From the Department of Biochemistry and Molecular
Biology, Pennsylvania State University,
University Park, Pennsylvania 16802, ¶ University of
Massachusetts Medical Center, Program in Molecular Medicine and
Department of Biochemistry and Molecular Biology,
Worcester, Massachusetts 01605, and ** National Center for
Biotechnology Information, National Library of Medicine, National
Institutes of Health, Bethesda, Maryland 20894
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ABSTRACT |
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There is currently no published report on the
isolation and definitive identification of histone H1 in
Saccharomyces cerevisiae. It was, however, recently shown
that the yeast HHO1 gene codes for a predicted protein
homologous to H1 of higher eukaryotes (Landsman, D. (1996) Trends
Biochem. Sci. 21, 287-288; Ushinsky, S. C., Bussey, H.,
Ahmed, A. A., Wang, Y., Friesen, J., Williams, B. A., and
Storms, R. K. (1997) Yeast 13, 151-161), although
there is no biochemical evidence that shows that Hho1p is, indeed,
yeast histone H1. We showed that purified recombinant Hho1p (rHho1p) has electrophoretic and chromatographic properties similar to linker
histones. The protein forms a stable ternary complex with a
reconstituted core di-nucleosome in vitro at molar
rHho1p:core ratios up to 1. Reconstitution of rHho1p with H1-stripped
chromatin confers a kinetic pause at ~168 base pairs in the
micrococcal nuclease digestion pattern of the chromatin. These results
strongly suggest that Hho1p is a bona fide linker histone. We deleted
the HHO1 gene and showed that the strain is viable and has
no growth or mating defects. Hho1p is not required for telomeric
silencing, basal transcriptional repression, or efficient sporulation.
Unlike core histone mutations, a hho1 strain does not
exhibit a Sin or Spt phenotype. The absence of Hho1p does not lead to a
change in the nucleosome repeat length of bulk chromatin nor to
differences in the in vivo micrococcal nuclease cleavage
sites in individual genes as detected by primer extension mapping.
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INTRODUCTION |
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The basic structural unit of eukaryotic chromatin is the nucleosome core, composed of an octameric complex of two copies of each of the core histones H2A, H2B, H3, and H4 onto which 146 bp1 of DNA is spooled as 1.75 turns of a left-handed superhelix (1-4), and is continued to adjacent nucleosome cores through a variable length of linker DNA. In a nucleosome the histone H1 is associated with the outside of the core and protects 168 bp or two full superhelical turns of DNA from MNase cleavage (5, 6). Histone H1 was first identified in 1951 as a lysine rich "subsidiary" histone in calf thymus (7) and was subsequently shown to be an extended family of histone isotypes or variants present in a wide variety of eukaryotes (8). Although the structural role of the nucleosome core and the spatial placement of the core histones within the octamer are well established (1, 3, 4, 9), the function and the precise location of the linker histone has been more elusive (reviewed in Ref. 10).
Electron microscopic and hydrodynamic studies have shown that histone H1 is required for the full salt-dependent condensation of chromatin in vitro (11-13). These observations have led to the proposal that the function of the linker histone is the partial neutralization of the negatively charged DNA backbone, allowing the close approach of the internucleosomal linker DNA that would normally coloumbically repel in the fully compacted 30 nm fiber (for a review, see Ref. 14 and references cited therein). This role of H1 is supported by the observed reduction in the size of mitotic chromatids and nuclear volume concurrent with the appearance of histone H1 during the mid-blastula transition of the developing Drosophila embryo (15). It is also consistent with the approximately 2-fold expansion of the Tetrahymena micro- or macronucleus in the absence of the four micronuclear-specific micLH polypeptides or macronuclear-specific H1 protein, respectively (16). This implied role of H1 in chromatin condensation is dynamic and is modulated by the cell cycle-dependent reversible phosphorylation of H1 by p34cdc2 (reviewed in Refs. 17 and 18). This modification presumably allows the interaction of additional factors such as the structural maintenance of chromosomes (SMC) class of proteins with chromatin, affecting full condensation into the mitotic chromosome (see Ref. 19 for a review).
There has been no definitive identification of a linker histone in Saccharomyces cerevisiae, although some studies have reported indirect evidence for the existence of an H1-like protein in yeast (20-23). However, the absence of a direct biochemical identification of the histone led some investigators to suggest that it is absent in yeast (16). This proposed lack is unusual, since eukaryotes that phylogenetically diverge both before (such as Tetrahymena) and after (such as Psammechinus) yeast have been shown to contain linker histones.
