Vol. 12, No. 11

MOLECULAR AND CELLULAR BIOLOGY, Nov. 1992, p. 5249-5259

0270-7306/92/115249-11$02.00/0 Copyright © 1992, American Society for Microbiology

Identification of a New Set of Cell Cycle-Regulatory Genes That Regulate S-Phase Transcription of Histone Genes in Saccharomyces cerevisiae HAIXIN XU, UNG-JIN KIM, TILLMAN SCHUSTER, AND MICHAEL GRUNSTEIN* Molecular Biology Institute and Department of Biology, University of California, Los Angeles, California 90024-1570 Received 20 April 1992/Returned for modification 9 June 1992/Accepted 11 August 1992

Histone mRNA synthesis is tightly regulated to S phase of the yeast Saccharomyces cerevisiae cell cycle as a result of transcriptional and posttranscriptional controls. Moreover, histone gene transcription decreases rapidly if DNA replication is inhibited by hydroxyurea or if cells are arrested in G1 by the mating pheromone a-factor. To identify the transcriptional controls responsible for cycle-specific histone mRNA synthesis, we have developed a selection for mutations which disrupt this process. Using this approach, we have isolated five mutants (hpcl, hpc2, hpc3, hpc4, and hpc5) in which cell cycle regulation of histone gene transcription is altered. All of these mutations are recessive and belong to separate complementation groups. Of these, only one (hpcl) falls in one of the three complementation groups identified previously by other means (M. A. Osley and D. Lycan, Mol. Cell. Biol. 7:4204-4210, 1987), indicating that at least seven different genes are involved in the cell cycle-specific regulation of histone gene transcription. hpc4 is unique in that derepression occurs only in the presence of hydroxyurea but not a-factor, suggesting that at least one of the regulatory factors is specific to histone gene transcription after DNA replication is blocked. One of the hpc mutations (hpc2) suppresses 8 insertion mutations in the HIS4 and LYS2 loci. This effect allowed the cloning and sequence analysis of HPC2, which encodes a 67.5-kDa, highly charged basic protein. transcribed divergently from common regulatory elements (19, 49). cis-acting regulatory elements, including three upstream activator sequence (UAS) repeats and a negative repressor element, have been identified in the region between the HTAI and HTB1 genes (34). The UAS elements are required for cell cycle-dependent activation of the HTAlHTB1 pair, while the negative element is necessary for repressing the transcription of this gene pair during stages other than S phase. Deletion of the negative element has resulted in the constitutive transcription of histone mRNAs during the yeast cell cycle (29, 34); however, histone mRNA accumulation is still S phase specific in these cells as a result of posttranscriptional degradation of histone mRNAs in stages other than S phase (34, 54). As an initial step toward unraveling the mechanism of cell cycle-dependent histone gene transcription, three genes (HIR1, HIR2, and HIR3) encoding factors that regulate histone mRNA synthesis have been identified (35, 44). Mutations in these genes were detected by including in a yeast strain an integrated HTA1 promoter-lacZ fusion gene and screening for mutants which overexpressed 1-galactosidase. These mutants, which also lost normal periodic transcription of HTA1 mRNA, did so as a result of a disruption in function through the repressor element (35). While these mutants altered the degree of repression of several other histone gene loci upon treatment of the cells with HU, other cell cycle-regulated genes such as the HO endonuclease (31), involved in switching of mating loci, and CDC9 (36), the structural gene for DNA ligase, were expressed normally. Interestingly, these hir mutations all suppress a wellcharacterized yeast mutant in which a solo 8 element, serving as the promoter of a yeast transposon (Ty), has been inserted at HIS4 and LYS2 loci his4-9128 and lys2-1288 (44). 8 insertions at these two loci have previously been shown to alter either transcriptional initiation (his4-9128) or termina-

Histone mRNAs are synthesized and accumulated preferentially in S phase of the eukaryotic cell cycle, partly as a result of transcriptional controls which cause the rate of synthesis of histone mRNAs to increase during S phase. Also, posttranscriptional regulation results in the preferential degradation of histone mRNAs outside of S (reviewed in reference 17). These two mechanisms of regulation function to decrease mammalian histone mRNA levels after DNA synthesis is blocked in the presence of hydroxyurea (HU) (17). In this report, we focus on cell cycle-specific, transcriptional control of histone mRNA synthesis. The human histone H4 gene is transcribed more efficiently (3- to 10-fold) in S-phase extracts than in extracts taken from cells at other stages of the cell cycle (18). Mammalian upstream promoter elements responsible for this regulation have been identified (1, 6, 16, 28, 42). Moreover, fusion of the 5' end of the mouse histone H3 gene to the bacterial neomycin resistance (neo) gene causes the bacterial mRNA to be synthesized in a cell cycle-specific manner (3). cisacting regulatory sequences and their trans-acting protein factors are likely responsible for regulating histone mRNA synthesis (7, 10, 11, 14, 50; reviewed in reference 33); however, the molecular details of the regulatory mechanism by which this occurs are still under investigation. The yeast Saccharomyces cerevisiae provides a system which allows the identification genetically of trans-acting factors that regulate cycle-specific histone mRNA synthesis. Yeast histone genes are organized into four genetically unlinked gene pairs: two copies each for the histone H2AH2B gene pair (HTA1-HTB1 and HTA2-HTB2) (20, 51) and two copies for the histone H3-H4 gene pair (HHT1-HHFI and HHT2-HHF2) (48). Each of the histone gene pairs is

*

Corresponding author. 5249

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XU ET AL.

tion (lys2-1288), producing a His- or Lys- phenotype (9, 47, 52). It is noteworthy that among the many loci which have been identified as SPT (suppressor of Ty), two of them, sptll and sptl2, have been characterized as mutations at the promoter region of the HTAI-HTBI locus. The wild-type HTAI-HTBI gene pair on a high-copy-number plasmid can also suppress his4-9128 and lys2-1288 (8). Furthermore, recessive mutations in three known SPT genes (SPT1, SPTIO, and SPT21) have also been found to derepress HTAI-HTB1 transcription upon the inhibition of DNA synthesis. SPT1 has been shown to be allelic to HIR2, while SPTIO and SPT21 are distinct from other histone-regulatory genes (44). Therefore, it seems that S. cerevisiae employs a complex system that involves two types of cis-regulatory elements and many trans-acting factors to coordinate histone synthesis with the cell division cycle. To isolate new regulatory mutants that alter periodic transcription of the yeast histone genes, we used a direct selection. This was done by fusing the HTAI promoter to the bacterial neo gene, which when transcribed at high levels causes yeast cells to become resistant to the neomycin analog G418. It is expected that mutations in the trans-acting factors repressing the HTAI promoter should result in constitutive high levels of neo synthesis. This analysis identified five mutations (hpcl, hpc2, hpc3, hpc4, and hpcS [hpc for histone promoter control]), each in a different complementation group. Four of these mutations belong to different complementation groups from those isolated previously as histone-regulatory mutations (35). In addition, hpc2 shows a very strong Spt- phenotype, suppressing both his4-9128 and lys2-1288 insertion mutations. This feature was used to clone the HPC2 gene. This gene encodes a 624-amino-acid, highly charged basic protein which can fully complement the Sptphenotype and restores normal cell cycle control of histone HTAJ gene expression in an hpc2 strain.

