The Yeast Protein Complex Containing Cdc68 and Pob3 Mediates Core-Promoter Repression Through the Cdc68 N-Terminal Domain

Corresponding author: Richard A. Singer, Department of Biochemistry, Sir Charles Tupper Medical Building, College St., Dalhousie University, Halifax, Nova Scotia, B3H 4H7 Canada., rasinger{at}is.dal.ca (E-mail).

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt^- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.

GENE expression in the eukaryotic nucleus takes place in the context of chromatin, a complex of DNA with proteins that play structural and regulatory roles. Transcription from promoter sequences by RNA polymerase II is stimulated by trans-activator proteins that become properly localized for this function through sequence-specific DNA binding; for many genes, trans-activator function is facilitated by chromatin-remodeling complexes such as the Swi/Snf complex (PETERSON and TAMKUN 1995 ; reviewed in KINGSTON et al. 1996 ). Conversely, in the absence of trans-activators or their binding sites along DNA, promoter sequences are generally unable to serve as efficient sites for transcription, in large part due to the repressive effects exerted by the chromatin environment of the DNA (KINGSTON et al. 1996 ). Several protein complexes have been shown genetically to be needed for global gene activation and/or for chromatin-mediated repression; among these is the recently described "CP" complex (BREWSTER et al. 1998 ).

The CP complex of the budding yeast Saccharomyces cerevisiae, an abundant nuclear dimer of the Cdc68 and Pob3 proteins, facilitates transcription at a number of genes. This finding stems in part from the effects of cdc68-1, a temperature-sensitive mutation that impairs gene expression at a restrictive temperature because of an alteration in Cdc68 protein structure (ROWLEY et al. 1991 ). In cdc68-1 mutant cells many mRNAs become depleted, including some that encode essential proteins; this finding accounts for the essential nature of both components of the CP complex (MALONE et al. 1991 ; WITTMEYER and FORMOSA 1997 ). The rates at which mRNAs disappear in cdc68 mutant cells correlate well with mRNA stabilities, suggesting that decreased mRNA abundance results from decreased transcription (ROWLEY et al. 1991 ).

The CP complex also mediates repression at promoters lacking DNA-bound trans-activators. For example, the effects of cdc68 mutations show that the CP complex not only facilitates transcription of the SUC2 and GAL1 genes (Q. XU, unpublished observations), but is also necessary for the repression of these genes when their UAS sequences (trans-activator binding sites) are deleted (MALONE et al. 1991 ; PRELICH and WINSTON 1993 ; XU et al. 1993 ; LYCAN et al. 1994 ). Similarly, the CP complex maintains the ho gene in a transcriptionally inactive state when the Swi4-Swi6 transcription activator is absent (LYCAN et al. 1994 ). The repressive effects of CP necessitate the actions of the Swi/Snf complex for the expression of SUC2 (MALONE et al. 1991 ). These findings indicate that the CP complex fosters chromatin repression as well as transcription. This dual role suggests that the CP complex may maintain chromatin in a configuration that facilitates proper gene regulation.

A chromatin role for the CP complex is also indicated by the effects of Cdc68 protein on the transcription of the reporter genes his4-912 and lys2-128. Each of these mutant alleles is normally unable to express functional mRNA because of the presence of the Ty1 retrotransposon long terminal repeat (-element) inserted within 5' sequences. The transcriptionally active -element, perhaps through a mechanism of promoter competition (HIRSCHMAN et al. 1988 ), changes the pattern of transcription (CLARK-ADAMS and WINSTON 1987 ; HIRSCHMAN et al. 1988 ; MALONE et al. 1991 ; SWANSON et al. 1991 ; reviewed in WINSTON 1992 ; WINSTON and CARLSON 1992 ). Functional transcription at these reporter genes can be restored either by a cdc68 mutation or by extra copies of the CDC68 gene (MALONE et al. 1991 ; ROWLEY et al. 1991 ; BREWSTER et al. 1998 ). This effect, termed the Spt^- phenotype (WINSTON 1992 ; WINSTON and CARLSON 1992 ), has been used to identify genetically many components that affect chromatin structure, including the histones. Indeed, the genes encoding the histones and Cdc68 are grouped, by Spt^- characteristics, in the same category of "SPT" genes; for several of these genes, mutations or increased gene dosage confer similar effects (CLARK-ADAMS et al. 1988 ; MALONE et al. 1991 ; ROWLEY et al. 1991 ; HIRSCHHORN et al. 1992 ; WINSTON 1992 ; PRELICH and WINSTON 1993 ; KRUGER et al. 1995 ; SANTISTEBAN et al. 1997 ). The POB3 gene in increased dosage also causes changes in gene expression (BREWSTER et al. 1998 ). The similar effects brought about by altered activity or abundance for the CP components Cdc68 and Pob3 and by the histones are other indications that the CP complex is involved in chromatin structure and/or remodeling.

Here we report initial structure/function studies of the Cdc68 component of the CP complex (ROWLEY et al. 1991 ). As one approach we have cloned a functional homolog of the CDC68 gene from the yeast Kluyveromyces lactis and show that the polypeptide encoded by this K. lactis gene is homologous to S. cerevisiae Cdc68 along its entire length. Deletion studies show that the N-terminal 30% of Cdc68, a region highly conserved between these two yeast homologs, is not required for essential transcription functions. In contrast, this N-terminal domain is necessary for effective chromatin-mediated repression and is partially responsible for the need for the Swi/Snf chromatin-remodeling complex. The N-terminal region of this CP component therefore mediates chromatin repression, while transcriptional activation is facilitated by the rest of the CP complex.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and media:
Yeast strains used in this study (Table 1) were grown as described (ROWLEY et al. 1991 ; XU et al. 1993 ). Recombinant DNA manipulations were carried out by standard procedures (SAMBROOK et al. 1989 ).

Plasmids:
The CDC68 and cdc68-197 plasmids pSC2-1, pBM13, and pBM46 have been described (PRENDERGAST et al. 1990 ; MALONE et al. 1991 ). The multicopy CDC68 plasmids p68-Ba-1A and YEpDE682 contain, respectively, the 5.2-kbp BamHI fragment and 4.75-kbp BglII-BamHI fragment from pSC2-1 in the BamHI site of YEp352 (HILL et al. 1986 ). The low-copy CDC68 plasmid pDE683 contains the 5.2-kbp KpnI-XbaI fragment from p68-Ba-1A between the KpnI and XbaI sites of pRS316 (SIKORSKI and HIETER 1989 ). The gal1UAS CEN LEU2 plasmid pAW638 was constructed by replacing the 2.6-kbp XhoI-SstI fragment of the SSA2-lacZ plasmid YCp102 (BARNES 1998 ) with the 2.7-kbp SmaI-SstI fragment from the gal1UAS-lacZ plasmid pJL638 (LI and HERSKOWITZ 1993 ; a gift from M. Dobson).

