MOLECULAR AND CELLULAR BIOLOGY, Sept. 1990, p. 4905-4911

Vol. 10, No. 9

0270-7306/90/094905-07$02.00/0 Copyright ©3 1990, American Society for Microbiology

A Conserved Sequence in Histone H2A Which Is a Ubiquitination Site in Higher Eucaryotes Is Not Required for Growth in Saccharomyces cerevisiae PAUL S. SWERDLOW,'* TILLMAN SCHUSTER,2 AND DAN FINLEY3

Division of Hematology-Oncology, Departments of Medicine and of Microbiology-Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 232981; Institut fur Molekulare Pathologie, A-1030 Vienna, Austria2; and Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 021153 Received 28 March 1990/Accepted 5 June 1990

Histones H2A and H2B

are

modified by ubiquitination of specific lysine residues in higher and lower

eucaryotes. To identify functions of ubiquitinated histone H2A, we studied an organism in which genetic analysis of histones is feasible, the yeast Saccharomyces cerevisiae. Surprisingly, immunoblotting experiments

using both anti-ubiquitin and anti-H2A antibodies gave no evidence that S. cerevisiae contains ubiquitinated histone H2A. The immunoblot detected a variety of other ubiquitinated species. A sequence of five residues in S. cerevisiae histone H2A that is identical to the site of H2A ubiquitination in higher eucaryotes was mutated to substitute arginines for lysines. Any ubiquitination at this site would be prevented by these mutations. Yeast organisms carrying this mutation were indistinguishable from the wild type under a variety of conditions. Thus, despite the existence in S. cerevisiae of several gene products, such as RAD6 and CDC34, which are capable of ubiquitinating histone H2A in vitro, ubiquitinated histone H2A is either scarce in or absent from S. cerevisiae. Furthermore, the histone H2A sequence which serves as a ubiquitination site in higher eucaryotes is not essential for yeast growth, sporulation, or resistance to either heat stress or UV radiation.

Covalent ligation of ubiquitin to acceptor proteins is involved in a number of cellular processes, such as DNA repair (16), progression through the cell cycle (7, 11), and a variety of stress responses (4). Coupling of ubiquitin to other proteins involves formation of an isopeptide bond between the carboxyl-terminal glycine residue of ubiquitin and the epsilon-amino group of a lysine residue in the acceptor protein. This reaction is catalyzed by a family of ubiquitinconjugating enzymes (also called E2 enzymes). In the yeast Saccharomyces cerevisiae, two of at least six such enzymes have been identified as products of the previously known genes RAD6, whose functions include DNA repair (16), and CDC34, which is required for transition from the Gl to the S phase of the cell cycle (11). It has been suggested that regulatory functions of the RAD6 and CDC34 gene products may be mediated through ubiquitination of histones (16). Purified histones are substrates for these enzymes in vitro, and in mammals and other eucaryotes, ubiquitinated H2A (uH2A) is apparently the most abundant ubiquitin-protein conjugate. Moreover, the processes ultimately dependent on RAD6 and CDC34DNA repair and synthesis-take place on a nucleoprotein template that contains histones, at least initially. Whether histones that have been assembled within nucleosomes can be ubiquitinated by RAD6 or CDC34 proteins is unclear, however. Previous studies have characterized ubiquitinated histones in terms of structure and metabolism but not function. Although one major function of ubiquitin is to mark proteins for selective elimination, there is no evidence that ubiquitin is involved in histone degradation (29). Thus, for histones, *

ubiquitination might serve to modify the structure or function of the acceptor protein rather than destabilize it metabolically. Consistent with this view, ubiquitination of histones is at least largely a reversible process. The ubiquitin moiety is removed from ubiquitinated histones at each metaphase (21) and, at least in Physarum sp., is restored within minutes after metaphase (25). In mammalian cells, ubiquitinated forms typically constitute 10% or more of H2A (at lysine 119) and 1 to 2% of H2B (at lysine 120). Decreases in the ubiquitination of H2A have been observed upon differentiation (26, 33). We have recently shown that uH2A levels are dramatically increased as mouse erythroleukemia cells begin to differentiate but then decline as differentiation progresses (12). Although higher eucaryotes have been used to characterize the structure and metabolism of ubiquitinated histones, lower eucaryotes, such as S. cerevisiae, are better suited for functional analysis. Moreover, the genetics of yeast histone H2A and H2B have been studied in some detail. The H2A and H2B genes in S. cerevisiae each exist in duplicate in the haploid genome and are organized in two nonallelic loci: the HTA-1 and HTB-1 locus and the unlinked HTA-2 and HTB-2 locus (13). The presence of at least one of these two H2A genes, together with either H2B gene, is required for yeast viability (17). Interestingly, deletion from the carboxyl terminus of histone H2A past a conserved five-residue sequence is not tolerated (28). This sequence, Leu-Leu-ProLYS-Lys, contains the site of ubiquitination in higher eucaryotes (shown in uppercase) and is conserved from Saccharomyces sp. through humans (the second Lys is replaced by either Thr or Gln in the two histone H2A genes of Schizosaccharomyces pombe) (6, 22). In this study, we tested whether elimination of the se-

Corresponding author. 4905

SWERDLOW ET AL.

