Mutations in SPT10 and SPT21 of Saccharomyces cerevisiae have been previously shown to cause two prominent mutant phenotypes: (1) defects in transcription of particular histone genes and (2) suppression of Ty and -insertion mutations (Spt^– phenotype). The requirement for Spt10 and Spt21 for transcription of particular histone genes suggested that they may interact with two factors previously shown to be present at histone loci, SBF (Swi4 and Swi6) and MBF (Mbp1 and Swi6). Therefore, we have studied swi4, mbp1, and swi6 mutants with respect to histone gene transcription and for interactions with spt10 and spt21. Our results suggest that MBF and SBF play only modest roles in activation of histone gene transcription. In addition, we were surprised to find that swi4, mbp1, and swi6 mutations suppress the spt21 Spt^– phenotype, but not the spt21 defect in histone gene transcription. In contrast, both swi4 and mbp1 cause lethality when combined with spt10. To learn more about mutations that can suppress the spt21 Spt^– phenotype, we performed a genetic screen and identified spt21 suppressors in seven additional genes. Three of these spt21 suppressors also cause lethality when combined with spt10. Analysis of one spt21 suppressor, reg1, led to the finding that hyperactivation of Snf1 kinase, as caused by reg1, suppresses the Spt^– phenotype of spt21. Taken together, these genetic interactions suggest distinct roles for Spt21 and Spt10 in vivo that are sensitive to multiple perturbations in transcription networks.

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THE insertion of Ty retrotransposons or Ty long terminal repeat sequences (LTRs or -elements) into or near the promoters of genes can cause transcriptional defects (BOEKE and SANDMEYER 1991). The isolation of suppressors of these transcriptional defects has identified a large set of genes referred to as SPT genes (WINSTON et al. 1984; WINSTON and SUDARSANAM 1998; YAMAGUCHI et al. 2001). Two functionally related genes, SPT10 and SPT21, were isolated in two different selections for spt mutants (FASSLER and WINSTON 1988; NATSOULIS et al. 1991). Studies of spt10 and spt21 mutants have shown that they share several related mutant phenotypes in addition to their Spt^– phenotype: impaired transcription of particular histone genes, suppression of the transcriptional defects caused by loss of upstream activating sequences or activators at particular genes, and defects in chromatin structure (DENIS 1984; NATSOULIS et al. 1991; MCKENZIE et al. 1993; PRELICH and WINSTON 1993; YAMASHITA 1993; DOLLARD et al. 1994; KAPLAN et al. 2003). Furthermore, Spt10 and Spt21 have been shown to physically interact (HESS et al. 2004). While mutant analysis has suggested that Spt10 and Spt21 overlap in some of their functions, other results indicate that each protein also has distinct roles, as spt10 mutants grow significantly more poorly and display broader transcriptional defects than spt21 mutants (DENIS and MALVAR 1990; NATSOULIS et al. 1994).

The regulation of histone synthesis is a critical aspect of eukaryotic cell division because histones play essential roles in all aspects of chromosome function. In Saccharomyces cerevisiae, altered histone levels have been shown to impair chromosome segregation, transcription, and other processes (OSLEY 1991). The regulation of histone gene transcription in S. cerevisiae is complex. Two sets of divergently transcribed gene pairs encode histones H2A and H2B and two other sets of loci encode histones H3 and H4 (HEREFORD et al. 1979; SMITH and MURRAY 1983). While transcription of all four histone loci is cell-cycle regulated, with peak expression during S phase (OSLEY 1991), the promoters at the four loci are highly divergent and are dependent on overlapping but distinct sets of transcription factors (OSLEY and LYCAN 1987; XU et al. 1992; DOLLARD et al. 1994; IYER et al. 2001; SIMON et al. 2001; HESS et al. 2004).