The sequencing of chromosome XVI in yeast (24) revealed the presence of a predicted open reading frame (YPL127C) with regions of significant sequence homology to the H1 histones of higher eukaryotes (25, 26). Theoretical model building2 suggested that these regions of homology may assume a structural conformation similar to that of the known single-winged helix structure of the chicken H5 (27) and H1.11L (28) globular domains. Ushinsky et al. (26) have shown that the predicted open reading frame is functional and that a fusion of the gene product with fluorescent green protein is located in the nucleus. These results contributed to the assignment of YPL127C as the HHO1 gene which was proposed to encode the yeast linker histone H1 (Hho1p) (25, 26). There are no other genes in the yeast genome that code for proteins with significant sequence similarities to H1 (25).
In this study, we biochemically address the assignment of Hho1p as the yeast H1 linker histone. In particular, we investigated the ability of this protein to stably associate with a reconstituted core di-nucleosome and to confer an MNase kinetic pause at approximately 168 bp in H1-stripped chromatin in vitro. The phenotype of a yeast strain in which the HHO1 gene has been deleted was also studied. We specifically investigated the effect of the absence of Hho1p on cell viability, growth rate, mating efficiency, basal transcription levels, telomeric repression, sporulation efficiency, chromatin structure, and Sin and Spt phenotypes.
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EXPERIMENTAL PROCEDURES |
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Construction of Plasmids and Yeast Strains-- Genomic DNA was isolated from yeast strain FY24 as described by Zhu et al. (29). A 1500-bp region between positions 308,523 and 310,024 on yeast chromosome XVI (24), incorporating the entire HHO1 open reading frame and 303 bp upstream and 420 bp downstream, was amplified with Taq DNA polymerase and template mismatched primers to introduce EcoRI and XbaI sites at the fragment ends, as described (30). The recovered fragment was digested with EcoRI and XbaI and ligated into the EcoRI and XbaI sites of the plasmid pRS413 (31) lacking the 2688-bp PmlI-EcoRV fragment. The resulting plasmid was denoted pHHO1, and the sequence of the insert confirmed by nucleotide sequencing. This plasmid was further modified by introducing a 1185-bp fragment containing the HIS3 gene into the BamHI and SphI sites of the HHO1 sequence, and the proper insertion of the HIS3 gene in the plasmid, denoted pHHO1-HIS3, was confirmed by restriction enzyme analysis. This manipulation removes the sequence coding for amino acids 63-187 of Hho1p, including the entire primary globular domain C-terminal to helix I, the lysine-rich interglobular region, and the assigned secondary globular domain N-terminal of helix II (25).
An Escherichia coli expression vector, pET20-HHO1, was constructed by ligating an NdeI/XhoI restricted 789-bp fragment containing the HHO1 coding sequence into the NdeI/XhoI sites of pET20b(+) (Novagen). This construct codes for the entire Hho1p protein and introduces six histidine residues at the Hho1p C terminus. The HHO1 deletion strain was constructed by electroporating strain YPH500
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Purification of Recombinant Hho1p--
A 1-liter culture of
E. coli strain BL21(DE3) containing pET20-HHO1 was induced
with 0.4 mM
isopropyl-1-thio--D-galactopyranoside for 2 h, and
the cells were recovered in 10 ml of 20 mM imidazole, 200 mM NaCl, and 20 mM Tris·Cl (pH 7.9) (binding
buffer). The cells were lysed by sonication (20% duty cycle at setting
6 on a Branson model 450 sonicator), and the cellular debris were
pelleted by centrifugation (18,000 rpm for 20 min at 4 °C in a
Sorval SS34 rotor). The recovered supernatant was loaded onto a
nickel-agarose column (1-ml bed volume), equilibrated in binding
buffer. The column was washed with 25 ml of binding buffer, followed by
15 ml of 40 mM imidazole, 200 mM NaCl, and 20 mM Tris·Cl (pH 7.9). The recombinant protein was eluted
from the column with 300 mM imidazole, 200 mM
NaCl, and 20 mM Tris·Cl (pH 7.9), and 500-µl fractions
were collected. Fractions enriched in recombinant Hho1p were identified
by SDS-PAGE electrophoresis, pooled, and loaded onto a CM-Sephadex
column (1-ml bed volume) equilibrated in phosphate buffer (10 mM sodium phosphate (pH 7.0), 0.25 mM EDTA, and
0.25 mM phenylmethylsulfonyl fluoride) containing 200 mM NaCl. The column was washed with 10 ml of the same
buffer and developed with 10 ml of a 200-1000 mM NaCl
linear gradient in phosphate buffer. Fractions (500 µl) containing
pure recombinant Hho1p were identified by SDS-PAGE, pooled, and
concentrated, and the buffer was replaced with 10 mM sodium
phosphate (pH 7.0), 0.25 mM EDTA, and 0.25 mM
phenylmethylsulfonyl fluoride with a P10 centricon device (Amicon). The
samples were stored frozen at
70 °C. The rHho1p concentration was
determined by the absorbance at 230 nm using an
E1% of 18.5 (34).
