From the Department of Biochemistry, Miyazaki Medical College, Kihara, Kiyotake, Miyazaki 889-1692, Japan
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ABSTRACT |
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Chromatin assembly factor-1 (CAF-1) is essential
for chromatin assembly in eukaryotes, and comprises three subunits of
150 kDa (p150), 60 kDa (p60), and 48 kDa (p48). We cloned and sequenced cDNA encoding the small subunit of the chicken CAF-1, chCAF-1p48. It consists of 425 amino acid residues including a putative initiation Met, possesses seven WD repeat motifs, and contains only one amino acid
change relative to the human and mouse CAF-1p48s. The
immunoprecipitation experiment followed by Western blotting revealed
that chCAF-1p48 interacts with chicken histone deacetylases (chHDAC-1
and -2) in vivo. The glutathione S-transferase
pulldown affinity assay revealed the in vitro interaction
of chCAF-1p48 with chHDAC-1, -2, and -3. We showed that the p48 subunit
tightly binds to two regions of chHDAC-2, located between amino acid
residues 82-180 and 245-314, respectively. We also established that
two N-terminal, two C-terminal, or one N-terminal and one C-terminal WD
repeat motif of chCAF-1p48 are required for this interaction, using
deletion mutants of the respective regions. These results suggest that chCAF-1p48 is involved in many aspects of DNA-utilizing processes, through alterations in the chromatin structure based on both the acetylation and deacetylation of core histones.
Understanding the process of chromatin assembly in eukaryotes is a
fundamental goal, because alterations in the chromatin structure have
been thought to be predominantly involved in DNA-utilizing processes,
such as replication, recombination, repair, and gene expression (1-9).
Chromatin assembly factor-1
(CAF-1)1 was originally
purified from human cells and promotes de novo chromatin
assembly on replicating SV40 DNA in the presence of a cytosol
replication system (10, 11). CAF-1 is a complex of three polypeptides
of 150 kDa (p150), 60 kDa (p60), and 48 kDa (p48) (12). During DNA
replication, CAF-1 assembles new nucleosomes through a two-step
reaction (10, 13). Coupled to DNA replication, as the first step,
histones H3 and H4 are deposited through a reaction that is
preferentially dependent upon CAF-1, but histones H2A and H2B are added
later to this immature nucleosome precursor, even in the absence of
CAF-1 (12, 14, 15). These results indicate that CAF-1 interacts
preferentially with H3 and H4, whereas NAP-1 binds to H2A and H2B (2,
16).
The WD protein family members, which are made up of highly conserved WD
repeating units, found in eukaryotes, but not in prokaryotes, are
involved in numerous biological processes such as signal transduction, RNA processing, gene expression, vesicular trafficking, and cell division (17-19). Thus, most of them seem to be regulatory, and none
is an enzyme. The consensus core of the repeating unit, the WD repeat,
usually ends with the characteristic sequence, Trp-Asp (WD), and such a
conserved unit occurs four to ten times within each polypeptide (19).
Each repeat comprises a region of variable length preceding a conserved
core of about 30 amino acids (maximum range, 23-41 amino acids),
ending with Gly-His (GH) and WD dipeptide residues. In addition, the
number of amino acids from WD to the next downstream GH is very
variable (6-94 amino acids), although shorter sequences are more
common (112 residues long). All of these WD repeat proteins have been
proposed to fold into propellers in which the internal CAF-1p48, with seven WD repeat motifs, is a member of this WD repeat
protein family. In recent years, knowledge concerning the
characteristics of CAF-1s in the DNA-utilizing processes has been
rapidly accumulated (9). For instance, CAF-1p48 was identified as a
polypeptide that is tightly associated with the catalytic subunit of
human histone deacetylase-1 (HDAC-1) (25). In addition, the smallest
subunit of Drosophila CAF-1, p55, is homologous to a
mammalian factor, RbAp48, associated with HDAC (26). Interestingly, Drosophila p55 was reported to be an integral subunit of the
nucleosome remodeling factor (NURF) (27). However, there was little
information concerning the detailed mechanisms for the protein-protein
interaction of CAF-1p48 in higher eukaryotes.
In this study we first cloned the cDNA encoding chicken CAF-1p48,
chCAF-1p48, and demonstrated that it tightly binds to chHDACs in
vivo and in vitro. We describe the in vitro
interaction of chCAF-1p48 with two regions of chHDAC-2, comprising
amino acid residues 82-180 and 245-314, respectively. We also
describe that this interaction requires two N-terminal, two C-terminal,
or one N-terminal plus one C-terminal WD repeat of chCAF-1p48, as
deletion of the respective regions results in a loss of the binding activity.