Spt10 and Spt21 play important roles in the transcription of histone genes. Previous work has shown that Spt10 and Spt21 are necessary for the transcription of two of the four histone loci and that they bind to the promoters of all four histone loci (DOLLARD et al. 1994; HESS et al. 2004). However, neither Spt10 nor Spt21 contains recognizable DNA-binding motifs, leading to the hypothesis that a DNA-binding factor may be required for Spt10 and Spt21 recruitment to histone gene promoters. Two candidates for such a factor are MBF (MCB-binding factor) and SBF (SCB-binding factor), which are known to regulate transcription during G1/S (BREEDEN and NASMYTH 1987; BREEDEN and MIKESELL 1991; DIRICK et al. 1992). Whole-genome binding studies of these factors (IYER et al. 2001; SIMON et al. 2001) have suggested that they are associated with the four histone loci during G1/S. In one of these studies, SBF was suggested to be associated with the HTA1-HTB1 and HTA2-HTB2 promoters (SIMON et al. 2001). In the second study, MBF and SBF were suggested to be associated with the HHT2-HHF2 promoter and MBF was associated only with HTA2-HTB2 (IYER et al. 2001). While these studies differ in their results, taken together they suggest that MBF and SBF are involved in histone gene regulation. Thus, MBF and/or SBF are candidates to help recruit Spt10 and Spt21 to histone gene promoters.

MBF and SBF are related and partially redundant transcription activation complexes. Each is a heterodimeric complex that contains Swi6. The complexes require different DNA-binding proteins, as MBF contains Mbp1 and SBF contains Swi4 (ANDREWS and HERSKOWITZ 1989; KOCH et al. 1993; MOLL et al. 1993). Although MBF and SBF bind to distinct sequences, each can activate transcription with reduced efficiency from the other's binding site (DIRICK et al. 1992). Genetic evidence for redundancy derives from the observation that swi4 and mbp1 single mutants have only mild phenotypes, while a swi4 mbp1 double mutant is inviable (KOCH et al. 1993). Surprisingly, swi6 single mutants are viable, but this has been attributed to the observation that Swi4 has a low level of activity as an activator in the absence of Swi6 (NASMYTH and DIRICK 1991; KOCH et al. 1993).

To address the roles of MBF and SBF in histone gene transcription and in possible interactions with Spt10 and Spt21, we have examined the levels of histone gene transcription in swi4, mbp1, and swi6 single mutants. Our results suggest that MBF and SBF serve redundant and modest roles in histone gene transcriptional activation. Surprisingly, we found that swi4, mbp1, and swi6 mutations suppress the Spt^– phenotype caused by an spt21 mutation, although they do not suppress the defect in histone gene transcription. These results prompted us to screen for additional suppressors of the Spt^– phenotype of spt21. We identified suppressors in seven genes, as well as one enhancer of the Spt^– phenotype. Unexpectedly, many of the spt21 suppressors cause lethality when combined with spt10. We investigated one spt21 suppressor in detail, a mutation in REG1, and discovered that suppression of spt21 by reg1 occurs via hyperactivation of the Snf1 kinase. Taken together, these genetic results suggest distinct roles for Spt10 and Spt21 that functionally interact with other transcriptional networks.

Yeast strains and genetic methods:

All S. cerevisiae strains used in this study (Table 1) are descended from a GAL2⁺ derivative of S288C (WINSTON et al. 1995). Standard methods for mating, sporulation, transformation, and tetrad analysis were used, and all media were prepared as previously described (ROSE et al. 1990). The null mutations for swi6, swi4, and mbp1 (swi6201::kanMX, swi4201::kanMX, and mbp1201::kanMX) were constructed using standard methods (BAUDIN et al. 1993; LORENZ et al. 1995), and spt10201::HIS3 and spt21201::HIS3 have been previously described (HESS et al. 2004). All null mutations are complete deletions of the open reading frame and are referred to by a -designation (GIAEVER et al. 2002). The genomic deletions were verified by PCR analysis. The Spt^– phenotype was tested using the insertion mutation lys2-128. In a wild-type background, lys2-128 causes a Lys^– (Spt⁺) phenotype and in spt mutants, lys2-128 causes a Lys⁺ (Spt^–) phenotype (SIMCHEN et al. 1984). Previous results have shown that these phenotypes correlate with transcription at lys2-128 (CLARK-ADAMS and WINSTON 1987).

View this table: In this window In a new window   TABLE 1 S. cerevisiae strains used in this study

 

RNA isolation and RT-PCR:

RNA isolation and reverse transcription were performed as previously described (HESS et al. 2004). Two dilutions of cDNA were subjected to quantitative radioactive PCR and resolved on acrylamide gels as previously described for chromatin immunoprecipitation analysis (HESS et al. 2004). The (experimental primer/ACT1) ratio was set at a value of 100 for wild type. All other strains are internally normalized to ACT1 and then expressed as a percentage of the wild-type ratio.