4906

MOL. CELL. BIOL. TABLE 1. Yeast strains and plasmids

Strain or plasmid

Yeast strains DKY116

PSH101 PSH107 PSDO04 PSDO05 PSDO06 PSDO12 PSDO08 PSDO13 PSDO16 TSY106 TSY107 TSY150 TSY153 TSY184 TSY185 TSY186 TSY187 TSY222a

Genotype orcharacteristics

a/a htal-JIHTAI HTA21hta2-1 ura3-521ura3-52 ade2IADE2 his3-lIHIS3-1 a htal-l hta2-1 ura3-52 his3(pHTA1) a htal-I hta2-1 ura3-52 his3(pHTA1-arg) a/a htal-llhtal-l hta2-llhta2-1 ura3-521ura3-52 his3/HIS3 ade2/ADE2(pHTA-1)(pHTA1-arg) a/a htal-llhtal-I hta2-J/hta2-1 ura3-521ura3-52 his3lHIS3 ade2/ADE2(pHTA-1)(pHTA1-arg) a/a htal-llhtal-I hta2-llhta2-1 ura3-521ura3-52 his3IHIS3 ade2/ADE2(pHTA-1)(pHTA1-arg) a/a htal-llhtal-J hta2-llhta2-1 ura3-521ura3-52 his3/HIS3 ade2/ADE2(pHTA-1)(pHTA1-arg) a/a htal-llhtal-I hta2-llhta2-1 ura3-521ura3-52 his3/HiS3 ade2/ADE2(pHTA1-arg)(pHTA1-arg) a/a htal-llhtal-J hta2-llhta2-1 ura3-521ura3-52 his3IHiS3 ade2/ADE2(pHTA1-arg)(pHTA1-arg) a/a htal-llhtal-J hta2-llhta2-1 ura3-521ura3-52 his3/HiS3 ade2/ADE2(pHTA1-arg)(pHTA1-arg) a htal-I hta2-1 ura3-52 his3(pHTA1 d4-20) a htal-I hta2-1 ura3-52 his3(pHTA1) a htbl-J htb2-1 ura3-52 ade5 ade2-101 lys2-801(pHTB2) a htbl-l htb2-1 ura3-52 met 13 lys 2-803(HTB2 d123-130) a HTAJ HTA2 ura3-52 ade2(pHTA1-arg) a HTA1 hta2-1 ura3-53 ade2(pHTA1-arg) a htal-J HTA2 ura3-52 ade2(pHTA1-arg) a htal-l hta2-1 ura3-52 ade2(pHTA1-arg) a htal-I hta2-1 ura3-52 his3(pTS29)

Source or reference

17 This study This study This study This study This study This study This study This study This study 28 28 28 28 This study This study This study This study This study

Plasmidsa

pHTA1 pHTA1 d4-20 pHTA1-arg pHTB2 pHTB2 d123-130

pTS29 a

Wild-type HTAI gene (also called pJC102; see reference 28) HTAI gene deleted for sequences encoding residues 4-20 (also called pTS2; see reference 28) HTAI gene with Lys-to-Arg mutations to preclude ubiquitination at conserved site (see Materials and Methods). Wild-type HTB2 gene (also called PTS4; see reference 28) HTB2 gene deleted from residues 123-130 (also called pTS5; see reference 28) Wild-type HTAI gene with GAL promoter in place of histone promoter and HIS3 (not URA3) marker.

Except as noted, all contain URA3 as a selectable marker and CEN and ARS sequences in addition to the histone genes noted.

quence which serves as a ubiquitination site in higher eucaryotes has functional consequences in S. cerevisiae.