MATERIALS AND METHODS Media, chemicals, and strain constructions. All media used have been described previously (43) except for the following. SG is identical to SD except that 2% galactose is used instead of glucose as the sole carbon source, and zinc chloride (20 ,g/ml) was added to the sporulation media to increase the ratio of tetrads to triads (4). The strains used in this study are shown in Table 1. Standard techniques for yeast genetics were followed (43). Mating-type conversion of MATa hpcl cells to MA4Tao was performed by transforming plasmid pGAL.HO (URA3+) (26) into hpcl cells, and Ura+ transformants were selected on SD-Ura plates. After growing in SD-Ura, the transformants were switched to YEPG medium, grown for 6 h, and plated on YEPD for singlecolony isolation. Each of the colonies grown on YEPD plates was tested for mating type by crossing with D585-4B1 (MATot hisl) and D587-11C (MA4Ta lysl) on YM plates. EMS (ethyl methanesulfonate) and HU were purchased from Sigma Chemical Co., a-mating pheromone was purchased from Peninsula Scientific Biochemicals, and G418 (Geneticine) was purchased from GIBCO. Construction of plasmids. Standard procedures for DNA manipulation (38) were used for construction of the plasmids described below. An approximately 800-bp HindIII-BamHI fragment including the region between the HTAI and HTBI genes was subcloned into the HindIII-BamHI sites of pUC8 (30). The plasmid was linearized by HindIII digestion, and the remaining HTAJ protein-coding region was removed by Bal 31 exonuclease. A SalI linker was added to both ends

MOL. CELL. BIOL.

TABLE 1. Yeast strains Strain

Relevant genotype

Source

YM259 YM214 UKY9 HXY100 HXY1O1 UKY24-1 UKY24-2 UKY35 UKY35-25 UKY36 UKY36-5D HXY103 HXY104 HXY116 21-1A 24-SB 30-9C FW1237 FW1238

AMTa ade2 his3 tyrl ura3 MA4Ta ade2 his3 iys2 ura3 MATa ade2 his3 tyrl ura3 hpcl AMTa ade2 his3 tyrl ura3 hpc2 MATTa ade2 his3 lys2 ura3 hpc2 MATa ade2 his3 Iys2 ura3 hpc3 MATa ade2 his3 tyrl ura3 hpc3 MATa ade2 his3 tyrl ura3 hpc4 MATa ade2 his3 lys2 ura3 hpc4 AlATa ade2 his3 tyrl ura3 hpcS MATa ade2 his3 Iys2 ura3 hpcS MATa ade2 hpc2 ura3 HIS3 LYS2 MATa ade2 hpc2 ura3 HIS3 LYS2 MATa ade2 lys2 HPC2/HPC2::URA3 MATa his3 his7 leu2 trpl ura3 hirl MATa his7 leu2 trpl ura3 hir2 MATa ade2 leu2 trpl ura3 hir3 MATa ura3 his4-9128 lys2-1288 MATa ura3 his4-9128 lys2-1288

L. Johnson L. Johnson This study This study This study This study This study This study This study This study This study This study This study This study M. A. Osley M. A. Osley M. A. Osley F. Winston F. Winston

and then digested by SaII to create sticky ends. The recircularized plasmids were screened by sequencing, and two deletions that removed HTA1 coding sequences up to 29 and 18 bp upstream of the ATG codon were picked to make the following two constructs. The XhoI-SaiI fragment from each of these two plasmids containing the HTA1-HTBI promoter region was subcloned into the SalI site of pSEYC58 plasmid (13) in such an orientation that the Sall site close to the EcoRI site is conserved and the direction of HTAI transcription is opposite that of the lacZ gene contained in pSEYC58. These constructs, after partial digestion with BamHI, were digested with Sal, and the large fragments were ligated to the 922-bp BgiII-SalI fragment of the neo gene from the bacterial transposon TnS (23). Colonies were screened on LB plates containing 10 ,ug of neomycin per ml after transformation into Eschenichia coli. Then a HindIII-EcoRI DNA fragment of pAB120 containing the yeast transcriptional termination sequence of the CYCI gene (obtained from Fred Sherman's laboratory) was inserted at the 3' end of the neo gene. BamHI fragments containing the slightly different HTA1 promoter-neo gene-CYCI terminator fusions were subcloned into the BamHI site of pTSA (a CEN3 URA3+ plasmid [41]). The two resultant plasmids were designated pTS38b and pTS39b, respectively. pTS42b and pTS43b were constructed as follows. A SacI-BamHI-SacI linker was inserted into the unique SacI site located at the center of the HTAl-HTBJ intergenic region. The approximately 400-bp BamHI fragment including the repressor element and two UASs was removed by partial BamHI digestion and religation. The large BamHI fragments now including the truncated intergenic regions fused to neo-CYCl terminator constructs were also ligated into the BamHI site of pTSA, resulting in plasmids pTS42b and pTS43b. Screening and analysis of regulatory mutants. Yeast strain YM259 was transformed with either pTS38b or pTS39b, and the transformants were mutagenized by EMS. Five milliliters of the cells in late log phase was pelleted, the cells were resuspended in 1.5 ml of 50 mM phosphate buffer (pH 7.0), 50 ,ul of EMS was added, and the cells were shaken at 30°C for 1 h. After addition of 8 ml of 5% sodium thiosulfate, the cells were pelleted by centrifugation, resuspended in 1.5 ml of YEPD, and shaken at 30°C for 2 h. Aliquots (200 ,ul) of the mutagenized cells were plated on YEPD containing 300 ,ug of

