The N-terminal tails of all four core histones are subject to
several post-translational modifications including the reversible
acetylation-deacetylation of
-amino groups of specific lysine
residues. Three distinct roles have been proposed for histone
acetylation. One is in gene activation and the regulation of
transcription(1, 2) . A clear correlation exists
between a high degree of acetylation and a high level of transcription (3) but whether the acetylation is a cause or an effect of gene
activation is unclear. Another role proposed for histone acetylation is
during histone synthesis and deposition. It is thought that a specific
transient acetylation of histones may be necessary for their deposition
during DNA replication(4, 5) . Finally, histone
acetylation appears to play a role in histone replacement during germ
cell maturation(6) .
Histone acetyltransferases have been
purified and characterized from a number of mammalian sources (7, 8) as well as yeast(9, 10) .
Fractionation studies suggest that there are at least two such enzymes,
one cytoplasmic that acetylates free histones, and one nuclear that
perhaps acetylates histones in chromatin(11) . It is still not
clear, however, whether different enzymes exist for each histone or
even for specific lysine residues on a given histone. To date, no
histone acetyltransferase genes or mutants have been reported. In order
to learn more about the in vivo roles of histone acetylation
and about the specificities of the enzymes, we looked for a yeast
mutant defective in this process. A collection of yeast
temperature-sensitive mutants was screened by an in vitro enzymatic assay that measured acetylation of an H4 peptide
corresponding to amino acids 1-28. In this report, we describe
the identification of a mutant specifically defective in the
acetylation of lysine 12 in this peptide. The cloning of the wild type
gene is also described.
MATERIALS AND METHODS
Yeast Strains, Media, and Strain
Manipulations
The following yeast strains were used: W303-1a: MATaade2 ura3 leu2 trp1 his3 can1;
W303-1b: MAT
, otherwise isogenic to W303-1a; W303:
diploid from W303-1a and W303-1b; SK56: isogenic to W303-1a plus hat1-2::TRP1; SK57: isogenic to W303-1b plus hat1-2::TRP1; A364a: MATaade1
ade2 ura1 tyr1 lys2 his7 gal1. (This is the parent strain for the
collection of temperature-sensitive mutants.) The four centromere
mapping strains CSH87L, CSH89L, X3144-11A, and X3382-3A have been
described(12) . Yeast cells were grown at 30 °C in YPD or
SC medium (13) plus appropriate supplements unless otherwise
stated. Strain constructions, genomic DNA isolation, sporulation,
tetrad dissection, and analysis were performed as
described(13) . The hat1-1 mutation was followed
through genetic crosses by the HAT (
)enzyme assay. Yeast
transformations were carried out by the lithium acetate
method(14) .
Preparation of Yeast Cell Extracts
Yeast cells
were grown at 23 °C in 20-40 ml of liquid medium to a cell
density of 2-8
10
cells/ml, shifted to 37
°C for 1 h, spun down, washed with 5 ml of H
O, and
resuspended in 200 µl of 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 500 mM KCl, 10% glycerol, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride.
Cell suspensions were transferred to microcentrifuge tubes containing
100 µl glass beads (300-µm diameter), chilled, and sonicated
for 12 s three times using a Heat Systems Ultrasonics cell disrupter
with a microtip at 2.0 output. After centrifugation for 15 min at
30,000
g, the supernatants were dialyzed into buffer A
(20 mM Tris-HCl, pH 8.3, 100 mM NaCl, 10% glycerol, 1
mM phenylmethylsulfonyl fluoride, 5 mM 2-mercaptoethanol) using a microdialysis chamber (Bethesda
Research Laboratories).
Fractionation of Extracts
The dialyzed extracts
were fractionated in small batches in microcentrifuge tubes by loading
175 µl of extract on 200 µl of Q-Sepharose FF resin
(Pharmacia), equilibrated with buffer A, and mixed gently for 15 min at
4 °C. After the resin had settled, the supernatant was discarded,
washed with 400 µl of buffer A, and eluted with 200 µl of
buffer B (same as buffer A but 625 mM NaCl). The supernatant,
referred to as the Q380 fraction, was used in the histone
acetyltransferase (HAT) assays. Protein concentrations were determined
by a microassay (Bio-Rad) using bovine serum albumin as a standard;
5 µg of protein were used per assay.
Histone Acetyltransferase Assay
The assays were
carried out in 50 µl of 75 mM Tris-HCl, pH 8.7, 0.1 mM EDTA, pH 8.0, and a final concentration of 150 mM NaCl.
The peptide concentration in the assay was 50 µM, and
[
H]acetyl coenzyme A was added to a final
concentration of 3.0 µM at 2.3 Ci/mmol. The reaction
mixture was preincubated for 30 s at 37 °C, the Q380 fraction was
added, and the incubation continued at 37 °C for 15 min. The
reaction was stopped by the addition of 1.5 ml of cold 20%
trichloroacetic acid. Precipitated peptide and proteins were collected
on glass fiber filters (Schleicher & Schuell), washed twice with 5%
trichloroacetic acid, once with methanol, dried, and counted in a
scintillation counter.