Generation of nested deletions in the CDC68 gene:
Unidirectional nested deletions in p68-Ba-1A were prepared using ExoIII and mung bean nucleases. The SstI and SmaI multiple cloning sites were used to generate 5' deletions; for 3' deletions the multiple cloning sites PstI and XbaI were used. All deleted clones were restriction mapped and most insert-vector junctions were sequenced.

Isolation of cdc68-1 mutant sequences by gap repair:
YEpDE682 was cleaved with SpeI and BstEII, generating a 2.4-kbp gap spanning the region to which the cdc68-1 mutation was localized (see RESULTS), and then transformed into the cdc68-1 strain ART68-1. The recircularized, gap-repaired plasmid YEpDE684 recovered from a Ura⁺ transformant was verified by restriction mapping with EcoRI and sequenced through open reading frame (ORF) nucleotides 19–535 to identify the cdc68-1 mutation.

Confirmation of cdc68-1 and cdc68-197:
The cdc68-197 (spt16-197) mutation was identified by sequencing a 1.2-kbp BglII-EcoRI fragment from pBM46 (MALONE et al. 1991 ). The equivalent fragment from pBM13, the unmutagenized precursor of pBM46, was also sequenced and found to be identical to that previously described (ROWLEY et al. 1991 ). The 2.1-kbp BamHI-ClaI fragment from pBM46 (containing the cdc68-197 sequence alteration) was used to replace the equivalent fragment of CDC68 in pDE683 and the resultant plasmid, pDE47, was tested after plasmid shuffling (SIKORSKI and BOEKE 1991 ).

Plasmid shuffling:
Test plasmids carrying the URA3 marker were introduced into cells of strain DE13a and Ura⁺ transformants were selected on medium lacking uracil. To determine if a URA3 plasmid could substitute for the resident CDC68 TRP1 plasmid, pBM13, transformants were then grown to stationary phase in YM1 medium, spread on YEPD medium for colony formation, and replica-plated to selective medium lacking leucine (to confirm the presence of the chromosomal cdc68::LEU2 [spt16-101::LEU2] disruption allele) or tryptophan (to test for the presence of pBM13), or uracil.

In vitro mutagenesis and targeted deletion of CDC68 coding sequences:
Mutant alleles deleted for CDC68 ORF sequences but expressed from the native CDC68 promoter were constructed by in vitro mutagenesis. The 1.8-kbp SstI-EagI fragment from p68-Ba-1A encompassing the promoter region and 5' end of the CDC68 ORF was first inserted into pBSII KS⁺ (Stratagene, La Jolla, CA) and a ClaI site was created at ORF nucleotides 16–21 by oligonucleotide-mediated site-directed mutagenesis (KUNKEL et al. 1987 ), using single-stranded DNA (RUSSEL et al. 1986 ) and Sequenase version 2.0 (United States Biochemical, Cleveland) for primer extension. The mutagenic primer, 5'-AGCTGAATATCGATTTTGAC, contains a single mismatch to ORF nucleotide 18 and creates a ClaI restriction site. The desired product of the mutagenesis, pDE-68M, was identified by ClaI cleavage and DNA sequencing. To generate a 0.9-kbp in-frame deletion between the new ClaI site and a second ClaI site (ORF nucleotide 920), pDE-68M was cleaved with ClaI and recircularized by ligation at low concentration. The 0.9-kbp KpnI-EagI fragment containing this deletion was then used to replace the 1.8-kbp KpnI-EagI fragment of p68-Ba-1A, generating the cdc68-922 allele and YEpDE-MC4. The 3.75-kbp SstI-SphI cdc68-922 fragment was moved to YEp351 to create YEpSH922. Low-copy cdc68-922 plasmids pDE-MC41 and pDE-141 were constructed by inserting into pRS316 the YEpDE-MC4 4.3-kbp KpnI-XbaI fragment and the 3.8-kbp BglII-XbaI fragment, respectively.

An in-frame deletion of the SalI-HpaI fragment was constructed by cleaving pDE-MC41 with SalI and HpaI, filling in the 5'-protruding end of the SalI site with Klenow enzyme and recircularizing using T4 ligase, generating pDE-MC43, which contains both 5' and central in-frame ORF deletions. The 2.9-kbp EagI-XbaI fragment from p68-BA-1A was then replaced with the 1.8-kbp EagI-XbaI fragment from pDE-MC43 to generate YEpDE-MC43, which contains an in-frame deletion of the SalI-HpaI ORF fragment. Using the ClaI site at ORF nucleotide 923, an in-frame deletion of the ClaI-SalI ORF fragment was constructed by first inserting the 5.2-kbp KpnI-XbaI fragment from p68-Ba-1A into pRS316. The resultant plasmid, pDE-683, was then cleaved with ClaI and SalI, the 5'-protruding ends were made flush using Klenow enzyme and the DNA was recircularized using T4 ligase, generating pDE-CS8, whose 4.5-kbp KpnI-XbaI fragment was transferred to YEp352 to generate plasmid YEpDE-CS8. Similarly, using the ClaI site created at ORF nucleotide 17 an in-frame ClaI-SalI deletion was constructed in plasmid QX681, creating plasmid QX681-CS. Plasmid p68d915-1035 was constructed by inserting an XbaI double-stranded oligonucleotide 5'-CTAGTCTAGACTAG (New England Biolabs, Beverly, MA) into the HpaI site of CDC68 in p68-Ba-1A, creating a truncated ORF in which a single phenylalanine (only) is encoded following W913.

Frameshift mutations:
A +2 frameshift mutation was generated by cleaving pDE-683 (prepared from the dam^- Escherichia coli strain BW58) with ClaI, filling in 5' protruding ends with Klenow enzyme and ligating at low concentration, generating pDE-921. The resultant polypeptide contains the C-terminal extension AIHLKKWPTTTIFY following I307. The 5.2-kbp KpnI-XbaI fragment of pDE-921 was then transferred to YEp352, generating plasmid YEpDE-921. A similar +2 frameshift mutation was introduced at the 5' end of the CDC68 ORF in pDE-68M that had been prepared from the dam⁺ E. coli strain DH5 to block cleavage at the ClaI site at nucleotide position 923, generating YEpDE-45. Both frameshift mutations were verified by sequencing.

Nucleotide sequencing:
Nucleotide sequencing was carried out using Sequenase version 2.0 (United States Biochemicals, Cleveland, OH) and [³⁵S]dATP (New England Nuclear, Boston). The junctions between deleted inserts and the YEp352 vector in plasmids of the p68-Exo1 series were sequenced directly using the M13–40 primer, while insert-vector junctions for plasmids of the p68-Exo2 series were sequenced using Reverse Primer. Plasmid DNA was sequenced directly (MIERENDORF and PFEFFER 1987 ).