Materials--
In this study the XL1-Blue MRF' Escherichia
coli strain, E. coli SORL strain (Stratagene), and
E. coli BL-21 strain (Amersham Pharmacia Biotech) were used.
pBluescript II SK( Cloning and Sequencing of cDNA Encoding
chCAF-1p48--
Based on conserved amino acid sequences (LVMTHALEWP
and PNEPWVICSV) in the mouse and human CAF-1p48s deduced from their
cDNAs (15, 25), sense and antisense degenerate oligonucleotide
primers containing sequences 5'-YYTGGTBATGACCCATGCYCTKSAGTGGCCC-3' and 5'-AYACDGARCAAATSACCCAAGGYTCATTGGG-3', respectively, were
constructed. A PCR product of 1082 base pairs, corresponding to a part
of the coding regions of mammalian CAF-1p48s, was prepared from the
chicken DT40 cDNAs using the two degenerate primers. To obtain
full-length chCAF-1p48 cDNAs, using the resultant PCR product as a
probe, we screened the DT40 Immunoprecipitation and Western Blotting--
To construct a
vector, designated ptetHAchCAF-1p48, expressing chCAF-1p48 under the
control of the tetracycline operator (tetO) and cytomegalovirus minimal
promoter, cDNA encoding influenza HA epitope-tagged chCAF-1p48 was
inserted into pUHD13-3 plasmid (29), concurrently replacing its
constituent luciferase gene. To construct ptTA-bleo, a cassette of the
bleomycin-resistant gene driven by
Cells (1.5 × 107) were lysed in 1 ml of RIPA buffer
(25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1%
Nonidet P-40, 1 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride) with occasional inversion. After
standing for 30 min at 4 °C, the lysate was centrifuged at 15,000 rpm for 10 min, and the supernatant obtained was divided into two equal
portions. To each portion, 5 µg of anti-HA antibody (12CA5, Roche
Molecular Biochemicals) or anti-FLAG antibody (Eastman Kodak Co.) were
added. After standing for 60 min on ice, protein G-Sepharose beads (40 µl) were added to the incubation mixture. Following gentle rotation
for 5 h at 4 °C, the protein G-Sepharose beads were pelleted by
centrifugation, washed with 1 ml of RIPA buffer 4 times, and boiled in
SDS sample buffer for 5 min. The resultant immunoprecipitated proteins
were resolved by 12% SDS-PAGE, essentially as described (28). Upon
transfer to a nitrocellulose membrane, proteins were probed with
anti-HA antiserum or rabbit anti-chHDAC-1 and -2 antisera (against,
respectively, recombinant chHDAC-1 and -2 C-terminal peptide-GST fusion
proteins),2 using an ECL kit according to the
manufacturer's protocol (Amersham Pharmacia Biotech). Horseradish
peroxidase-conjugated anti-mouse IgG for HA antibody (Dako) and
horseradish peroxidase-conjugated anti-rabbit IgG for chHDACs antibody
were used as secondary antibodies.
Plasmid Construction--
To construct the pGEX-2TKchCAF-1p48
plasmid, the NcoI/ScaI fragment containing the
full-length chCAF-1p48 cDNA was excised from the parental chimeric
plasmid (pB(II)SKp48), blunt-ended with T4 polymerase, and then
subcloned into the SmaI site of the pGEX-2TK plasmid in
frame. We also constructed the pGEX-2TKchHDAC-2 plasmid as follows. The
parental chimeric plasmid (pB(II)SKchHDAC-2) carrying the full-length
chHDAC-2 cDNA2 was digested with KpnI,
followed by treatment with T4 polymerase. From the resultant
blunt-ended material, the KpnI blunt-end/XbaI fragment containing the full-length chHDAC-2 cDNA was excised and
subcloned between the BamHI (blunt-ended by T4 polymerase) and XbaI sites of the pGEX-2TK plasmid.