VOL. 12, 1992

G418 per ml and incubated at 30°C. The selected colonies were picked and grown for further analysis. Of the five mutants picked, all but hpc3 were obtained from the transformation of YM259 cells with plasmid pTS38b. hpc3 was obtained by the use of pTS39b. HU arrest was done as described previously (29), with some modifications. Cells were grown to approximately 5 x 10' cells per ml in YEPD. HU was added to a final concentration of 0.3 M, and the culture was shaken at 30°C for 40 min. Arrest of cells was examined by microscopy. Synchronization of cells with the yeast pheromone a-factor was done as described previously (25). The length of the cell cycle was found to vary somewhat in these experiments, possibly because of variability in recovery from a-factor arrest. Northern (RNA) blot analysis. Total yeast RNA was isolated by hot phenol extraction (12), with minor modifications. About 10 ml of yeast log-phase culture (optical density at 600 nm of 0.3 to 0.5) was harvested and resuspended in 1 ml of 100 mM sodium acetate (pH 5.3)-10 mM EDTA-1% sodium dodecyl sulfate. The cell suspension was vortexed and then extracted with an equal volume of phenol at 65°C for 10 min with vigorous shaking. The probes were prepared and Northern blot experiments were performed as described previously (12). Films were preflashed and exposed at -70°C with intensifying screens (Dupont Cronex Lightning-Plus). Cloning and sequencing of the HPC2 gene. HXY104 (hpc2 his4-9128 lys2-1288 ura3) was used as the recipient strain for transformation of a yeast genomic library in plasmid YCp5O (37). Two plasmids with overlapped inserts which fully complemented both Spt- and hpc2 mutations were isolated. Further subcloning was carried out to limit the DNA fragment that is able to complement the mutations. The restriction fragment pairs ClaI-ClaI, BamHI-BamHI, SalI-ClaI, BamHI-BamHI, and HindIII-EcoRI were subcloned into shuttle vector pRS316 (46), resulting in plasmids pHX104, pHX105, pHX107, pHX111, and pHX113, respectively. These plasmids were then transformed into HXY104 to examine their ability to rescue the mutation. pHX111 was chosen for sequencing in both directions by the dideoxychain termination method, using Sequenase (U.S. Biochemical Corp.) (39). Primer extension analysis. Total RNA was extracted from the wild-type yeast strain YM259 grown in YEPD as described above. Primer extension procedures used were as described elsewhere (38). The primer used for the reaction was 5'-GCGTTIGCATT-ITGCCACTCG-3', corresponding to the region from positions 52 to 73. Nucleotide sequence accession number. The nucleotide sequence reported in this article has been submitted to the GenBank sequence data base under accession number M94207. RESULTS Selection for mutants that alter cell cycle-specific expression of histone genes. To select for mutants which cause deregulation of cell cycle-specific histone mRNA synthesis, we constructed the plasmids shown in Fig. 1. pTS39b contains the bacterial neo gene, which encodes aminoglycoside phosphotransferase (23), under the control of the histone HTA1 promoter (up to 18 bp upstream of the HTAJ initiation codon). When active, the fusion gene should allow cell growth in the presence of the neomycin analog G418 (21, 27). Since the HTA1 promoter is normally active only during S phase (35), we reasoned that neo activity controlled by the HTA1 promoter in a wild-type genetic background should

HISTONE CELL CYCLE-REGULATORY GENES neo

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FIG. 1. Restriction map of plasmid pTS39b. The coding region of the bacterial neo gene from TnS, the HTA1 promoter, and the transcriptional termination sequence from the CYCI gene are fused so that neo transcription is under the control of the histone HTAI promoter. The plasmid also contains URA3, CEN3, and ARS1 sequences for stable maintenance in yeast cells. pTS39b and pTS38b are identical except that pTS38b contains HTAI promoter sequences up to -29 bp from the ATG codon, while pTS39b has the same sequences up to -18 bp (see Materials and Methods).

allow resistance to a moderate G418 level, while constitutive synthesis resulting from a mutation in a cycle-specific regulatory protein should allow resistance to higher levels of G418. We found that control YM259 yeast cells could not grow on rich medium (YEPD plates) containing 100 ,ug of G418 per ml. In contrast, YM259 cells transformed with plasmid pTS39b showed resistance to G418 at 100 jig/ml. However, these cells could not grow on YEPD plates containing G418 at 300 ,ug/ml. To determine whether constitutive synthesis of aminoglycoside phosphotransferase would allow resistance to the higher level of G418, YM259 cells were transformed with plasmid pTS43b, in which the repressor element through which cell cycle-specific HTAJ regulation is controlled was deleted from the promoter construct of pTS39b (see Materials and Methods). This procedure results in constitutive activity of the HTAJ promoter throughout the cell cycle (35). We found that at 300 ,ug of G418 per ml, approximately 30% of the YM259 cells containing pTS43b survived. In a parallel set of experiments, we also used an analogous plasmid construction which contained the HTAI promoter sequence deleted up to 29 bp upstream of the ATG initiation codon. This plasmid, pTS38b, and its derivative, pTS42b, whose repressor element was also deleted, were transformed in YM259 cells. The strains containing these plasmids responded in the same manner to G418 as did those containing pTS39b and its repressor element deletion derivative pTS43b. Therefore, treatment of yeast cells with 300 ,ug of G418 per ml should allow for the selection of yeast mutants in which neo activity has become constitutive. YM259 cells transformed with pTS38b or pTS39b were mutagenized with EMS, and approximately 200 G418-resistant colonies were picked. Since normal histone mRNA synthesis is repressed as a result of blocking DNA synthesis or at stages outside of S phase, the only mutant strains of interest to us were those whose endogenous histone mRNA synthesis was no longer repressed after treatment of the cells with HU or upon arrest in G1 with yeast a-mating factor. Therefore, mutant strains were arrested by either HU or a-mating factor and subjected to Northern blot analysis. From this secondary screen, five mutants, hpcl, hpc2, hpc3, hpc4, and hpcS, which showed a reproducible derepression of HTA1 mRNA synthesis were chosen (Fig. 2). The DNA probe used for detecting RNA levels in the Northern blot

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XU ET AL.

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MOL. CELL. BIOL.

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FIG. 2. Identification of histone-regulatory HTAJ mutants. The levels of histone HTA1 transcript in response to the presence of HU and a-factor arrest (a) are shown by Northern blot analysis. After EMS mutagenesis, five G418-resistant mutant strains in different complementation groups were isolated. These mutant and wild-type (HPC+) strains were grown in YEPD medium, and aliquots of the culture were arrested by 0.3 M HU for 40 min or arrested at G1 by a-factor (10 pg/ml) for 3 h. Total RNA was then extracted from harvested cells, and 7 ,ug was electrophoresed in each lane. A 1.4-kb HindIII fragment of the TRTI region containing HTA1 and PRTI! AKY2 coding sequences (20) was used as a probe. An hpc mutant phenotype is seen as the continuous transcription of the HTA1 gene in the presence of HU and/or a-factor.

analysis contains both HTAJ and AKY2IPRTI DNA seThe latter is a contiguous gene which codes for an mRNA that is not cell cycle regulated and serves as an internal control (32, 35). RNA taken from unarrested wildtype cells (HPC+) produces an HTAI band which is considerably darker than the PRTJ mRNA band (lane 1). However, after either HU or a-factor arrest, the intensity of the HTAI band decreases so that its signal is equal to or less than that of PRT1. hpcl, hpc2, hpc3, and hpcS all cause a similar derepression in HTAI mRNA synthesis, since the level of the HTAI mRNA was significantly higher after either HU or a-factor arrest compared with that of the HPC+ cells undergoing the same treatments. In contrast, hpc4 shows little if any effect on HTAl derepression after at-factor treatment but strongly derepresses HTAJ transcription after HU arrest. Identification of four new complementation groups involved in cycle-specific HTAI regulation. To estimate the number of genes involved in the regulation of HTAJ transcription, complementation tests were done (Fig. 3). All of the mutants were crossed in pairwise combinations, and the resultant diploids were tested for the ability to repress HTAI transcription after HU arrest. When crossed to each other, all of the hpc mutants complemented each other's defect in HUmediated repression. However, when mated to form homozygous diploids, none of the five hpc mutants complemented. Therefore, these mutations comprise five different complementation groups. Figure 3 also demonstrates that diploids generated from the matings between hpc mutants and YM214 (HPC+) showed wild-type phenotypes with respect to HU-mediated repression of HTA1 transcription. Therefore, hpc mutations are all recessive to their wild-type homologs and are likely in genes encoding trans-acting factors. The trans-acting nature of hpc mutations was further demonstrated by restoring the G418 resistance of the mutants after plasmid pTS39b was lost and retransformed into quences.