Replacement of the chromosomal CDC68 gene with cdc68-922:
The cdc68-922 allele was integrated into the chromosome by a modification of the two-step gene-replacement method (SHERER and DAVIS 1979 ). The URA3 plasmid pDE-1410, comprising the 3.8-kbp BglII-XbaI cdc68-922 fragment in pRS306 (SIKORSKI and HIETER 1989 ), was cleaved at a unique HpaI site in cdc68-922 sequences and transformed into the cdc68::LEU2/+ ura3-52/ura3-52 diploid strain BM64. Ura⁺ transformants were patched onto medium containing 5-fluoro-orotic acid and Ura^- recombinants were tested for leucine auxotrophy. Ura^- Leu^- recombinants were selected as candidate strains in which the pDE-1410 DNA had initially integrated at the chromosomal cdc68::LEU2 locus and from which recombination had excised cdc68::LEU2 sequences. Strains in which replacement of cdc68::LEU2 by the cdc68-922 allele had occurred were identified by Southern analysis of BamHI-digested genomic DNA and one such strain, DE4B, was selected for further study.

Cloning the K. lactis CDC68 homolog:
Genomic sequences from the budding yeast K. lactis that complement the temperature sensitivity of cdc68-1 were identified in a low-copy (CEN) genomic library (STARK and MILNER 1989 ); the active region was localized by subcloning and sequenced on both strands. The ORF so identified is named KlCDC68 (accession number U48701).

RNA analysis:
RNA was prepared, resolved, and subjected to Northern analysis as described (XU et al. 1993 ). The suc2UAS probe was from pRB58 (CARLSON and BOTSTEIN 1982 ; a gift from F. Winston), while the HTA1+HTB1+ADK1 probe was as described (XU et al. 1993 ).

Protein stability assay:
Cells were inhibited with cycloheximide and extracts were prepared, resolved electrophoretically, and analyzed by immunoblot as described (XU et al. 1995 ), using polyclonal anti-Cdc68 antibodies (BREWSTER et al. 1998 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Deletions of the CDC68 gene:
Many proteins consist of multiple domains, which in some cases can function independently. This modular feature of proteins is particularly evident for proteins involved in transcription (FRANKEL and KIM 1991 ). To assess potential domains within the Cdc68 component of the CP complex we constructed deletions of the CDC68 gene using ExoIII exonuclease and tested these deleted clones for the restoration of high-temperature growth of temperature-sensitive cdc68 mutant cells. These studies (Figure 1A) suggested that C-terminal coding sequences of Cdc68 are essential for CP function. A more striking observation was that six of the clones deleted for 5' flanking and N-terminal coding sequences alleviated the temperature sensitivity of cdc68 mutations, suggesting that these plasmids supply essential Cdc68 function (Figure 1A).

View larger version (34K): In this window In a new window Download PPT slide   Figure 1. Deletion analysis of the CDC68 gene. The indicated sequences, with shaded areas marking the ORF, were tested in multicopy plasmids for 37° complementation of the cdc68-1 and cdc68-197 mutations; similar results were found for both mutations. +, good growth; (+), some growth; -, no growth; (P), papillae containing temperature-resistant cells. (A) 5' and 3' deletions, with the junction nucleotide of the remaining CDC68 sequences indicated flanking the sequence. (B) In-frame deletions and frameshifts; coding sequences following frameshifts are lightly shaded. (C) Restriction fragments, generated by the enzyme indicated in the plasmid name. The fragments in p68-Hp-2B and p68-Ec-2B also contain YEp24 vector sequences to the left of the indicated sequence. Restriction-site symbols and cleavage positions: B, BamHI, -788, 3879; C, ClaI, 923; boxed C, ClaI site created by site-directed mutagenesis, 17; S, SalI, 1663; Ec, EcoRI, 475, 1655, 3750; Hi, HindIII, 2288; Hp, HpaI, 532, 2739.

The complementation by N-terminally deleted CDC68 plasmids caused us to determine if the CDC68 ORF inferred from the nucleotide sequence accurately predicts the Cdc68 protein. Two frameshift mutations, one 6 codons downstream of the 5' ATG of the ORF and the other at codon 308, each abolished gene function (Figure 1B). These findings suggest that Cdc68 is in fact encoded by the entire ORF. The sizes of CDC68 mRNA (3.2 kb; ROWLEY et al. 1991 ) and the Cdc68 protein itself (BREWSTER et al. 1998 ) are entirely consistent with this conclusion.

An internally deleted Cdc68 polypeptide lacking N-terminal sequences supplies essential function:
In the six functional but 5'-deleted clones described above the CDC68 promoter and the first ATG of the ORF are missing. (The truncated genes are presumably expressed from a fortuitous vector promoter.) To generate truncated forms of Cdc68 that are expressed from the authentic CDC68 promoter, and thus with known structure and expression, we made in-frame deletions within the CDC68 ORF. One of these deletion alleles, termed cdc68-922 (Figure 1B, plasmid YEpDE-MC4), is missing nucleotides 19 to 921 of the ORF (up to the ClaI site), more than those removed by the largest functional 5' deletion generated by ExoIII digestion (Figure 1A, Figure P68-Exo2-4). The cdc68-922 ORF contains the first 5 CDC68 codons (including the putative translation initiation codon) fused in-frame to codon 307.

A plasmid-borne cdc68-922 allele alleviated the 37° temperature sensitivity of cdc68-1 and cdc68-197 mutant cells (Figure 1B); this effect was also seen for cdc68-922 on a low-copy CEN vector (data not shown). The cdc68-922 allele could also support the growth of cells lacking a functional CDC68 chromosomal locus. To show this we used the diploid strain BM64, in which one CDC68 homolog is disrupted by a LEU2 insertion and thus unable to support growth (MALONE et al. 1991 ); Leu⁺ segregants were recovered after sporulation of strain BM64 harboring a low-copy cdc68-922 plasmid, indicating that cdc68-922 supplies essential Cdc68 function.

A larger in-frame deletion removing additional sequences down to codon 555 generated an N-terminally truncated Cdc68 polypeptide without detectable function (Figure 1B, plasmid pQX681-CS). This deletion and cdc68-922 therefore roughly demarcate the extent of Cdc68 necessary for function. The essential nature of sequences downstream of codon 306 was confirmed by the elimination of central or C-terminal sequences through in-frame deletions, ExoIII deletions, or frame-shift mutation: all of these deleted versions of Cdc68 were nonfunctional in complementation assays (Figure 1A and Figure B).

