From the Departments of Medicine,
§ Biochemistry, and ¶ Microbiology and Immunology,
Royal Victoria Hospital, McGill University, Montréal,
Québec H3A 1A1, Canada
Received for publication, December 12, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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Chromatin plays an important role in regulating
eukaryotic gene expression. Chromatin is composed of DNA wrapped around
a nucleosome core (consisting of two copies of the well conserved histones H2A, H2B, H3, and H4) and a more variable linker histone H1.
Various in vitro and in vivo studies have
implicated histone H1 as a repressor of gene expression or as an
activator, but its exact role is still unclear. Sequencing of the yeast
genome has led to the identification of a putative histone H1 gene.
Biochemical studies demonstrated that yeast does indeed possess a
bona fide histone H1. However, deletion of the unique yeast
H1 gene is not associated with any phenotypes, and it was questioned
whether it plays any role. To address this issue, we performed
whole-genome microarray analysis to identify genes that are affected by
H1 removal. Surprisingly, deletion of the gene encoding histone H1 does
not result in increased gene expression but rather in a modest reduction. Northern blot analysis of selected genes confirmed the
results obtained with the microarray analysis. A similar effect was
observed with an integrated lacZ reporter. Thus, our
data demonstrate that removal of yeast histone H1 only results in
decreased gene expression.
In recent years, numerous studies have shown a functional role of
chromatin in regulating eukaryotic gene expression. Nucleosomes are the
basic repeating unit of chromatin. The nucleosome core is composed of
DNA wrapped around an octamer composed of two copies of each of the
core histones H2A, H2B, H3, and H4 (1, 2). In addition, a linker
histone H1 is thought to interact with the DNA located between
nucleosome cores (3). Core histones are highly conserved between
species ranging from yeast to human. Histones have been shown to
repress transcription by preventing access of transcription factors to
target DNA sequences. Core histones are subject to various
modifications including acetylation. A number of complexes with histone
acetyltransferase activity have been characterized (reviewed in Refs.
4-6). How histone acetylation results in increased transcription is
not clear. One model states that acetylation of core histone tails
loosens the interaction with DNA, resulting in easier access of the
transcriptional machinery and leading to increased gene
transcription (5). However, there is also growing evidence that histone
acetylation (and other modifications) may serve as signals for
interaction with other proteins (reviewed in Ref. 7). Conversely, other complexes have been shown to possess histone deacetylase activity and
are involved in repression of gene expression (6). Thus, a relatively
clear picture of the role of core histones along with complexes
involved in core histone modification has emerged in recent years.
Linker histones are less conserved throughout evolution when compared
with core histones. Moreover, the location of linker histone H1 and its
role in regulating gene expression are less clear (reviewed in Ref. 8).
The addition of H1 has been shown to inhibit transcription in
vitro (9), but no effect of H1 was observed in other studies (10,
11). The deletion of Tetrahymena H1 does not result in
global alteration of transcription but rather in selective decreased
and increased gene expression in vivo (12). Similarly,
deletion of the unique H1 gene in Aspergillus
nidulans does not result in any apparent phenotype (13). In
Xenopus oocytes, overexpression of H1 results in repression
of the oocyte 5 S rRNA gene but has no effect on somatic 5 S rRNA (14,
15). Mice lacking the testis-specific histone H1t do not show any
defect in spermatogenesis. H1t-deficient germ cells show a normal H1 to
nucleosome ratio, perhaps because of compensation by other H1 isoforms
(16), a fact that could account for the absence of a phenotype.
Deletion of the genes encoding other H1 isoforms does not lead to any
apparent phenotype in mice or chicken cells (17-19).
For many years, there has been no evidence for the existence of histone
H1 in yeast. This was in agreement with the unusually short nucleosomal
repeat length observed in this organism (20). However, sequencing of
the yeast genome revealed a unique open reading frame
(ORF)1 encoding a putative H1
gene (21, 22). Recombinant yeast H1 was shown to behave like H1 found
in higher eukaryotes because it forms a stable ternary complex with a
reconstituted core dinucleosome in vitro (23). Moreover, a
fusion of H1 and the green fluorescent protein is localized in the
nucleus (22). Deletion of the H1 gene is not lethal in yeast and does
not result in any apparent phenotype such as slower growth, alteration
in telomeric silencing, mating, sporulation, or induction of gene
expression (22-24). In addition, upon removal of histone H1, the
nucleosomal repeat length is unchanged in yeast and in A. nidulans (13, 23).
