SPN1, a Conserved Gene Identified by Suppression of a Postrecruitment-Defective Yeast TATA-Binding Protein Mutant

Corresponding author: Laurie A. Stargell, Colorado State University, Fort Collins, CO 80523-1870., lstargel{at}lamar.colostate.edu (E-mail)

Communicating editor: M. HAMPSEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Little is known about TATA-binding protein (TBP) functions after recruitment to the TATA element, although several TBP mutants display postrecruitment defects. Here we describe a genetic screen for suppressors of a postrecruitment-defective TBP allele. Suppression was achieved by a single point mutation in a previously uncharacterized Saccharomyces cerevisiae gene, SPN1 (suppresses postrecruitment functions gene number 1). SPN1 is an essential yeast gene that is highly conserved throughout evolution. The suppressing mutation in SPN1 substitutes an asparagine for an invariant lysine at position 192 (spn1^K192N). The spn1^K192N strain is able to suppress additional alleles of TBP that possess postrecruitment defects, but not a TBP allele that is postrecruitment competent. In addition, Spn1p does not stably associate with TFIID in vivo. Cells containing the spn1^K192N allele exhibit a temperature-sensitive phenotype and some defects in activated transcription, whereas constitutive transcription appears relatively robust in the mutant background. Consistent with an important role in postrecruitment functions, transcription from the CYC1 promoter, which has been shown to be regulated by postrecruitment mechanisms, is enhanced in spn1^K192N cells. Moreover, we find that SPN1 is a member of the SPT gene family, further supporting a functional requirement for the SPN1 gene product in transcriptional processes.

INITIATION of RNA polymerase II (Pol II) transcription in eukaryotic organisms requires the well-orchestrated involvement of a large number of proteins at specific sites on the DNA template (ORPHANIDES et al. 1996 ; HAMPSEY 1998 ). TFIID, a general transcription factor (GTF) containing TATA-binding protein (TBP) and TBP-associated factors (TAFs), nucleates the preinitiation complex by binding the TATA element in a sequence-specific manner. A competent preinitiation complex also requires other GTFs, including TFIIA, -B, -E, -F, and -H, as well as Pol II. In vivo, several of these GTFs and other factors such as Srb/Mediator are thought to preassemble with Pol II in a multicomponent complex (termed the holoenzyme) prior to binding TFIID and DNA (KIM et al. 1994 ; KOLESKE and YOUNG 1994 ; OSSIPOW et al. 1995 ).

TBP occupancy at a promoter correlates strongly with transcriptional output (KURAS and STRUHL 1999 ; LI et al. 1999 ) and recruitment of TBP to the promoter can be a rate-limiting step for transcription initiation (COLGAN and MANLEY 1992 ; TANSEY et al. 1994 ; CHATTERJEE and STRUHL 1995 ; KLAGES and STRUBIN 1995 ; XIAO et al. 1995 ), which is enhanced by the presence of an activator (KLEIN and STRUHL 1994 ). Additionally, transcriptional activators function via protein-protein interactions to enhance recruitment to the promoter of other GTFs (STRINGER et al. 1990 ; INGLES et al. 1991 ; LIN et al. 1991 ; CHOY and GREEN 1993 ; DRYSDALE et al. 1998 ), certain coactivators (DRYSDALE et al. 1998 ; UTLEY et al. 1998 ), the holoenzyme (KOLESKE and YOUNG 1994 ; DRYSDALE et al. 1998 ; NATARAJAN et al. 1999 ), and chromatin remodeling or modifying enzymes (NATARAJAN et al. 1999 ; NEELY et al. 1999 , NEELY et al. 2002 ). In this way, activators contribute positively to the assembly and stabilization of a productive preinitiation complex (HAHN 1993 ; ORPHANIDES et al. 1996 ). In addition to stimulating transcription at the initiation step, certain activators can influence subsequent steps in the transcriptional process. Activators can enhance promoter clearance (KUMAR et al. 1998 ), aid in release of Pol II pausing (BROWN et al. 1996 ), and increase the elongation rate of Pol II (YANKULOV et al. 1994 ). Thus, activators appear to exert influence on both recruitment and postrecruitment steps in the transcriptional process.

One way to bypass the requirement for an activation domain is through artificial recruitment of a member of the preinitiation complex. For example, when wild-type TBP is tethered to DNA via a DNA-binding domain (LexA) located upstream of a reporter gene, an activator is no longer required and LexA-TBP activates transcription of the gene directly (CHATTERJEE and STRUHL 1995 ; KLAGES and STRUBIN 1995 ; XIAO et al. 1995 ). Under these artificial recruitment circumstances, interactions that occur prior to TBP binding to the TATA element, such as interaction with an activation domain, can be bypassed. However, functions that are required after TBP is recruited to the promoter (such as the recruitment of Pol II or promoter clearance) cannot be bypassed with this assay. Although activation-defective alleles of TBP that exhibit postrecruitment defects have been described (STARGELL and STRUHL 1996 ), little is known about the functions of TBP after it binds the TATA element.