The above experiments used cells in which the chromosomal CDC68 ORF is simply disrupted (MALONE et al. 1991 ; ROWLEY et al. 1991 ), and which may as a consequence contain Cdc68 fragments synthesized from intact N-terminal sequences of cdc68::LEU2. We therefore showed that cdc68-922 is functional in the absence of any other CDC68 sequences. The cdc68::LEU2 allele in diploid strain BM64 was replaced with the cdc68-922 allele, generating the cdc68-922/CDC68 strain DE4B. Sporulation of this diploid showed that in each of the 20 tetrads analyzed all four spores were viable; spore germination and mitotic growth can thus be supported entirely by the Cdc68-922 polypeptide. In liquid culture at 22° the growth rate of cdc68-922 mutant segregants was the same as that of CDC68 wild-type cells (data not shown). In these cdc68-922 mutant cells the truncated Cdc68 protein is present at normal abundance, as shown by immunoblot analysis of cdc68-922 and CDC68 cell extracts (data not shown), and is still associated with the Pob3 protein in the CP complex (BREWSTER et al. 1998 ). Therefore the N-terminally deleted polypeptide encoded by the cdc68-922 mutant allele provides all Cdc68 functions necessary for the transcription of essential genes (ROWLEY et al. 1991 ).

N-terminal sequences are conserved in K. lactis Cdc68:
The growth of cdc68-922 mutant cells raised questions concerning the importance of the N-terminal portion of the Cdc68 polypeptide and suggested that a Cdc68 protein from another organism would provide useful structural information. We therefore cloned a CDC68 homolog from the yeast K. lactis by functional complementation of the S. cerevisiae cdc68-1 temperature sensitivity. Sequencing showed that the complementing K. lactis genomic DNA encodes a 1033-residue polypeptide highly homologous (72.3% identical) to S. cerevisiae Cdc68 over its entire length (Figure 2). This homology is especially evident in the 443-residue stretch from amino acids 530 to 972 (85.6% identity). Moreover, the N-terminal domain missing from the Cdc68-922 polypeptide is also highly conserved between the S. cerevisiae and K. lactis versions of Cdc68 (70.6% identity for residues 27–308). This conservation of the N-terminal domain of Cdc68 suggests that the entire protein is important for function.

View larger version (69K): In this window In a new window Download PPT slide   Figure 2. The K. lactis Cdc68 protein (top), aligned with Cdc68 from S. cerevisiae. Identities are indicated by double dots in the S. cerevisiae sequence, while dashes mark gaps introduced to maximize alignment. The glycine residue at position 132 that is replaced in temperature-sensitive mutant forms of S. cerevisiae Cdc68 is in bold type, while the analogous glycine in the K. lactis version is highlighted. Residues not encoded by the S. cerevisiae cdc68-922 allele are overlined.

The Cdc68 N-terminal domain affects transcription—The Spt^- effect:
The his4-912 and lys2-128 mutant alleles cause histidine and lysine auxotrophy, respectively, because the sequence inserted in the 5' region of each gene alters the pattern of transcription (WINSTON 1992 ; WINSTON and CARLSON 1992 ). Both increased Cdc68 activity through increased gene dosage and decreased activity caused by temperature-sensitive cdc68 mutations can affect transcription and restore histidine and/or lysine prototrophy to his4-912 lys2-128 mutant cells (the Spt^- phenotype; MALONE et al. 1991 ; ROWLEY et al. 1991 ; BREWSTER et al. 1998 ).

Like other cdc68 mutations, cdc68-922 confers an Spt^- phenotype; lys2-128 cdc68::LEU2 haploid cells harboring a low-copy cdc68-922 plasmid were Lys⁺, whereas the same recipient cells harboring a low-copy CDC68 plasmid were not (Figure 3). Similarly, for each of 20 tetrads from a cdc68-922/CDC68 diploid that was homozygous for both his4-912 and lys2-128, the Spt^- phenotype of His⁺ Lys⁺ growth segregated 2:2 and cosegregated with the temperature sensitivity that we show below is due to cdc68-922 (data not shown). Thus the Cdc68-922 polypeptide alters transcription at the his4-912 and lys2-128 loci. This alteration was seen not only at 30° as found for other cdc68 mutant alleles (MALONE et al. 1991 ; ROWLEY et al. 1991 ), but also at 22°. At all temperatures the effects of cdc68-922 were recessive to those of the wild-type CDC68 gene (data not shown). Thus the CP complex containing the Cdc68-922 protein has a constitutively decreased activity.

View larger version (68K): In this window In a new window Download PPT slide   Figure 3. cdc68-922 confers an Spt- phenotype. From transformants of the cdc68-101::LEU2/CDC68 heterozygous diploid strain BM64, cdc68-101::LEU2 his4-912 lys2-128 segregants harboring multicopy cdc68-922 or CDC68 plasmids, or low-copy (CEN) cdc68-922 or CDC68 plasmids (YEpDE-MC4, p68-Ba-1A, pDE-MC4, and pDE-683, respectively), were identified, spread on synthetic complete media lacking either histidine or lysine and incubated at 23°.

The Cdc68 N-terminal domain helps bring about the need for the Swi/Snf complex:
Chromatin can be remodeled by a large protein complex termed the Swi/Snf complex (reviewed in PETERSON and TAMKUN 1995 ). This complex can be isolated from yeast and mammalian cells as a distinct multiprotein assembly (CAIRNS et al. 1994 ; KWON et al. 1994 ; PETERSON et al. 1994 ). In cells containing structurally normal CP complex the Swi/Snf complex facilitates the expression of several genes. For example, mutations that disable the Swi2/Snf2 or Snf5 components of the Swi/Snf complex prevent effective SUC2 expression and cause a Suc^- phenotype (ABRAMS et al. 1986 ; HIRSCHHORN et al. 1992 ). However, Suc⁺ growth by swi2 mutant cells and snf5 mutant cells is restored at 30° by the cdc68-197 mutation (MALONE et al. 1991 ), suggesting that Swi/Snf chromatin-remodeling activity can be bypassed to some extent by altering the CP complex.

The Swi/Snf complex is also less important for gene expression in cdc68-922 mutant cells. We showed this genetically by crossing a cdc68-922 strain with a snf5::URA3 strain and analyzing the growth of meiotic segregants (and parental strains); cdc68-922::LEU2, a cdc68-922 mutant allele with an adjacent integrated LEU2 gene, was used to allow unambiguous assignment of genotype. All CDC68 SNF5 and cdc68-922 SNF5 segregants (13 and 10, respectively) were Suc⁺, as indicated by fermentative growth (NEIGEBORN et al. 1986 ) using raffinose as carbon source, and all 10 CDC68 snf5 segregants were Suc^-. Nine of the 14 cdc68-922 snf5 segregants were weakly Suc⁺, showing that cdc68-922 can allow SUC2 expression in the absence of Swi/Snf function. Stronger evidence for this effect was provided by scoring inositol auxotrophy: INO1 gene expression is also Swi/Snf dependent (PETERSON et al. 1991 ). All of the 14 cdc68-922 snf5 segregants were Ino⁺, as were all of the cdc68-922 SNF5 and CDC68 SNF5 segregants, while all of the CDC68 snf5 segregants were Ino^- (data not shown). Elimination of the Cdc68 N-terminal domain from the CP complex can therefore decrease the reliance on Swi/Snf activity for gene expression. These findings suggest that the CP complex, through the Cdc68 N-terminal domain, affects chromatin structure to inhibit transcription, and that Swi/Snf activity counteracts this inhibition.