We constructed the pCiteHDAC-1, -2, and -3 plasmids as follows. The
three parental plasmids, pB(II)SKchHDAC-1, -2, and -3,2
were digested with ClaI, blunt-ended with T4 polymerase, and then digested with NotI. The resultant blunt-ended
ClaI/NotI fragments, carrying the full-length
chHDAC-1, -2, and -3 cDNAs, respectively, were subcloned
between the MscI and NotI sites of the
pCite4a(
Deletion mutants of chHDAC-2 were constructed as follows. The
StuI/PstI fragment encoding amino acids 82-488,
the HincII/PstI fragment encoding amino acids
115-488, the XcmI/PstI fragment encoding amino
acids 181-488, the NdeI/PstI fragment encoding amino acids 315-488, and the FspI/PstI fragment
encoding amino acids 371-488 of chHDAC-2, respectively, were deleted
by digestion of pCiteHDAC-2 with the corresponding enzymes and
religated after being blunt-ended with T4 polymerase to generate
pCiteHDAC-2-(1-81), pCiteHDAC-2-(1-114), pCiteHDAC-2-(1-180),
pCiteHDAC-2-(1-314), and pCiteHDAC-2-(1-370). pCiteHDAC-2-(82-370),
pCiteHDAC-2-(162-370), and pCiteHDAC-2-(245-370), respectively, were
generated by digestion of pCiteHDAC-2-(1-370) with NcoI
plus StuI, NcoI plus XcmI, and NcoI
plus HincII, followed by religation. pCiteHDAC-2-(315-488) was generated by digestion of pCiteHDAC-2 with NcoI plus
NdeI to delete the NcoI/NdeI fragment
encoding amino acids 1-314, followed by religation.
pCiteHDAC-2-(82-180) and pCiteHDAC-2-(245-314), respectively, were
generated from pCiteHDAC-2-(82-370) and pCiteHDAC-2-(245-370) by
digestion with XcmI plus BamHI and
NdeI plus BamHI, followed by religation after
being blunt-ended with T4 polymerase.
We constructed the pCiteHAp48 plasmid, carrying both the full-length
chCAF-1p48 cDNA and the HA fragment, as follows. The NcoI/ScaI fragment carrying the full-length
chCAF-1p48 cDNA was first excised from the parental pB(II)SKp48
plasmid, blunt-ended with T4 polymerase, and then ligated into the
SmaI site of pGBT9 to yield pGBT9p48. The
NcoI/SalI fragment carrying the full-length chCAF-1p48 cDNA was excised from the resultant plasmid and
introduced between the NcoI and SalI sites of the
pCiteHA plasmid, carrying the HA sequence derived from pAS.1 (kindly
provided by Dr. C. M. Tiree), which we constructed.
We generated deletion mutants of chCAF-1p48 as follows.
pCiteHAp48-(1-267) and pCiteHAp48-(1-375), respectively, were
generated by excision of the XcmI/SalI fragment
encoding amino acids 268-425 and the EcoRV/BamHI
fragment encoding amino acids 376-425 from pCiteHAp48 by digestion
with XcmI plus SalI and EcoRV plus
BamHI, followed by religation after blunt-ending with Klenow
polymerase. To generate pCiteHAp48-(1-328), the
NcoI/BsaI fragment of chCAF-1p48 cDNA was
excised from pCiteHAp48 and introduced between the NcoI and
SalI sites of the same plasmid. pCiteHAp48-(55-425),
pCiteHAp48-(139-425), and pCiteHAp48-(268-425), respectively, were
generated by digestion of pCiteHAp48 with NcoI plus
StuI, NcoI plus NsiI, and
NcoI plus XcmI, and blunt-ended with T4
polymerase before religation. To generate pCiteHAp48-(181-425), we
first constructed sense and antisense primers, containing sequences
5'-TATGGGTTGTCATGGAACCCAAACC-3' and 5'-CATGGCCATATGACCACCCAAGCTA-3',
respectively, corresponding to amino acids 181-188 of chCAF-1p48 and
amino acids 12-19 of HA. Next we prepared the DNA fragment without
that encoding amino acids 1-180, by PCR using pCiteHAp48 as a template
with these two primers and then ligated the resultant PCR product to
yield pCiteHAp48-(181-425). pCiteHAp48-(55-375) and
pCiteHAp48-(139-375) were generated by excision of the
EcoRV/BamHI fragment encoding amino acids
376-425 from pCiteHAp48-(55-425) and pCiteHAp48-(139-425), respectively, and religated after blunt-ending with T4 polymerase. Each
end point of the deletions was determined by sequence analysis involving the dye terminator method.
Expression and Purification of GST Fusion Proteins--
E.
coli BL-21 cells were transformed with pGEX-2TKchCAF-1p48 and
pGEX-2TKchHDAC-2, respectively, harboring the full-length chCAF-1p48
and chHDAC-2 cDNAs and grown to A600 nm = ~0.2 in 400 ml of LB medium supplemented with 200 µg/ml ampicillin.