the same mutant strains. To determine whether the deregulation of histone gene transcription in the five hpc mutants was the result of mutations in single genes, we sporulated each heterozygous diploid and monitored the segregation of hpc and HPC genes by using the HU-mediated test. We found that while two spore colonies in each tetrad dissected contained the wildtype HPC homologs, each of the other two contained an hpc mutant allele (data not shown). This 2:2 segregation pattern is the expected outcome for mutations in single nuclear

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FIG. 3. hpc mutant complementation tests. hpc and hir haploid mutant strains were crossed in pairwise combinations, and the resultant diploids were tested for the inability to repress the HTA1 transcript level after HU arrest. Successful complementation resulted in a significantly reduced level of HTAI mRNA, comparing levels before (-) and after (+) HU treatment. Diploids formed by mating hpc strains to wild-type cells (HPC+) or to themselves were included as controls. All hpc mutations complement hir mutations except hpcl, which falls in the same complementation group as does

hir3. genes. Therefore, all hpc mutants appear to have mutations in single chromosomal genes. Three different complementation groups (hirl, hir2, and hir3) identified previously (35, 44) have mutant phenotypes similar to those of hpcl, hpc2, hpc3, and hpcS. To determine whether any of the five hpc and three hir mutants were in the same complementation groups, these mutants were crossed with each other. As shown in Fig. 3, all hpc mutations complemented hir mutations except for hpcl, which did not complement hir3. This finding indicates that hpcl and hir3 belong to the same complementation group. Therefore, we conclude that there are at least seven different gene products involved in the cycle-specific repression of histone HTA1 mRNA synthesis (occurring after HU arrest). hpcl, hpc2, hpc3, and hpc5 alter periodic synthesis of HTAI mRNA but not DNA polymerase I mRNA. To examine the effect of hpc mutations on periodic synthesis of HTAJ mRNA, we compared HTAJ mRNA levels during synchronous growth after a-factor arrest of hpc mutant strains (Fig. 4). In HPC+ cells, the expression of HTAJ mRNA peaks during S phase (35 to 55 min in the first cell cycle and 100 to 120 min in the subsequent cell cycle). In contrast, in comparison with the level of HTAJ mRNA in the wild-type strain, all hpc strains except hpc4 showed a two- to fourfold increase in the level of accumulation of HTA1 mRNA outside of S phase (normalized to the PRTJ control message level) (Fig. 4; data not shown), indicating that the repression of HTAJ mRNA synthesis in G1 and G2 phases has been disrupted in these mutant strains. While periodicity in HTAI mRNA levels can still be noticed in these mutant strains, the peak level of HTA1 mRNA accumulation in hpc3 was delayed for approximately 25 min compared with the result for the wild-type control strain. Consistent with the results showing that the hpc4 mutation does not prevent HTAI mRNA accumulation after a-factor arrest (Fig. 2), the hpc4 mutation shows little if any effect on the periodicity of the HTAJ message level during the cell cycle (Fig. 4). We then wished to determine whether mutations in HPC

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HISTONE CELL CYCLE-REGULATORY GENES

VOL. 12, 1992

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FIG. 4. Effects of hpc mutations on the periodic syntheses of HTA1 and POLI mRNAs. HPC+ and hpc cells were synchronized in G1 by a-factor. After release from this block, aliquots of the cultures were removed during the subsequent cycles of synchronous growth. The levels of mRNA were measured in these samples by Northern analysis by probing with a 0.5-kb EcoRI-SaII DNA fragment of pLJ23 containing the POLI coding region and a 1.4-kb HindlIl DNA fragment of the TRTJ region for HTAJ and PRT1 messages (20). Numbers indicate times in minutes after release of the cell cultures from a-factor arrest. None of the hpc mutations prevent cycle-specific synthesis of POLl mRNA compared with the cycling of HTA1 mRNAs.

genes disrupt cycle-specific synthesis of a cell cycle-regulated nonhistone mRNA. Therefore, we tested the effects of these mutations on the transcription of the DNA polymerase I (POLl) gene, which is cell cycle regulated in yeast cells (22). Our results demonstrate that this gene is generally under normal cell cycle regulation in all hpc mutant strains when we compare peak accumulation times with that of HTAJ mRNA (Fig. 4). In hpc3, both H7TA and POLl accumulation peaks lag in a similar manner compared with their analogous peak in the HPC+ strain. This lag may result from a nonspecific effect of the hpc3 mutation in allowing recovery from a-factor release. Previous studies with hir mutations have suggested that cell cycle regulation of histone genes is distinct from that of CDC9 and HO genes (35). These and our data indicate that histone genes utilize regulatory factors which are distinct from those of other cell cycle-regulated genes. Effect of hpc mutations on the cell cycle regulation of other histone gene loci. All four core histone mRNAs are accumulated specifically in S phase, as determined by Northern analysis (24, 35). hirl, hir2, and hir3, which eliminate HTA1 HU-mediated repression, also eliminate repression of genes coding for histones H2B2 (HTB2), H3-1 (HHT1), and H3-2 (HH72) after HU treatment (35). Therefore, we wished to determine whether hpc mutations affected these histone gene classes (Fig. 2 and 5). We observed the following. (i) The HTB2 gene is not affected by any of the hpc mutations in strains undergoing HU treatment. In addition, none of the mutations appear to cause the accumulation of HTB2 mRNA during a-factor arrest. This result is similar to that obtained previously with the use of hir mutations (35). These data indicate that the HTA2-HTB2 gene pair utilizes a cell cycle-

regulatory mechanism distinct from that of the other histone gene pairs. In support of this view, two newly isolated recessive mutations (sptlO and spt2l) appear to have a direct effect on HTA2-HTB2 transcription only (44). (ii) All of the mutations except hpc3 prevent HU-mediated repression of the HTAJ, HHT1, and HHT2 loci as well as HTB1 (data not shown). (iii) The effect of the hpc3 mutation appears to be restricted to the HTAI gene and does not affect the responses of two HHT genes to treatment with either HU or

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FIG. 5. Effects of hpc mutations on the periodic synthesis of histone mRNA other than HTAI. RNA was isolated from wild-type (HPC+) and hpc mutant cells with or without HU or a-factor (a) treatment. The levels of histone mRNAs were measured by Northern analysis by probing with the following labeled DNA fragments: a 0.7-kb EcoRI fragment from pMH201 (15) containing HTB2, a 0.5-kb EcoRV-SmaI fragment of pMS203 containing HHT1, and a 0.45-kb BamHI-EcoRV fragment of pMS191 containing HHT2 (47). In the case of HHTI and HHT2, the probes cross-hybridized to the two classes of transcripts because of the strong homology between these two copies, and thus fainter secondary bands were generated.