Identification of spt21 suppressors:

Strain FY2199 was mutagenized by transformation with an insertional mutagen library based on the transposon Tn3::lacZ::LEU2 (BURNS et al. 1994). Transformed cells were plated on SC-Leu medium at a density empirically determined to yield 250 colonies per plate, and plates were incubated for 2 days at 30°. The plates were replica plated to SC-Lys-Leu medium to screen for colonies that were Lys^–, indicating suppression of the Spt^– (Lys⁺) phenotype. Candidates that retested as Lys^– after colony purification were then tested for linkage of the Leu⁺ and Lys^– phenotypes by crossing each candidate to strain FY2268. For those candidates that displayed linkage, the exact position of the insertion element was identified via the Vectorette PCR-based technique and DNA sequence analysis (RILEY et al. 1990). For each gene identified in this way, the complete deletion from the systematic deletion collection was then crossed to FY2268 to test for its ability to suppress (GIAEVER et al. 2002). Further analysis of the suppressors was carried out using these complete deletions.

Introduction of spt10 into the suppressor strains:

Following the crosses between the deletion set and FY2268 described above, spores that contained the following genotype were isolated: MAT SPT21⁺ lys2-128 sup::kanMX. These strains were then crossed to the spt10 mutant, FY2191. Tetrads from these crosses were grown on medium lacking uracil to maintain the wild-type copy of SPT10 on a URA3-marked plasmid (pFW217) (NATSOULIS et al. 1994). Before testing for mutant phenotypes, these strains were grown on medium containing 5-flouroorotic acid (5-FOA) to select for cells that no longer maintained pFW217 (BOEKE et al. 1984).

MBF and SBF play modest roles in activation of histone gene transcription:

Whole-genome binding studies have suggested that the histone gene promoters are bound by MBF and/or SBF (IYER et al. 2001; SIMON et al. 2001). To examine the transcriptional dependence of the histone loci on MBF and SBF, we measured the mRNA levels for all eight histone-encoding genes in wild-type, mbp1, swi4, and swi6 strains. An spt21 mutant was included for comparison. Mbp1 is a component of MBF and Swi4 is a component of SBF, while Swi6 is a component of both complexes. The mRNA levels were measured by RT-PCR analysis (MATERIALS AND METHODS).

Our results show an overlapping requirement for MBF and SBF (Table 2). First, mbp1 causes no significant reduction in histone gene mRNA levels, suggesting little dependence upon MBF. Second, swi4 causes a mild reduction in histone gene mRNA levels at all four loci, suggesting a modest requirement for SBF. To test for possible redundancy between MBF and SBF, we also tested swi6. Loss of both complexes via swi6 reduces all eight histone gene mRNA levels, with the greatest effects at HHT1-HHF1. These data suggest that both MBF and SBF contribute to activation of histone gene transcription, with SBF playing a more prominent role. Even with the loss of both complexes in swi6, the effects at HTA2-HTB2 and HHT2-HHF2 are not as severe as the defect observed in an spt21 mutant. However, in contrast to spt21, loss of MBF and SBF causes transcriptional defects at HTA1-HTB1 and HHT1-HHF1. These results suggest that MBF and SBF act independently of Spt10 and Spt21 in transcription of histone genes.

View this table: In this window In a new window   TABLE 2 Analysis of histone gene mRNA levels in MBF and SBF mutants

 

mbp, swi4, and swi6 suppress the Spt^– phenotype of the spt21 mutant:

To test for possible redundancy between Spt21, SBF, and MBF, we constructed three double mutants, spt21 mbp1, spt21 swi4, and spt21 swi6, and tested them for an Spt^– phenotype and for histone gene mRNA levels. Surprisingly, we found that swi4, mbp1, and swi6 each suppress the Spt^– phenotype caused by spt21, with mbp1 and swi4 being moderate suppressors and swi6 being a strong suppressor (Figure 1).

View larger version (121K): In this window In a new window Download PPT slide   FIGURE 1.— Suppression of spt21 Spt– phenotype by mbp1, swi4, and swi6. The Spt phenotype of each strain was assessed by spotting a dilution series (from 108 to 103 cells/ml) on either SC medium lacking lysine (SC – lys) to test for Spt– phenotype or SC medium to control for the general growth of cells. The most informative dilutions are displayed for each medium (108–106 cells/ml for SC – lys and 105–103 cells/ml for SC). The SC – lys plate was incubated for 3 days at 30°. On SC – lys, spt21 displays strong growth indicative of an Spt– phenotype, but this phenotype is suppressed when combined with mbp1, swi4, or swi6.