Upon induction with 50 µM isopropyl
GST Pulldown Affinity Assay--
To produce
[35S]Met-labeled full-length chCAF-1p48 and chHDAC-1, -2, and -3, a set of truncated mutants of chCAF-1p48, and a set of
truncated mutants of chHDAC-2, the TNT-coupled
transcription-translation system (Promega) was used. In
vitro binding assays were performed using 5 µl of
[35S]Met-labeled protein fractions and 4 µg of the GST
fusion proteins or 6 µg of GST, prepared as described, in 200 µl of
bead-binding buffer (50 mM potassium phosphate buffer, pH
7.5, 450 mM KCl, 10 mM MgCl2, 10%
glycerol, 1% Triton X-100, 1% BSA). After 1 h of standing, the
reaction mixture was added to 20 µl of a 50% slurry of
glutathione-agarose beads, containing 10 mg/ml BSA and 4 µg of EtBr,
followed by gentle rotation for 1 h at 4 °C. The affinity beads
were collected by centrifugation at 3,500 rpm for 2 min and then washed
with 1 ml of the bead-binding buffer without BSA and EtBr but
containing 0.1% phenylbenzosulfonyl fluoride five times. The beads
were suspended in 30 µl of 2× SDS sample buffer and then boiled for
5 min. Aliquots (15 µl) of the resultant eluates were analyzed by
12% SDS-PAGE, and then the gels were stained, dried, and subjected to fluorography.
Cloning of cDNA Encoding chCAF-1p48--
To determine
unequivocally the identification of chCAF-1p48 as a novel subunit of
CAF-1, we cloned and sequenced its cDNA. Based on conserved amino
acid sequences (the corresponding sequences in the chicken homolog are
underlined in Fig.
1A) in the mouse and human
CAF-1p48s (15, 25), we prepared the 1082-base pair PCR fragment,
corresponding to a part of cDNAs encoding the mammalian p48
subunits, by PCR using cDNAs from DT40 cells with degenerate primers, i.e. a sense primer and an antisense primer. Our
screening, using the resultant PCR product, of a DT40
Therefore, chCAF-1p48 is a member of the WD repeat protein family and
possesses seven copies of the WD motif, a motif of 37-61 amino acid
residues, including a WD, FD, or WN dipeptide (Fig. 1B).
Like most proteins containing WD repeats, chCAF-1p48 is expected not
only to physically associate with other proteins but also to act as a
scaffold upon which multimeric complexes can be built.
In Vivo Interaction of chCAF-1p48 with chHDACs--
To determine
whether or not chCAF-1p48 binds to these chHDACs in vivo,
Western blotting, using anti-chHDAC-1 and -2 antisera, against,
respectively, recombinant chHDAC-1 and -2 C-terminal peptide-GST fusion
proteins was carried out. DT40 cells were first co-transfected with
ptet-HAchCAF-1p48 and ptTA-bleo, and the transfected cells that express
the HA-tagged chCAF-1p48 were selected with phleomycin. A cell line
(tetHAp48) overexpressing the HA-tagged protein in the absence of
tetracycline was established. Proteins in the lysate of tetHAp48 cells
were immunoprecipitated with anti-HA or anti-FLAG antiserum. The
immunoprecipitated samples, together with the tetHAp48 lysate and the
DT40 cell lysate, were subjected to Western blotting.
As shown in Fig. 2A, anti-HA
antiserum recognized a protein species of the same size as the
HA-tagged chCAF-1p48 present in the tetHAp48 cell lysate and the
anti-HA antiserum-precipitated sample, but not the anti-FLAG
antiserum-precipitated sample or the DT40 cell lysate. In the case of
the immunoprecipitation with anti-HA antiserum, the band corresponding
to the HA-tagged chCAF-1p48 migrated very closely to that of mouse IgG
heavy chain present in the sample immunoprecipitated with anti-FLAG
antiserum.