These results raise the question of whether histone H1 plays any role
in regulating gene expression in yeast. To address this issue, we
performed whole-genome microarray analysis of yeast cells carrying a
deletion of the H1 gene. Strikingly, our analysis did not provide any
evidence for increased expression of any gene because of the removal of
histone H1. Unexpectedly, we observed rather an overall (but modest)
decrease of gene expression with 27 genes affected by a factor of 2 or more.
Strains, Media, and Growth Conditions--
The
Saccharomyces cerevisiae strains used in this study are
derived from BY4741 or BY4742 (25) and were obtained from H. Bussey
(Saccharomyces deletion project, Research Genetics strains 2125 and 12125, hereafter referred to as Microarray Analysis--
Total RNA was isolated with the hot
phenol procedure (27). RNA was further purified with Qiagen columns
according to the manufacturer's protocol. cDNA labeling and
hybridization were performed exactly as described (28). Custom-made
yeast whole-genome microarrays (>6200 yeast ORFs) were obtained from
the Microarray Center at the Ontario Cancer Institute (Toronto,
Canada). Scanning and quantification were performed exactly as
described (29). Briefly, chip A was hybridized with a mixture of wild
type cDNA labeled with Cy3 (WT-Cy3) and Cy5 (WT-Cy5); chip B, a
mixture of WT-Cy3 and Southern and Northern Blot Analysis--
Genomic DNA was
isolated according to Ref. 30. Southern and Northern blot analysis were
done according to standard procedures (31). Hybridizations were
performed at 42 °C in 50% formamide, 1 M NaCl, 2.8×
Denhardt's solution, 0.5% SDS, and 10% dextran sulfate. The probe
for Southern blot analysis of the deletion of the HHO1 gene
was obtained by PCR amplification of the promoter region of the
HHO1 gene with genomic DNA and the primers
CGGGATCCTTATTGCCGGTTACTGAACT and GGAATTCATCAGGTGCCCATAAATAAC. The PCR
product was cut with EcoRI and BamHI and
subcloned into Bluescript KS+ (Stratagene) cut with the same enzymes. A
HpaI-HpaI fragment of the lacZ gene was used as a probe to verify proper integration of the EFT1
reporter (see below). Probes for Northern blot analysis were obtained
by PCR amplification of specific open reading frames using genomic DNA
and the following oligos: SSE1 gene,
CGGGATCCATGAGTACTCCATTTGGTTTAG and GGAATTCCAGCAGCAGTTAGAATTCTGT;
PMA1 gene, CGGGATCCCCAAGCTGGTTCTATTGTCG and
AGACAGGGTCAAATGGATGGAATT; EFT1 gene,
CGGGATCCATGGTTGCTTTCACTGTTGA and GGAATTCTCTTAGCATATCTGGTGGCG.
The PCR products were cut with EcoRI and
BamHI and subcloned into Bluescript KS+ (Stratagene) cut
with the same enzymes. Fragments were gel-purified and randomly labeled
(31) for Northern blot analysis.
The PCR product was cut with XhoI and BamHI
and subcloned into plasmid p178MB (a high copy plasmid with a URA3
selection marker (33) cut with BamHI and XhoI to
give EFT1-lacZ-2 µm. The reporter contains about 700 base pairs of promoter sequences relative to the ATG. The ATG of the promoter is used to initiate translation of the
lacZ gene. A reporter for chromosomal integration was
obtained by deleting 2-µm sequences by cutting with
HindIII and religating the backbone to give
EFT1-lacZ-IP. The resulting plasmid was linearized by
cutting at the unique ApaI site located in the
URA3 marker. DNA was transformed into BY4741 or
Strains BY4741 and In our study, we used a wild type yeast haploid strain and strains
(either MATa or MAT
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
hho1a and
hho1
). Yeast cells were grown in rich medium (YPD
(26)) to an A600 of 0.8-1.0. for RNA isolation.
hho1-Cy5; chip C, a mixture of
hho1-Cy3 and WT-Cy5; chip D, a mixture of
hho1-Cy3 and
hho1-Cy5. The ratio of
hho1/WT for each ORF obtained from chips B and C was normalized with the corresponding ratio of the same ORF from chips A and D. Because each
ORF is duplicated on the same chip, four ratios obtained from chips B
and C were normalized individually with the four ratios obtained from
chips A and D; i.e. 16 values were obtained for each ORF.