Phenotypic Characterizations-- The 5-fluoroorotic acid (5-FOA) sensitivity of a strain was determined by diluting 1 ml of an overnight culture to an A600 of 1.0, and 10 µl of this culture, serially diluted 10-fold, was applied to a complete synthetic medium (CSM, 6.7 g/liter yeast nitrogen base without amino acids, 20 g/liter glucose, 0.7 g/liter amino acid supplement (Bio 101), and 20 g/liter bacto agar) plate lacking the appropriate amino acid in the absence or presence of 0.1% (w/v) 5-fluoroorotic acid (Jersey Laboratories). The plates were incubated at 30 °C for 2-3 days.
The sporulation efficiency of a strain was determined by inoculating a single diploid colony into 10 ml of pre-sporulation medium (0.25% (w/v) glucose, 1% (w/v) potassium acetate, 0.6% (w/v) yeast nitrogen base without amino acids, 0.5% (w/v) yeast extract, and 0.5% (w/v) bacto peptone) and incubating the culture with vigorous shaking at 30 °C to an A600 of 0.5-0.6. The cells were pelleted, washed in sporulation medium (1% (w/v) potassium acetate, all auxotrophic supplements at 0.25 × shown in Ref. 33), resuspended in 5-ml volumes of sporulation medium to an A600 of 0.2, and incubated at 22 °C for 5 days. The sporulation efficiency of a culture was quantified by counting the number of tetrads relative to the total number of cells and tetrads in a hemocytometer. TheChromatin Reconstitution and Nuclease Digestions-- The 390-bp 32P-labeled fragment (10 ng) containing a tandem dimer of the sea urchin 5 S rRNA gene was incubated with 40 µg of H1-stripped HeLa chromatin (a gift from M. Vignali and T. Owen-Hughes) in a 100-µl volume of reconstitution buffer (10 mM Tris·Cl (pH 8.0), 0.25 mM EDTA and 0.25 mM phenylmethylsulfonyl fluoride) containing 1000 mM NaCl at 37 °C for 30 min. The ionic strength was reduced to 800 and 600 mM NaCl by the sequential addition of reconstitution buffer at 30-min intervals at 37 °C. Where rHho1p was added, the sample was split into aliquots following the dilution to 600 mM NaCl, and rHho1p was added in the amounts indicated in the text. The dilution of fractions in the presence or absence of rHho1p was continued to 400 mM NaCl after 30 min, followed by a stepwise 2-fold dilution with reconstitution buffer at 37 °C at 30-min intervals to a final concentration of 12.5 mM NaCl. Aliquots (20 µl) were run on a 0.7% (w/v) agarose gel in 0.5× TBE at 100 V, and the gel was dried and autoradiographed. The reconstitution of H1-stripped HeLa chromatin with Hho1p was performed exactly as described, except that the 5 S rRNA gene dimer fragment was omitted, and rHho1p was added at a molar ratio of 1:1 (Hho1p:core). This and a similarly treated sample lacking Hho1p were digested with concentrations of MNase indicated in the text. The digestion products were purified and electrophoresed on an 8% (w/v) polyacrylamide gel in 1× TBE at 150 V. The preparation of nuclei, digestion of chromatin and free DNA, and the primer extension of the recovered DNA template were performed as described previously (30).