In contrast, anti-chHDAC-1 antiserum recognized the protein band
corresponding to chHDAC-1 in the cell lysates of both tetHAp48 and DT40
cells (Fig. 2B). Furthermore, as expected, the same
(chHDAC-1) band showed up for the sample immunoprecipitated with
anti-HA antiserum but not that immunoprecipitated with anti-FLAG
antiserum. Similarly, anti-chHDAC-2 antiserum recognized the band
corresponding to chHDAC-2 in the lysates of both tetHAp48 and DT40
cells, as well as the sample immunoprecipitated with anti-HA antiserum
(Fig. 2C). Reasonably, no band showed up for the sample
immunoprecipitated with anti-FLAG antiserum. These results indicate
clearly that both chHDAC-1 and -2 were present in the sample
immunoprecipitated with anti-HA antiserum, suggesting that the two
enzymes bind tightly to chCAF-1p48 in vivo.
Expression and Purification of GST-chCAF-1p48 Fusion Protein in E. coli--
To construct a chimeric plasmid, pGEX-2TKchCAF-1p48,
expressing the GST-chCAF-1p48 fusion protein, chCAF-1p48 cDNA was
subcloned into the pGEX-2TK plasmid in frame. GST fusion proteins were
synthesized in E. coli, extracted, and purified essentially
as described above. As shown in Fig. 3,
the electrophoretic patterns on SDS-PAGE of whole cell lysates before
and after the induction with IPTG revealed that GST-chCAF-1p48 fusion
proteins of approximately 74 kDa were dramatically accumulated in
E. coli BL-21 cells containing the pGEX-2TKchCAF-1p48
plasmid. In addition, the GST-chCAF-1p48 fusion proteins were purified
to more than 95% homogeneity, using glutathione-agarose beads (see
lane 3 in Fig. 3).
In Vitro Interaction of chCAF-1p48 with chHDAC-1, -2, and
-3--
We cloned and sequenced three cDNAs, encoding chHDAC-1,
-2, and -3, respectively, which comprise 480, 488, and 428 amino acid residues, including a putative initiation Met.2 Compared
with chHDAC-1 and -2, the C-terminal region of chHDAC-3 is about 50 amino acids shorter. However, in the corresponding regions all three
chHDACs exhibit extensive homology (95%). Furthermore, we recently
developed E. coli systems with which all of chHDAC-1, -2, and -3 could be dramatically synthesized. These recombinant chHDAC-1,
-2, and -3, like recombinant maize HDAC-2 expressed in E. coli (30), exhibited little enzymatic activity in vitro (data not shown). As discussed previously (30), the problem may be due
to (i) the fact that the chicken and maize enzymes are active only when
assembled as correct complexes, (ii) the incorrect folding of
recombinant HDACs, or (iii) the requirement of certain
post-translational modifications, e.g. phosphorylation, for
the active enzymes. Despite their lack of enzymatic activities, recombinant chHDAC-1, -2, and -3 probably have maintained the correct
conformations to interact with chCAF-1p48.
To determine whether or not the p48 subunit binds to these three
chHDACs, therefore, the GST pulldown affinity assay was carried out.
chHDAC-1, -2, and -3 were translated in vitro in the
presence of [35S]Met and then assayed as to their
abilities to interact with chCAF-1p48. Each sample was separated by
12% SDS-PAGE, and proteins were stained with Coomassie Blue, followed
by fluorography. As shown in Fig. 4, the
GST-chCAF-1p48 fusion protein bound to all of chHDAC-1, -2, and -3, whereas under the same conditions Two Regions of chHDAC-2 Required for in Vitro Interaction with
chCAF-1p48--
To determine the putative binding region(s), which
should be conserved in the three chHDACs, we first constructed a series of C-terminal deletion mutants of chHDAC-2 and studied their in vitro interaction with the GST-chCAF-1p48 fusion protein (Fig. 5). Three deletion mutant proteins,
Next we constructed a series of mutants with simultaneous deletion of
both the N-terminal and C-terminal regions of chHDAC-2 and assayed
their binding activities essentially as described above. Fig. 5 shows
that
These results suggested that two possible binding regions of chHDAC-2
as to chCAF-1p48 are located between amino acids 115-180 and amino
acids 245-314, respectively, and that either is enough as the binding
region. Finally, to confirm these results we constructed two mutant
proteins, Two WD Repeats of chCAF-1p48 Required for in Vitro Interaction with
chHDAC-2--
To clarify the putative binding domain(s) of chCAF-1p48
as to chHDAC-2, we first constructed a series of C-terminal truncated mutants of HA-tagged chCAF-1p48 and studied the in vitro
interaction with the GST-chHDAC-2 fusion protein, essentially as
described above. Two truncated proteins,
Next we constructed a series of N-terminal truncated mutants of
HA-tagged chCAF-1p48 and studied their binding abilities as to
chHDAC-2, essentially as described above. As shown in Fig. 6, the
abilities of two truncated mutant proteins,
Moreover, we constructed two truncated mutants, respectively, with
simultaneous deletion of both the N-terminal and C-terminal regions of
chCAF-1p48 and assayed their binding activities. The results obtained
indicated that
Our analyses, like these, of a series of deletion mutant proteins of
chCAF-1p48 revealed several noticeable features regarding its binding
ability as to chHDAC-2 as follows. First the finding that
Eukaryotic HDACs, like acetylpolyamine amidohydrolases, are
members of a deacetylase superfamily of proteins that not only recognize an acetylaminopropyl group and catalyze the removal of an
acetyl group by cleaving a non-peptide amide bond but also that share
nine blocks, 1 to 9, exhibiting considerable sequence homology (31).