MATERIALS AND METHODS Cell culture and growth conditions. The yeast strains and plasmids used are listed in Table 1. Yeast media were prepared as previously described (30). Yeast cells were grown in liquid medium or on plates at 30°C unless otherwise indicated. Growth on alternative carbon sources was done on minimal defined media, as S. cerevisiae can use amino acids as carbon sources. Cell counts were performed in triplicate with a hemacytometer. Budded cells were counted as one cell unless the bud was of a size equal to that of the mother. Yeast strains PSH101 and PSH107 were created by transformation of TSY222a with plasmids pHTAl and pHTAl-arg, respectively, and selection for Ura+ His- colonies. Strains PSD004 and PSDO16 were made by mating PSH101 and PSH107 to TSY187. For UV radiation studies, yeast cells were plated at low density and the plates were exposed to a General Electric G30T8 lamp (254-nm peak) for the times indicated. For heat stress studies, yeast cells were plated at low density and the plates were incubated at 530C for the times indicated. In either case, the plates were then incubated at 300C. The number of colonies that grew after several days was taken as the number of cells surviving the radiation or heat stress. All platings were done in triplicate. Cell extracts and immunoblotting. Yeast extracts were prepared by washing log-phase cells twice with deionized water and suspending the pellet in boiling 2% sodium dodecyl sulfate-30 mM ,-mercaptoethanol. The samples were boiled for 5 min, vortexed for 1 min while still hot, and then

centrifuged at 1,600 x g for 10 min. The supernatant was transferred to a microcentrifuge tube, spun at 12,000 x g for 10 min, and transferred to a fresh tube. Glycerol was then added to 10%. HeLa cell extracts were prepared similarly, except that log-phase cells were washed with phosphatebuffered saline instead of water. Each milliliter of yeast extract was prepared from 2 x 109 cells, while each milliliter of HeLa extract was prepared from 107 cells. Antiubiquitin immunoblotting used autoclaved filters as previously described (31). The anti-H2A immunoblotting procedure differed only in that filters were baked (not autoclaved) for 2 h at 80°C in a vacuum oven and blocked with 10% fetal bovine serum.

Site-specific mutagenesis of yeast histone H2A. The HTAJ gene was cloned into M13mp8. Bacteriophage DNA was isolated (14) and mixed with EcoRI-digested replicative form DNA of M13mpl8 in 10 mM Tris hydrochloride (pH 8)-i mM EDTA. The mixture was denatured with alkali and neutralized to generate gapped duplexes. A mutagenic oligo-

nucleotide, CTTGGGAGAT(T/C)TT(T/C)TTGGCAACAA, was synthesized, 32P end labeled with T4 polynucleotide kinase and [.y-32P]ATP, and sequenced. The oligonucleotide was annealed to the gapped duplex. The gapped strand was completed and circularized by using Klenow fragment and T4 ligase (34). The duplex was immediately used to transform Escherichia coli JM101, which contains the amber suppressor required by M13mp8. Transformants were plated onto a lawn of E. coli JM105, which lacks the suppressor function required by M13mp8 but not by M13mpl8. Thus, only the Mpl8-derived strands, which would be expected to contain the mutation, were able to produce plaques. Plaques were replicated onto nitrocel-

VOL.

10, 1990

CONSERVED H2A SEQUENCE

lulose filters (BA85; Schleicher & Schuell, Inc., N.H.) and positive clones were detected by hybridization the end-labeled oligonucleotide. DNA was isolated plaque-purified phage grown as described above and quenced in the area of the mutation (27). Sequencing HTAl-arg revealed that both lysine codons had changed to arginines as intended, as well as two additional changes at threonine 125 (to alanine) and alanine 127 proline). The latter changes are 6 and 4 residues carboxy terminus of HTA-1 and in a region that can deleted without producing a detectable phenotype

NOT REQUIRED

2

1

Keene,

IN

S.

3

H2A

to

wt

from

CEREVISIAE

del

wt

4

H2B

4907

5 wt

del

se-

of clone been

39--

(to

from

the

be

(28).

Transformation and sporulation of S. cerevisiae. Yeast transformed by the lithium acetate method (15). cells were sporulated by growth on presporulation plates 2 days and then transfer to liquid sporulation medium Alternatively, sporulation was done directly in 0.1 M sium acetate. Sporulation frequencies were determined counting sporulated and unsporulated cells after staining with malachite green (30). Asci were digested with yase (0.1 mg/ml) in a buffer containing 1 M sorbitol, cells

were

I

Yeast

17

for

(20).

potas-

by

H2A._

Zymol2

P-mercaptoethanol,

10 mM MgCl2, and phosphate (pH 7.0) and dissected with a

25

mM

mM

potassium

micromanipulator.

DNA preparation, sequencing, and blotting. DNA isolated from S. cerevisiae (30) and from M13 previously described. Sequencing was performed dideoxy technique (27) for M13 DNA and by the MaxamGilbert technique (23) for oligonucleotides and rescued mids. DNA was isolated from haploid strains derived random spores (30), cut with HindIll, and electrophoresed on an agarose gel. DNA was blotted to nitrocellulose hybridized as previously described (20) to a 32P-radiolabeled

was

(24)

as

by

the

plas-

from

and

yeast HTA-1 probe.