Amino Acid Sequence Analysis
In order to obtain
enough peptide for sequence analysis, several HAT assays of an extract
were pooled. Reactions were terminated by incubating them at 65 °C
for 5 min instead of by trichloroacetic acid precipitation. The samples
were bound to Immobilon PVDF membranes (Millipore) and sequenced on an
automatic protein Sequencer. The eluates from each cycle were
collected, dried, and counted for 10 min in a scintillation counter.
Cloning, Sequencing, and Null Mutation
The
preparation of
-DNA and cloning of the genomic inserts from the
-DNA into the shuttle vector pBM2384 were done as
described(15) . Subclones from the initial plasmid, p5538, were
cloned into YEp352 or M13mp18 using standard protocols(16) .
Nucleotide sequence was determined from M13 single-stranded DNA by the
dideoxy method. The hat1-2::TRP1 null mutant plasmid was
generated by cloning the EcoRI-BglII fragment from
the upstream region of the HAT1 gene into the EcoRI
and BamHI of the disruption vector pRS304(17) , and
the StuI-NruI fragment from the 3` end of the gene
into a blunt-ended XhoI site of the same vector. The resulting
plasmid, pSK2, was linearized with SalI, transformed into a
wild type diploid (W303), which after sporulation and dissection gave
rise to strains SK56 and SK57. The same plasmid was also used to
transform strain MX1-4c, which resulted in strain SK55. The correct
gene replacements were verified by Southern blot. The hat1-2::TRP1 null mutation deletes 50 bp upstream of the HAT1 open
reading frame (ORF) as well as amino acids 1-266.
HAT1 Expression in Escherichia coli
The HAT1 gene was cloned into the T7 expression vector pET3b (18) to yield pSTT21. This plasmid encoded a fusion protein
consisting of the first 11 amino acids of T7 gene 10 protein, 4 linker
amino acids, and the entire 374 amino acids of HAT1, all downstream of
a T7 RNA polymerase promoter. E. coli strain BL21-DE3
containing pSTT21 was grown in LB plus ampicillin medium, induced with
isopropyl-1-thio-
-D-galactopyranoside for 2 h, and
harvested. Cells were resuspended in 200 µl of cold buffer B,
sonicated, and centrifuged for 1 min in a desk top centrifuge. The
supernatant was used directly for HAT assays.
RESULTS AND DISCUSSION
Identification of the hat1-1 Mutant
The hat1-1 mutant was found by screening a collection of
temperature-sensitive (ts) yeast mutants (19, 20) for
histone H4 acetyltransferase activity in vitro. Previous
studies in our laboratory (21) as well as published partial
purification protocols for acetyltransferases from yeast suggested the
presence of more than one such activity in yeast (10) . Since
multiple activities contributing to an in vitro assay might
obscure the identification of a mutation in only one activity, we
attempted to gain specificity by making two improvements: first,
instead of detecting the acetyltransferase activity in crude extracts,
we fractionated the extracts over a Q-Sepharose anion exchange resin
and assayed a high salt eluate (this fraction is referred to as Q380);
second, we used as a specific substrate a peptide corresponding to
amino acids 1-28 of yeast histone H4 (a gift from B. Alberts,
University of California, San Francisco). This peptide was incubated in
the presence of [
H]acetyl coenzyme A with the
Q380 fractions made from the temperature-sensitive strains, and
incorporation of
H into the peptide was measured. In order
to check that the assay was not measuring N-terminal acetylation, known
yeast N-terminal acetyltransferase mutants, including nat1, ard1, nat2, and mak3 strains (21, 22, 23) were assayed and found to have
the same level of activity as a wild type strain. Additional evidence
that the assay is measuring acetylation of
-amino groups of lysine
residues on the peptide is described below.After screening about
250 mutants, a strain was found which reproducibly showed a 40%
reduction in histone acetyltransferase (HAT) activity (see Table 1for typical results). When this mutant was outcrossed to a
wild type strain, the resulting tetrads segregated
2
:2
for the decreased HAT activity,
indicating that the decrease was caused by a single nuclear mutation,
which we called hat1-1. We also noticed that the decrease did
not cosegregate with the ts phenotype of the original strain. Strains
carrying the hat1-1 mutation alone are not ts and show no
obvious phenotypes.