We have focused on one particular postrecruitment-defective allele of TBP, F237D (in which the phenylalanine at position 237 is mutated to aspartic acid). To identify factors important in these postrecruitment functions, the F237D allele was used in a genetic screen in which spontaneous mutations that allow LexA-F237D to activate transcription from a reporter gene containing a LexA operator were isolated. Thus, we targeted directly the postrecruitment defect. Here, we describe the initial cloning and characterization of the first of these genes, termed SPN1 (suppresses postrecruitment functions gene number 1). SPN1 is essential in yeast and conserved throughout evolution. Our functional characterization of this gene shows that a mutation in SPN1 alters transcription by Pol II and confers Spt^- phenotypes. Taken together, these results suggest an important role for SPN1 in the mechanisms of transcriptional regulation in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains and DNA:
Strains are shown in Table 1. Most are derived from the FT5 strain, with genotype MAT ura3-52 trp1-63 his3-200 leu2::PET56 (TZAMARIAS and STRUHL 1994 ). A SPN1 deletion strain JF11 was created using a one-step knockout (via LEU2) and a URA3-marked cover plasmid with a genomic EcoRI-HindIII fragment containing the SPN1 gene. Genomic PCR confirmed LEU2 integration at the appropriate locus. In the yeast strain JF12, the upstream region of the HIS3 gene was repaired to wild type by recombination with a wild-type fragment of this region. TRP1-marked plasmids based on YCp22 were constructed, bearing full-length wild-type or K192N mutant SPN1, as well as the N-terminal deletions and C-terminal truncations of SPN1. These were made with and without the myc epitope tag at the N terminus. These plasmids were transformed into JF12, followed by growth on 5-fluoroorotic acid (5-FOA) to cure the strain of the URA3-marked cover plasmid, yielding JF13 (wild-type SPN1), JF14 (spn1^K192N), JF15 (myc-SPN1), and JF16 (myc-spn1^K192N). Immunoprecipitation studies employed the SK1, JF15, and RYP5 strains. RYP5 is derived from BY2 (CORMACK et al. 1991 ). The strains with various activator-binding sites upstream of the HIS3 gene (VI47, VI48, VI66, and VI64) were derived from KY2002 (IYER and STRUHL 1995A ). A one-step genomic knockout of SPN1 was created in each strain, using the same method as described above. The knockout strains were transformed with the YCp22-based SPN1 plasmids (wild type or K192N) and then cured of the URA3-marked cover plasmid to yield the strains that were used for transcriptional analysis: JF17, JF18, JF19, and JF20 (all wild-type SPN1) and JF21, JF22, JF23, and JF24 (all spn1^K192N). Elongation and inositol phenotypes were assayed using strains derived from BY4741 (ResGen). A one-step genomic knockout of SPN1 was created in BY4741 as described above for other strains. To make the LZ1 and LZ2 strains, the knockout strain LZ0 was transformed with HIS3-marked SPN1 plasmids (wild type or K192N) and then cured of the URA3-marked cover plasmid. The LZ1 and LZ2 strains and the control BY4741 spt4 strain were made URA3⁺ prior to spotting for growth on 6-azauracil and mycophenolic acid. Spt^- phenotypes were tested using strains FY120 and FY241 (gift of Fred Winston) and the FY120-derived JF25. The JF25 strain was derived from FY120 by making a one-step genomic knockout of SPN1 using LEU2 and a URA3-based cover plasmid containing spn1^K192N. The phenotypes of various TBP alleles were tested by making a genomic one-step knockout of TBP via LEU2 in the SK1 and JF10 strains. The strains were then transformed with various TBP alleles on TRP1-marked plasmids and cured of the URA3-marked cover plasmid by growth on 5-FOA.

Genetic screen for spontaneous suppression of F237D TBP:
SK10 cells, containing a LexA operator in place of the Gcn4-binding site upstream of the HIS3 gene and harboring the fusion protein LexA-F237D on a URA3-marked plasmid, were spread at 10⁶ cells/plate onto solid medium containing 15 mM 3-aminotriazole (AT) and incubated at 30° until AT-resistant colonies appeared. Strains were analyzed to determine if growth on AT was due to extragenic suppression. First, the LexA-F237D plasmid was shuffled out of each strain by growth on 5-FOA; strains that retained AT resistance were discarded. The remaining strains were then retransformed with the LexA-F237D plasmid and checked for growth on AT. The strains that remained sensitive to AT were discarded. In addition, the shuffled strains were transformed with a plasmid encoding the LexA DNA-binding domain only, and only the strains that remained AT sensitive were retained. Of 758 strains originally isolated, 11 suppressors fit the above criteria.

Cloning by complementation:
The suppressor strain JF10, which has a recessive temperature-sensitive (ts) phenotype, was transformed with a yeast genomic library (no. 95-02A, YCp50 CEN, ATCC). Cells in mid-log growth phase were transformed by the standard lithium acetate method with library DNA (2 µg per 10⁸ cells) and plated to medium lacking uracil at 10⁸ cells/plate. Plates were left at room temperature for 1 hr and then incubated at 38° until colonies appeared. Calculated transformation efficiency was 1650 colonies per microgram of DNA. The library was covered at least six times. Five independent plasmids were able to confer temperature resistance when retransformed. These five plasmids were found to contain an overlapping region of genomic DNA, which contained eight open reading frames (ORFs). A series of plasmids was constructed with various sections of the genomic DNA to determine which ORF was responsible for complementation of the suppressor strain phenotype. The ORF found to confer temperature resistance was YPR133c. The plasmid containing the YPR133c ORF was cut with restriction enzymes and transformed into the suppressor strain to recover the genomic allele of YPR133c by gap repair. Phenotypic analysis of several gapped plasmids for their ability to complement the ts phenotype of the suppressing strain served to map the location of the suppressing mutation. This region was sequenced, and the mutation was identified.