The Cdc68 N-terminal domain strengthens repression in the absence of trans-activators:
Deletion of SUC2 upstream activating sequences impairs growth on sucrose or raffinose (suc2UAS; SAROKIN and CARLSON 1984 ), but this growth can be restored by cdc68 mutations (MALONE et al. 1991 ; PRELICH and WINSTON 1993 ). Thus a second effect of the CP complex is to repress transcription in the absence of DNA-bound trans-activators. To determine if the Cdc68 N-terminal domain is involved in this repression we crossed the Suc⁺ strain DE4B-17b (cdc68-922 SUC2) with the Suc^- strain BM404 (CDC68 suc2UAS) and scored the growth of meiotic segregants as above. As expected, temperature sensitivity (cdc68-922) segregated 2:2 for the 23 tetrads tested, whereas Suc⁺ segregants were in excess (in 14 tetrads Suc⁺:Suc^- segregated 3:1, and 6 others were 4:0), indicating that some segregants were Suc⁺ due to increased expression of suc2UAS. All temperature-sensitive (cdc68-922) segregants were Suc⁺ (even though the CDC68 and SUC2 genetic loci assort independently), indicating that cdc68-922 can mediate this increased suc2UAS expression. Suc⁺ growth was seen not only at 30°, as found for cdc68-197 (MALONE et al. 1991 ), but also at 22°. Increased cdc68-922 gene dosage did not restore suc2UAS repression in cdc68-922 mutant cells, and cells remained Suc⁺ (data not shown). Thus the CP complex lacking the Cdc68 N-terminal domain is unable to maintain effective chromatin repression.

We also measured cdc68-922 effects on expression from another core promoter lacking trans-activation sequences, that of the GAL1 gene deleted for UAS_GAL. Transcription from this gal1UAS promoter was monitored as ß-galactosidase activity expressed by a gal1UAS-lacZ reporter gene on a centromeric plasmid. This ß-galactosidase activity was greater in cdc68-922 mutant cells than in CDC68 cells (0.7 Miller units for cdc68-922 mutant cells growing on raffinose medium at 23° vs. <0.1 Miller units for CDC68 cells under the same growth conditions). Therefore, for at least two core promoters (lacking trans-activation) the CP complex lacking the Cdc68 N-terminal domain is unable to maintain full repression, suggesting that the CP complex has a general role in repression.

Core-promoter repression by the CP complex is not mediated through histone gene expression:
Transcriptional repression at suc2UAS and other loci is maintained by normal histone abundance and stoichiometry, as shown by the fact that altered histone gene expression can activate suc2UAS (CLARK-ADAMS et al. 1988 ). The CP complex facilitates expression of the HTA1-HTB1 gene pair encoding histones H2A and H2B, respectively (XU et al. 1993 ), raising the possibility that impaired repression shown above for suc2UAS may result indirectly from cdc68 effects on histone gene expression. We therefore determined if the activation of suc2UAS in cdc68 mutant cells depends on histone gene expression. For these experiments we used the cdc68-197 gene, whose activity can be rapidly decreased by temperature shift. Populations of actively growing cells were aligned at the G₂/M cell-cycle boundary by treatment with nocodazole (JACOBS et al. 1988 ), a situation that allows virtually no histone gene expression (Figure 4). After the cells had become aligned at the nocodazole blockpoint, the blocked culture was transferred to 37° for further incubation. Northern analysis showed that this 37° incubation resulted in a significant increase in suc2UAS mRNA levels within 30 min of transfer in cdc68-197 mutant cells, while there was no increase in CDC68 cells similarly treated (Figure 4A). During the 37° incubation in this protocol histone mRNA levels remained low (Figure 4B), consistent with the absence of cdc68 effects on the timing of histone gene expression (XU et al. 1993 ). These findings suggest that altered histone stoichiometry through altered histone biosynthesis may not be necessary for the activation of the suc2UAS core promoter by modified CP-complex function.

View larger version (73K): In this window In a new window Download PPT slide   Figure 4. Core-promoter derepression upon depletion of Cdc68 activity is independent of histone gene expression. To cells growing in glucose medium, nocodazole (15 µg/ml) was added to block the cell cycle in G2/M phase. After 4–5 hr further incubation, when each population had accumulated with the characteristic large-bud morphology, the arrested cells were transferred to complex rich medium containing nocodazole and 2% sucrose instead of glucose, and incubation was continued for a further 90 min. One portion of each culture was then supplemented with an additional 10 µg nocodazole/ml and transferred to 37° for further incubation. At the indicated times samples were removed for the preparation of total RNA, which was then resolved and probed for mRNAs. Each population remained arrested for the duration of the experiment, as indicated by cell morphology. (A) RNA from the cdc68-197 suc2UAS strain BM403 and the CDC68 suc2UAS strain BM404 probed for suc2UAS mRNA; rRNA levels serve as loading controls. (B) RNA from the cdc68-1 strain 68507A and the CDC68 strain 21R, and from control cells growing at 23° in the absence of nocodazole (lane C), probed for HTA1+HTB1 (histones H2A and H2B) mRNA and the internal-control ADK1 mRNA that is visualized by the same probe. Equal loading was verified by rRNA levels (not shown).

The Cdc68 N-terminal domain is altered by temperature-sensitive point mutations:
In cdc68-1 and cdc68-197 cells certain noncomplementing plasmids harboring only fragments of the CDC68 gene gave rise to temperature-resistant papillae (Figure 1) consisting of cells that remained temperature-resistant after plasmid loss (data not shown). Thus a functional CDC68 gene results from recombination between the mutant chromosomal locus and plasmid-borne wild-type DNA. Plasmids with this effect all contain sequences upstream of the EcoRI site within codon 159, localizing mutations responsible for the temperature sensitivity of cdc68-1 and cdc68-197 to the 5' portion of the gene.

Nucleotide sequencing showed that the cdc68-1 and cdc68-197 mutant alleles each have only one sequence alteration within the ORF upstream of the codon-159 EcoRI site, a G-to-A substitution at nucleotide 395 causing a glycine-to-aspartate substitution at residue 132 (G132D). This mutation generates an EcoRV site; Southern analysis revealed that both the cdc68-1 strain ART68-1 and the cdc68-197 strain L577 (distinguishable by auxotrophies) contain this site, which was absent from wild-type genomic DNA (data not shown). A fragment from cdc68-197 encoding the G132D substitution was used to replace the homologous wild-type fragment in a low-copy CDC68 plasmid; the resultant recombinant plasmid was shown, by plasmid shuffling in a cdc68::LEU2 strain, to confer the temperature sensitivity and Spt^- phenotype of cdc68-197 (data not shown). We conclude that the cdc68-1 and cdc68-197 mutant effects are due to a G132D substitution in Cdc68.