RESULTS

S. cerevisiae does not contain uH2A in amounts comparable to those of higher eucaryotes. We attempted to identify in whole-cell extracts of S. cerevisiae by immunoblotting. Proteins were fractionated by sodium dodecyl sulfate-polyuH2A

acrylamide gel electrophoresis, transferred to nitrocellulose filters, and probed with an antibody to yeast antibody did not detect any protein in the molecular H2A.

This

weight

expected of uH2A,

although some minor seen in addition to that of H2A (Fig. 1). To cross-reactive proteins from true modified H2A range

bands

were

distinguish species,

immunoblotted extracts from yeast cells containing tional but amino-terminally truncated histone malian H2A is ubiquitinated near its carboxyl terminus.) deletion results in a substantial mobility shift band (Fig. 1, lane 2). Should a significant histone H2A exist in a modified form, a minor band either shift position or possibly disappear in sample. The deletion had no effect on the minor

we

func-

H2A.

in

(Mam-

the

The

H2A

proportion

of

should

the

mutant

bands

1, lanes 1 and 2; data not shown). To ensure further minor bands did not represent H2A species whose

(Fig.

that

the

migration

not affected by the deletion, antibody was the bands and used to reprobe parallel nitrocellulose was

eluted

No antibody from any of the minor bands had reactivity similar to that from the H2A band. antibody reacted most strongly to the band

a

from

strips.

pattemn

Each

from

which

isolated and to other minor bands, suggesting reactivity of non-H2A proteins to the antibody. The of sufficient sensitivity to suggest that no more than was

is ubiquitinated.

This is

not a definitive

limit, since we cannot exclude the possibility that clonal H2A antibody detects H2A more strongly

the

it

cross-

test

yeast H2A

of

eluted

was

1%

of

upper

poly-

than uH2A.

FIG. 1. Immunoblot of yeast extracts probed with anti-H2A antibody. Yeast extracts from strains with wild-type (wt) or deleted (del) H2A genes were run on 18% polyacrylamide-sodium dodecyl sulfate gels and blotted with an anti-yeast H2A antibody (5). No bands were seen with mobility greater than that of histone H2A, so only the region of the gel above histone H2A, where uH2A might be expected, is shown. Lanes: 1, TSY107 (htal-I hta2-1[pHTA1]); 2, TSY106 (htal-i hta2-1[pHTA1 d4-20]); 3, TSY150 (HTAI HTA2

htbl

-I

htb2-1[pHTB2]);

I[HTB2 d123-130]);

4, TSY153 (HTAI HTA2 htbl-J htb2-

5, TSY184 (HTAI HTA2[pHTA1-arg]). Note the dramatic shift in H2A mobility with H2A deletion strain TSY106. Note the absence of a higher-molecular-weight band corresponding to uH2A, even with strain TSY153, which lacks the mammalian H2B ubiquitination site. The intense band at the top is cross-reactive material not identified. It is notH2A-related, as it did not shift position with the H2A deletions. The numbers on the left indicate molecular sizes in kilodaltons.

Since ubiquitination of mammalian histone H2B has also been reported (32), we looked to see whether yeast cells missing a sequence analogous to the mammalian consensus site for ubiquitination of histone H2B might ubiquitinate histone H2A (Fig. 1, lanes 3 and 4). Such yeast cells were previously reported and are able to grow despite the deletion (28). No additional band corresponding to ubiquitinated H2A was found, arguing that ubiquitinated histone H2A is not formed when the consensus site of ubiquitination of histone H2B is deleted. Figure 2 shows a similar set of immunoblotting experiments carried out with a polyclonal antibody to ubiquitin. This affinity-purified antibody detects not only ubiquitin but also a number of other cellular proteins which are presumably ubiquitin-protein conjugates. Parallel extracts with mammalian uH2A added either before yeast lysis or after extract preparation gave identical signals on immunoblots (data not shown), ruling out destruction of uH2A during extract preparation. To test whether any of the observed bands represented uH2A, we again screened for a band whose mobility was increased in samples from H2A deletion mutants. Strains TSY106 (Fig. 2A, lane 5, and B, lane 2) and TSY107 (Fig. 2A, lane 6, and B, lane 3) had H2A histones with widely differing mobilities (Fig. 1), but despite this difference, no major band shifted position or disappeared on the antiubiquitin immunoblot. Overexposures gave the same result for a

4908

MOL. CELL. BIOL.

SWERDLOW ET AL.