All of them are located in the approximately two-thirds N-terminal
region of each of three chHDACs.2 Two binding domains,
designated as BD1 and BD2, comprising amino acids 82-180 (in fact,
probably 115-180) and amino acids 245-314 of chHDAC-2 (Fig. 5),
respectively, contain blocks 3, 4, and 5 plus approximately half of the
N-terminal region of block 6 and approximately half of the C-terminal
region of block 8 plus block 9. BD1 and BD2 are also both located in
the corresponding regions of chHDAC-1 and -3 as in those of other
mammalian HDAC homologs (25, 32, 33). To determine how this interaction
of BD1 and/or BD2 with chCAF-1p48 is involved in DNA-utilizing
processes, such as replication, recombination, repair, and gene
expression, further studies must be performed.
The amino acid sequence of chCAF-1p48 shows that it is a member of the
superfamily of WD repeat proteins (Fig. 1). All of the seven WD repeats
found in it well match those in the small subunits of CAF-1s in most
eukaryotes (15, 25, 27). chCAF-1p48 exhibits binding ability as to
chHDAC-2 (and chHDAC-1 and 3) when the second WD dipeptide motif and
the shorter region of 13 amino acids preceding it remain, but the p48
subunit loses the ability when both the first and second WD repeats
with the variable region of 27 amino acids just behind the second WD
dipeptide motif are lacking (Fig. 6). On the other hand, the binding
ability of chCAF-1p48 disappears when the region of 16 amino acids
preceding the sixth WD dipeptide motif, together with this motif and
the seventh one, is lacking. Conversely, the ability does not change
even when the shorter region of 6 amino acids preceding the seventh WD
dipeptide motif, together with both this motif and the C-terminal
region of the protein, is lacking. Interestingly, even when the first WD repeat plus the region of 44 amino acids just behind the first FD
dipeptide motif, or the shorter region of 6 amino acids preceding the
seventh WN dipeptide motif plus the C-terminal region is independently deleted, no influence is observed on the binding ability of chCAF-1p48; however, the simultaneous deletion of the two N-terminal and C-terminal portions abolishes the binding ability of chCAF-1p48 (Fig. 6). The
findings made based on the in vitro assay system most likely reflected what happens in vivo because chCAF-1p48 also
interacted with chHDAC-1 and -2 in the in vivo experiment
(see Fig. 2).
Based on the results obtained for a series of truncated mutant proteins
of chCAF-1p48, we propose a model for its interaction with chHDAC-2. As
in most WD proteins (19), the WD repeat motifs of chCAF-1p48 should
form a The overall picture concerning the in vivo interaction of
chCAF-1p48 with chHDACs will be clarified, using gene targeting techniques, with the DT40 chicken B cell line, which incorporates foreign DNA by targeted integration at frequencies similar to those for
random integration (34) for a number of different genomic loci,
including genes encoding histones (35-40) and histone deacetylases.2
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-strands form
a rigid skeleton that is fleshed out on the surface by specialized
loops to which other proteins bind (19-22). The amino acid residues in
the WD repeats of Tup1, the yeast repressor, that are required for the
interaction of Tup1 with homeodomain protein
2 have been genetically
identified (23). Point mutations in the WD40 domains of the Eed
(embryonic ectoderm development)
protein block its interaction with Ezh2, a mammalian homolog of the
Drosophila enhancer of zeste [E(z)] (24).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and pCite4a(
) were purchased from Promega. The
pGEX-2TK gene fusion vector and glutathione-Sepharose beads were
products of Amersham Pharmacia Biotech.