Artificial recruitment assay:
Plasmids encoding LexA fused to various TBP alleles were transformed into the SK1 and JF10 strains, and the resulting transformants were streaked onto solid medium containing 5–15 mM AT. Growth was generally scored after 5 days of incubation at 30°. Failure to grow on AT indicates that the TBP allele has a postrecruitment defect in transcription.

Phenotypic studies:
To assay the Spt^- phenotype, strains FY120, JF25, and FY241 (spt4-3) were spotted in 10-fold serial dilutions onto medium lacking histidine or lysine and scored after 3 days of growth. Growth on both types of media indicates suppression of the insertion mutations. Elongation phenotypes were tested with BY4741-derived strains (LZ1, LZ2, and BY4741spt4) on synthetic complete medium lacking uracil and supplemented with 75 µg/ml 6-azauracil or 20 µg/ml mycophenolic acid. These strains were also tested on synthetic complete medium lacking inositol.

Co-immunoprecipitation:
Co-immunoprecipitation experiments were performed essentially as described previously (MOQTADERI et al. 1996 ) with a few modifications. Cultures were grown to an optical density (600 nm) of 1.0 in rich medium containing 2% dextrose. Cell extracts (300 µg) were used immediately following preparation and were precleared by incubation with 50 µl plain protein A-sepharose beads (Pharmacia) for 1 hr at 4°. A small sample was taken after the preclear step to provide a load control. Anti-myc antibodies were coupled to protein A-sepharose beads, and the remaining extract was incubated with 50 µl of these coupled beads for 2 hr at room temperature. After six washes, the beads were boiled in loading buffer and 15 µl was loaded for SDS-PAGE, followed by immunoblot analysis.

Acid phosphatase assays:
Assays were performed as described (HAGUENAUER-TSAPIS and HINNEN 1984 ). Cultures of JF13 and JF14 in midlog phase were harvested and resuspended in medium lacking inorganic phosphate (-P_i) to induce the phosphatase genes. For the assay at 38°, cultures in standard (+P_i) medium were shifted to 38° for 15 min, then allowed to recover at 30° for 1 hr, and finally shifted to 38° for 1 hr. After this hour at 38°, the medium was replaced with -P_i medium, and the assay was otherwise conducted in the same way. All cultures were assayed in duplicate at each time point, and assays were performed on at least three independent cultures.

Transcriptional analyses:
S1 nuclease analyses were conducted as described (IYER and STRUHL 1996 ). When a temperature shift was involved, the cultures were incubated at 38° for 15 min, then 30° for 1 hr, and finally 38° for 1 hr. RNA was prepared by hot phenol extraction and quantitated spectrophotometrically at 260 nm. For activated transcription of the HIS3 and HIS4 genes, AT was added to a final concentration of 10 mM and cells were incubated one additional hour. For PHO5 analysis, cells grown in +P_i medium were resuspended in -P_i medium and incubated for several additional hours. Similarly, for GAL7 analysis, the cells were grown in raffinose medium, then diluted in galactose medium, and incubated several hours. For CYC1 induction, cultures grown overnight in rich medium containing 2% glucose were washed three times in medium lacking glucose, diluted into medium containing 3% ethanol, and cultured at 30° for 6 hr. For all analyses at 38°, the temperature shift was done 1 hr prior to supplementing or changing the medium. Hybridizations with excess probe were normally done with 25–30 µg of RNA overnight at 55°. S1 nuclease digestion was performed on the hybridized samples for 30–45 min at 37°. Band intensity was normalized to the intensity of the tRNA^w band, which was unaffected by spn1^K192N. For analysis using the panel of strains with various upstream activator-binding sites, cultures were induced by means appropriate for the activator: Gal4 by culturing in galactose-containing medium for 3 hr and heat-shock factor (Hsf) by shifting to 38° for 15 min. Strains containing poly(dA:dT) or no activator-binding site were grown in rich medium.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and identification of a suppressor of a postrecruitment-defective TBP allele:
The TBP allele F237D was isolated in a previous genetic screen for TBP mutants able to support transcription by RNA polymerase III but defective for some aspect of RNA polymerase II transcription (STARGELL and STRUHL 1996 ). Further characterization of the F237D mutant revealed a defect in its response to activators. Moreover, the activation defect of F237D cannot be bypassed through artificial recruitment to the promoter, indicating that this TBP mutant is defective in one or more postrecruitment steps. These observations do not preclude additional defects in recruitment steps, but offer an approach to target the postrecruitment steps in vivo.

Therefore, a genetic screen was conducted to obtain extragenic suppressors of the postrecruitment-defective TBP allele F237D (Fig 1). The suppressor screen was performed in yeast strain SK10, containing a LexA operator located upstream of the HIS3 gene and a URA3-marked plasmid encoding the F237D TBP allele fused at its N terminus with a LexA DNA-binding domain. Despite this DNA-binding domain, and unlike wild-type TBP, the F237D allele is unable to activate transcription in this context. Thus, transcriptional output from the HIS3 reporter gene is insufficient to support viability on medium containing 3-aminotriazole (AT), a competitive inhibitor of the HIS3 gene product. We selected 758 spontaneous suppressors that formed colonies in the presence of AT. All strains were analyzed to determine if growth on AT was due to extragenic suppression. First, the LexA-F237D plasmid was shuffled out of each strain by growth on 5-FOA, and the cured strains were tested for growth on AT. In the absence of LexA-F237D, 563 strains maintained AT resistance. These were eliminated since they are likely to possess mutations that potentiate basal transcription in a manner that is not dependent on F237D function. The remaining 195 strains were retransformed with the LexA-F237D plasmid and checked for growth on AT. We eliminated 163 strains that remained sensitive to AT, as their suppressing phenotype is likely due to a mutation linked to the original LexA-F237D plasmid. In addition, the shuffled strains were transformed with a plasmid encoding the LexA DNA-binding domain without the F237D fusion. Those 21 strains that grew on AT in the presence of LexA were rejected because the suppression could have been due to mutations that potentiate weak activators. Eleven suppressor strains fit all of the above criteria. Here we describe the cloning and characterization of the gene SPN1.