5254

XU ET AL.

a-factor. Moreover, hpc3 affects the response of the HTBI gene to both HU and a-factor arrest (data not shown). These data suggest that the hpc3 mutation affects a trans-acting factor unique to the cycle-specific control of the HTAlHTB1 locus only. (iv) The hpc4 mutation does not cause accumulation of mRNAs synthesized from the HTA1 and two HHT genes under a-factor arrest. In contrast, the hpc4 mutation derepresses transcription from these loci when treated with HU, suggesting that its role is specific to histone gene transcription after DNA replication is blocked. hpc2 suppresses 8 insertion mutations at the HIS4 and LYS2 loci. To further understand how HPC genes are involved in histone gene regulation, we attempted to isolate HPC genes by complementing the G418 resistance phenotype in hpc mutant strains. However, this method did not succeed because of the presence of a large number of false positives. Since both changes in histone gene dosage and hir mutations suppress 8 insertion mutations his4-9128 and lys2-1288 (8, 44), we decided to examine the effects of hpc mutations on these two 8 insertion alleles. If an hpc mutation suppresses the 8 insertion mutations (his4-9128 and lys2-1288), then we expect that segregation of His+/His- and Lys+/Lys- phenotypes will deviate from 2:2 when the resultant diploid strain is sporulated. By crossing hpc2, hpc3, hpc4, and hpcS strains which are HIS4 LYS2 with FW1238 (HPC+ his4-9128 lys2-1288), we found that hpc4 and hpcS do not suppress 8 insertion mutations, as the His+/His- and Lys+/Lys- phenotypes segregated 2:2 in 9 of 11 tetrads dissected for hpc4 analysis and 14 of 14 tetrads dissected for hpcS analysis. The suppression pattern of hpc3 could not be determined since sporulation of the diploid strain hpc3/FW1238 was extremely poor. In 10 of 14 tetrads dissected from a diploid strain generated by crossing hpc2 and a 8 insertion strain (FW1238), there was an excess of His+ and/or Lys+ spore colonies grown in media lacking either histidine or lysine, resulting in a 3:1 ratio of His+/Hisand/or Lys+/Lys-. The excess His+ and/or Lys' spore colonies which grew slowly were anticipated to arise as a result of suppression of the his4-9128 or lys2-1288 allele by hpc2. All of these segregants were found to contain the hpc2 mutation, as determined by derepression of HTAJ transcription in the presence of a chromosome replication block (data not shown). These results suggest that among the hpc strains tested, only hpc2 shows a strong Spt- phenotype and may suppress the two 8 insertion mutations. To confirm that hpc2 does suppress 8 insertions, we backcrossed a segregant which was hpc2 his4-9128 lys2-1288 (HXY104) to the original HPC2 his4-9128 lys2-1288 strain, FW1237. The hpc phenotype should cosegregate tightly with His' and Lys+ only if the hpc2 mutation causes the suppression. Ten tetrads were dissected and tested for both hpc and auxotrophic phenotypes. Our results show that the His+/His-, Lys'/Lys-, and HPC+/hpc phenotypes segregated in the ratio of 2:2. In addition, all His+ Lys+ spores were hpc and all His- Lys- spores were HPC+, as determined by the HU-mediated test (Fig. 6). These results confirm that hpc2 suppresses both the his4-9128 and lys21288 alleles. All spt mutations as well as the three hir mutations that have been examined suppress 8 insertions at the transcriptional level. To test whether this was also true in the hpc2 strain, we used Northern gel analysis to compare transcription in both hpc2 and HPC2 strains at the HIS4 locus. Our results demonstrate that hpc2 suppresses the his4-9128 insertion by partially restoring HIS4 transcription at its normal start site (data not shown).

MOL. CELL. BIOL.

HPC+

1A

A

1B

lC

1D

2A 2B 2C [ _ [

2D

J-

L..

SD complete

-f 1'-

SD-histidrne

SD-lysine +

D

+

4-

+

+

+

+

+

HU - PRT1 - HTA1

40 a 40 a FIG. 6. Suppression of 8 insertion mutations by hpc2. Meiotic

progeny from two tetrads of a cross between FW1237 (HPC2 his4-9128 lys2-1288) and HXY104 (hpc2 his4-9128 lys2-1288) were tested for suppression of the his4-9128 and lys2-1288 alleles by replica plating onto SD complete (row A), SD-histidine (row B), and SD-lysine (row C) media. An Spt- phenotype is manifest as growth on all three media. The HPC2 phenotype of the same spore colonies from the cross described above was determined by the HU-mediated test as described in Materials and Methods (row D). The first two lanes, included as controls, are wild-type (HPC') cells treated with (+) or without (-) HU. An hpc2 phenotype is indicated as precisely cosegregating with the Spt- phenotype.

Cloning and sequence analysis of the HPC2 gene. To clone the HPC2 gene, HXY104 (hpc2 his4-9128 lys2-1288 ura3) was transformed with a yeast genomic library constructed in plasmid YCp5O (37). The recipient strain, HXY104, is His' Lys+ because of the suppression of his4-9128 and lys2-1288 by hpc2. Since hpc2 is recessive, Ura+ transformants which carry the wild-type HPC2 gene on a plasmid should be HisLys-. Therefore, the Ura+ transformants were screened for those that acquired a His- Lys- phenotype. Two candidates were obtained out of approximately 25,000 transformants screened that complemented the Spt- as well as the hpc mutant phenotype. They contained 9 and 14 kb, respectively, of genomic DNA insert in YCp50. These two clones were shown to have common restriction fragments (data not shown). The 9-kb insert, named pHXl1Ob, was chosen since it demonstrated full HPC2 function by the criteria described above (Fig. 7A). To characterize the HPC2 gene in greater detail, several subclones were constructed and tested for HPC2 function. pHXlll, which contains a 5-kb BamHI fragment, was found to be sufficient to perform fully both HPC+ and SPT+ functions (Fig. 7B), suggesting that the HPC2 gene resides in this region. We verified that the clones contained the authentic HPC2 gene by demonstrating that the cloned DNA directed integration of a plasmid into the HPC2 locus. Plasmid pHX116, an integrating plasmid which contains the 5-kb HindIIIEcoRI fragment of cloned DNA, was linearized at a unique SpeI site within the cloned DNA and introduced into YM259 (HPC2 his3 tyrl ura3). A Ura+ transformant (HXY116) was crossed to strain HXY104 (hpc2 lys2 ura3). In eight tetrads dissected, the HPC+ and URA+ phenotypes cosegregated in every tetrad, indicating that pHX116 DNA was tightly linked to the HPC2 locus. Therefore, we conclude that the hpc2complementing DNA fragment that we have isolated most likely contains the HPC2 gene. The nucleotide sequence of the 5.0-kb BamHI DNA fragment that contains the hpc2-complementing activity was determined by dideoxy sequencing in both orientations (38). Within this region, two large open reading frames (ORF1 and ORF2) which are transcribed in opposite directions were found (Fig. 7A). To determine which of the two ORFs is able to encode the HPC2 function, we constructed several sub-