 
To determine if mbp1, swi4, and swi6 also suppress the spt21 histone gene transcription defect, we measured mRNA levels for HTB2 in the double mutants (Table 3). This analysis revealed that the spt21 defect in histone gene transcription is not suppressed by any of these mutations. These results strongly suggest that the loss of MBF or SBF suppresses a defect in spt21 mutants other than the defect in transcription of HTA2-HTB2.

View this table: In this window In a new window   TABLE 3 Analysis of HTB2 mRNA levels in double-mutant strains

 

The loss of MBF or SBF in spt10 mutants causes inviability:

Since spt21 and spt10 mutants share many mutant phenotypes, we set out to test the phenotypes of spt10 when combined with mbp1, swi4, and swi6. To do this, we constructed double mutants that also contained wild-type SPT10 on a URA3 CEN plasmid and then tested the ability of strains to grow on medium containing 5-FOA. Our results revealed that, in contrast to spt21, spt10 causes lethality when combined with mbp1 or swi4 (Figure 2). Diploids heterozygous for both spt10 and swi6 failed to sporulate. This striking difference between spt10 and spt21 when combined with mbp1 or swi4 is explored in the DISCUSSION.

View larger version (104K): In this window In a new window Download PPT slide   FIGURE 2.— spt10 is synthetically lethal with mbp1 and swi4. Strains with the noted genotypes were constructed such that they also contained a plasmid that contained wild-type SPT10 on a URA3 CEN plasmid. The phenotype of each strain was assessed by spotting a dilution series of cells (from 108 to 103 cells/ml) on either SC medium containing 5-FOA to test for viability or SC medium to control for the general growth of cells. The most informative dilutions are displayed for each medium (108–106 cells/ml for 5-FOA and 105–103 cells/ml for SC). The 5-FOA plate was incubated for 4 days at 30°. On 5-FOA plates, spt10 displays slow growth, but spt10 mbp1 or spt10 swi4 double mutants are unable to grow and thus are synthetically lethal. Diploids containing both spt10 and swi6 in the heterozygous state failed to sporulate; thus the spt10 swi6 phenotype was not assayed.

 

Isolation of new suppressors of the Spt^– phenotype of the spt21 mutant:

We have discovered two classes of Spt^– suppressors of spt21 mutants. The first class is defined by two alleles of SPT10 that suppress spt21 with respect to both the Spt^– phenotype and the defect in HTA2 and HTB2 transcription (HESS et al. 2004). The second class, defined by the mbp1, swi4, and swi6 mutations, suppresses the Spt^– phenotype but does not suppress the defect in HTA2 and HTB2 transcription. Many members of this second class, including swi6 and reg1 (described later), are stronger suppressors of the Spt^– phenotype than the SPT10 suppressor alleles described previously (HESS et al. 2004). Therefore, there is not a correlation between the strength of suppression of the spt21 Spt^– phenotype and suppression of the spt21 histone gene transcription defect.

As the spt21 suppressors studied so far resulted from analysis of only four specific genes (SPT10, MBP1, SWI4, and SWI6), it seemed likely that suppressors of spt21 could be obtained in other genes, possibly revealing additional insights into Spt21 function. Therefore, we performed a screen to identify additional suppressors of spt21. We used a transposon library (BURNS et al. 1994) as described in MATERIALS AND METHODS and screened 20,000 candidates for suppression or enhancement of the spt21 Spt^– phenotype using the lys2-128 insertion allele. We identified seven suppressors and one enhancer of the Spt^– phenotype that are stable and segregate 2:2 in crosses, indicating that suppression in each case was caused by a single mutation.

For the seven suppressor mutations and one enhancer mutation, the mutant genes were identified (Table 4; MATERIALS AND METHODS). For each gene identified, we tested whether the suppression phenotype was due to loss of function. To do this, we crossed strains that contain complete deletions of each gene to an spt21 mutant. In all but two cases, GTR1 and UBR2, the deletion allele caused an equivalent or stronger suppression phenotype than did the original insertion mutation (Table 4). We observed a range of suppression phenotypes that were scored as weak, moderate, or strong (Table 4, Figure 3). To determine if any of these mutations also suppress the spt21 HTB2 transcription defect, we performed RT-PCR analysis. Our results show that none of the suppressors significantly alter this phenotype (Table 5). Thus, we have identified a set of mutations that suppress the Spt^– phenotype of spt21 but do not suppress the defect in HTA2 and HTB2 transcription, similar to mbp1, swi4, and swi6.

