From the Department of Biochemistry and Molecular
Genetics, University of Virginia, Charlottesville, Virginia 22908, the
§ Department of Biology, University of Rochester, Rochester,
New York 14627, and the ¶ Department of Microbiology and
Immunology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Linker histone phosphorylation has been suggested
to play roles in both chromosome condensation and transcriptional
regulation. In the ciliated protozoan Tetrahymena, in
contrast to many eukaryotes, histone H1 of macronuclei is highly
phosphorylated during interphase. Macronuclei divide amitotically
without overt chromosome condensation in this organism, suggesting that
requirements for phosphorylation of macronuclear H1 may be limited to
transcriptional regulation. Here we report the major sites of
phosphorylation of macronuclear H1 in Tetrahymena
thermophila. Five phosphorylation sites, present in a single
cluster, were identified by sequencing 32P-labeled peptides
isolated from tryptic peptide maps. Phosphothreonine was detected
within two TPVK motifs and one TPTK motif that
resemble established p34cdc2 kinase consensus sequences.
Phosphoserine was detected at two non-proline-directed sites that do
not resemble known kinase consensus sequences. Phosphorylation at the
two noncanonical sites appears to be hierarchical because it was
observed only when a nearby p34cdc2 site was also
phosphorylated. Cells expressing macronuclear H1 containing alanine
substitutions at all five of these phosphorylation sites were viable
even though macronuclear H1 phosphorylation was abolished. These data
suggest that the five sites identified comprise the entire collection
of sites utilized by Tetrahymena and demonstrate that
phosphorylation of macronuclear H1, like the protein itself, is not
essential for viability in Tetrahymena.
In eukaryotes, the association of DNA with histones in nucleosomes
and the folding of nucleosomal filaments within chromatin can restrict
the accessibility of DNA sequences to factors required for gene
expression and DNA replication (1, 2). Crystallographic analyses have
led to detailed appreciation of the arrangement of core histones within
nucleosomes and the histone-histone and histone-DNA interactions
responsible for the stability of nucleosomal structure (3, 4). Many
aspects of nucleosome structure are expected to be common to all
eukaryotes because core histones are among the most highly conserved
proteins known (1, 2). However, the structure and occurrence of
proteins, collectively referred to as H1 or linker histones that bind
the outer surface of nucleosomes and portions of the linker DNA
extending between adjacent nucleosomes, are more variable (1, 2, 5,
6).
Data from recent biochemical and molecular genetic analyses have firmly
established that chromatin structure plays a fundamental role in the
regulation of gene expression. Acetylation of conserved lysine residues
within the amino termini of core histones, for example, is a major
pathway for modulating transcriptional activity (7, 8). In contrast,
the function of linker histones and their various post-translational
modifications in vivo remain unclear (5, 6, 9). Previous
notions that H1 acts globally to repress transcription (10, 11) and
that H1 phosphorylation is involved in mitotic chromosome condensation
(9) are in contrast to evidence that linker histones can affect
transcription positively or negatively in gene-specific fashion
in vivo (12, 13) and that linker histones are not required
for mitotic chromosome condensation in vitro (14) or
in vivo (15). Furthermore, recent observations suggest a
role for H1 phosphorylation in transcriptional regulation (16, 17).
Tetrahymena thermophila is a model organism that offers
several advantages for investigations of the function and metabolism of
linker histones. Like other ciliated protozoa, vegetative
Tetrahymena contain two nuclei, a transcriptionally
inactive, germ-line micronucleus, which divides mitotically, and a
transcriptionally active, somatic macronucleus, which divides
amitotically (18). Macronuclear H1 shares the overall amino acid
composition and acid solubility characteristics of eukaryotic H1, in
general, but lacks the central globular domain found in H1 of metazoans
(6, 19, 20). In contrast to the multiplicity of H1 genes found in most
eukaryotes, the Tetrahymena haploid genome contains only one
copy of the macronuclear H1 gene (20), facilitating methods of genetic
transformation we have utilized previously to demonstrate that although
macronuclear H1 is not required for viability, it is required for
normal compaction of interphase chromatin (15) and can function as a
positive or negative regulator of expression in a gene-specific fashion in vivo (12). Like H1 of multicellular eukaryotes,
macronuclear H1 is a substrate for p34cdc2 in vitro,
and we have presented evidence previously that suggests that this H1 is
phosphorylated by a homolog of Cdc2 in vivo (21, 22).