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RESULTS |
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Is Hho1p the Yeast Linker Histone H1?-- We investigated the assigned identity of Hho1p as a linker histone in yeast by studying the properties of a recombinant Hho1p (rHho1p). Histidine-tagged rHho1p was overexpressed in E. coli cells and isolated from the sonicated cell lysate by passage over a nickel-agarose column (Fig. 1A). The identity of the band indicated as rHho1p (Fig. 1A) was confirmed by its absence in identically performed mock isolations from E. coli containing only the vector without the HHO1 coding sequence. Although the amino acid sequence of Hho1p predicts a molecular mass of approximately 28 kDa, the protein migrates at a size equivalent to approximately 33 kDa during SDS-PAGE (Fig. 1, A and B). This anomalously slow electrophoretic behavior closely resembles that of linker histones from other species (36) and is likely due to the basicity of the protein, which has a predicted isoelectric point of 10.2. The recovered rHho1p was next purified from contaminating nickel-agarose binding E. coli proteins by passage over a cation exchange resin, eluted by a linear salt gradient. Recombinant Hho1p eluted off the cation exchange column at approximately 700 mM NaCl (Fig. 1B). This chromatographic trait of rHho1p is also very similar to that of linker histones from higher eukaryotes (37). These properties, however, reflect only the amino acid composition of the protein and not its proposed function as a linker histone. We therefore tested the ability of the purified recombinant protein to conform to two common characteristics of a linker histone: (i) to form a stable ternary complex with a reconstituted nucleosome core in vitro, and (ii) to produce a kinetic pause at approximately 168 bp in the MNase digestion pattern of H1-stripped native chromatin.
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rHho1p Forms a Stable Ternary Complex with a Reconstituted Core Di-nucleosome in Vitro-- Several investigators have shown that both the full-length and globular domain of recombinant and native linker histones can be reconstituted with nucleosome cores in vitro (13, 38). Since the proposed role of histone H1 is the neutralization of the charge of the internucleosomal linker DNA, we chose to investigate the association of rHho1p with a reconstituted core di-nucleosome, which contains a short length of linker DNA and is expected to resemble a natural H1 substrate more closely. A 390-bp radiolabeled fragment containing a tandem repeat of sea urchin 5 S DNA (39) was reconstituted into a di-nucleosome in the presence of a range of rHho1p concentrations, and the reconstitution products were electrophoretically resolved on an agarose gel (Fig. 2). Addition of rHho1p to the core di-nucleosome resulted in the formation of two slower migrating species. The faster migrating of these two species (N1) appears at lower molar Hho1p input ratios and decreases toward higher input ratios, concurrent with an increase in the slower migrating complex (N2). At a molar input ratio of one molecule of rHho1p per core, essentially all of the core di-nucleosome exists as the N2 species. At higher molar input ratios or at ionic strengths in excess of approximately 100 mM NaCl, the reconstitute aggregates and does not enter the gel matrix. This result clearly demonstrates that rHho1p forms a stable ternary complex with a reconstituted core di-nucleosome. The apparent conversion of the N1 to the N2 species as a function of rHho1p input suggests that at lower rHho1p ratios a single molecule of rHho1p binds to the core di-nucleosome forming the N1 complex. At higher rHho1p:core ratios a second molecule of rHho1p binds to the N1 complex, resulting in the appearance of the N2 complex.
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MNase Digestion of H1-stripped Chromatin Reconstituted with rHho1p Shows a 168-bp Kinetic Pause-- The presence of histone H1 in chromatin protects an additional 10 bp of DNA on either side of the nucleosome core from exonucleolytic MNase digestion, resulting in the appearance of a digestion intermediate of approximately 168 bp prior to trimming to 146 bp and subsequent sub-nucleosomal length fragments (5, 6). The appearance of the kinetic pause at ~168 bp was shown to require the basic amino acid residues Lys-40 and Arg-42 between helix I and helix II and Lys-52 on helix II (40) in the proposed secondary DNA-binding site of the globular domain of H5 (27). The alignment of the proposed primary globular domain of Hho1p (25) with that of several H1 isotypes from different species (Fig. 3) shows that all three of the predicted DNA-binding residues in the secondary DNA-binding site (27) that are highly conserved among the different H1 proteins are also conserved in yeast Hho1p. Also, at least two of the three most conserved basic residues in the proposed primary DNA-binding site (27) are conserved in Hho1p, as is also the case for the pea H1.