View this table: In this window In a new window   TABLE 4 Suppressors of spt21 mutant Spt– phenotype

 

View larger version (98K): In this window In a new window Download PPT slide   FIGURE 3.— Suppression of spt21 Spt– phenotype. The phenotype of each strain was assessed by spotting a dilution series of cells (from 108 to 103 cells/ml) on either SC medium lacking lysine (SC – lys) to test for Spt– phenotype or SC medium to control for the general growth of cells. The most informative dilutions are displayed for each medium (108–105 cells/ml for SC – lys and 106–103 cells/ml for SC). The SC – lys plate was incubated for 3 days at 30°. On the SC – lys plate, spt21 displays strong growth indicative of an Spt– phenotype, but this phenotype is suppressed when combined with the indicated suppressor.

 

View this table: In this window In a new window   TABLE 5 Analysis of HTB2 mRNA levels in spt21 suppressors

 

The spt21 suppressors cause distinct interactions with spt10:

Strains with either spt21 or spt10 share many phenotypes, including Spt^– and a defect in histone gene transcription. To determine if some of the spt21 suppressors genetically interact with spt10, we constructed double mutants. Our results show that the suppressors fall into distinct groups with respect to interactions with spt10, and they generally differed from the interactions with spt21 (Table 4). Of the five spt21 suppressors tested, three (reg1, gtr1, and gup1) cause synthetic lethality when combined with spt10, thus behaving similarly to mbp1 and swi4. In addition, ubr2 suppresses the spt10 growth defect but not the Spt^– phenotype, and spf1 suppresses the Spt^– phenotype of spt10. These data further support the hypothesis that although Spt10 and Spt21 are both required to activate histone gene transcription, they also have distinct roles.

A bcy1 mutation enhances the Spt^– phenotype of spt21:

The one identified enhancer of spt21 was determined to be an insertion in the 3' end of the BCY1 coding region. This mutant displayed phenotypes previously identified for bcy1 mutants, such as the inability to grow on media containing galactose or glycerol as the sole carbon source (TODA et al. 1987; data not shown). Unfortunately, our mutant displays the inability to enter stationary phase and loses viability when stored at 4° or –70°. This phenotype, observed previously for bcy1 mutants (PAZ et al. 1999), prevented further analysis of this mutant.

Constitutive activation of the Snf1 protein kinase suppresses the spt21 Spt^– phenotype:

The identification of reg1 as a strong suppressor of spt21 suggested an interaction of Spt21 with the Snf1 kinase. Reg1 controls Snf1 activity by controlling the Snf1 phosphorylation state, as Reg1 is the regulatory subunit of Glc7, the phosphatase that dephosphorylates Snf1. Snf1 is an important signaling molecule whose direct targets include cytoplasmic proteins, transcription factors, and histone H3 (HARDY et al. 1994; JIANG and CARLSON 1997; SANZ et al. 2000; LO et al. 2001). Snf1 is activated by phosphorylation, as this modification of Snf1 controls its association with Snf4, a Snf1 stimulatory factor (CARLSON 1999; HONG et al. 2003). Therefore, in a reg1 mutant, Snf1 is hyperphosphorylated, constitutively bound to Snf4, and highly active (JIANG and CARLSON 1997; SANZ et al. 2000).

The identification of reg1 as an spt21 suppressor suggested that Snf1 hyperactivation causes suppression of the Spt^– phenotype of spt21. To test this idea we first tested if Snf1 is required for suppression of spt21 by reg1. Our results (Figure 4) demonstrate that indeed, a snf1 mutation abolishes reg1 suppression of spt21. Therefore, Snf1 is required for suppression by reg1. We then tested whether hyperactivation of Snf1 by a different mutation, SNF4-204 (SHIRRA and ARNDT 1999), will suppress spt21. As expected, SNF4-204 suppresses spt21. These two results strongly suggest that Snf1 hyperactivation suppresses the Spt^– phenotype of spt21.