However, unlike many eukaryotes in which high levels of H1
phosphorylation occur mainly during mitosis, the majority of macronuclear H1 molecules are highly phosphorylated in unsynchronized, growing cultures of Tetrahymena (19, 21-23). Given the
absence of mitotic chromosome condensation in the amitotically dividing macronucleus (18), these differences likely reflect the high percentage
of the macronuclear genome that is transcribed (24). Thus, in
Tetrahymena macronuclei, potential relationships between macronuclear H1 phosphorylation and chromatin transcription can be
investigated apart from effects related to the hyperphosphorylation of
linker histones at mitosis that occurs in other eukaryotes. In pursuit
of this goal, we have mapped the major sites in macronuclear H1 that
are phosphorylated in vivo in T. thermophila to
enable a molecular genetic approach employing mutagenesis of sites to investigate the function of macronuclear H1 phosphorylation.
Cell Culture and Strains Employed--
T. thermophila
strains CU427 (Mpr/Mpr [6 mp-s VI]) and CU428 (Chx/Chx-[cy-S]VII)
were grown in 1% enriched proteose peptone (SPP) as described
previously (25). The creation of strain Metabolic Labeling--
Cultures were labeled continuously
during growth overnight (14-16 h) in SPP containing 5-25 µCi/ml
[32P]orthophosphate (ICN Catalog No. 64014) and harvested
at a density of 2.5-3.0 × 105 cells/ml.
Preparation of Macronuclear H1--
Incubation of
Tetrahymena at 4 °C rapidly induces dephosphorylation of
macronuclear H1.2
Accordingly, except where noted, cells were collected by centrifugation at room temperature (1000 × g for 5 min) prior to
disruption and extraction at 4 °C as described below. Macronuclei
were isolated as described previously (27) using 0.1 mM
p-chloromercuriphenylsulfonic acid in the
homogenization and wash buffers to inhibit phosphatases (28).
Macronuclei were extracted with 0.2 M
H2SO4, and crude macronuclear H1 was prepared
by selective precipitation of other proteins with 5.4% (w/v final)
perchloric acid (PCA).3 Crude
macronuclear H1 was recovered from the PCA-soluble fraction by
precipitation with trichloroacetic acid (20% w/v final), and these
precipitates were washed once with acetone containing 0.1% (v/v) HCl
and twice with acetone. Alternatively, crude macronuclear H1 was
prepared by direct extraction of cells in 5.4% (w/v) PCA. Cells
collected by centrifugation were washed twice at room temperature in 10 mM Tris-HCl, pH 7.4, prior to sonication in 5 volumes of ice-cold 5.4% (w/v) PCA. Four to six 10-s bursts of sonication (Branson model 250 with semi-micro tip, output = 5, continuous duty) were required to disrupt the majority of cells. Sonicates were
incubated in an ice water bath between bursts so that the temperature
did not exceed 6 °C. Sonicates were clarified by centrifugation at
10,000 × g for 20 min, and crude macronuclear H1 was
recovered from the supernatant by trichloroacetic acid precipitation.
Purified macronuclear H1 was prepared from extracts of isolated
macronuclei or whole cells by reverse-phase HPLC (RP-HPLC) using a C8
column (Aquapore RP-300, 4.6 mm x 250 mm, Perkin-Elmer Applied
Biosystems) and a gradient of acetonitrile (1% (v/v) per min) in 0.1%
(v/v) trifluoroacetic acid. Macronuclear H1 was recovered from RP-HPLC eluates by solvent evaporation.
Gel Electrophoresis--
Proteins were analyzed in 12% (w/v)
acrylamide gels (15 cm long) containing SDS (29) or 15% (w/v)
acrylamide gels (30 cm long) containing 6 M urea and 0.9 N acetic acid (30).