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Yeast Lacking Hho1p Are Viable and Have Normal Growth and Mating
Properties--
We constructed a haploid yeast strain (YHGP101) in
which the single HHO1 coding sequence was partially replaced
with the HIS3 selectable marker. The absence of the
poly(A)+ HHO1 mRNA transcript, and therefore
the Hho1p protein, was confirmed in the YHGP101 strain by Northern
analysis (data not shown). There was no detectable difference in the
growth rate of the hho1 strain compared with the
WT strain. Similarly, yeast cells lacking Hho1p mated as
efficiently as WT cells, suggesting that both silent mating
type loci and the appropriate cell type-specific genes are properly
repressed in the absence of Hho1p.
Repression of Basal Transcription--
The repression of basal
transcription by nucleosomes is well established (reviewed in Ref. 43).
Several studies have also shown that the basal transcription of
chromatin templates in vitro is reduced in an
H1-dependent manner (44-46). We investigated the possible
involvement of Hho1p in basal transcriptional repression in
situ using a reporter gene that consists of a minimal
PHO5 promoter fused to the URA3 coding sequence
(47). Cells that harbor this plasmid do not express URA3 and
are resistant to the drug 5-FOA which kills cells that express the
URA3 gene product. If basal transcription is elevated,
however, the cells become sensitive to 5-FOA. The transcriptional
activity of this episomal reporter gene was tested in the WT
and hho1 strains by measuring the growth of transformants
in the presence of 5-FOA (Fig.
5A). At equivalent dilution of
cells, comparable numbers of WT and hho1
cells
survive in the presence of 5-FOA. This result demonstrates that in
contrast to the core histones (43), Hho1p does not appear to be
involved in the general repression of basal polymerase II transcription
in situ. This finding does not, however, exclude the
possible involvement of Hho1p in specialized regulatory mechanisms at
specific genes.
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Hho1p Is Not Required for Telomeric Repression--
Several
studies have demonstrated the transcriptionally repressive nature of
heterochromatin-like structures at yeast telomeres and at the silent
mating-type loci (Ref. 48 and references cited therein). The
involvement of chromatin in the establishment of these repressive
structures was clearly shown by the requirement for the N terminus of
histone H4 for full repression at both loci (49) and histone H3 for
repression at the telomeric ends and at a partially crippled silent
mating-type locus (50). Since Bedoyan et al. (51) have also
shown that telomeric chromatin fragments isolated from rat liver nuclei
contain H1, we investigated whether Hho1p was required for telomeric
silencing in yeast. We constructed an isogenic pair of WT
and hho1 strains that contained a URA3 gene
integrated at a telomeric locus. In these strains, telomeric repression
of the URA3 gene allows the cells to grow on 5-FOA medium,
whereas defective repression results in an increased sensitivity to
5-FOA. As shown in Fig. 5B, the URA3 gene was
strongly repressed in both the WT and hho1
cells. In contrast, a strain bearing a sir3 deletion,
previously implicated in telomeric silencing (49), was highly sensitive
to 5-FOA, indicating a loss of repression of the telomeric
URA3 gene. These results indicate that Hho1p does not play a
detectable role in telomeric silencing.
SIN Phenotype-- SWI/SNF, which is believed to function as a chromatin remodeling complex, is required for the activated transcription of a set of yeast genes (35). Previous studies have shown that the reduced transcription of the HO gene in swi/snf mutants is partially relieved by SIN mutations (52). This class of mutation appears to function at the level of chromatin, since the SIN2 gene was subsequently shown to be identical to HHT1, one of two copies of the gene coding for histone H3. Mutations in either of the two histone H3 genes or substitution of two highly conserved amino acid residues in the histone fold domain of histone H4 was shown to result in a Sin phenotype (53). We therefore asked whether the absence of Hho1p would similarly result in a Sin phenotype.
The SWI1-dependent activity of the HO promoter was investigated in strains containing an HO-lacZ gene fusion integrated at the ho locus (54). In the presence of theAnalysis of a hho1 sin1 Double Mutant--
The SIN1
gene was identified as a mutation that alleviated transcriptional
defects due to inactivation of the SWI·SNF complex. The predicted
Sin1p protein has an amino acid composition similar to the
chromatin-associated mammalian non-histone HMG1 protein, and it has
been suggested to be involved in the creation of a proper chromatin
context for transcription (55). Strains containing a deletion of the
SIN1 gene are viable and show no detectable growth or
transcriptional defects. We asked whether the absence of Hho1p causes a
synthetic phenotype in conjunction with a deletion of SIN1.