View larger version (28K): In this window In a new window Download PPT slide   Figure 1. Genetic screen for spontaneous suppressors of the postrecruitment defects of the TBP allele F237D. (A) The starting strain contained a LexA operator (LexA Op) and the LexA-F237D derivative on a URA3-marked plasmid. When plated on media containing AT, a competitive inhibitor of the HIS3 gene product, the strain fails to grow (ATS). Spontaneous suppressors were selected that were resistant to AT (ATR). These were subsequently cured of the LexA-F237D plasmid by growth on 5-FOA. (B) Strains that maintained ATR in the absence of LexA-F237D were discarded. ATS strains were retransformed with the LexA-F237D plasmid. (C) Those that remained ATS were discarded, as their suppressing phenotype was likely due to a mutation on the original LexA-F237D plasmid. Those that regained ATR in the presence of LexA-F237D were further characterized by transforming the cured strain with LexA alone (not shown) to test for dependence on the TBP portion of the fusion derivative.

In addition to suppressing LexA-F237D, spn1 cells exhibit a recessive ts phenotype (data not shown). Cloning by complementation of the ts phenotype was used to identify the suppressing gene as the yeast open reading frame YPR133c. SPN1 is an essential gene and encodes a 410-amino-acid polypeptide (data not shown). The spn1 allele present in JF10 was recovered by plasmid gap repair. Sequencing revealed a point mutation at residue 192, resulting in the substitution of an asparagine for a lysine (spn1^K192N). All phenotypes observed for JF10 were recapitulated in a strain with the genomic copy of SPN1 disrupted by LEU2 and covered by spn1^K192N on a plasmid (data not shown), indicating that this single substitution confers the suppressing and ts phenotypes.

SPN1 is highly conserved throughout evolution:
Yeast SPN1 has homologs in a wide range of eukaryotic organisms, including human, mouse, plants, fly, and worm (ALTSCHUL et al. 1997 ). Sequence alignment reveals a central conserved region of 113 amino acids in length, in which the human sequence is 42% identical and 63% similar to the yeast sequence (Fig 2). Notably, the suppressing substitution, K192N, occurs in an invariant residue within this conserved region. N-terminal deletions and C-terminal truncations of SPN1 were constructed, assayed for expression, and tested for their ability to cover a genomic knockout of the gene (Fig 3). These studies demonstrate that the nonconserved N-terminal and C-terminal regions are dispensable, while the entire conserved core is necessary for the essential functions of SPN1 in yeast cells. Interestingly, none of the homologs has been functionally characterized, and Spn1 protein (Spn1p) contains no known functional protein motifs. Thus, yeast SPN1 is the first characterized member of this class of genes.

View larger version (66K): In this window In a new window Download PPT slide   Figure 2. SPN1 has a highly conserved central domain. Identical amino acids are shown in blue; similar amino acids are shown in pink; and the lysine 192 that is mutated in the suppressor strain is highlighted in yellow and with an asterisk. Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Hs, Homo sapiens; Mm, Mus musculus; At, Arabidopsis thaliana; Os, Oryza sativa; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans. Numbers indicate the starting and ending amino acid positions shown for each homolog, followed by the total number of amino acids in the homolog. Alignment was produced with the aid of the BLAST search engine (ALTSCHUL et al. 1997 ).

View larger version (43K): In this window In a new window Download PPT slide   Figure 3. The central conserved region of SPN1 is necessary for the essential functions of SPN1. (A) N- and C-terminal residues of each truncation construct are labeled. The conserved region is denoted by the solid box. Viability denotes the growth (+) or no growth (-) phenotype of cells when the indicated derivative is tested for its ability to cover a genomic knockout of the SPN1 gene in a plasmid shuffle experiment. (B) Immunoblot probed with -myc antibodies showing protein expression of myc-tagged truncations corresponding to the constructs in A. The residues included in each construct are shown above each lane. The blot was stripped and reprobed for TBP as a load control.

spn1^K192N suppresses the postrecruitment defect of two additional TBP alleles:
We next tested whether the suppression of the postrecruitment defect of LexA-F237D by spn1^K192N is allele specific. The strain harboring the suppressing mutation was transformed with a panel of LexA-TBP derivatives, some of which exhibit a postrecruitment defect in a wild-type SPN1 background (F237D, E236P, and K201I). As shown, the suppressor enabled growth of all of the postrecruitment-defective TBP alleles (Table 2), which are defective in a variety of interactions (YAMAMOTO et al. 1992 ; LEE and STRUHL 1995 ; STARGELL and STRUHL 1996 ). Given that spn1^K192N is not allele specific in its suppression ability, it is unlikely to represent a compensatory mutant for F237D TBP.