Moreover, the results presented in Table I are an average of two
independent experiments performed with independent RNA preparations.
-Galactosidase Assays--
An EFT1 reporter
was constructed by amplifying its promoter using genomic DNA isolated
from strain YPH499 (32) and the following oligos:
ATCGACTCGAGTCACGTCCAACATGATTATC and CGGGATCCCATTTTTATCTGTTATTAAAAATTCTTGGG.
hho1a and colonies selected on plates lacking uracil. Proper
integration of the reporter was verified by Southern blot analysis.
hho1a were also transformed with
EFT1-lacZ-2 µm. Colonies were grown overnight in YPD and
diluted 2000-fold in minimal medium (SD, Ref. 26) supplemented with the
appropriate amino acids and adenine.
-Galactosidase assays were
performed with permeabilized cells (34). Values are the average of at least three independent experiments performed at least in duplicate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) carrying a
deletion of the entire open reading frame of the HHO1 gene
encoding histone H1. The deletions were confirmed by Southern
blot analysis (Fig. 1). Wild
type and
hho1 strains were grown in rich medium, and RNA
was isolated for microarray analysis. An analysis was done on 6216 genes and is an average of two independent experiments performed with
duplicate genomes. Although H1 is thought to be a repressor of
transcription, no significant increase (more than 50%) in gene
expression was detected in cells lacking H1 (Fig.
2). Similar results were obtained with a
strain of the opposite mating type (
hho1
,
data not shown). These results are in agreement with the fact that no
changes in telomeric silencing were observed in a
hho1
strain (23). Thus, our whole-genome analysis does not provide any
evidence for the involvement of yeast H1 in repressing RNA polymerase
II transcription. Our data point rather to a slight and general
decrease of transcription in cells lacking histone H1 (Fig. 2). This
effect is modest because most genes show a less than 2-fold reduction
in mRNA levels. Only about 27 genes showed reduced mRNA
levels of more than 2-fold (Table I). The
modest effect of H1 removal on mRNA levels may explain the absence of
any growth defect in cells lacking H1.
View larger version (19K):
[in a new window]
Fig. 1.
Southern blot analysis of the HHO1
gene deletions. Top, schematic map of the region
encompassing the HHO1 gene. The numbering in base
pairs (bp) is relative to the initiator codon. The
black rectangle corresponds to the ORF of the
HHO1 gene; striped rectangle, probe used for
Southern blot analysis; open rectangle, G418 resistance
gene, which was used for selection of the deletion mutants (see
"Material and Methods"). Bottom, Southern blot of the WT
and the deletion strains hho1a and
hho1
,
which are isogenic to strains BY4741 and BY4742 (25), respectively. The
relevant sizes of DNA fragments are shown in kilobases (kb)
on the left.
View larger version (18K):
[in a new window]
Fig. 2.
Effect of histone H1 removal on overall
mRNA levels. A summary of the microarray analysis performed
with strains BY4741 and hho1a (see "Material and
Methods"). The number of genes is given on the left
relative to their expression in the
hho1a strain as
compared with the wild type strain (mRNA levels
hho1a/WT strain)
List of the genes affected by removal of histone H1
hho1 strain as compared to the wild type strain
BY4741.
Table I provides a list of the genes that were most sensitive to H1
removal. We could not assign these genes to a common functional class.
For example, genes with decreased mRNA levels in the
hho1 strain include EFT1, an elongation
factor; PFK2, a phosphofructokinase; APE3, a
vacuolar aminopeptidase; YLR232W, a gene of unknown
function. In addition, there was no correlation between the level of
expression and the genes affected by the HHO1 knockout. For
example, PMA1 has 43 mRNA molecules/cell;
PFK2, 6.8 molecules/cell; and PGM2, 0.3 molecules/cell (35). Moreover, there was no apparent clustering of the
genes with reduced mRNA levels in the
hho1 strain.
For example, there was no clustering for these genes near the telomeres
or the centromeres of the chromosomes (data not shown). In summary, our
whole-genome microarray analysis showed that removal of histone H1 has
modest effects on steady-state mRNA levels with about 27 genes
being affected by a factor of 2 or more.
