The resultant diploid strain was sporulated to give yJTW5, which expresses C-terminally TAP-tagged Ifh1 and C-terminally myc-tagged Fhl1 and also contains a hmo1 mutation. yJTW4 was used as a control for these experiments.
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Genome-wide analysis of Hmo1 association. Hmo1 targets were identified in strain yDH419. Microarrays containing duplicate spots of 6,528 PCR products corresponding to nearly all yeast intergenic regions were hybridized with a mixture of amplified immunoprecipitated (labeled with Cy5 fluorescent dye) and input (labeled with Cy3 dye) samples as described previously (28). A control experiment was performed using the untagged strain BY4742, and all values of Hmo1 association are normalized to the values in this control experiment. Conserved DNA motifs were identified using AlignACE (33). WebLogo was used to generate the IFHL motif logo (7).
Transcriptional analysis. Total RNA was purified using QIAGEN RNeasy columns with DNase I treatment. For analyzing transcription of individual genes, first-strand cDNA was synthesized using a random hexamer, and quantitative PCR in real time was performed on the resulting first-strand cDNA using primers specific to the gene of interest (42). RNA levels were determined relative to the ACT1 control gene. For whole-genome transcriptional analysis, single-stranded cDNA was copied to make second-strand cDNA using DNA polymerase and RNase H (32). DNA was then amplified and hybridized to microarrays containing duplicate spots of PCR products corresponding to nearly all yeast open reading frames, as described previously (32).
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RESULTS |
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The microarray analysis identified a small number of non-RP promoters as Hmo1 targets, including the HMO1 promoter itself. Three out of four non-RP promoters (the exception being the SUC2 promoter) identified by the microarray analysis show significant occupancy by Hmo1 (Fig. 1B), indicating that the majority of non-RP targets are genuine. Additionally, several non-RP promoters that bind Rap1 show low but significant Hmo1 occupancy (Fig. 1C). In contrast, Hmo1 does not associate with telomeric regions that bind Rap1 (Fig. 1C). Taken together, these observations suggest that Hmo1 association can be significantly influenced, but not strictly determined, by Rap1.
RP genes are among the most highly transcribed genes of all organisms. Hence, Hmo1 is associated with the promoters of many highly transcribed genes. We determined the relationship between transcription and Hmo1 occupancy for non-RP promoters. Interestingly, non-RP promoters associated with high levels of Hmo1 tend to be more highly transcribed (Fig. 1D), suggesting a role for Hmo1 in transcription activation.
Hmo1 associates with the IFHL motif at RP promoters. Using AlignACE, we identified three sequence motifs strongly overrepresented among the genomic regions bound by Hmo1. These include an A-rich motif known to be present at many RP promoters (10), the Rap1 binding site, and the IFHL motif (Fig. 2A). AlignACE did not identify the IFHL motif in the RP promoters not bound by Hmo1, whereas it did identify the A-rich motif and the Rap1 binding site. The IFHL motif has previously been linked to the association of Fhl1 and Ifh1 with RP promoters (42), and it is also present at a number of the non-RP promoters bound by Hmo1.
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Hmo1 associates at many locations throughout the rRNA gene locus. Genetic analysis indicates that Hmo1 affects Pol I-directed transcription of the rRNA gene (9), although there is no evidence for a direct role. Although we did not identify the rRNA gene locus as a target of Hmo1 in the microarray analysis, both of the microarray probes corresponding to the rRNA gene ranked in the top 2% of genomic regions with respect to Hmo1 association. Direct analysis indicates that Hmo1 association is high (25- to 50-fold) across the entire Pol I-transcribed region of the rRNA gene, whereas Hmo1 association is lower at the Pol III-transcribed 5S RNA gene (Fig. 3E). Rap1 does not associate with the rRNA gene region, and this indicates that Hmo1 association at the rRNA gene locus is independent of Rap1. It is unclear whether Hmo1 association is due to the presence of multiple IFHL motifs throughout the Pol I-transcribed region or is due to association with Pol I itself.