B

A 3

1

5

6

8

7

2

3

27--

_

_

z

~~~~~-uH2A

uH2A--

--17

17---39 Sa_ wS -

-

Ub

-4

FIG. 2. Immunoblot of yeast extracts probed with anti-ubiquitin antibody. Whole-cell extracts from yeast and HeLa cells were run on 18% polyacrylamide-sodium dodecyl sulfate gels and immunoblotted with an antibody to ubiquitin. Molecular size standards are indicated in kilodaltons. (A) Comparison of yeast strains containing an H2A ubiquitination site mutation, H2A deletion mutations, and HeLa cells. Extracts from 4 x 107 yeast or 8 x 104 HeLa cells were loaded in each lane. Lanes: 1, TSY184 (HTAJ HTA2 [pHTA1-arg]); 2, TSY185 (HTAI hta2-J[pHTA1-arg]); 3, TSY186 (htal-i HTA2[pHTA1-arg]); 4, TSY187 (htal -I hta2-J[pHTA1-arg]); 5, TSY106 (htal-1 hta2-1[pHTA1 d4-20]); 6, TSY107 (htal-I hta2-l[pHTA1]); 7, calf thymus uH2A marker; 8, HeLa cell extract containing an amount of histones equal to that in lanes 1 to 7. Ub, Ubiquitin. (B) High-resolution analysis at the 17- to 27-kilodalton region of an anti-ubiquitin immunoblot. Extracts from TSY106 (lane 2) and TSY107 (lane 3), along with the calf thymus uH2A marker (lane 1), were run on a 32-cm 18% polyacrylamide-sodium dodecyl sulfate gel. The gel was cut, and the low-molecular-weight region was immunoblotted as described for panel A.

number of minor bands (data not shown). Lane 8 contained similar extract from HeLa cells. uH2A was easily detected in the HeLa sample, although the HeLa and yeast samples were loaded in amounts calculated to give equivalent amounts of H2A in each lane. On the basis of a graded series of autoradiographic exposures, we estimated that yeast bands containing as little as 1% of the intensity of the HeLa signal would have been detected. H2A mutants lacking a sequence ubiquitinated in higher eucaryotes (see below) also showed a wild-type pattern of bands on antiubiquitin immunoblots (Fig. 2, lanes 1 to 4). Construction of a histone H2A mutant lacking a sequence ubiquitinated in higher eucaryotes. Despite our inability to detect uH2A in S. cerevisiae, it remained possible that S. cerevisiae might ubiquitinate histone H2A at the site used by higher eucaryotes, particularly since there is remarkable conservation of the sequence neighboring this site from mammals to S. cerevisiae. Accordingly, we constructed mutations in the HTAJ gene which result in Lys-to-Arg substitutions at residues 119 and 120 (see Materials and Methods). We call this altered H2A gene HTAI-arg (see Materials and Methods) and the URA3-containing yeast centromere plasmid carrying the gene pHTA1-arg. Strain DKY116 (htal-JIHTAI hta2-JIHTA2; htal-i and hta2-1 are frameshifted null alleles) was transformed with pHTA1-arg and sporulated. DNAs were prepared from haploid strains derived from random Ura+ spores, Southern blotted, and probed with radiolabeled H2A DNA (Fig. 3). Four strains were picked for further study, each containing plasmid pHTA1-arg and both wild-type H2A genes, HTAJ and HTA2, HTAI with hta2-1, HTA2 with htal-i, or htal-i with hta2-1. The plasmid was rescued from S. cerevisiae by transformation of E. coli, and the presence of the Lys-to-Arg mutations was confirmed by sequencing. Characterization of HTAl-arg mutants. The four strains containing the pHTA1-arg plasmid and 2, 1, or 0 copies of the wild-type H2A genes all grew normally. Table 2 summaa

rizes the phenotypic tests carried out on these strains. The cells were streaked on SC-URA plates and incubated at different temperatures, as indicated. The colony sizes of the four strains were indistinguishable at each temperature. Heat-induced killing was also assayed with yeast cells spread on plates and incubated at 53°C for 0 to 6 h. No significant survival differences were seen. Osmotic sensitivity was tested on SC-URA plates containing 0, 0.5, 1, or 1.5 M KCI. No differences in colony size were noted. Use of glycerol and maltose as alternative carbon sources resulted in no detectable growth differences among the four strains. These studies were done in defined minimal media to ensure that no other compounds could function as carbon sources. Growth rates in liquid medium were also examined by using diploid strains congenic except for HTA-1. PSDO04 contains a wild-type HTA-1 gene, while PSDO16 does not; both contain pHTA1-arg. The strains were grown in liquid medium (YPD) at 30°C. The growth curve from one such experiment is shown in Fig. 4. No significant growth differences were found in any of four such experiments. Recently, a 20-kilodalton ubiquitin-conjugating enzyme was shown to be a product of the RAD6 gene, mutants of which are sensitive to UV light and X-ray irradiation (16). To test whether the H2A mutants had a similar phenotype, haploid strains were subjected to UV radiation at 254 nm for various times. No significant differences in UV sensitivity were noted among the three strains tested (Fig. 5). The effect of the HTAI-arg mutation on sporulation was evaluated by using congenic strains PSD004 and PSDO16 (Table 3). Strain PSD016 has only plasmid pHTA1-arg (containing the ubiquitination site mutation), while PSD004 has both plasmids pHTA1 and pHTA1-arg. It is, of course, possible that either strain could lose one of its two plasmids with prolonged growth. Sporulation, however, revealed that all four spores were nearly always viable. Only two would have been viable if only a single plasmid had been present. The copy number of the plasmids was not directly deter-

CONSERVED H2A SEQUENCE NOT REQUIRED IN S. CEREVISIAE

VOL. 10, 1990

4909

TABLE 2. Yeast phenotypes

1

3

2

_

4

Condition