Phosphorylation Site Mapping--
32P-Labeled
macronuclear H1 (1-10 mg/ml) in 50 mM
NH4HCO3 was digested overnight (typically
14 h) at 37 °C with tosylphenylalanyl chloromethyl
ketone-treated trypsin (Sigma T-8642) using a 1:10 (w/w) trypsin:H1
ratio. The next morning, digests were supplemented with 50% more
trypsin and incubated for 6 h. Excess
NH4HCO3 was removed by repeated drying under
vacuum, and digests were analyzed on 40% (w/v) acrylamide gels
(resolving segment 10 cm long) at alkaline pH (31). Gels were dried
onto chromatography paper (Whatman No. 3MM) immediately following
electrophoresis and exposed to autoradiographic film (Kodak X-Omat).
Using autoradiograms as templates, phosphopeptide bands of interest
were excised from the dried gels, and most of the backing paper was
removed using a razor blade. Each phosphopeptide band was then cut into
approximately 3 mm x 3 mm pieces, transferred to a 15-ml polypropylene
centrifuge tube, and extracted with 5-10 volumes of distilled water
for 4-6 h at 4 °C using a laboratory rotator. Following
sedimentation of gel pieces by centrifugation, the supernatant was
removed and the extraction was repeated overnight. The supernatants
from both extractions were pooled and lyophilized or used directly for
phosphopeptide purification by RP-HPLC.
Phosphopeptides were recovered in salt-free form by RP-HPLC.
Lyophilisates were dissolved in 0.1% (v/v) trifluoroacetic acid, and
all samples were adjusted to pH 2.5 with trifluoroacetic acid prior to
injection onto a C18 column (Vydac 218TP52, The Separations Group).
After washing with 0.1% (v/v) trifluoroacetic acid to return the base
line to the preinjection level, the column was eluted with a shallow
gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid. Peptide
elution was monitored at 214 nm and by liquid scintillation counting.
Following solvent evaporation under vacuum, phosphopeptides were
analyzed by amino-terminal sequencing.
Macronuclear H1 or phosphopeptides were coupled to Immobilon-AA
(Millipore) according to the manufacturer's instructions prior to
sequencing. Sequencing was performed on an Applied Biosystems 477A
system using standard techniques except that 90% (v/v) methanol containing 15 µl of 85% (w/v) phosphoric acid/100 ml was employed for residue extraction because this gave better recovery of
phosphorylated derivatives. An in-line split diverted a portion of each
sequencing cycle to a fraction collector to determine 32P
content by liquid scintillation counting while the remaining portion
was employed for residue identification by RP-HPLC.
Macronuclear H1 prepared from Tetrahymena labeled with
[32P]orthophosphate during vegetative growth was resolved
into five distinct bands upon electrophoresis in polyacrylamide gels
containing acetic acid and urea (AU-PAGE) and in gels containing SDS
(SDS-PAGE) (Fig. 1A).
Essentially all the electrophoretic heterogeneity associated with
macronuclear H1 in this species appears to be due to phosphorylation because it is abolished by incubating extracts with alkaline
phosphatase prior to electrophoresis (19, 21, 23, 32). Careful
alignment of gels and corresponding autoradiograms established that the fastest migrating species in each gel system represented
nonphosphorylated macronuclear H1 (arrowheads, Fig.
1A). The observation that the AU-PAGE mobility of H1
proteins is inversely related to the degree of phosphorylation (33)
suggests that the most slowly migrating form in AU-PAGE represented
tetraphosphorylated macronuclear H1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1-5 will be described in
detail elsewhere.1 Briefly,
site-directed mutagenesis was employed to convert the five
phosphorylation sites to alanines in a construct containing the
HHO1 gene coding region as well as 5'- and 3'-flanking
sequences, with a single copy of the neo2 cassette (26)
containing the drug resistance marker neo inserted within
the HHO1 gene 3'-flanking sequence. Complete replacement of
the wild type HHO1 gene in polyploid macronuclei was
obtained by homologous recombination following biolistic transformation
and selection of transformants with paromomycin.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
T. thermophila macronuclear H1 is
phosphorylated at multiple sites in vivo.