To confirm these results, we performed Northern blot analysis on a few
selected genes. RNA was isolated from a wild type strain and a strain
carrying a deletion of the HHO1 gene. As expected from the
microarray analysis, decreased mRNA levels for the genes EFT1, SSE1, and PMA1 were observed in
the hho1 strain (Fig. 3). The EFT1 and the SSE1 probes cross-hybridized
with ribosomal RNA, thus providing an internal control for equal
loading and transfer of RNA isolated for the wild type and knockout
strains (Fig. 3). PhosphorImager analysis showed that mRNA
levels are reduced about 2-fold for SSE1, EFT1,
and PMA1 in cells lacking H1. These values are in good
agreement with those obtained by microarray analysis (Table I).
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The reduced mRNA levels of these specific genes could be because of
mRNA destabilization or decreased transcription. To test whether
the effect is at the transcriptional level, we constructed a reporter
plasmid containing about 700 base pairs of promoter sequences of the
EFT1 gene (including the natural ATG) fused to the
lacZ gene. The activity of the integrated EFT1
reporter was reduced about 3-fold in the hho1 strain
(Table II), in agreement with the
microarray analysis (Table I). Even though the lacZ fusion
contains the 5'-untranslated region of the EFT1 mRNA, it is very likely that the reduced activity is because of decreased transcription and not mRNA destabilization. Moreover, the data show
that the effect of histone H1 on activity of the EFT1
promoter does not depend on a specific chromosomal location.
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Interestingly, no significant difference of the EFT1
promoter activity could be detected between the wild type and
hho1 strains when assayed with a high copy episomal
reporter (Table II). This difference may be due to a variation in the
copy number of the 2-µm plasmid between wild type and
hho1 strains or a difference in the chromatin structure
of the integrated and episomal EFT1 reporters.
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DISCUSSION |
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In this study, we have performed whole-genome analysis of yeast cells carrying a deletion of the HHO1 gene that encodes histone H1. Results show a slight decrease of expression for the majority of the yeast genes. However, only a small fraction of the genes (27 of 6216) had mRNA levels reduced by 2-fold or more. This subtle (but significant) effect of histone H1 likely explains why previous analyses have failed to detect any phenotype (22-24). Similarly, no variation in the expression of a limited number of tested genes was observed with the exception of CYC1 (see below) (22-24). Importantly, we observe only a slight increase (less than 50%) in expression for a small number of genes. Thus, in yeast, H1 is not a general repressor of gene expression.
Studies performed in Tetrahymena thermophila have shown previously that deletion of the gene encoding histone H1 is not lethal and does result in global changes of transcription as assayed by measuring the overall RNA content. However, both increases in basal expression (but not activated transcription) and decreases in gene expression were observed in cells lacking H1 (12). Similarly, no phenotype is observed in A. nidulans lacking H1 (13). In contrast, H1 in Ascobulus immersus is essential for long life span but not early vegetative growth (36).
Finding a role for H1 in higher eukaryotes may be complicated by the presence of many isoforms. For example, knockout of the testis-specific H1t does not result in an H1 imbalance, because of the presence of other H1 subtypes, and does not result in any obvious phenotype (16). In contrast, the nonessential histones H1 in yeast and Tetrahymena are more divergent than their mammalian counterparts. For example, Tetrahymena has an unusually small H1 without a globular domain. In contrast, yeast H1 has two putative globular domains unlike mammalian H1 (21). Nonetheless, although A. nidulans has a typical H1, its absence does not result in any apparent phenotype (13). Thus, H1 may perform more specialized functions in these organisms. For example, a majority of yeast genes are expressed under normal growth conditions. This may explain the differences in the role and the primary structure of H1 in yeast as compared with higher eukaryotes.
Our studies point to an effect of yeast H1 on a rather limited number
of genes. We do not observe any obvious common feature for these genes.