~~~plasmid

hta1-1 *

* hta2-1

I HTAI HTA2 * FIG. 3. Isolation of haploid strains containing the ubiquitination site mutation. DNAs were isolated from haploid strains resulting from the sporulation of strain DKY116 containing plasmid pHTA1arg. DNA samples were cut with HindlIl. The hta-l and hta-2 null alleles were originally generated by destroying the HindIll sites within the coding sequences of these genes. Thus, the fragments containing frameshifted loci are larger than those containing wildtype genes. DNA was run on a 1% agarose gel, blotted to nitrocellulose, and probed with 32P-labeled HTA-J DNA. Bands corresponding to plasmid, wild-type HTA-1, wild-type HTA-2, frameshifted HTA-1, and frameshifted HTA-2 are marked at the right. Strains TSY184 to TSY187 correspond to lanes 1 through 4 and contain the plasmid plus 2, 1, 1, and 0 wild-type genes, respectively.

mined. On microscopic examination of the spores stained with malachite green, morphologically mature asci of both strains were seen. Sporulation frequencies were assessed in five additional independent strains, each equivalent to either PSDO04 or PSDO16. Overall, yeast strains with two wildtype plasmids had slightly higher sporulation efficiencies, although the differences were modest (PSDO04, 61.4%; PSDO05, 50%; PSDO12, 52.8%; and PSDO06, 56% versus PSDO16, 32%; PSDO08, 36%; and PSDO13, 47.8%). None of the strains having only HTAI-arg histones sporulated as well as the strains with wild-type HTAI. Differences between the means were statistically significant for all, except for the difference between PSDO13 and PSDO05. Furthermore, the strains containing only pHTA1-arg made a smaller percentage of four-spored asci. Dissection of spores was performed. Even 5 days after sporulation, viability of spores exceeded 95% and was no different between strains with the wild-type gene and those with the HTAJ-arg gene. In contrast, the ubi4 mutant, which lacks the yeast polyubiquitin gene, loses viability shortly after sporulation (10). The ade2 marker present in TSY187 segregated properly. The H2A null alleles used in this study resulted from

Result with the following no. of wild-type H2A genesa: 2 1 0

Temperature, °C 11 16 20 23 28 35 37

+ + + + + + +

+ + + + + + +

+ + +

Minimal medium (SD) With auxotrophic nutrients Without auxotrophic nutrients

+

+

+

-

-

-

+ +

+ +

+

+

+ + +

+ + +

+ + +

+ +

+

+

+

1O-3

1O-3

1o-8b

Alternative carbon source (minimal defined medium) Glucose Glycerol Maltose Ionic strength 0.5 M KCI

1.0MKCl 1.5 M KCI Expression of mating type Frequency of resistance to 5fluoro-orotic acid

+ + + +

+

a +, Growth; -, no growth. b All survivors retained the Ura+ plasmid.