To this end, basal expression from an episomal CYC1-lacZ fusion gene that contains only a minimal promoter (56) was measured in
a WT, hho1
, sin1, and a
hho1
sin1 strain. The hho1
sin1 strain was viable and showed no detectable growth
defects (data not shown). Consistent with the basal transcription
results presented above, neither the hho1
,
sin1, nor the hho1
sin1 strain
exhibited any elevation in the basal transcription levels of the
reporter gene as assayed by
-galactosidase activity (Table
II). The repressed state of a
URA3 gene integrated at a telomeric locus was also unaffected in the hho1
sin1 strain (data not
shown).
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SPT Phenotype--
The SPT genes
(suppressor of Ty) were
identified by selection for extragenic suppressors of the
transcriptional defects caused by and Ty insertions in
the 5' regions of the HIS4 and LYS2 genes (57,
58). Distinct classes of SPT genes have been identified, one
of which includes SPT11/HTA1 and SPT12/HTB1
encoding histones H2A and H2B, respectively (59). To determine whether loss of Hho1p causes Spt phenotype-like mutations of these core histones, we investigated the effect of the hho1
mutation
on transcription of the his4-912
and
lys2-128
alleles. In an Spt+
his4-912
strain, the predominant HIS4 transcript is
initiated in the solo
insertion, producing an abnormally long
HIS4 transcript in which the normal HIS4
translation start is not used, resulting in a His
phenotype. In an Spt
his4-912
strain, a
wild-type HIS4 transcript is present in addition to the solo
-mediated transcript, resulting in histidine prototrophy (His+). Similarly, for Spt+
lys2-128
, LYS2 transcription initiates at the
sequence within the 5'-exon of the LYS2 coding region,
resulting in a nonproductive short transcript and lysine auxotrophy.
The presence of an spt mutation results in transcription
initiation at the wild-type start site, resulting in lysine
prototrophy. The ability of an SPT mutation to modulate the
transcriptional activation of the solo
insertion and adjacent gene
is not currently understood at a mechanistic level.
Hho1p Is Not Required for Efficient Sporulation--
Since we have
shown above that the rate of growth of a mitotically dividing yeast
cell is unaffected by the absence of Hho1p, we asked whether Hho1p may
be required for meiosis. This was investigated by determining the
sporulation efficiency of diploid strains. The sporulation
efficiencies, expressed as the percentage of tetrads relative to the
total number of cells, is shown in Table
III. Referring to Table III, it is seen
that approximately 83% of wild-type diploid cells sporulated. In the
case of the homozygous hho1/hho1
strain, approximately 60% of the diploids sporulated over the same period. To
investigate whether this decrease was due to the absence of Hho1p or
due to a genetic difference between the two congenic diploid strains, a
wild-type copy of the HHO1 gene was reintroduced at the
URA3 locus in the hho1
/hho1
diploid strain. This strain sporulated at approximately 72% efficiency
(see Table III). Although these results suggest a minor involvement of
Hho1p in sporulation, a WT/hho1
strain
sporulated at only approximately 37% efficiency. Thus, we cannot
exclude the contribution of genotypic differences between the compared
congenic strains. A similar result was obtained with a sin1/sin1
hho1
/hho1
double mutant strain (see Table III). In all cases examined, the spores germinated and grew normally (data
not shown). These data suggest that although Hho1p may make a minor
contribution to the efficiency of sporulation, it is not absolutely
required for meiosis or spore germination.
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There Is No Detectable Change in the Chromatin Structure of a
hho1 Strain--
The association of H1 with chromatin, in addition
to changing the degree of compaction, has also been shown to change the spacing between adjacent nucleosomes (60). We therefore compared the
nucleosome repeat length of bulk chromatin in the isogenic WT and hho1
strains. No differences were
detected (data not shown). To ensure that minor structural differences
are not overlooked, we investigated the chromatin structure of selected
regions at single nucleotide resolution. In Fig.