HISTONE CELL CYCLE-REGULATORY GENES

VOL. 12, 1992

5255

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i

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pHXI 13

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+

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--HTA1

FIG. 7. Cloning and characterization of the HPC2 gene. (A) Restriction map of the HPC2 locus. The top line (pHX100b) represents the 9-kb insert containing the HPC2 locus and its restriction sites. The thin portion of the line represents vector sequences. DNA fragments that were subcloned to test for HPC/SPT function are shown below. ORFi and ORF2 are represented by filled and hatched boxes, respectively. Directions of transcription are indicated by arrows. (B) Evidence that the HPC2 gene resides in a 5-kb HindIII-EcoRI fragment. The ability to complement the hpc2 mutation by different subclones was determined by HU treatment. Northern analysis was used to measure both HTA1 and PRTI mRNA levels. An hpc2 strain (HXY103) was used as the recipient strain for transformation by all of the subclones as well as the 9-kb insert (pHX100b). Both HPC2 and hpc2 strains were also included as controls.

clones which delete part of either one of these two coding sequences. pHX105, which removes 245 putative amino acid codons from ORFi but leaves ORF2 intact, was no longer capable of rescuing either the Spt- or hpc2 mutant phenotype (Fig. 7B). In contrast, pHX113, which deletes 185 putative amino acid codons from ORF2 but leaves ORFi intact, was fully able to perform SPT+ and HPC+ functions. Therefore, we conclude that the HPC2 gene resides in ORF1. Defining the HPC2 transcriptional unit. There are several ATGs found in frame which could serve as the translation start codon within the presumed HPC2 ORE (Fig. 8). To determine which one may be utilized as the start codon to encode the putative HPC2 polypeptide, we mapped the 5' transcriptional initiation site of the HPC2 gene by primer extension analysis. An adenine at position -37 was mapped as the major start site of HPC2 gene transcription (Fig. 9A).

Two minor bands appeared as a result of primer extension, suggesting that two guanine residues at positions -34 and -35 may also serve as minor initiation sites. Thus, the adenine residue in the first in-frame ATG has been numbered as nucleotide 1. The HPC2 transcript was identified by probing a Northern blot of total yeast RNA with a ClaI-NheI DNA fragment within the HPC2 coding region (Fig. 8). This analysis revealed a major transcript of about 2.1 kb (Fig. 9B). Allowing for the 3' untranslated sequence and a poly(A) tail, the mRNA size is consistent with the size of the HPC2 coding region (1,872 bp) (see below). Shown in Fig. 8 is the nucleotide sequence of the HPC2containing DNA fragment. This sequence reveals that ORFi encodes a protein of 624 amino acids with a calculated molecular weight of 67,490. The predicted protein is highly charged (30% charged amino acid groups) and basic, as the estimated pI is approximately 10.05. No substantial homol-

MOL. CELL. BIOL.

XU ET AL.

5256

GGATOGCAATCC -480

-360

-240

ATGATATCAATTGTTTCTTGTAC'TGGAAAAAAAGTCAGAAATATGTCAGCAAGCCACA

-120

AAACAGATGTATAATTTGCA&TTCTGAAACGATTTAACAACAGTATTAA

ATCA

1 M I

A

I V

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D NS

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55S

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AAAAAGATATTAGTTTCAAATTAAAAAAGAAGAAACACTGTAGATGGAACGACAGAAGTC

121

KE T GS

D SE D LFN KF

S NK K TN RK

I

P N

IA E ELA KN RN Y VKG A

80

Clax TAAAAAATTGAA 241TCCGCCCTAATTTGTCCTACTTCTAGCCCTCCATCACCAGGAACAGAC PBSP I II S GS S STS PS G P SS S STN P MG I P TN R FNKXNT V EL

361TACGACACTCCGCAGCATAA Y

P

Q HSP

VMNT

TNK

CTAAAAGAAAGCAAATAATTGTAAAAACCGAGGACTTCTTT S SFA P E RG T DT E EKRQ N N RNMD N KN T

120

160

CTACAAAATTTCTCTACAACCCAGATCAAATTCTT;CACTTGCAAAAATATCCCGATAATA

481

A

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SL

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S

S

NE

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SKT

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TCCTTCGATCTCGCGATGATATATAAACCCACCCCGATATTTAGCAATACTCACCGAAGA N M D I A Y A E L H T E G N E S L I K P P s S. P R N K S L T P K V I L P TQ

601

GAAT;AAGAc

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320

Nhel AGC1RAACAAATAGTACTACAATCAACACAGCAGCTAAAAC=ACAATGGTCACCAAAACC K K A A N T R K K N A A I L P K P T T T K T K S A T E K A K D

360

TGAAaTCTA,CTAGAGGCATCGCCAATACCGTAAGAATGTTTTTCACAGCCGGAGAAGTA E E K D K G K K N L I K A T K K N A L N T V K E S N K T T A I E

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440

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480

841

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GACATCGACACCACCCGAAGATGACTTAAG N T T I K K S H P E T T V T P K M K G

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AAATGTGTATTAGTAGTCTCTGTATGATATTGAGAACTCGCCAGAGTTTTTTCTGTCTAT K N LI GK Y DV ED PF I D DBSE LL W EEQRAA T K DG FF V YFGPL I

600

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2041TCCTAAATTTACTCTTACCGACATTGAGTATTTGTGATAAAATAACTATTTCATAAATATG 2161

AGALTTCGTT;TTTATCCACA&TTTCCTTATACTTCGGAAG;TTTATCAGGTTCGACTGACTTCAAATTACGAATCAATC2%GA& AsuXX

predicted amino acid sequence of its gene product. Nucleotides are numbered on the (the adenine residue of the first ATG codon downstream from the transcription start site was defined as 1); amino acids are numbered on the right. The termination codon is represented by an asterisk. The major transcriptional start site is indicated by a triangle, and minor start sites are marked by carets. The primer (OLIGO) used in primer extension is underlined. The Clal-Nhel fragmnent used in measuring HPC2 FIG. 8. Nucleotide sequence of the HPC2 gene and

left

mRNA size is also indicated.

ogy

was

detected when the deduced amino acid sequence of

existing sequences in the GenBank data base or in the extensive private data base of Mark Goebl (University of Indiana).