A, macronuclear H1 prepared from cultures labeled with
[32P]orthophosphate in vivo was
electrophoresed in polyacrylamide gels containing acetic acid and urea
or SDS. Only the portions of the stained gels containing macronuclear
H1 and corresponding autoradiograms are shown. Arrowheads
denote the position of nonphosphorylated macronuclear H1 in each gel
system. B, 32P-labeled macronuclear H1 was
digested extensively with trypsin, and the resulting peptides were
resolved on polyacrylamide gels at alkaline pH. Nine peptide species
(labeled 1-9 on the left) were consistently
observed (lanes marked digest). Aliquots of the
eight peptide fractions recovered from bands excised from preparative
gels were resolved in the same gel system to assess their purity prior
to microsequencing (lanes 1-7 and 9). The recovery of
peptide 8 was insufficient for subsequent analysis. Only the
autoradiogram is shown.
Although 6 serine + threonine residues occur within the first 11 residues of this H1 (Fig. 3), phosphorylation was not detected between residues 1 and 12 when macronuclear H1 prepared from cultures grown overnight in the presence of [32P]orthophosphate was sequenced (data not shown). Due to the absence of potential sites between residues 12 and 30, we inferred that no phosphorylation occurs within the first 30 residues of this protein. We have shown previously that all phosphorylation in this species occurs in the amino-terminal fragment liberated upon CNBr cleavage of the methionine residue at position 692 (21, 23) (Fig. 3). Together, these data suggest that all sites of phosphorylation can be localized to the segment extending from Thr-31 to Thr-68. Eleven potential sites of phosphorylation, comprising 2 serine and 9 threonine residues, together with numerous lysine residues are found within this 37-residue segment.
To prepare phosphopeptides for microsequencing, 32P-labeled macronuclear H1 was digested extensively with trypsin, and the resulting peptides were resolved on polyacrylamide gels at alkaline pH (31). Following alignment of the autoradiograms and corresponding gels, bands of interest were excised from the dried gels and the phosphopeptides eluted by extracting excised gel pieces with water. Phosphopeptides were recovered from these eluates in salt-free form using RP-HPLC. This procedure enabled the recovery of eight phosphopeptides, purified to apparent homogeneity, representing all the major species and all but one of the minor species detected in the original digest (Fig. 1B). Single peaks of radioactivity were detected for all eight peptides when RP-HPLC eluates were monitored by liquid scintillation counting, suggesting that each band in the original gels was homogeneous (data not shown).
Each of these eight phosphopeptides was analyzed by microsequencing.
Plots of the 32P cpm released (y axis) against
the major amino acid released in each sequencing cycle (x
axis) unequivocally identified 2 serine and 3 threonine residues as
sites of phosphorylation (Fig. 2). Remarkably, the eight phosphopeptides represented only three peptide "families" in which either the same sequence was phosphorylated to
different extents (e.g. phosphopeptides 1 and 7, phosphopeptides 3 and 6) or the same phosphorylation site was contained
within both a limit tryptic peptide and a slightly longer peptide
resulting from incomplete digestion (e.g. phosphopeptides 2 and 4, phosphopeptides 5 and 9, phosphopeptides 6 and 7). Three
phosphorylation sites, Ser-42, Ser-44, and Thr-46, were identified
during the sequencing of phosphopeptides 1, 3, 6, and 7, which are all
related to the limit tryptic peptide spanning residues 40-49.
Phosphorylation at Thr-34 was detected during sequencing of the
peptides spanning residues 30-37 (phosphopeptide 4) and residues
29-39 (phosphopeptide 2). Phosphorylation at Thr-53 was detected
during sequencing of the peptides spanning residues 51-56
(phosphopeptide 9) and residues 51-60 (phosphopeptide 5). These data
are summarized and compared with findings for T. pyriformis
(34) in Fig. 3. Although the analysis of
macronuclear H1 phosphorylation in T. pyriformis did not
provide unambiguous localization of phosphorylation to single residues
in every instance, comparison of the two macronuclear H1 sequences
suggests that three of the T. thermophila sites identified here, Thr-34, Ser-44, and Thr-46, are conserved as Thr-35, Ser-46, and
Thr-48, respectively, in T. pyriformis (Fig. 3). Given these similarities, it is surprising that these species differ with regard to
phosphorylation in their carboxyl-terminal domains.