ZAP II cDNA library constructed by
us,2 using poly(A) mRNAs
prepared from the chicken DT40 cell line, essentially as described
(28). The entire nucleotide sequences of both strands of the largest
cDNA insert were sequenced by the dye terminator method (Applied
Biosystems Division, Perkin-Elmer).
-actin promoter was inserted into
the pUHD15-1 plasmid that contains tet-transactivator gene controlled
by cytomegalovirus promoter (29). DT40 cells were co-transfected with
ptet-HAchCAF-1p48 and ptTA-bleo, and the DT40 cell lines that express
the HA-tagged chCAF-1p48 were selected by incubation in medium
containing 0.3 mg/ml phleomycin (Sigma). Of these subclones, the clone
(designated tetHAp48) that overexpresses the HA-tagged chCAF-1p48 in
the absence of tetracycline was further selected.
) plasmid.
-D-thiogalactopyranoside (IPTG) overnight at 20 °C,
the cells were collected by centrifugation and suspended in 10 ml of
lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 1 mM EDTA) containing 0.1% (v/v) phenylbenzosulfonyl
fluoride and 1 mg/ml lysozyme, in liquid N2 for 2 min,
followed by ultrasonication for 3 min. Continuously, the cell lysate
was added to Sarkosyl to a final concentration of 1%. After standing
for 60 min, Triton X-100 was added to a final concentration of 1%, and
then the cell lysate was allowed to stand for an additional 30 min. The
cell homogenate thus prepared was subjected to centrifugation at 15,000 rpm for 30 min. The supernatant obtained was mixed with 1 ml of a 50%
slurry of glutathione-agarose beads for 4 h with gentle rotation.
The GST fusion protein-bound beads were collected by centrifugation at
3,500 rpm for 2 min and washed with the lysis buffer containing 1%
Triton X-100 and 0.1% phenylbenzosulfonyl fluoride four times, then
with phosphate-buffered saline buffer once. The GST fusion proteins
were eluted with 4 ml of 20 mM glutathione in 50 mM Tris-HCl, pH 8.5, and the resultant eluate was
concentrated with a Millipore membrane, followed by the addition of
glycerol to a final concentration of 20%. The samples obtained were
resolved by 10% SDS-PAGE, essentially as described (28).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP II
cDNA library2 yielded 11 positive cDNA clones.
Sequence analysis of the largest cDNA insert of 2405 base pairs of
the 11 cDNA clones revealed that the clone designated as 5 contained
both an initiation codon and a 3' poly(A) tail and thus appeared to
contain the full-length sequence of the chicken CAF-1p48 cDNA. The
nucleotide and deduced amino acid sequences are presented in Fig.
1A. The chicken protein comprises 425 amino acid residues
including a putative initiation Met and contains only one amino acid
alteration relative to the human and mouse CAF-1p48s (15, 25). The
alteration in the amino acid sequence is Asn, instead of Tyr, at
position 409. Thus, this protein exhibits 99.8%, 93.9%, and 90.4%
identity in amino acid sequence to the human and mouse CAF-1p48s, human
and mouse CAF-1p46s (15, 25), and Drosophila CAF-1p55 (or
NURF-55) (26, 27), respectively. Therefore, it is the chicken homolog
of mammalian CAF-1p48s and is designated as chCAF-1p48
(GenBankTM accession number AF097750).
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Fig. 1.
Nucleotide and deduced amino acid sequences
of chicken chromatin assembly factor-1p48. A,
nucleotides are numbered in the 5' to 3' direction with the
adenosine of the translation initiation codon designated as +1. The
translation stop codon is indicated by an asterisk. The
predicted amino acid sequence of chCAF-1p48 is presented in a
single-letter code. The seven WD (including FD
and WN) dipeptide motifs are boxed. Two regions
corresponding to those in the human and mouse CAF-1p48s (15, 25), used
as primer sequences, are underlined. B, the WD
repeat motifs of chCAF-1p48. The characteristic GH and WD dipeptide
motifs are boxed. Each WD repeat contains a variable region
of 5-29 amino acid residues and a core of 32 amino acid
residues.
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Fig. 2.
Western blotting of cell lysates and samples
immunoprecipitated with anti-HA or anti-FLAG antiserum. Aliquots
of the lysates of DT40 and tetHAp48 cells and tetHAp48 samples
immunoprecipitated with anti-HA or anti-FLAG antiserum were subjected
to 12% SDS-PAGE. After being transferred to a nitrocellulose membrane,
proteins were probed with anti-chHDAC-1 or -2 antiserum
(middle and lower panels) or anti-HA
antiserum (upper panel).