the HPC2 gene

product

was

compared

with

DISCUSSION

cycle-specific histone gene transcription in yeast cells regulated by cis-acting regulatory elements. In the HTAlHTBI gene pair, such sequences include three UAS repeats and a negative repressor element (34). The UAS elements are utilized for cell cycle-specific activation of the H-TAlHTBI pair, while the negative element is necessary for repressing its transcription outside of S phase. Deletion of the negative element or mutations in trans-acting factors functioning through this element results in the constitutive Cell

is

transcription of histone mRNAs during the yeast cell cycle (29, 34). Therefore, we have introduced a new selection scheme for yeast mutations whose regulation of cell cyclespecific histone mRNA synthesis has been disrupted. This method employs an HTAI promoter-neo reporter gene in mutagenized yeast strains and predicts that mutations in trans-acting repressor regulatory factors will result in constitutive HTA1 promoter function and therefore resistance to

Using this (HPCJ, HPC2, HPC3, HPC4, and HPCS) encoding trans-acting factors whose mutation causes cell cycle regulation of histone gene transcription to be disrupted. All of these genes except HPCI differ from previously identified HIR (HIRI, HIR2, and HIR3) and SPT (SPTJO and SPT'21) genes whose mutations also disrupt histone cell cycle regulation (35, 44). HPCJ high

concentrations of the

assay,

we

neomycin analog

G418.

have identified five different genes

VOL. 12, 1992 A

A'

G G

.\ \

HISTONE CELL CYCLE-REGULATORY GENES

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FIG. 9. Evidence that the HPC2 gene encodes a 2.1-kb transcript. (A) Mapping of the HPC2 transcription start site by primer extension. RNA was extracted from exponentially growing wildtype YM259 cells. The primer extension reactions were monitored as described previously (38). Products of dideoxy sequencing of plasmid pHX113, using 35S-ATP as the labeling agent, were loaded next to the primer extension reaction mixture. The same oligonucleotide used for both primer extension and sequencing reactions is indicated in Fig. 8. The sequencing lanes and the primer extension lane were derived from the same gel exposed for different periods of time. (B) Northern analysis of HPC2 mRNA. Total RNA was isolated from YM259 cells, and 20 and 10 pg were run in lanes 1 and 2, respectively. A ClaI-NheI DNA fragment within the HPC2 coding region was used to probe the Northern blot (see also Fig. 8). Numbers on the left indicate the positions and sizes (in kilobases) of the molecular size markers.

and HIR3 are in the same complementation group. One of the HPC genes, HPC2, has been cloned by complementation and shown to encode a highly charged basic protein. Our work and that published previously (35, 44) describing the ease in identifying new complementation groups whose mutation alters cycle-specific histone mRNA synthesis suggest that more such genes have yet to be identified. Therefore, it is likely that S. cerevisiae utilizes a complex system involving a large number of regulatory factors to regulate histone mRNA synthesis during the cell cycle. A distinct regulatory mechanism controls histone gene

5257

expression. A number of nonhistone genes are also regulated in a cycle-specific manner in yeast cells (2). Among these genes are those encoding proteins involved in DNA replication (CDC9 and POLl) or mating-type switching (HO). Interestingly, the four new HPC genes described here and three HIR genes identified previously comprise a regulatory mechanism unique to the cycle-specific regulation of histone genes. In support of this conclusion, none of the hpc or hir mutations affects cell cycle regulation of genes other than histone genes (35; this study). Conversely, we could not find any evidence suggesting that regulatory mutations of the HO gene (swil, swi4, swi5, and sinl) (30) altered cycle-specific transcription of histone genes (41). In addition, the consensus activation sequences found at the histone promoter region differ from those at the promoter of other cell cyclecontrolled genes, such as the HO gene (40). The existence of distinct regulatory mechanisms for histone gene expression may reflect the finding that histone genes appear to be autoregulated and are transcribed later in the yeast cell cycle (late G1/early S) than are the CDC9, POL1, and HO genes, which are also expressed in G1 in a cell cycle-dependent manner (2). Multiple regulatory pathways are involved in cell cyclespecific HTA1 transcription. The hpcl/hir3, hpc2, and hpcS mutations display phenotypes similar to those of hirl and hir2 mutations reported previously (35). All of them cause derepression of histone gene transcription after DNA synthesis is inhibited as well as accumulation of mRNAs at G1 and G2 phases from HTAl-HTB1 and two HHT loci. The similar phenotypes of mutants defective in HPCJ/HIR3, HPC2, HPCS, HIR1, and HIR2 gene products could possibly be explained if such genes regulate the synthesis of others in this group. However, none of the HPC gene products regulate HPC2 transcription (53). In addition, HIRJ and HIR2 do not regulate each other's transcription (45). Therefore, these gene products may contribute to the same pathway regulating cycle-specific transcription (Table 2). Mutations in the HPC3 and HPC4 genes reveal that at least two additional pathways may be involved in cyclespecific histone gene regulation. First, unlike the hpc and hir mutations described above, hpc3 shows a stringent locusspecific derepression, disrupting only the HTAI-HTB1 locus. Moreover, a newly identified dominant mutant (HIR9) also affects the HTAI-HTB1 locus only (33). Second, hpc4 shows strong derepression of HTAJ transcription upon treatment with HU but not with a-factor. This finding suggests for the first time that certain factors are distinct to the

TABLE 2. Properties of histone-regulatory mutants Histone mRNA expression

Strain(s)r Wild typec hpcl/hir3, hpc2, hirl, hir2/sptl hpcS hpc4 hpc3, HIR9 sptlO, spt2l

After a-factor

After HU

Cell cycle

Suppression of 8

affest

arrest

specific

msertionsb

No Yes Yes No Yes ND

No Yes Yes Yes Yes Yes

Yes No No Yes No ND

No Yes No No

Histone genes affected

NDd

HTAl-HTBI, HHTI, HHT2 HTAI-HTBI, HHTI, HHT2 HTAI-HTBI, HHTI, HHT2 HTAI-HTBI

Yes

HTA2-HTB2

a Data for hir/spt mutations were derived from references 33, 35, and 44. b Strains FW1237 and FW1238, containing 8 insertion mutations his4-9128 and lys2-128B, were used to cross with hpc/hir/spt mutants. c Strain YM259, from which all hpc mutant strains were obtained. d ND, not determined.