View larger version (70K): In this window In a new window Download PPT slide   FIGURE 4.— reg1 suppression of spt21 occurs via Snf1 kinase hyperactivation. The phenotype of each strain was assessed by spotting a dilution series of cells (from 108 to 103 cells/ml) on either SC medium lacking lysine (SC – lys) to test for Spt– phenotype or SC medium to control for the general growth of cells. The most informative dilutions are displayed for each medium(108–106 cells/ml for SC – lys and 105–103 cells/ml for SC). The SC – lys plate was incubated for 3 days at 30°. On SC – lys, spt21 displays strong growth indicative of an Spt– phenotype that is suppressed by reg1. On SC – lys, reg1 do not display an Spt– phenotype (data not shown). This suppression requires Snf1 as demonstrated by the Spt– phenotype of the snf1 reg1 spt21 triple mutant combined with the Spt+ phenotype of snf1. spt21 is also suppressed by SNF4-204, an allele of SNF4 that hyperactivates Snf1 kinase activity.

 

Previous studies of Spt21 and Spt10 suggested that they have closely related functions, one of which is the transcriptional activation of particular histone genes (NATSOULIS et al. 1991, 1994; DOLLARD et al. 1994; HESS et al. 2004). Results in this article have provided new information about the roles of Spt21 and Spt10 in histone gene transcription and have provided strong support that Spt21 and Spt10 have distinct roles in vivo in addition to their related roles in controlling histone gene transcription. First, the finding that mbp1, swi4, and swi6 mutations have only mild effects on histone gene transcription shows that Spt21 and Spt10 are not strongly dependent upon MBF and SBF for this process. Second, the demonstration that several suppressors of the spt21 Spt^– phenotype, including swi4, mbp1, and swi6, cause synthetic lethality with spt10 suggests that some aspects of Spt21 and Spt10 functions are distinct from each other. Third, the identification of Snf1 kinase activity as important in some aspect of Spt21 function, as well as the identification of several other suppressors of spt21, indicates that the defects in spt21 mutants are sensitive to a broad set of perturbations.

Previous analysis suggested that Spt21 is required for a subset of Spt10 functions, as spt10 mutants had been shown to have more extensive phenotypes than spt21 mutants, including a slower growth rate and other transcriptional defects (DENIS 1984; NATSOULIS et al. 1991; YAMASHITA 1993). In addition, the identification of spt10 mutations that partially suppress spt21 suggested that Spt21 may play a less direct role than Spt10 in their common functions, such as transcription of histone genes (HESS et al. 2004). The results in this article alter this view of Spt21 and Spt10 to one in which their functions overlap in some cases, such as histone gene transcription, but are distinct in other cases. If Spt21 were simply serving as an auxiliary factor for a subset of Spt10 functions, then the suppressors of spt21 would be expected to also suppress spt10 or be genetically neutral. Instead, in many cases the spt21 suppressors are synthetically lethal with spt10.

The spt21 suppressors identified in this work suppress the spt21 Spt^– phenotype, but do not affect the spt21 histone gene transcription defect, raising the question of whether suppression is related to histone levels and chromatin structure. We propose a model for suppression that addresses this issue and accounts for the wide range of suppressors identified. As previously described, spt21 mutants have reduced histone levels and are likely to have altered chromatin structure, resulting in suppression of insertion mutations such as lys2-128. However, they have only a slight growth defect on rich media. If an additional mutation, such as mbp1, swi4, or swi6, lengthened S phase, this change might result in sufficient time for the assembly of a normal chromatin structure even with reduced histone levels, resulting in suppression of the spt21 Spt^– phenotype. This model can explain why such a wide range of suppressors was identified, as many pathways feed into S-phase processes. Furthermore, this model can also explain why mutations that suppress spt21 are lethal in combination with spt10. Unlike spt21, spt10 has a severe growth defect that may activate S-phase checkpoints. Thus, mutations such as mbp1, swi4, or swi6, which may impair checkpoint activation (SIDOROVA and BREEDEN 1997, 2002, 2003) or lengthen S phase, may be lethal when combined with the severe spt10 defects.

Several additional issues remain to be answered regarding Spt21 and Spt10. Prominent among them is their mechanism of recruitment to histone gene promoters, as well as the target(s) of the putative Spt10 acetyltransferase. In addition, more extensive analysis of the functional interaction between Spt21 and Snf1, a key signaling molecule, is likely to uncover interesting aspects regarding the function of both of these proteins and may identify Snf1 targets relevant to the function of Spt21.

We are grateful to Karen Arndt and Peggy Shirra for providing the SNF4-204 allele and reg1 alleles. We thank Krista Dobi, Jenny Wu, and Rolf Sternglanz for helpful comments on the manuscript. This work was supported by National Institutes of Health grant GM32967 to F.W.

¹ Present address: Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544.

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Communicating editor: M. D. ROSE

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