To investigate further the link between spn1^K192N and TBP function, we looked for suppression of TBP mutant phenotypes by spn1^K192N outside the context of the artificial recruitment assay. The genomic copy of TBP was knocked out in SK1 (SPN1) and JF10 (spn1^K192N) strains and covered by a variety of TBP alleles. Unfortunately, when not tethered to a DNA-binding domain, the F237D allele of TBP exhibits no mutant phenotypes for spn1^K192N to suppress (Table 3). The same is true for the E236P derivative. The final postrecruitment-defective allele, K201I, did exhibit mutant phenotypes in this strain background, and we observed that two phenotypes ascribed to the K201I allele were suppressed in the spn1^K192N background (Table 3). This result parallels the finding that the K201I allele, which is postrecruitment defective in a wild-type SPN1 strain, is competent for artificial recruitment in the context of spn1^K192N (Table 2). In contrast, the S118L/S128T allele, which is postrecruitment competent, gains exacerbated phenotypes in the spn1^K192N context (Table 3). Thus, a mutation in SPN1 specifically suppresses a TBP mutant with known defects in postrecruitment functions, whereas another TBP mutant, which is functional in a postrecruitment assay, is not suppressed.

Spn1 protein does not stably associate with the TFIID complex in vivo:
It is possible that direct interaction of Spn1p with the TFIID complex could mediate suppression of postrecruitment-defective TBP alleles. Co-immunoprecipitation experiments were performed to determine if Spn1p is associated with TFIID in vivo. Whole-cell extracts were prepared from strains expressing myc-tagged versions of TBP or Spn1p and from a control strain. Antibodies against the myc epitope were used for immunoprecipitation from these extracts. The precipitated complexes were analyzed by immunoblot, employing polyclonal antibodies against TBP, Spn1p, and several TAFs (Fig 4). Immunoprecipitation of myc-TBP shows association of TBP with TAFs, indicating the presence of intact TFIID, but Spn1p was not detected. Moreover, in the reciprocal experiment, when myc-Spn1p was immunoprecipitated, TBP was not detected. These data indicate that Spn1p does not stably associate with the TFIID complex in vivo. This result is not surprising given the ability of spn1^K192N to suppress the postrecruitment defects of multiple TBP alleles, each of which may possess unique defects in postrecruitment functions.

View larger version (69K): In this window In a new window Download PPT slide   Figure 4. Spn1p is not stably associated with TFIID in vivo. Co-immunoprecipitation was performed on cell extracts from strains expressing myc-TBP, myc-Spn1p, or untagged cells (non-myc). Immunoprecipitated complexes (IP) were resolved by SDS-PAGE and probed by immunoblot analysis using polyclonal antibodies specific to TBP, Spn1p, scTAF1, scTAF5, and scTAF10. Input from each extract (Load) is indicated.

spn1^K192N cells are mildly defective for constitutive transcription:
To analyze the effect of a mutation in SPN1 on transcription, RNA was isolated from cultures of SPN1 and spn1^K192N strains at the permissive and restrictive temperatures, and S1 nuclease experiments were performed. When transcript levels from the constitutively expressed genes HIS3, DED1, HTA2, RPS4, and ENO2 were measured, the mutant strain was observed to have only mild reductions in RNA levels compared to wild type (Fig 5). At 30°, we observed 15–35% reductions in transcriptional output of the spn1^K192N strain. At 38°, levels of transcripts from spn1^K192N show changes similar to those observed at 30°, with the exception that DED1 exhibits a more significant reduction. Thus, the spn1^K192N allele has modest effects on the expression of constitutively transcribed genes.

View larger version (67K): In this window In a new window Download PPT slide   Figure 5. Constitutive transcription is mildly affected in spn1K192N cells. Mutant (K192N) and wild-type (WT) SPN1-containing cells were grown under steady-state conditions and harvested in log phase at 30° or shifted to the restrictive temperature (38°) for 1 hr. Total RNA harvested before and after the temperature shift was hybridized with a 100-fold excess of the indicated probe, treated with S1 nuclease, and resolved on a denaturing gel; tRNAw served as a load control.

SPN1 plays an important role in activated transcription at a subset of inducible genes:
Transcriptional output from a constitutively expressed gene in vivo is likely to reflect the maintenance of activated transcription, which might differ on a mechanistic level from the induction of transcription. Hence, we tested whether the spn1^K192N mutant is competent for inducible gene transcription. Several different inducible systems were tested at their endogenous responsive genes. Total RNA was isolated from cells grown under inducing conditions appropriate for activation of GAL7 (growth on galactose), SSA4 (heat shock), PHO5 (absence of phosphate), or HIS3 and HIS4 (growth on aminotriazole). The results reveal defects that are dependent on the temperature and the particular gene examined. GAL7 transcription displayed no significant reduction in the mutant strain at either 30° or 38° (Fig 6A). Similarly, SSA4 transcription in the spn1^K192N background upon induction by heat shock was also robust (Fig 6B). In contrast, PHO5 in the spn1^K192N cells induced nearly as well as wild type at 30° but failed to activate at 38° compared to a two- to threefold induction in wild type (Fig 6C). Thus, transcriptional induction of PHO5 is temperature dependent. The results for both temperatures were confirmed with acid phosphatase colorimetric assays on wild-type and spn1^K192N strains (data not shown). In contrast, the induction of HIS3 at 30° was significantly affected by spn1^K192N, with induced transcription levels in the mutant strain at one-half of those observed in the wild-type strain (Fig 6D). This effect is not due to reduced levels of Gcn4 in the spn1^K192N strain, since the HIS4 gene, which is also regulated by Gcn4, induces with equal robustness in wild-type and mutant strains at 30°. Thus, the HIS3 transcriptional defects are not likely to be due to a reduction of Gcn4 in the mutant strain. Like the PHO5 gene, both HIS3 and HIS4 induce poorly at 38° in the spn1^K192N strain, reaching only 25 and 15% of wild-type induced levels, respectively. As determined by immunoblot analyses, the amount of Spn1p in wild-type and mutant strains does not vary with temperature (data not shown), suggesting that temperature-dependent effects on transcription are likely to be due to loss of activity of Spn1p rather than to a reduction in protein level.