Restriction analysis showed that another allele, cdc68-11 (LYCAN et al. 1994 ), also contains the EcoRV site diagnostic of the G132D substitution, while the parent strain in which cdc68-11 was isolated is missing this EcoRV site (data not shown). Thus three independently isolated cdc68 mutant alleles each encode the same amino acid substitution in the N-terminal region.

CP complex lacking the Cdc68 N-terminal domain has temperature-sensitive function:
Although the Cdc68-922 protein can supply all essential CP functions, cdc68-922 mutant cells grew poorly at 37° (Figure 5). Diploid cells heterozygous for cdc68-922 were temperature resistant, indicating that the temperature sensitivity caused by cdc68-922 is recessive. This finding indicates decreased Cdc68-922 protein function at elevated temperatures.

View larger version (60K): In this window In a new window Download PPT slide   Figure 5. The cdc68-922 mutant allele supports growth in a temperature-sensitive and dosage-dependent manner. Cells of the cdc68-922 strain DE4B-17c harboring either the multicopy cdc68-922 plasmid YEpSH922 or the control vector YEp351 were spread on solid medium and incubated at the indicated temperatures.

The presence in cdc68-922 cells of plasmid YEpDE-921, encoding the N-terminal 307 residues (only) of Cdc68 (the same segment missing from Cdc68-922), had no effect on temperature sensitivity (data not shown). On the other hand, 37° growth is restored by increased gene dosage: cdc68-922 mutant cells harboring a cdc68-922 low-copy plasmid grew at 37° (data not shown), while a cdc68-922 multicopy plasmid allowed even better growth (Figure 5). Therefore, the CP complex containing the Cdc68-922 polypeptide functions at 37°, but too weakly (at normal levels of expression) to support high-temperature growth.

San1 mediates cdc68-922 temperature sensitivity but not other effects:
The San1 protein (SCHNELL et al. 1989 ) can antagonize CP activity: san1 mutations alleviate the temperature sensitivity (at 35°) and reverse the Spt^- phenotype of cdc68 point-mutant cells (XU et al. 1993 ). To determine the effects of a san1 mutation in cdc68-922 mutant cells, a strain containing a cdc68-1 allele identifiable by a URA3 gene integrated downstream (cdc68-1::URA3; ROWLEY et al. 1991 ) and san1-3, a mutant allele that alleviates cdc68-1 temperature sensitivity, was crossed with a cdc68-922 SAN1 strain, and the diploid was sporulated. In the 12 tetrads tested, 2:2 segregation was seen for Ura⁺ (cdc68-1::URA3) and for temperature resistance; some Ura^- (cdc68-922) segregants were temperature resistant. Thus the temperature sensitivity of both cdc68-1 and cdc68-922 is alleviated by san1-3. Southern analysis and transformation with a low-copy SAN1 plasmid confirmed that the temperature resistance of the cdc68-922 cells is due to san1-3 (data not shown). The san1::URA3 deletion/replacement allele (XU et al. 1993 ) also alleviated cdc68-922 temperature sensitivity (data not shown). San1 therefore antagonizes high-temperature function of the CP complex lacking the Cdc68 N-terminal domain.

At 33° a low-copy SAN1 plasmid markedly inhibited the growth of cdc68-922 mutant cells (Figure 6A), with no effect on wild-type CDC68 cells as previously noted (XU et al. 1993 ). Increased SAN1 gene dosage therefore exacerbates the temperature sensitivity of cdc68-922 mutant cells, as it does for cdc68 point-mutant cells (XU et al. 1993 ). SAN1 gene dosage had negligible effect in cdc68-922 mutant cells at 22° (Figure 6A), a finding analogous to that for cdc68-1 or cdc68-197 mutant cells at a permissive temperature (XU et al. 1993 ).

View larger version (68K): In this window In a new window Download PPT slide   Figure 6. San1 mediates certain Cdc68-922 functions. (A) Increased SAN1 gene dosage exacerbates cdc68-922 temperature sensitivity. Wild-type (strain DE4B-17a) and cdc68-922 mutant cells (strain DE4B-17b) harboring the low-copy SAN1 plasmid pBE3 (XU et al. 1993 ) or the control vector pRS316 were spread on selective medium and incubated at 33°. (B) san1 mutations do not affect the Spt- phenotype of cdc68-922. Cells of the lys2-128 strains DE4B-17c (cdc68-922), DE81-2b (cdc68-922 san1-3), DE1N-15d (cdc68-922 san1::URA3), and DE4B-17a (CDC68 SAN1) were spread on synthetic complete medium lacking lysine and incubated at 22°.

San1 function did not affect the cdc68-922 Spt^- phenotype: cdc68-922 lys2-128 cells containing either san1-3 or san1::URA3 grew on medium lacking lysine (Figure 6B). This finding contrasts with the effective suppression of the Spt^- phenotype of cdc68-1 and cdc68-197 mutant cells by san1 mutations (XU et al. 1993 ). Eliminating San1 protein also fails to restore chromatin repression to cdc68-922 mutant cells: san1::URA3 did not alter the Suc⁺ growth of cdc68-922 suc2UAS mutant cells (data not shown), whereas san1 cdc68-1 suc2UAS triple-mutant cells are Suc^- due to san1 suppression (XU et al. 1993 ). Thus the mutant effects of cdc68-922 cannot be completely alleviated by eliminating San1 function, suggesting that the Spt^- and repression effects and the temperature sensitivity of cdc68-922 mutant cells may reflect different aspects of CP-complex function.

sug1 mutations that suppress cdc68-1 do not alleviate cdc68-922 temperature sensitivity:
The temperature sensitivity caused by cdc68-1 can also be alleviated by sug1 mutations (XU et al. 1995 ). The Sug1 protein is part of the 26S proteasome complex for ubiquitin-targeted protein degradation (GHISLAIN et al. 1993 ; RUBIN et al. 1996 ), and a sug1 mutation identified by cdc68-1 suppression has other effects consistent with impaired protein degradation (Q. XU, G. C. JOHNSTON and R. A. SINGER, unpublished results). Therefore protein degradation is implicated in this suppression. Tetrad analysis showed that sug1 mutant alleles that alleviate the temperature sensitivity of cdc68-1 do not affect the temperature sensitivity of cdc68-922 (data not shown). In fact, a sug1 mutant allele (GHISLAIN et al. 1993 ) that does not affect cdc68-1 (XU et al. 1995 ) further impaired the growth of cdc68-922 mutant cells at 35°, so that sug1-3 cdc68-922 double-mutant cells showed less growth than single-mutant segregants (data not shown). Similarly, sug1 mutant alleles did not alter the gene-dosage effects of cdc68-922 (data not shown). These observations suggest that protein degradation may not be an important feature of cdc68-922 temperature sensitivity.