Rap1 is important for Hmo1 association at RP promoters, whereas the IFHL motif plays a minor role. To specifically determine the role of the Rap1 binding site and the IFHL motif in association of Hmo1 with RP promoters, we analyzed two classes of artificial promoters. First, we analyzed derivatives of the RPS11B promoter in which the Rap1 sites are deleted or replaced either by the upstream activating sequence (UAS) of another RP promoter, RPS8A, a minimal UAS containing just the Rap1 binding sites from RPS8A, and a mutated version of this minimal UAS in which the Rap1 binding sites are destroyed (Fig. 4A). Hmo1 only associates with the promoters that contain functional Rap1 binding sites, strongly suggesting that Rap1 is required for association of Hmo1 with RP promoters. Second, we analyzed artificial promoters consisting of the Rap1 binding site from the PGK1 promoter and either a consensus or a mutated IFHL motif (Fig. 4B). Hmo1 occupancy is high at both promoters, with little if any significant difference in the level of association. Taken together, these results suggest Rap1 is sufficient for Hmo1 association with RP promoters, and that the IFHL motif plays a limited role. In support of this, Hmo1 did not associate with constructs containing multimerized IFHL motifs (data not shown).
Effect of heat shock on Hmo1 binding. Transcription of RP genes decreases rapidly following a heat shock. We therefore determined the level of Hmo1 association with RP and non-RP targets before and after a heat shock (Fig. 5). Hmo1 association with RP promoters drops approximately twofold following a heat shock, although binding is still high. Hmo1 association also decreases slightly at the TYE7 and HMO1 promoters. In contrast, at the TSL1 promoter, which is transcriptionally activated upon heat shock, Hmo1 binding increases slightly following a heat shock. Thus, in response to heat shock, Hmo1 association at target promoters correlates with transcriptional activity, but the magnitude of this effect is subtle.
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The above experiments do not identify RP genes as being significantly regulated by Hmo1. We confirmed this observation by showing that there is no significant difference in RNA levels between the wild-type and hmo1 deletion strains for any of the six RP genes tested (Fig. 6A). Furthermore, the wild-type and hmo1 deletion strains behave indistinguishably with respect to the rapid reduction of RP RNA levels in response to heat shock (data not shown). In addition, the wild-type and hmo1 deletion strains show similar levels of 5S RNA, a gene transcribed by Pol III (Fig. 6B). As expected (9), the hmo1 strain shows twofold reduced levels of 18S and 25S rRNAs, which are transcribed by Pol I.
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Cooperative binding of Hmo1 and Fhl1 to RP promoters in vivo. Fhl1 and Hmo1 have remarkable similarities with respect to their ability to bind RP promoters. Both proteins bind to a large subset of RP promoters in a Rap1-dependent manner, and they preferentially associate with the IFHL motif (42). Furthermore, two large-scale interaction studies identified a physical interaction between Fhl1 and Hmo1 (12, 14). We therefore determined whether Hmo1 and Fhl1 affect each other's binding at RP promoters. Hmo1 binding at the two RP promoters tested was significantly reduced (three- to fivefold) in the fhl1 deletion strain relative to the wild-type strain, whereas binding to non-RP targets was either unchanged or significantly increased (Fig. 7A). Interestingly, Hmo1 association at the rRNA gene locus (RDN18) was increased threefold, suggesting that the rRNA gene and RP loci compete for binding Hmo1 in a manner influenced by Fhl1. Conversely, at all RP promoters tested, Fhl1 binding was virtually eliminated in the hmo1 deletion strain (Fig. 7B). As expected from the observation that Fhl1 recruits Ifh1 to RP promoters (25, 34, 35, 42), Ifh1 binding was also drastically reduced in the hmo1 deletion strain (Fig. 7C). These results strongly suggest Fhl1 and Hmo1 bind cooperatively to RP promoters in vivo. The observation that loss of Hmo1 abolishes Fhl1 binding but does not significantly affect RP transcription is very unexpected, and it challenges current views (25, 34, 35, 42) on the regulation of RP transcription (see Discussion).