View larger version (34K): In this window In a new window Download PPT slide   Figure 6. Analyses of inducible gene expression in spn1K192N. (A) To examine the response to the transcriptional activator Gal4, cells were grown to log phase in raffinose-containing medium and then transferred into galactose-containing medium for the time indicated and probed for expression of GAL7. Strains contained either wild-type SPN1 (WT) or spn1K192N (Mt). (B) Response to heat-shock factor was assayed by transferring cells to 38° for 15 min prior to harvest and probing for expression of SSA4. (C) Activation by Pho4 was induced by growing cells to log phase and then transferring cells into medium lacking inorganic phosphate for 6 hr (30°) or 4 hr (38°) prior to harvest, as indicated. (D) Gcn4 responsiveness was determined by treating cells with 10 mM AT for 1 hr prior to harvest and by examining the levels of HIS3 (with +1 and +13 start sites indicated) and HIS4 transcripts. For all treatments, 25 µg of total RNA was hybridized with excess probe, treated with S1 nuclease, and resolved on a denaturing gel.

Various factors could account for the gene-specific differences in transcriptional output seen under inducing conditions in the spn1^K192N strain. A gene-specific effect may reflect dependence either on a given activator or on a particular gene context (i.e., promoter sequence, chromatin structure, and/or the transcribed region of the gene). To investigate this further, we assayed HIS3 transcription from a set of strains in which different sequence elements were cloned upstream of the promoter (IYER and STRUHL 1995A ). As such, each element must function in the same gene context. As expected for constitutive transcription, in this case due to intrinsic DNA structure (IYER and STRUHL 1995B ), the poly(dA:dT) upstream sequence stimulated transcription of HIS3 equally well in spn1^K192N and in wild-type cells (Fig 7). We next tested Gal4- and Hsf-binding sites using inducing conditions appropriate for each activator. We observed robust activation of the HIS3 gene in wild-type cells driven from the Gal4- and Hsf-binding sites, whereas SPN1 mutant cells exhibited reproducible 30 and 50% reductions from these promoters, respectively (Fig 7). Thus, in the context of the HIS3 gene, Hsf and, to a lesser extent, Gal4, show dependence on Spn1p for normal levels of induced transcription. And yet, no dependence on Spn1p was observed in the context of their native responsive promoters (Fig 6). Thus, the HIS3 gene is particularly sensitive to perturbations in normal Spn1p function, since activators that are SPN1 independent at their normal sites become dependent in the HIS3 gene context. Whether this is due to promoter sequence, chromatin structure, and/or the transcribed region of the HIS3 gene remains to be determined.

View larger version (61K): In this window In a new window Download PPT slide   Figure 7. Transcriptional output from a variety of activators at HIS3 in wild-type and spn1K192N cells. RNA from strains containing various activator-binding sites upstream of the HIS3 locus was subjected to quantitative S1 analysis. Cells were grown under conditions appropriate for the activator of interest and harvested in log phase. For Gcn4, 10 mM AT was added for 1 hr. For Gal4, cells were grown in galactose-containing medium for 3 hr. For Hsf, cells were exposed to 38° for 15 min. Other strains were grown in rich medium under normal conditions. Strains contained either wild-type SPN1 (WT) or spn1K192N (Mt). Thirty micrograms of total RNA was hybridized with excess probe, treated with S1 nuclease, and resolved on a denaturing gel. tRNAw is a load control.

spn1^K192N aids in transcription from the postrecruitment regulated CYC1 promoter:
At the majority of promoters, recruitment of the transcription machinery is the primary mechanism by which transcription is regulated (KURAS and STRUHL 1999 ; LI et al. 1999 ). However, the CYC1 promoter is occupied by both TBP and Pol II when in a transcriptionally inactive state (LUE et al. 1989 ; CHEN et al. 1994 ; KURAS and STRUHL 1999 ; MARTENS et al. 2001 ). Activation in this case is not limited by TFIID or Pol II recruitment but is regulated in a postrecruitment step. One would predict that if Spn1p functions to regulate CYC1 expression, then transcription should be enhanced in spn1 mutant cells. After a 6-hr incubation at 30° in 2% glucose (partially repressing) or 3% ethanol (activating), total RNA was isolated from wild-type and spn1^K192N cells, and CYC1 transcription was analyzed by S1 nuclease assay. The spn1^K192N cells had CYC1 transcript levels significantly greater than the corresponding wild-type levels, under both partially repressing and activating conditions (Fig 8). This result indicates that transcription at a postrecruitment regulated promoter is facilitated by spn1^K192N and further supports our previous data suggesting that Spn1p acts at a postrecruitment step in transcription.