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Based on microsequencing of 32P-labeled tryptic peptides,
phosphorylation at five sites accounts for all of the major species resolved on the phosphopeptide maps, suggesting that these five sites
comprise the entire collection of sites utilized in vivo. To
test this hypothesis, we analyzed macronuclear H1 phosphorylation in a
Tetrahymena strain, 1-5, expressing macronuclear H1 in
which alanine was substituted for Ser/Thr at the identified
phosphorylation sites (Fig.
4A). A detailed description of
the creation, molecular characterization, and phenotypic analysis of
this strain will be given elsewhere.1
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Initial analyses of macronuclear H1 from growing 1-5 cells on SDS
gels revealed normal amounts of macronuclear H1 completely lacking the
electrophoretic heterogeneity associated with macronuclear H1 from
growing wild type cells, suggesting that phosphorylation of
1-5
macronuclear H1 did not occur in vivo or occurred at a limited number of sites in a manner that was not sufficient to retard
migration of the protein in SDS gels. To determine whether
1-5
macronuclear H1 was phosphorylated to any extent in vivo, we
labeled
1-5 and wild type cells in parallel with
[32P]orthophosphate during growth overnight, prepared H1
from whole cell extracts, and analyzed it by autoradiography of SDS
gels. Even though phosphorylation was readily detected in macronuclear H1 from wild type cells, phosphorylation was not detected when an
equivalent amount of macronuclear H1 from
1-5 cells was analyzed in
parallel, even after lengthy autoradiographic exposures (Fig. 4B). Because macronuclear H1 from the wild type cells (but
not that from
1-5 cells) was prepared using a method that enriched less phosphorylated forms, we conclude that macronuclear H1 is not
detectably phosphorylated in growing
1-5 cells under the conditions
employed here.
Taken together, these data suggest that the five sites identified by
microsequencing represent the complete set of macronuclear H1
phosphorylation sites utilized by T. thermophila.
Alternatively, a hierarchy of sites in macronuclear H1 may exist such
that the abolition of phosphorylation (or possibly the alanine
substitution itself) at one or more of the sites mutated in 1-5
macronuclear H1 precludes phosphorylation at unidentified secondary
sites. It should be noted that our analyses of phosphopeptides 1, 3, 6, and 7 indicate that, in wild type cells, phosphorylation occurs hierarchically, first at Thr-46, then at Ser-42, and last at Ser-44, in vivo (Fig. 2). Whether this hierarchy extends to other
sites is not known at present. Regardless of whether we have identified all of the sites of phosphorylation, it is clear that eliminating the 5 sites identified here results in cells completely lacking detectable
phosphorylation of macronuclear H1. Our findings that macronuclear H1
phosphorylation is not essential and that
1-5 strain cells are
viable without marked alterations in growth,1 suggest
either that macronuclear H1 phosphorylation does not have major, global
effects on transcription or that mechanisms exist that are capable of
compensating for the absence of macronuclear H1 phosphorylation in
1-5 cells. Further analyses of the
1-5 strain, in comparison
with wild type Tetrahymena and to strains constructed to
mimic constitutive phosphorylation or dephosphorylation of all or some
of the five sites identified here, should define the function of
macronuclear H1 phosphorylation in vivo.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM40922 (to C. D. A.) and GM21793 (to M. A. G.).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 Genetics, University of Virginia Health
Sciences Center, Box 440, Charlottesville, VA 22908. Tel.:
804-243-6048; Fax: 804-924-5069; E-mail: allis{at}virginia.edu.
1 Y. Dou, C. A. Mizzen, C. D. Allis, and M. A. Gorovsky, manuscript in preparation.
2 C. A. Mizzen and C. D. Allis, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PCA, perchloric acid; AU-PAGE, acetic acid-urea-polyacrylamide gel electrophoresis; RP-HPLC, reverse-phase high performance liquid chromatography.
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