A wild type strain and the identified hat1-1 mutant were compared using an N-terminally blocked peptide as the
substrate. This peptide corresponds to amino acids 1-20 of
histone H4 and contains a tertiary butoxycarbonyl group at its N
terminus (H4 aa
1-20 in Table 1). Both strains
exhibit a slight reduction in activity compared to the longer,
unblocked H4 aa 1-28 peptide, but the hat1-1 mutant
still exhibits a 40% reduction in activity compared to wild type with
this substrate. We attribute the overall reduction in HAT activity in
both strains with the blocked peptide substrate to a lower affinity of
the HAT enzyme(s) for this substrate. As another test for the
specificity of the assay, we used as a substrate a 21-amino acid
peptide, corresponding to the N terminus of histone H3 (kindly provided
by D. Allis, Syracuse University). The wild type and hat1-1 strains showed the same amount of activity with this substrate (Table 1), but this amount corresponds to only 15% of the
activity found with the 28-amino acid H4 substrate. Apparently, the
Q380 fraction of the yeast extract contains only a minor activity that
recognizes the H3 substrate, but this activity is not affected by the hat1-1 mutation. A different fraction of the yeast extract,
containing much more acetylation activity specific for H3, also is
unaffected by the hat1-1 mutation. (
)The Q380
subfraction used here seems enriched for an H4 specific activity.
HAT1 Is a Heat-sensitive Acetyltransferase
Activity
The hat1-1 mutant still showed 60% HAT
activity when compared to a wild type strain (Table 1). This
remaining activity could be attributed either to a leaky phenotype of
the hat1-1 allele or to a second activity that is not affected
by the mutation. To distinguish between these two possibilities, we
examined the heat inactivation profile of the HAT activity by
subjecting the Q380 fraction to 45 °C for various periods of time. Fig. 1shows that the wild type activity has a biphasic heat
inactivation profile, with about 25% of the activity being rapidly
inactivated and the remaining 75% being inactivated much more slowly.
On the other hand, all the activity remaining in the mutant (60% of the
initial wild type activity) is very heat-resistant. Reproducibly, the
activity remaining in the mutant after heating is always lower than
that remaining in the wild type fraction (Fig. 1). We interpret
these data to show that the Q380 fraction has at least two HAT
activities, one heat-sensitive and one or more heat-resistant. The hat1 mutant lacks the heat-sensitive activity and also appears
to have partially lost the heat-resistant activity. Perhaps the HAT1
protein is a component of two different HAT enzymes, one heat-sensitive
and one heat-resistant. The Q380 fraction clearly contains a
significant heat-resistant HAT activity unaffected by the hat1 mutation.
Figure 1:
HAT activity after heat inactivation
of Q380 fractions from wild type and a hat1 mutant. Q380
fractions prepared from a wild type strain (
-
)
and a hat1-1 mutant (
- - -
)
were incubated at 45 °C for the indicated times and then assayed
for HAT activity in the standard assay.
The HAT1 Activity Acetylates Lysine 12 of Histone
H4
In order to determine which lysine residues of the H4 peptide
were being acetylated, a standard assay with
[
H]acetyl coenzyme A was done using the Q380
fraction from both wild type and the hat1-1 mutant. The
peptide substrate was then subjected to amino acid sequence analysis,
and the amount of radioactivity at each position was determined. As is
shown in Fig. 2, the wild type fraction incorporates
H mainly into lysines at positions 5, 8, and 12 with a
small amount into lysine 16. The majority of acetylation by the Q380
fraction is clearly on lysine 12. The hat1 mutant-labeled
peptide exhibits a strikingly different pattern (Fig. 2). The
amount of
H incorporated into lysines 5, 8, and 16 is
approximately the same as seen for the wild type extract, but the
amount of label in lysine 12 is greatly reduced. Thus it appears that
the hat1 mutation affects a histone acetyltransferase activity
specific for lysine 12 of H4.
Figure 2:
Distribution of [
H]
in the H4 peptide substrate after assay with wild type or hat1 mutant. The H4 peptide was labeled with
[
H]acetyl coenzyme A in the standard HAT assay,
using either the wild type (
-
) or the hat1-1 mutant Q380 fraction (
- -
-
). The peptide was subjected to microsequence analysis,
and the amount of radioactivity in each cycle (amino acid) was
determined. Since the amount of radioactivity in each of the two
samples differed (wild type, 4107 cpm; mutant, 1022 cpm), the
counts/min were arbitrarily normalized to be equal at amino acid 8. No
matter how the results are graphed, they clearly demonstrate that the hat1 mutant is deficient at acetylation of lysine
12.