View larger version (72K): In this window In a new window Download PPT slide   Figure 8. Transcription is enhanced by spn1K192N at a postrecruitment-regulated promoter. RNA was isolated from wild-type (WT) and spn1K192N (K192N) strains grown in medium containing either 2% glucose (glu) or 3% ethanol (eth) for 6 hr. Glucose causes partial repression of CYC1, while ethanol causes activation. The RNA was subjected to S1 nuclease analysis using a probe for CYC1. tRNAw is the load control.

SPN1 is a member of the SPT gene family:
The TBP gene, SPT15, was cloned using a genetic screen for suppressors of Ty insertions in the 5' regions of HIS4 and LYS2 (EISENMANN et al. 1989 ; HAHN et al. 1989 ). The SPT family of genes, each capable of suppressing Ty insertions when mutated, represents a large collection of genes involved in an array of transcriptional functions. In addition to TBP, this family includes histones and histone modifying or remodeling complexes, members of the Pol II mediator complex, and proteins involved in elongation steps of transcription (WINSTON 1992 ). In addition to the genetic interactions described herein between SPN1 and SPT15 (TBP), a further link to the SPT gene family arises from the recent observation that Spn1p was found to associate with Spt6p, using a large-scale proteomics approach (GAVIN et al. 2002 ). Spt6p has been shown to bind histones and assemble nucleosomes in vitro and to remodel chromatin in vivo (BORTVIN and WINSTON 1996 ). In addition, Spt6p, in association with Spt4p and Spt5p, is thought to function in elongation (HARTZOG et al. 1998 ; ANDRULIS et al. 2000 ; KAPLAN et al. 2000 ). These connections to SPT genes prompted us to test directly whether the SPN1 mutant suppresses Ty insertions. We constructed a one-step genomic knockout of SPN1 in the yeast strain FY120, which possesses the insertion mutations his4-912 and lys2-128 (SWANSON et al. 1991 ). The genomic knockout of SPN1 was covered by spn1^K192N and assayed on medium lacking histidine or lysine (Fig 9A). Like the spt4-3 control cells, the spn1^K192N cells grow well on these media, indicating the ability to suppress the insertion mutations. Thus, SPN1 falls under the umbrella of the SPT gene family.

View larger version (91K): In this window In a new window Download PPT slide   Figure 9. The spn1K192N strain has Spt- phenotypes. (A) The indicated strains were spotted onto medium lacking histidine (-His), medium lacking lysine (-Lys), or complete medium (Control) to assay for an Spt- phenotype. The spn1K192N strain was derived from the FY120 strain (WT), which contains his4-912 and lys2-128 insertion mutations. The strain harboring the spt4-3 allele has the same insertions. Growth was scored after 3 days. A Spt- phenotype corresponds to growth on media lacking histidine and on media lacking lysine, indicating suppression of the insertion mutations. (B) BY4741-derived wild-type (WT), spn1K192N, or spt4 strains were spotted onto medium containing 75 µg/ml 6-azauracil (+6-AU), 20 µg/ml mycophenolic acid (+MPA), or control medium (Control). Growth was examined after 3 days. (C) Strains were also spotted onto synthetic complete medium lacking inositol (-Ino) or rich medium (Control). Growth was examined after 3 days.

SPT6 has been implicated in elongation processes, along with SPT4 and SPT5 (HARTZOG et al. 1998 ; ANDRULIS et al. 2000 ; KAPLAN et al. 2000 ). Since elongation is clearly a postrecruitment function, we tested the SPN1 mutant for sensitivity to the drugs 6-azauracil (6-AU) and mycophenolic acid (MPA). Such sensitivity generally indicates the presence of a defect in elongation, presumably due to a decrease in the pools of free nucleotides, which results in increased pausing and arrest by Pol II (EXINGER and LACROUTE 1992 ; UPTAIN et al. 1997 ). In contrast to spt4 cells, which are sensitive to 6-AU and MPA (HARTZOG et al. 1998 ), spn1^K192N cells grow robustly on media containing these agents (Fig 9B). Inositol auxotrophy is another phenotype commonly shared by mutations in SPT genes (WINSTON and CARLSON 1992 ; GRANT et al. 1998 ). Unlike spt4 cells, which display impaired growth in the absence of inositol, again the spn1^K192N cells grow robustly (Fig 9C). As such, although a mutation in SPN1 confers some phenotypes consistent with an SPT designation, the observed differences in growth on media with 6-AU, MPA, and minus inositol suggest a role that may be, at least in part, functionally distinct from that of SPT4-SPT5-SPT6.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

SPN1 is involved in postrecruitment functions of TBP:
A spontaneous suppressor screen of a postrecruitment-defective allele of TBP (F237D) has identified SPN1. SPN1 is an essential gene in yeast that is conserved across species. A single substitution of a conserved lysine to an asparagine (spn1^K192N) is able to suppress the postrecruitment defects of F237D. The fact that the spn1^K192N mutant can suppress additional postrecruitment-defective TBP alleles, with presumably a range of mechanistic defects, indicates that spn1^K192N is not a compensatory mutant for F237D. However, in the case of S118L/S128T, a postrecruitment competent TBP allele, mutant phenotypes are not suppressed but rather exacerbated by spn1^K192N. Thus the spn1^K192N mutant is not a general suppressor of TBP defects. Although a transient or DNA-dependent association cannot be ruled out, Spn1p is not detected in association with TFIID by co-immunoprecipitation. Taken together, the data suggest a broader involvement of Spn1p in the transcription process, rather than an involvement via direct association with TBP.