For example, they perform different functions such as translation
(e.g. EFT1, EFT2, SUI3,
YEF3), metabolism (PFK1, PFK2,
GPH1, GAD1), and heat shock response
(HSP82, SSE1). Reduced mRNA levels are
observed for the gene products of the secretory pathway like
SEC26, HAC1 (a transcription factor required for
the unfolded protein response pathway), SEC23,
SEC27, and SEC61. However, we did not observe
increased sensitivity of a hho1 strain to stress induced
by the addition of dithiothreitol to plates or to the glycosylation
inhibitor tunicamycin.2
Similarly, reduced mRNA levels of heat shock genes HSP82
and SSE1 at normal growth temperature (30 °C) does not
result in sensitivity of the
hho1 strain to high
temperatures as assayed at 37 °C and 45 °C.2
Moreover, the expression levels of genes affected by H1 vary over a
wide range (0.3-43 molecules/cell) (35). Thus, H1 is not specifically
implicated in the activation of genes with, for example, a low
transcription rate. In addition, there is no correlation between
dependence on the chromatin remodeling complexes SAGA (Spt-Ada-Gnc5-acetyltransferase) or SWI/SNF (35) and H1. Finally, there
is no specific location of these genes near the telomeres or the
centromeres, unlike what is observed for H4 (37). This finding
is in agreement with our observation that a lacZ reporter driven by the EFT1 promoter shows decreased
-galactosidase activity in
hho1 cells when integrated
at the URA3 locus.
What is the mechanism of action of histone H1? Our study did not
include genes transcribed by RNA polymerases I and III. However, given
the absence of growth differences in minimal media (24), it is unlikely
that altered transcription by RNA polymerase I or III could account for
the decreased expression of the genes observed in a HHO1
knockout strain. Yeast studies have shown that H1 has no effect on
nucleosome positioning on the POT1 and the YIL161W genes (38), which are not affected by H1 removal
according to our microarray analysis. Similarly, nucleosome positioning is independent of histone H1 in Tetrahymena (39). We
observed decreased expression of EFT1 irrespective of its
chromosomal location (Table II). However, the effect of H1 deletion was
not observed with an EFT1-lacZ reporter present
on a 2-µm plasmid (Table II). This finding is somewhat surprising
because high copy reporters generally behave like endogenous genes (2).
We do not know the basis for the difference between the results
obtained with integrated and episomal reporters. No difference in
expression of the CYC1 gene was observed with our microarray
analysis. However, using a low copy episomal
CYC1-lacZ reporter, increased -galactosidase activity (2.5-fold) was observed in a
hho1 strain (22).
Thus, the CYC1 promoter present on an episomal plasmid may
not behave like the endogenous gene, as observed for EFT1.
Decreased activity with the EFT1 gene is not due to ORF
sequences because the EFT1 promoter linked to the
lacZ gene and integrated at the URA3 locus (or
episomal reporters) behaved like the natural gene.
It is possible that the affected genes have an unusual
nucleosomal ladder in their promoter regions making them sensitive to
H1 removal. Deletion of the H1 gene in Tetrahymena results in selectively decreased and increased gene expression (12). It was
proposed that the removal of H1 increases the accessibility of
trans-acting protein complexes that act as activators or repressors of
transcription (40). In yeast, a high level of acetylation of core
histones is observed (41). Thus, the removal of histone H1 may affect
the acetylation state of core histones of some genes. For instance,
yeast genes affected by the removal of histone H1 may be more
accessible to histone deacetylases resulting in decreased gene
expression. This would explain the overall decrease of gene expression
observed in yeast cells lacking histone H1 (Fig. 2). In conclusion, our
studies provide additional evidence that histone H1 has a more
specialized function than the core histones. Importantly, yeast H1 is
involved in the general activation of genes, with a limited number of
genes being more affected by its removal. Thus, in yeast, H1 is not
involved in the repression of gene expression. Our studies were
performed with cells grown under rich medium. Testing the effect of H1
removal under more stringent conditions may identify a larger number of
genes that are affected by histone H1.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Deming Xu and Al Edwards (Best Microarray Center, University of Toronto) for microarray analysis. We also thank Dr. Howard Bussey and Nicholas Pagé for providing yeast strains. We thank Dr. M. Featherstone for critical review of the manuscript. We also thank Drs. J. J. Lebrun and H. Zingg for advice.
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FOOTNOTES |
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* This work was supported by grants from the Medical Research Council of Canada (Genomics) and the National Sciences and Engineering Research Council of Canada.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.
A scholar from the Fonds de la recherche en santé du
Québec. To whom correspondence should be addressed. Tel.:
514-842-1231, ext. 5046; Fax: 514-982-0893; E-mail:
turcotte@lan1.molonc.mcgill.ca.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011196200
2 K. Hellauer, E. Sirard, and B. Turcotte, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ORF, open reading frame; WT, wild type; PCR, polymerase chain reaction.
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