5258

XU ET AL.

regulatory mechanisms functioning in HTA1-HTB1 cyclespecific transcription after DNA replication is blocked or the cell cycle is arrested at GI. It is interesting that hpc4 and hpcS also appear unique in that they do not suppress the 8 insertion mutations at the HIS4 and LYS2 loci, in contrast to the effects of hpc2, hirl, hir2, and hpcllhir3 mutations (44). We do not know whether hpc3 has a similar suppression function due to its poor sporulation rate when crossed to a strain containing the 8 insertion mutations. Suppression is likely to result from changes in histone stoichiometry which affect nucleosome structure at the promoter regions of his4-9128 and lys2-1288, leading to altered promoter usage at these two loci (8). While it is likely that hpcS causes overexpression of histone dimers, judging from its constitutive histone mRNA synthesis throughout the cell cycle (Fig. 4), it is possible that the magnitude of imbalanced histone synthesis in hpc4 and hpcS is not sufficient to cause alterations of transcription at HIS4 and LYS2 promoter regions. Alternatively, these HPC genes may have unique functions in the regulation of histone synthesis. For example, the HPC4 gene product is required only for replication-dependent transcription of histone genes. The functions altered in hpc4 or hpcS may be intrinsically incapable of suppressing 8 insertion mutations. Why these additional levels of regulation are important when HTAJ and HTBI are already subject to temporal and autogenous regulation (33) remains to be determined. It is likely that mutations distinguishing between the various pathways will be useful in determining the nature of these various pathways. HPC/HIR proteins may function not by direct DNA binding but by assisting a dedicated repressor(s). The repressor element present within the HTA1-HTB1 intergenic region and probably also in the two HHT-HHF intergenic promoters (5, 33) is necessary to repress transcription in the G1 and G2 phases of the normal cell cycle as well as in S phase after the inhibition of DNA replication (29, 34). It is unclear whether all the HPC gene products function through the repressor element; however, several lines of evidence suggest that this may be the case. The hpcllhir3 mutation eliminates the capability of the repressor element to repress the transcription of a construct containing the CYCI promoter fused to the lacZ reporter gene in G1 and G2 phases (35). In addition, the hpcllhir3, hpc2, hpc3, and hpcS mutants all cause an accumulation of the HTA1 and HTBJ mRNAs in G1 and G2 phases of the cell cycle, indicating a deficiency of repression of histone gene transcription. The hpc4 mutation disrupts one of the two essential functions of the repressor element, demonstrated by derepressing replication-dependent histone gene transcription. Therefore, all five hpc as well as three hir mutations are likely to affect genes encoding proteins that disrupt repression of the HTA1-HTB1 promoter. Consistent with this view, the HTA2-HTB2 locus whose regulation remains intact in all five hpc mutants is the only locus among the four histone gene loci which does not contain the repressive element in its promoter region (5, 33). Sequence analysis indicates that none of the known HIRI SPT or HPC genes contain any motifs associated with DNA binding, nor do they show significant homology to existing genes in the GenBank data base, suggesting that they may fall into a novel class of genes encoding specific regulatory factors. It is possible most of these function through proteinprotein interactions. By using partially purified yeast extracts from either hpc mutant or wild-type HPC strains, we did not detect any difference in the DNA footprint at the HTAI-HTB1 promoter region (53). These data suggest that HPC gene products are unlikely to be a group of DNA-

MOL. CELL. BIOL.

binding proteins. Rather, they may play a catalytic role in stimulating some other regulatory factor(s) to modulate histone gene expression during the cell cycle. The hpc mutations described here should help in deciphering these interactions. ACKNOWLEDGMENTS We thank Jef Boeke, Lianna Johnson, Mary Ann Osley, and Fred Winston for yeast strains and many helpful discussions. We are grateful to Mark Goebl for searching his private data base for sequence homologies. We are also thankful to Lianna Johnson and Bo Thomsen for reading the manuscript and for helpful comments. This work was supported by Public Health Service grant GM23674 from the National Institutes of Health. REFERENCES 1. Alterman, R.-B., S. Ganguly, D. H. Schulze, W. F. Marzluff, and A. I. Skoultchi. 1984. Cell cycle regulation of mouse histone H3 mRNA metabolism. Mol. Cell. Biol. 4:123-132. 2. Andrews, B. J., and I. Herskowitz. 1990. Regulation of cell cycle-dependent gene expression in yeast. J. Biol. Chem. 265: 14057-14060. 3. Artishevsky, A., A. Grafsky, and A. S. Lee. 1985. Isolation of a mammalian sequence capable of conferring cell cycle regulation to a heterologous gene. Science 230:1061-1063. 4. Bilinski, C. A., and J. J. Miller. 1980. Induction of normal ascosporogenesis in two-spored Saccharomyces cerevisiae by glucose, acetate, and zinc. J. Bacteriol. 143:343-348. 5. Breeden, L. 1988. Cell cycle-regulated promoters in budding yeast. Trends Genet. 4:249-253. 6. Capasso, O., G. C. Bleecker, and N. Heintz. 1987. Sequences controlling histone H4 mRNA abundance. EMBO J. 6:18251831. 7. Capasso, O., and N. Heintz. 1985. Regulated expression of mammalian human histone H4 genes in vivo requires a transacting transcription factor. Proc. Natl. Acad. Sci. USA 82:56225626. 8. Clark-Adams, C. D., D. Norris, M. A. Osley, J. Fessler, and F. Winston. 1988. Changes in histone gene dosage alter transcription in yeast. Genes Dev. 2:150-159. 9. Clark-Adams, C. D., and F. Winston. 1987. The SPT6 gene is essential for growth and is required for b-mediated transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 7:679-686. 10. Dailey, L., S. M. Hanly, R. G. Roeder, and N. Heintz. 1986. Distinct transcription factors bind specifically to two regions of the human histone H4 promoter. Proc. Natl. Acad. Sci. USA 83:7241-7245. 11. Dailey, L., S. B. Roberts, and N. Heintz. 1988. Purification of the human histone H4 gene-specific transcription factors H4TF-1 and H4TF-2. Genes Dev. 2:1700-1712. 12. Domdey, H., B. Apostol, R.-J. Lin, A. Newman, E. Brody, and J. Abelson. 1984. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell 39:611-621. 13. Emr, S. D., A. Vassorotti, J. Garrett, B. L. Geller, M. Takeda, and M. G. Douglas. 1986. The amino terminus of the yeast F1-ATPase-subunit precursor functions as a mitochondrial import signal. J. Cell Biol. 102:523-533. 14. Fletcher, C., N. Heintz, and R. G. Roeder. 1987. Purification and characterization of OTF-1, a transcription factor regulating cell cycle expression of a human histone H2b gene. Cell 51:773-781. 15. Han, M., M. Chang, U.-J. Kim, and M. Grunstein. 1987. Histone H2B repression causes cell-cycle-specific arrest in yeast: effects on chromosome segregation, replication, and transcription. Cell 48:589-597. 16. Hanley, S. M., G. C. Bleecker, and N. Heintz. 1985. Identification of promoter elements necessary for transcriptional regulation of a human histone H4 gene in vitro. Mol. Cell. Biol. 5:380-389. 17. Heintz, N. 1991. The regulation of histone gene expression during the cell cycle. Biochim. Biophys. Acta 1088:327-339. 18. Heintz, N., and R. G. Roeder. 1984. Transcription of human histone genes in extracts from synchronized Hela cells. Proc.

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