6 we show the primer extension mapping of
the micrococcal nuclease scissions in the STE6 gene and the
centromeric region of chromosome III in vivo. A comparison of the MNase cleavage sites in chromatin and free DNA at the
STE6 locus (Fig. 6A) slows a clear repetitive,
nucleosomal pattern in both the WT and the
hho1
strain. There is no readily detectable change in the
MNase accessibility at the pseudo-dyad axis or within the short
internucleosomal linker in the absence of Hho1p. Nor is there evidence
of a change in the extent of the nucleosome footprint or the nucleosome
repeat length throughout the STE6 gene. The primer extension
footprinting of the centromeric region of chromosome III also shows a
nucleosomal organization abutting the centromeric region which remains
relatively nuclease-resistant in the hho1
strain. To
address the possibility that Hho1p is degraded during nuclei isolation,
we repeated the mapping of these regions using the more rapid procedure
of preparing permeabilized spheroplasts (61). Identical results were
obtained (data not shown).
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DISCUSSION |
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Is Hho1p the Yeast Linker Histone H1?-- We have shown above that recombinant Hho1p has electrophoretic and chromatographic properties similar to the linker histones of higher eukaryotes. Although there are several other lysine-rich proteins in the yeast genome with expected biochemical properties similar to Hho1p, we have also shown that rHho1p forms a stable ternary complex with a core di-nucleosome in vitro. This association appears to be specific, since the titration of the core di-nucleosome with rHho1p leads to the stepwise appearance of two well defined supershifted complexes, most likely a core di-nucleosome containing one and two rHho1p molecules, respectively. These complexes are formed at rHho1p:core ratios similar to those found for the linker histones of higher eukaryotes (38). Although we have not shown direct chromatin association in vivo, Ushinsky et al. (26) have shown that a fusion of Hho1p with the fluorescent green protein is located in the nucleus, strongly suggesting that the nucleosome core binding Hho1p is chromatin-associated.
The predicted secondary structure of the assigned globular domain of Hho1p suggests a single-winged helix protein fold similar to that of the chicken H5 (27) and H1.11L (28) globular domains. Several other DNA-binding proteins such as HNF-3What Is the Role of Histone H1?--
A substantial body of
evidence exists that implicates histone H1 in chromatin condensation
(reviewed in Ref. 14). We have systematically investigated an extensive
list of possible phenotypes that may reflect the aberrant organization
or improper condensation of chromatin in the hho1 strain
and have shown that a yeast cell lacking Hho1p appears to function as
efficiently as a WT cell. This result is not unexpected,
since mice, homozygous for an H10 gene disruption, were
found to grow and reproduce normally with no anatomical or histological
abnormality, although other H1 variants may have compensated for the
absence of H10 (64). Similarly, Gorovsky and colleagues
(16, 65) have shown in Tetrahymena that vegetative growth,
general polymerase I, II, and III transcription, protein synthesis, and
general nucleosome repeat length are all unaffected by the absence of
the four micronucleus-specific micLH peptides or macronucleus-specific
H1. Linker histone-dependent changes were, however,
observed in the nuclear volume, the transcriptional regulation of
specific genes, and the efficiency of meiotic division (65). Although
we did observe a difference in the sporulation efficiency of a WT
versus a hho1
yeast strain, we could not exclude the possible
contribution of minor genetic background differences to this
observation. Thus, although Hho1p may have an effect, it is not
required for sporulation and spore formation.
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ACKNOWLEDGEMENTS |
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We thank Marissa Vignali and Tom Owen-Hughes for a generous supply of H1-stripped HeLa chromatin; Michael Grunstein for the PHO5-URA3 plasmid; Virginia Zakian for the URA3::ADH4 and sir3URA3::ADH4 strains; Fred Winston for the SPT tester strains and the optimized sporulation method; Jonathan McLeod for technical assistance; and colleagues for helpful discussions and suggestions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants 1 RO1 GM54096 (to C. L. P.) and 1 RO1 GM52399 (to R. T. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 308 Althouse Laboratories, Pennsylvania State University, University Park, PA 16802. Tel.: 814-863-0332; Fax: 814-863-0099; E-mail: hgp1{at}psu.edu.
Supported by a postdoctoral fellowship from the American
Cancer Society. Current address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Mailstop A1-162, Seattle, WA
98109-19204.
Leukemia Society of America Scholar.
1 The abbreviations used are: bp, base pair(s); MNase, micrococcal nuclease; PAGE, polyacrylamide gel electrophoresis; 5-FOA, 5-fluoroorotic acid; CSM, complete synthetic medium.
2 A. D. Baxevanis and D. Landsman, unpublished data.
3 H. G. Patterton, C. C. Landel, D. Landsman, C. L. Peterson, and R. T. Simpson, unpublished data.
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