frameshift mutations (17). Reversion of these mutations could potentially mask any phenotype produced by HTAJarg. To assay for such reversion, we attempted to select against the pHTA1-arg plasmid. 5-Fluoro-orotic acid selects against yeast strains which are Ura+ (3) and thus against the URA3 gene on the pHTA1-arg plasmid. As expected, strains containing the mutated plasmid and two disrupted H2A genes were sensitive to 5-fluoro-orotic acid, while yeast strains containing the plasmid plus one wild-type gene were resistant at a frequency of l0-3. DISCUSSION The finding that ubiquitinated histones are either scarce in or absent from S. cerevisiae is unexpected for a number of reasons. (i) Ubiquitin and the histones are among the most conserved eucaryotic proteins known. (ii) Yeast ubiquitinconjugating enzymes have been shown to ubiquitinate histones H2A and H2B in vitro (11, 16). (iii) Yeast histone H2A is identical to mammalian H2A near the mammalian ubiquitination site. (iv) This site is contained within a region essential for H2A function in S. cerevisiae as determined by deletion mutations. Nonetheless, our results argue from two distinct experimental approaches that ubiquitination of histone H2A at the mammalian ubiquitination site is not required for growth in S. cerevisiae. uH2A is an abundant protein in many organisms, including lower eucaryotes, such as Physarum sp. and Tetrahymena sp. The lowest amount yet reported is in Chironomus tetans, in which 1% of histone H2A is ubiquitinated (8). Our data appear to set an upper limit for the uH2A/H2A ratio in S.

SWERDLOW ET AL.

4910

MOL. CELL. BIOL. TABLE 3. Studies of congenic strains PSDO04 and PSD016 Characteristic PSDO16 PSDO04

tau-3

1000

Survival of heat stress Sporulation Segregation of ade2 Late germination

-

E

+ + +

+ + +

>95%

>95%

0n

0

u

I0)

l

50-

0

l

2

3

4

5

7

6

Hours FIG. .4. Identical growth rates of congenic strains differing only in the ubiquitination site mutation. Yeast strains were grown on YPD a]nd counted as described in Materials and Methods. Symbols: 0, stratin PSD004 (HTAI); A, PSD016 (HTAI-arg).

cerevi.!siae at 1/100 of that in HeLa cells, which contain appro.ximately 10% of histone H2A as uH2A (18). Thus, S. cerevi!siae may have less than 0.1% of histone H2A ubiquitinatedl. Therefore, there does not seem to be an absolute physic)logical requirement in S. cerevisiae for histone H2A to be iubiquitinated at the mammalian site. Since nearly 40% of the yeast genome is transcribed, the amount of ubiquitinated histone H2A that could be present in the cell seems insuffiicient to account for any general involvement in transcriptiional processes. However, in other eucaryotes, transcribe d chromatin appears to be enriched in ubiquitinated histon e H2A (1, 2, 19, 26a). The site of ubiquitination of mammalian histone H2B has been ireported (32), and yeast strains lacking the analogous sequelnce of yeast H2B have been previously created and found to be viable (28). This lack of a requirement for the

0

a-

1 100

a)

\

0-

T

1 0 < 1 \ T

Q-

-T

0

0)

:3>)

1040

10

20

30

40

50

UV exposure (seconds) FIG 5. Equal sensitivities of wild-type and HTAI-arg strains to UV ra,diation. Strains PSH101 (0), PSH107 (0), and TSY153 (A) were sitreaked on YPD plates and exposed to UV radiation at 254 nm for the times shown. PSH101 and PSH107 are congenic except for the prcDsence of plasmids containing HTAJ and HTAI-arg, respectively. TSY153 lacks the putative ubiquitination site of histone H2B. Standard error bars are shown only where larger than symbol size. The daLta are plotted as logs of cells surviving so that parallel lines are eqiually sensitive to radiation. Starting numbers of cells were deliberrately distinct so that the parallel nature of the lines could be more easily seen. Slopes of linear regressions performed on the above--described data (r values of 0.999 or better) are very similar: PSH1C)1, 0.028; PSH107, 0.030; and TSY153, 0.031.

mammalian site of ubiquitination for growth in S. cerevisiae is similar to the results of this study. Since such yeast strains might be more likely to ubiquitinate histone H2A, we examm ined extracts for modified histone H2A by immunoblotting. No ubiquitinated or modified histone H2A was found, arguing that uH2A is not formed to compensate for a lack of will be ceeiie uH2B. Further studies needed to determine the role, if any of ... if any, of uiitnHBnS. H2B in S. cerevisiae. ubiquitin By using genetic techniques, we altered the putative ubiquitination site of yeast histone H2A. Since arginines were substituted for the ubiquitinatable lysines, the histone maintained charge and size but could not be ubiquitinated at the substituted site. Such mutants grew normally under a variety of temperatures, ionic strengths, and carbon sources. They carried out UV repair and germinated as efficiently as yeast strains containing wild-type H2A genes. Thus, the HTAI-arg mutant showed none of the phenotypic changes seen in rad6, cdc34, or ubi4 (polyubiquitin gene) mutants. The phenotypes of these latter mutants cannot be based on an inability to ubiquitinate lysine residue 119 or 120. Nevertheless, we cannot rule out the possibility that small amounts of ubiquitinated histones exist in S. cerevisiae, that substantial amounts may be formed under physiological conditions that we have not examined or with overexpression of ubiquitin, or that yeast histones may be ubiquitinated at typical rates but deubiquitinated much more rapidly than those of mammalian cells. Similarly, we cannot exclude the possibility that a low level of ubiquitination of histone H2A is required for RAD6, CDC34, or UBI4 function, although such ubiquitination cannot be required at the identified mammalian rate. The modest change in sporulation efficiency may reflect such a function. Our results raise the question of whether modification of histone H2A by ubiquitination is an important regulator of the structure or function of chromatin. However, the chromatin structure of S. cerevisiae differs from that of higher eucaryotes in aspects other than ubiquitination, most notably in the apparent absence of histone Hi in S. cerevisiae and in a relatively short nucleosome repeat length. The difference in ubiquitination patterns between S. cerevisiae and higher organisms may be related to these distinct properties of S. cerevisiae chromatin. A low abundance of uH2A is not necessarily peculiar to S. cerevisiae, however. It is possible that ubiquitination of histone H2A functions in higher eucaryotes as one element of a mechanism that regulates chromatin condensation, particularly during mi-