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DISCUSSION |
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As HMG proteins have intrinsically low sequence specificity for DNA, the specific association of Hmo1 across the yeast genome must be due to recruitment by other proteins with higher DNA specificity. At RP promoters, a functional DNA binding site for Rap1 is required for recruitment of Hmo1, suggesting that Rap1 recruits Hmo1 (Fig. 4A). Furthermore, an isolated Rap1 site is sufficient to recruit to Hmo1 (Rap1-X construct in Fig. 4B). Rap1-dependent Hmo1 binding might be due to a direct interaction between Rap1 and Hmo1 or a more complex interaction involving a bridging protein(s) such as Fhl1. Although an isolated Rap1 site is sufficient to recruit Hmo1, Rap1 binding at natural genomic locations does not necessarily result in Hmo1 association (Fig. 1C). For example, Hmo1 is not detected at PGK1, even though the Rap1 site in the PGK1 promoter efficiently recruits Hmo1 when isolated in the Rap1-X construct. In addition, Hmo1 is not detected at the telomere, which is strongly bound by Rap1. Taken together, these observations suggest that Rap1 is sufficient to recruit Hmo1, but that other Rap1 interaction proteins can block Hmo1 recruitment. Such putative blocking proteins might include Gcr1 (and Gcr2), which interacts with Rap1 at glycolytic promoters such as PGK1 (22, 40), and Rif1 (or Rif2), which interacts with Rap1 at the telomere and is important for silencing (11, 44).
Hmo1 also binds to several regions not bound by Rap1, suggesting that proteins other than Rap1 are important for Hmo1 recruitment. Of particular interest, Hmo1 associates throughout the rRNA gene locus, which is not bound by Rap1. The rRNA gene locus appears to have multiple copies of the IFHL motif, but it seems unlikely that this motif is sufficient for Hmo1 recruitment (see below). Given that Hmo1 does not associate at a specific location but rather throughout essentially the entire rRNA gene locus, we speculate that RNA polymerase I, or an associated factor, may be important for Hmo1 recruitment.
Cooperative binding of Hmo1 and Fhl1 at the IFHL motif.
The IFHL motif is enriched at Hmo1 target regions, including RP promoters and the rRNA gene, suggestive of a role in Hmo1 recruitment (Fig. 2A). The IFHL motif plays an important role in transcription from RP promoters (42), and we show here that Hmo1 binding at RP promoters is centered on the IFHL motif rather than the Rap1 site (Fig. 3A to D). However, mutation of the IFHL motif has little effect on the level of Hmo1 binding (Fig. 4B). Interestingly, binding of Fhl1 and Ifh1 at RP promoters mirrors that of Hmo1: binding of Fhl1 and Ifh1 depends on Rap1 and is centered on the IFHL motif but is barely affected by mutation of this motif (42). To account for these observations, we propose that Hmo1, Fhl1, and associated proteins (e.g., Ifh1) are recruited to RP promoters by Rap1 and are then positioned by the IFHL motif. In this view, the IFHL motif is preferred over flanking sequences and contributes to overall affinity for Hmo1 and Fhl1, but it does not impart sufficient specificity to permit binding in the absence of Rap1. In support of this theory, the IFHL motif is typically located 50 to 200 bp downstream of the Rap1 binding site, and the Rap1 binding sites at promoters with high Hmo1 occupancy are typically 150 bp further upstream of the translation start site than RP promoters with low Hmo1 occupancy. As the IFHL motif is required for maximal transcription from RP promoters (42), it seems likely that the relative locations of the Rap1 binding site, the IFHL motif, and the core promoter region are functionally important.
Hmo1 binding is significantly reduced in the absence of Fhl1, and association of Fhl1 (and Ifh1) is essentially abolished in the absence of Hmo1 (Fig. 7). As Fhl1 and Hmo1 physically interact in vivo (12, 14), the combined results strongly suggest that Fhl1 and Hmo1 bind cooperatively to RP promoters via a physical interaction between the two proteins. It is unknown which, if either, of these proteins directly interacts with the IFHL motif, but we favor the view that Hmo1 has a more direct role. Hmo1 can bind to some extent in the absence of Fhl1, whereas Fhl1 fails to bind in the absence of Hmo1. Hmo1 associates with the rRNA gene locus, which contains multiple IFHL motifs, whereas Fhl1 does not, and Hmo1 can bind DNA in vitro via its two HMG domains (16).