Functional role of Spn1p:
Functional studies indicate that spn1^K192N has little effect on constitutive transcription at the permissive and restrictive temperatures. However, significant transcriptional defects are observed in the induction of activated transcription, although these defects depend on the gene assayed. Whereas induction of the GAL7 (via Gal4) and SSA4 (via Hsf) genes is unaltered in the SPN1 mutant background, induced transcription of HIS3 (via Gcn4) is significantly reduced at both temperatures. Thus, the HIS3 gene is highly dependent on Spn1p for maximum transcriptional output. On the basis of the robust activation of HIS4 (via Gcn4), a reduction in the activator level is not likely to be the cause of the HIS3 defects. Moreover, when Gal4 and Hsf act upstream of the HIS3 gene at engineered binding sites, they become dependent on Spn1p function, whereas they are independent at their native promoters. As such, the negative effect of Spn1p^K192N on activation appears to be more dependent on the context of the gene (promoter sequence, chromatin structure, or transcribed region of the gene) and less dependent on the particular activator.

Analysis of transcription at the CYC1 promoter supports a postrecruitment function for Spn1p. Both Pol II and TBP have been shown to occupy the transcriptionally inactive form of this promoter (LUE et al. 1989 ; CHEN et al. 1994 ; KURAS and STRUHL 1999 ; MARTENS et al. 2001 ). Thus, quite atypically, the recruitment of the transcription machinery is not the primary means of regulation at CYC1. The finding that CYC1 transcription in the spn1^K192N strain is significantly enhanced as compared to the wild-type strain suggests that Spn1p^K192N aids transcription by overcoming some postrecruitment barrier to transcription of CYC1. Clearly, the sum of Spn1p^K192N functional defects, together with the genetic and transcriptional evidence that SPN1 suppresses postrecruitment functions, indicates that Spn1p plays an important role in transcription.

Classification of SPN1 as a member of the SPT gene family:
The identification of SPN1 as an SPT gene places it in a broad family of genes, which includes chromatin components and remodeling complexes, general transcription factors and mediator/holoenzyme subunits, and elongation factors. Recently, a large-scale proteomics approach has detected an association between Spn1p and Spt6p (GAVIN et al. 2002 ). Spt6p has been shown to bind histones, assemble nucleosomes and remodel chromatin (BORTVIN and WINSTON 1996 ), and function in elongation (HARTZOG et al. 1998 ; ANDRULIS et al. 2000 ; KAPLAN et al. 2000 ). Our characterizations of SPN1 are supportive of a role in transcription that appears to mesh well with the types of influence and regulation likely to be exhibited by Spt6p-associated proteins. However, spn1^K192N cells do not exhibit growth defects associated with inositol metabolism [whereas SPT mutants often display these phenotypes (WINSTON and CARLSON 1992 ; GRANT et al. 1998 )], nor do they show an elongation defect by phenotypic testing. Thus, it seems likely that Spn1p, in concert with Spt6p or other proteins, has additional or distinct roles in transcriptional processes.

Potential mechanisms of Spn1p in transcriptional processes:
The emergence of spn1^K192N in a screen for suppressors of a postrecruitment-defective TBP allele and the ability of spn1^K192N to enhance transcription from CYC1 suggests that wild-type Spn1p functions negatively. However, such a role appears to be at odds with our results demonstrating that activated transcription at a subset of genes is diminished in the spn1^K192N background, implying a positive role for Spn1p. We can reconcile these opposing effects by considering that Spn1p may function in both postrecruitment and recruitment steps of transcription. In the genetic screen and at CYC1, TBP is already recruited. As such, the focus is on interactions centered around the core promoter (i.e., the TBP-binding site and the transcriptional initiation site) and on functions that are important after TBP is recruited (postrecruitment functions). Thus, wild-type Spn1p may have negative effects in the context of core promoter and/or postrecruitment functions. Activated transcription is a complex process in which multiple steps, including recruitment, are influenced by activators bound at upstream promoter elements. In this context, Spn1p appears to positively affect transcription in a gene-specific manner. Thus, the positive and negative effects of Spn1p might reflect differing functions of the factor depending on the step in the transcriptional process (i.e., recruitment vs. postrecruitment). A growing body of evidence supports the idea that certain factors can influence multiple steps of the transcriptional process. For example, the Paf1/Pol II complex (MUELLER and JAEHNING 2002 ), the FACT complex (ORPHANIDES et al. 1998 ; FORMOSA et al. 2001 ), and the Spt4-5-6 complex (SWANSON and WINSTON 1992 ; HARTZOG et al. 1998 ) have each been implicated in initiation (recruitment) and elongation (postrecruitment) steps of transcription. These observations serve to underscore the complexity and interplay between the components of the Pol II machinery.

In summary, we describe the cloning and initial characterization of Spn1p, a new player involved in transcriptional mechanisms in vivo. The SPN1 gene, an essential gene conserved throughout evolution, was identified in a screen for suppressors of a TBP mutant. The SPN1 gene also shares phenotypes with the SPT family of transcription factors, of which TBP is a member. In addition, SPN1 function is required for normal gene expression at a subset of genes. Taken together, these results support a functional conservation of the role(s) of Spn1p in the mechanism of transcription in vivo.

	ACKNOWLEDGMENTS

We thank Fred Winston for the FY120 and FY241 strains, Michael Green for TAF antibodies, Lei Zhang for the use of BY4741-derived strains, and Michael Place and Laurel Respicio for technical assistance. This work was supported by National Institutes of Health grant no. GM-56884 to L.A.S.

Manuscript received August 27, 2002; Accepted for publication September 30, 2002.

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