cycles. S. cerevisiae has a small genome and does not substantially condense its chromatin during metaphase (9). Histone Hi has been strongly implicated in the regulation of chromatin condensation; its apparent absence from S. cerevisiae (5) is consistent with the minimal chromatin conden-

totic

sation in this organism. The rapid and complete deubiquiti-

nation of histones which occurs at the onset of mitosis, as well as the prompt reestablishment of ubiquitination patterns after metaphase, suggests that changes in histone ubiquitination actively participate in condensation and decondensation processes. Such questions may be explored in other

CONSERVED H2A SEQUENCE NOT REQUIRED IN S. CEREVISIAE

VOL. 10, 1990

eucaryotes, possibly through genetic analysis of Schizosaccharomyces pombe, a yeast whose chromatin more closely resembles that of higher eucaryotes. ACKNOWLEDGMENTS We thank Alexander Varshavsky and Michael Grunstein for initial encouragement and support and Mike Ellison for helpful discussions. C. Paige Wirt and Marianna Clougherty provided expert technical assistance. This work was supported by the American Cancer Society, by Public Health Service grant K08 HL01788 (P.S.) from the National Heart, Lung, and Blood Institute, and by Public Health Service grant GM43601-01 (D.F.) from the National Institutes of Health. LITERATURE CITED 1. Barsoum, J., L. Levinger, and A. Varshavsky. 1982. On the chromatin structure of the amplified, transcriptionally active gene for dihydrofolate reductase in mouse cells. J. Biol. Chem. 257:5274-5282. 2. Barsoum, J., and A. Varshavsky. 1985. Preferential localization of variant nucleosomes near the 5'-end of the mouse dihydrofolate reductase gene. J. Biol. Chem. 260:7688-7697. 3. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197:345-346. 4. Bond, U., and M. J. Schlesinger. 1985. Ubiquitin is a heat shock protein in chicken embryo fibroblasts. Mol. Cell. Biol. 5:949956. 5. Certa, U., M. Colavito Shepanski, and M. Grunstein. 1984. Yeast may not contain histone Hi: the only known 'histone Hi-like' protein in Saccharomyces cerevisiae is a mitochondrial protein. Nucleic Acids Res. 12:7975-7985. 6. Choe, J., T. Schuster, and M. Grunstein. 1985. Organization, primary structure, and evolution of histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe. Mol. Cell. Biol. 5:3261-3269. 7. Ciechanover, A., D. Finley, and A. Varshavsky. 1985. Mammalian cell cycle mutant defective in intracellular protein degradation and ubiquitin-protein conjugation. Prog. Clin. Biol. Res. 180:17-31. 8. Ericsson, C., I. L. Goldknopf, M. Lezzi, and B. Daneholt. 1986. Low degree of ubiquitination of histone 2A in the dipteran Chironomus tentans. Cell Differ. 19:263-269. 9. Fangman, W. L., and V. A. Zakian. 1981. Genome structure and replication, p. 27-58. In J. N. Strathern, E. W. Jones, and J. R. Broach (ed.), The molecular biology of the yeast Saccharomyces, life cycle and inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 10. Finley, D., E. Ozkaynak, and A. Varshavsky. 1987. The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 48:1035-1046. 11. Goebl, M. G., J. Yochem, S. Jentsch, J. P. McGrath, A. Varshavsky, and B. Byers. 1988. The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme. Science 241: 1331-1335. 12. Hensold, J. O., P. S. Swerdlow, and D. E. Housman. 1988. A transient increase in histone H2A ubiquitination is coincident with the onset of erythroleukemic cell differentiation. Blood

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