A potential role for Hmo1 in coordinating expression of rRNA and ribosomal protein genes. Hmo1 is required for maximal transcription of the rRNA gene by Pol I (9), a result we confirm here, and it also is important for efficient rRNA maturation (Fig. 6B). Hmo1 associates strongly with essentially the entire rRNA gene locus (Fig. 3E), strongly suggesting that it directly influences transcription and maturation of rRNA. Based on the role of Hmo1 in rRNA synthesis and the weak sequence similarity between Hmo1 and UBF, a component of the mammalian Pol I transcription machinery, it has been suggested that Hmo1 functions analogously to UBF (9). Our results provide strong support for this idea, because UBF is also associated with the entire rRNA gene locus in mammalian cells (30). It has been proposed that both UBF and Hmo1 define an rRNA gene-specific chromatin structure, but the mechanisms by which Hmo1 affects rRNA transcription or maturation are unknown.
Ribosomes are composed of rRNAs and ribosomal proteins, and the syntheses of these components are coordinately regulated in response to growth stimuli and environmental stress (43). Our observation that Hmo1 associates strongly with many RP promoters and with the rRNA gene locus indicates that Hmo1 is involved in multiple aspects of ribosome biogenesis. Interestingly, Hmo1 interacts with the NuA4 histone acetylase complex (18), which is recruited to RP promoters in a Rap1-dependent manner (31). In addition, Ifh1 may also connect RP gene and rRNA transcription, as it forms a complex with the rRNA processing factor Utp22 (15; J. T. Wade and K. Struhl, unpublished data). Thus, Hmo1 might be part of the mechanism that coordinates the synthesis of ribosome components synthesized by the Pol I and Pol II transcription machineries, although the role of Hmo1 in RP transcription is unclear (see below).
Implications for the coordinate regulation of RP genes. Rap1 is essential for coordinate regulation of RP genes (17, 20, 26, 27), but it has many other biological functions (29, 37) and hence is not the specific regulator that controls RP transcription. A set of recent studies has strongly implicated Fhl1, and particularly Ifh1, as being the key regulator of RP genes (25, 34, 35, 42). Specifically, Fhl1 and Ifh1 associate almost exclusively with RP promoters, and Fhl1 recruits Ifh1 in a regulated manner that directly correlates with RP transcription. This view is challenged by the striking observation that loss of Hmo1 virtually abolishes Fhl1 and Ifh1 association with RP promoters yet causes no detectable effect on RP transcription (Fig. 6A). While formally possible, it is highly unlikely that the very low levels of Fhl1 and Ifh1 at RP promoters in the hmo1 deletion strain can support normal levels of RP transcription. Strains expressing low levels of Ifh1 via a heterologous promoter show significantly reduced levels of RP transcription (25, 34, 35, 42). Furthermore, the lethal phenotype of an ifh1 deletion is suppressed by an fhl1 deletion, indicating that some RP transcription can occur in the absence of both Fhl1 and Ifh1 (6).
Taken together, these observations strongly suggest that yeast cells have a parallel pathway that regulates RP transcription. This parallel pathway(s) involves proteins other than Hmo1, Fhl1, and Ifh1, but it presumably requires Rap1, because RP promoters lacking Rap1 sites do not respond to environmental stimuli that regulate RP gene expression (17, 20, 26, 27). This parallel pathway may involve proteins associating with the IFHL motif, because deletions that remove this motif show significantly reduced levels of RP transcription (42). It is possible that Sfp1 may play a role in this parallel pathway, because Sfp1 associates with RP promoters in vivo, and its nuclear localization is regulated by environmental conditions that correlate well with RP transcription (15, 24). In any event, our results indicate that the Hmo1-Fhl1-Ifh1 pathway is not essential for control of RP transcription. Instead, this pathway is likely to play a direct, but more subtle, role in regulating the complex biological process of ribosome synthesis in response to environmental conditions.
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ACKNOWLEDGMENTS |
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D.B.H. was supported by a Helen Hay Whitney postdoctoral fellowship, and J.T.W. was supported by a Charles A. King Trust Postdoctoral Fellowship, Bank of America, Co-Trustee (Boston, MA). This work was supported by a research grant to K.S. from the National Institutes of Health (GM30186).
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: Radiation Monitoring Devices, Inc., Watertown, MA 02472-4699.
These authors contributed equally to the work.
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