Polypeptide stability:
Cdc68 is a stable protein, while that encoded by cdc68-1 is inherently unstable and is degraded more rapidly at high temperature (XU et al. 1995 ). To assess directly the stability of the Cdc68-922 mutant protein, cycloheximide was added to growing cells to halt new protein synthesis and the abundance of preexisting Cdc68 protein was determined over time by immunoblotting with polyclonal antiserum (XU et al. 1995 ). In this assay the Cdc68-922 polypeptide was stable at 35° and unaffected by san1-3 (Figure 7). Thus neither cdc68-922 temperature sensitivity nor its alleviation by san1-3 is accounted for by altered abundance or stability of the Cdc68-922 polypeptide. In contrast, the unstable Cdc68-1 polypeptide was indeed stabilized by the san1-3 suppressor mutation (Figure 7), suggesting that decreased protein degradation may contribute to the effects of san1-3.

View larger version (20K): In this window In a new window Download PPT slide   Figure 7. The Cdc68-922 protein is stable, whereas San1 destabilizes the Cdc68-1 mutant protein. Cells of strains DE4B-17b (cdc68-922), DE81-6c (cdc68-922 san1-3), 68507A (cdc68-1), and QX3 (cdc68-1 san1-3) growing at 23° were treated with cycloheximide to inhibit further protein synthesis, and then immediately transferred to 35° for further incubation. Extracts were prepared at the indicated times and immunoblotted with antibodies directed against Cdc68 and ß-tubulin as an internal control.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The CP complex is necessary both for gene activation by trans-activators and for repression at core promoters (BREWSTER et al. 1998 ). These functions are revealed by the effects of mutations that affect the Cdc68 component of the CP complex. A transfer of cdc68 mutant cells to 37° causes a rapid inhibition of transcription for a wide spectrum of genes, indicating that the CP complex facilitates transcription (ROWLEY et al. 1991 ; XU et al. 1993 ). At 30°, however, cdc68 mutations have little effect on gene activation but allow transcription from core promoters (lacking trans-activators or their binding sites) that are otherwise subject to the repressive effects of chromatin (MALONE et al. 1991 ; PRELICH and WINSTON 1993 ; XU et al. 1993 ; LYCAN et al. 1994 ). Until now the relationship between these positive and negative activities of the Cdc68 protein and the CP complex has not been investigated; we show here using the newly created cdc68-922 mutant allele that only a portion of the Cdc68 protein is needed for the transcription function of the CP complex and that this activation domain can be distinguished structurally from a Cdc68 domain that is necessary for CP-mediated repression.

The cdc68 point mutation:
Previous evidence for Cdc68 function was provided by the effects of three independently derived temperature-sensitive cdc68 mutations (MALONE et al. 1991 ; ROWLEY et al. 1991 ; LYCAN et al. 1994 ). Remarkably, each of these mutant alleles, as shown here, contains the same base pair substitution that is sufficient for temperature sensitivity. This mutation destabilizes the mutant Cdc68 polypeptide (XU et al. 1995 ; Figure 7); thus a decrease in Cdc68 protein abundance may be sufficient to compromise CP-mediated chromatin repression and gene activation. This suggestion is supported by two findings. First, mutations that enfeeble the Sug1 protein, a component of the nuclear 26S proteasome that mediates targeted protein degradation (RUBIN et al. 1996 ), can alleviate the effects of the cdc68 point mutation on chromatin repression and cell growth (XU et al. 1995 ); indeed, a suppressing sug1 mutation stabilizes the mutant Cdc68 polypeptide (XU et al. 1995 ) and exerts other effects consistent with decreased protein degradation (Q. XU, G. C. JOHNSTON and R. A. SINGER, unpublished results). Second, gene-dosage studies show that the Cdc68-197 mutant protein retains function even at 37°, suggesting that much of the effects of the cdc68 point mutation, including impaired core-promoter repression, may be due to inadequate Cdc68 mutant protein (and CP complex) abundance (XU et al. 1993 ). The localization of the point mutations to the N-terminal portion of the CDC68 ORF may be indicative of a surface of the Cdc68 protein that can be a sensitive target for ubiquitin-mediated protein degradation. Alternatively, the unexpected identity of the three independent temperature-sensitive mutations may reflect specificity in N-terminal functions, an idea consistent with the conservation of the mutated glycine-132 in the Cdc68 polypeptide of K. lactis (Figure 2). In any event, the complicating factor of polypeptide degradation makes the effects of these cdc68 point mutations difficult to interpret mechanistically.

The cdc68-922 allele is also temperature sensitive for function, but in this case Cdc68 polypeptide proteolysis is not a factor, for we show that the Cdc68-922 polypeptide is stable under our assay conditions. The N-terminal portion of Cdc68 may facilitate the 37° function of the C-terminal part of Cdc68 represented by the Cdc68-922 protein, perhaps by providing structural cues for polypeptide folding. Alternatively, the Cdc68-922 protein, with residues 1–5 of the full-length protein grafted onto residue 307, may be dysfunctional at high temperature due to a misfolding of these juxtaposed sequences. Regardless, at low growth temperatures the stability of the Cdc68-922 protein allows the functions of the N-terminal and C-terminal portions of the Cdc68 protein to be deduced from effects on gene expression.

N-terminal Cdc68 sequences in the CP complex facilitate chromatin-mediated repression:
Promoter sequences do not support transcription in the absence of trans-activator proteins or the DNA sequences that localize such trans-activators. The lack of promoter activity under these conditions is seen in vivo, and also in vitro for promoters complexed with histones and other proteins in the form of chromatin (reviewed in KINGSTON et al. 1996 ). Chromatin thus has a generally repressive effect on transcription. Maintenance of this chromatin-mediated repression depends on the CP complex (MALONE et al. 1991 ; PRELICH and WINSTON 1993 ; XU et al. 1993 ; LYCAN et al. 1994 ), and we show here that the N-terminal 300 residues of the Cdc68 component of CP are especially important for this repression.

The relief of chromatin repression at several promoters can be mediated by the Swi/Snf complex, a multisubunit protein assembly with nucleosome-reorganizing activity in vivo (HIRSCHHORN et al. 1992 ; reviewed in PETERSON and TAMKUN 1995 ; KINGSTON et al. 1996 ). We show here that one function of the Swi/Snf complex is to counteract the repressive effect of the Cdc68 N-terminal domain. The effects of the Cdc68 N-terminal domain—maintaining repression at core promoters and necessitating Swi/Snf function to relieve chromatin repression—may be manifestations of the same repressive function of the CP complex.