Cloning of the HAT1 Gene
Analysis of the initial
crosses between the hat1-1 mutant and a HAT1
strain indicated that the hat1-1 mutation was centromere-linked. This conclusion was based on the
observation that there was a paucity of tetratype tetrads between hat1-1 and trp1, a centromere-linked marker on
chromosome IV. In order to localize the hat1-1 mutation to a
particular chromosome, we crossed the hat1-1 mutant with
several mapping strains carrying many centromere-linked markers. In
these crosses, the hat1-1 mutation was followed by the
enzymatic assay. The results showed linkage of hat1-1 to aro7, a marker near the centromere and on the right arm of
chromosome XVI. A three-factor cross involving hat1, aro7, and rad1, a centromere-linked marker on the
left arm of chromosome XVI, mapped the hat1 mutation to the
interval between rad1 and aro7, close to the
centromere (data not shown). Next, we obtained three overlapping
phages containing yeast genomic DNA from the region spanning centromere
XVI(24) . The genomic inserts from these phages were introduced
into a specialized yeast shuttle vector developed for this
purpose(15) . We used the 2µ plasmid pBM2384 for these
constructions to avoid generating a plasmid with two centromeres, since
one of the phages contained centromere XVI. Q380 fractions from a hat1-1 mutant carrying a plasmid containing the genomic insert
from
phage 5538 (p5538) restored activity to the mutant to about
90% of the wild type level (data not shown). A 6-kb SalI
subclone from p5538 also restored activity to the mutant. We determined
the DNA sequence of short stretches from this subclone and aligned them
to a stretch of 10 kb of preliminary chromosome XVI DNA sequence,
kindly provided to us by H. Bussey, McGill University. ORFs within the
6-kb SalI fragment were identified and used to direct further
subcloning. A subclone with a 2.3-kb StuI-EcoRI
fragment (p16ORF4), containing only one ORF, was found to restore HAT
activity to the mutant. We named the gene corresponding to this ORF HAT1. The DNA sequence of HAT1 and the surrounding
region has recently been deposited in GenBank
as part of
the yeast genome sequencing project (accession number Z48483). Analysis
of the DNA sequence near HAT1 revealed that the gene is
immediately adjacent to the centromere on the left arm of chromosome
XVI, with its direction of transcription toward the centromere. The
374-amino acid sequence predicted for the HAT1 protein is shown in Fig. 3. The protein has an unusually large number (23) of phenylalanine residues and a molecular mass of 44 kDa.
A search of the GenBank
data base using the Blast
algorithm revealed no significant homology between HAT1 and other
proteins. Further analysis revealed that HAT1 does show homology to a
bipartite consensus sequence found previously in a group of N-terminal
acetyltransferases(23) . The two regions of homology to the
consensus are underlined in the HAT1 sequence shown in Fig. 3.
We speculate that these regions of homology may constitute an acetyl
coenzyme A binding site.
Figure 3:
Amino
acid sequence of the HAT1 protein. The sequence is deduced from the DNA
sequence (GenBank
accession number G48483). The alignment
with a bipartite consensus sequence derived recently (23) for a
group of N-terminal acetyltransferases is indicated (region A, single underline; region B, double underline). The
agreement with the region A consensus
(h..h.h..Y..[H/K]GI[A/G][K/R].Lh . . . h,
where h = hydrophobic) is almost perfect, as indicated by the bold letters. The agreement with the region B consensus
(h.h[D/E]. . . . N..A . . . Y . . . GF. . . . . . . .
Y..[D/E]G) is also quite good.
A hat1-2 null mutant was
constructed by deleting the ORF from amino acids 1-266 plus an
additional upstream 50 bp of 5`-untranslated region and replacing it
with the selectable marker TRP1. Strains carrying the hat1-2::TRP1 allele are viable and show the same amount of HAT
activity (60% of wild type) as the originally identified hat1-1 allele (data not shown). This result confirmed that the residual
activity seen in hat1-1 mutants is caused by a second activity
and is not due to a leaky allele. The hat1-2 null mutant has
no obvious phenotype; thus far, we have not found any differences
between the two hat1 alleles in in vitro assays or in
other phenotype studies.
HAT1 Expression in E. coli
The HAT1 gene
was cloned into a T7 expression vector (18) and transformed
into a suitable E. coli strain (see ``Materials and
Methods''). After induction, a large amount of soluble HAT1
protein was produced, as judged by the appearance of a band of the
expected mobility on an SDS-polyacrylamide gel (data not shown).
Extracts prepared from these cells had very high levels of HAT activity (Fig. 4). Even a 1:1000 dilution of the extract had detectable
activity. A control extract from an E. coli strain without the HAT1 plasmid had no activity. These results show that HAT1 is the structural gene for the enzyme and that no other subunits
are required for activity.
Figure 4:
Expression of HAT1 activity in E.
coli. HAT1 was expressed in E. coli and assayed for
enzymatic activity in vitro by the standard assay. Activity is
shown for various dilutions of the original
extract.
In summary, we have identified a mutant
and cloned the gene (HAT1) for a histone acetyltransferase
that acetylates lysine 12 of H4. It remains to be seen whether other
enzymes exist in yeast with the same or overlapping specificity. The
fact that the hat1 mutant has no obvious growth defect makes
the latter possibility quite likely.