The chromatin-repression effects of cdc68 mutations are reminiscent of those caused by impaired histone function (PRELICH and WINSTON 1993 ; KRUGER et al. 1995 ; LENFANT et al. 1996 ; SANTISTEBAN et al. 1997 ; WECHSER et al. 1997 ). Certain histone H4 mutations that alter interaction with the histone H2A-H2B dimer mimic the effects of cdc68-922, including the different degrees of SUC2 and INO1 expression in cells impaired for Swi/Snf function (SANTISTEBAN et al. 1997 ). These similarities suggest that the nucleosome may be a downstream effector of some aspects of CP function.

Transcription in the absence of localized trans-activators or under conditions of impaired Swi/Snf function can be brought about by alterations in histone stoichiometry through decreased histone-gene dosage (CLARK-ADAMS et al. 1988 ; HIRSCHHORN et al. 1992 ). Altered histone stoichiometry may also be caused by the cdc68-1 mutation, which affects expression of the HTA1-HTB1 gene set encoding histones H2A and H2B (XU et al. 1993 ). It is therefore significant that we find, for cells arrested at a cell-cycle position that precludes histone gene induction, that the suc2UAS gene is activated promptly after the impairment of CP function. This finding indicates that an altered histone stoichiometry through aberrant histone gene expression is not necessary for core-promoter activity in cdc68 mutant cells, and points to a more direct role for the CP complex in the maintenance of a repressive chromatin structure. The abundant nature of the CP complex (BREWSTER et al. 1998 ) is consistent with this suggestion.

Chromatin repression is mediated by several other proteins, including the Spt5 and Spt6 proteins. These proteins have effects on the his4-912 and lys2-128 reporter genes and can be mutated to activate core promoters and decrease the need for Swi/Snf activity (NEIGEBORN et al. 1986 , NEIGEBORN et al. 1987 ; CLARK-ADAMS and WINSTON 1987 ; SWANSON and WINSTON 1992 ; PRELICH and WINSTON 1993 ). Recent studies indicate a coordinate role for Spt5 and Spt6 in the elongation phase of transcription (HARTZOG et al. 1998 ; WADA et al. 1998 ). Mutations affecting the Spt6 protein can alter chromatin structure, and Spt6 physically interacts with histone H3 (BORTVIN and WINSTON 1996 ). Genetic evidence suggests that these Spt proteins and CP have overlapping but distinct functions (MALONE et al. 1991 ), while biochemical characterization suggests that the CP complex does not contain these Spt proteins (WITTMEYER and FORMOSA 1997 ; BREWSTER et al. 1998 ).

C-terminal Cdc68 sequences supply all essential functions, probably for gene activation:
The Cdc68-922 protein, although constitutively impaired for chromatin repression, is sufficient for cell viability. Among the essential functions supplied by the C-terminal 70% of Cdc68 is the facilitation of trans-activation, as evidenced by rapid declines in mRNA abundance for a variety of genes when the function and abundance of the Cdc68-1 mutant protein is decreased (XU et al. 1995 ; Figure 7). The deletion studies illustrated in Figure 1 show that several subregions of the essential Cdc68 C-terminal domain are themselves essential. Notable among these are the C-terminal 122 residues, which include a highly acidic region similar to that of the other CP component Pob3 (WITTMEYER and FORMOSA 1997 ) and analogous to the acidic regions of the Spt5 and Spt6 proteins (SWANSON et al. 1990 , SWANSON et al. 1991 ).

The eukaryotic CP complex:
There is homology along the entire lengths of the Cdc68 polypeptides from S. cerevisiae and the yeast K. lactis, including the N-terminal repression domain (Figure 2). Moreover, a database search, including the EST database containing animal and plant partial cDNA sequences, yielded several cDNAs or cDNA fragments encoding polypeptides structurally related to yeast Cdc68 protein (Figure 8). Similarities are found not only for the acidic C terminus and the region most highly conserved between the two yeast Cdc68 proteins, but also along the N-terminal repression domain as defined here. Even the glycine at position 132 that is substituted in the three cdc68 point-mutant alleles is conserved in cDNA-encoded polypeptides related to this region. These similarities, plus the existence of proteins structurally related to the Cdc68 partner protein Pob3 in the CP complex (WITTMEYER and FORMOSA 1997 ), suggest that the CP complex is found across the eukaryote kingdom, an inference consistent with the essential nature of both Cdc68 and Pob3 in yeast.

View larger version (13K): In this window In a new window Download PPT slide   Figure 8. Cdc68 schematic, showing the acidic domain (hatched), the dispensable N terminus (white), the GD mutation in cdc68-1, cdc68-11, and cdc68-197, and the region of maximal identity between the S. cerevisiae and K. lactis forms of Cdc68. Horizontal bars denote regions of similarity between Cdc68, the Drosophila dre4 protein (fly), and the polypeptides encoded by EST partial cDNAs from Caenorhabditis elegans (worm), human, rice, and Arabidopsis sources.

The relationship between the activation and repression effects of the CP complex is not clear. Despite these opposing effects the abundant CP complex may have a single function, imposing an orderly structure upon chromatin. This view hypothesizes that chromatin can be altered to a degree that actually inhibits transcription, and that this situation develops when CP function is grossly insufficient. Thus the relief of core-promoter repression and the inhibition of trans-activation, seen under different situations of altered CP function, might be manifestations of different degrees of chromatin malfunction. This view is consistent with the effects of certain histone H4 mutations that affect interactions within the nucleosome; these mutations at one growth temperature bypass the need for trans-activators and alter chromatin structure at some promoters, but under more severe conditions inhibit the expression of several genes (SANTISTEBAN et al. 1997 ). Alternatively, the CP complex may be bifunctional, maintaining chromatin repression but allowing trans-activator proteins to stimulate transcription.

	FOOTNOTES

¹ Present address: Friedrich Miescher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland.
² Present address: The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037.
³ Present address: Cell Biology Unit, Glaxo-Wellcome, Gunnels Wood Rd., Stevenage, Hertfordshire, United Kingdom SG1 2NY.
⁴ Present address: Department of Biochemistry, Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

	ACKNOWLEDGMENTS

We thank Chris Barnes, Linda Breeden, Marian Carlson, Melanie Dobson, Betsy Malone, and Fred Winston for strains and plasmids and David Carruthers, Sally Harvie, and Amy Wheeler for technical assistance. This work was supported by a grant to G.C.J. and R.A.S. from the Medical Research Council (MRC) of Canada. Q.X. was recipient of an Edward F. Crease Memorial Graduate Studentship in Cancer Research; A.R. was supported by an MRC Studentship. G.C.J. is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada.

Manuscript received June 11, 1998; Accepted for publication September 2, 1998.

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