IP:antiHA, the sample immunoprecipitated with
anti-HA antiserum; IP:antiFlag, the sample
immunoprecipitated with anti-FLAG antiserum; HA-p48,
HA-tagged chCAF-1p48; IgG-H, mouse IgG heavy
chain.
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Fig. 3.
SDS gel electrophoretic patterns of
GST-chCAF-1p48 fusion protein-containing fractions at different
purification steps. Protein samples prepared were subjected to
10% SDS-PAGE, followed by Coomassie Blue staining. Lane 1,
whole cell lysate of BL-21 cells containing the pGEX-2TKchCAF-1p48
plasmid without induction by IPTG; lane 2, the lysate with
induction by 50 µM IPTG; lane 3, the fraction
purified with glutathione-agarose beads; lane 4, GST;
lane M, molecular mass standards.
-galactosidase did not bind to it,
and GST alone also exhibited no interaction with these three chHDACs.
These findings were confirmed by an immunoprecipitation experiment
involving anti-chCAF-1p48 antiserum (data not shown), suggesting the
existence of a region(s) conserved in chHDAC-1, -2, and -3 to which
chCAF-1p48 binds.
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Fig. 4.
In vitro interaction of the
GST-chCAF-1p48 fusion protein with chHDAC-1, -2, and -3. chHDAC-1,
-2, and -3 were each labeled with [35S]Met with the
in vitro translation system, and then their binding
activities as to the GST-chCAF-1p48 fusion protein were examined by
means of the GST pulldown affinity assay. Each sample was resolved on
12% SDS-PAGE, and then the proteins were stained with Coomassie Blue
(upper panel), followed by fluorography (lower
panel). -Galactosidase (
-gal) was used as a control.
GST-p48, GST-chCAF-1p48 fusion protein.
chHDAC-2-(1-370),
chHDAC-2-(1-314), and
chHDAC-2-(1-180),
exhibited similar binding activity toward the parental chHDAC-2
protein. On the other hand, two other mutant proteins,
chHDAC-2-(1-114) and
chHDAC-2-(1-81), exhibited no binding
activity. These findings suggested that the region comprising amino
acids 115-180 of chHDAC-2 is necessary for its binding to chCAF-1p48.
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Fig. 5.
In vitro interaction of
truncated mutant proteins of chHDAC-2 with the GST-chCAF-1p48 fusion
protein. A, a series of mutant proteins, respectively,
devoid of the N-terminal, C-terminal, and both the N-terminal plus
C-terminal regions of chHDAC-2 were constructed and their binding
activities as to the GST-chCAF-1p48 fusion protein were examined as in
Fig. 4. B, the results obtained for the in vitro
interaction of chHDAC-2 and its truncated proteins with chCAF-1p48 are
schematically presented.
chHDAC-2-(245-370), like
chHDAC-2-(82-370) and
chHDAC-2-(162-370), exhibited binding activity, although it even
lacks the region comprising amino acids 115-180. In addition,
chHDAC-2-(315-488), lacking the N-terminal region comprising amino
acids 1-314, exhibited no activity, whereas
chHDAC-2-(1-314) bearing the same N-terminal region exhibited the binding ability.
chHDAC-2-(82-180) and
chHDAC-2-(245-314), and studied
their binding activities. As expected, the two regions definitely
interacted with chCAF-1p48, indicating that either is enough for the
in vitro interaction with chCAF-1p48. Moreover, the findings
that both regions were extensively conserved within chHDAC-1 and
-3,2 as in homologs from other organisms, and that
chCAF-1p48 bound to the two chHDACs (see Fig. 4) suggest the possible
involvement of the two corresponding domains of all HDACs in their
in vitro interactions with CAF-1p48.
chCAF-1p48-(1-328) and
chCAF-1p48-(1-267), exhibited no binding activity, although the
other one (
chCAF-1p48-(1-375)) exhibited binding activity similar
to that of the parental chCAF-1p48 protein (Fig.
6). These findings suggested that the
deletion of the region comprising amino acids 329-375, in addition to
the C-terminal region comprising amino acids 376-425, resulted in the
loss of the binding activity.
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Fig. 6.
In vitro interaction of truncated
mutant proteins of chCAF-1p48 with the GST-chHDAC-2 fusion
protein. A, a series of mutant proteins, respectively,
devoid of the N-terminal, C-terminal, and both the N-terminal plus
C-terminal regions of HA-tagged chCAF-1p48 were constructed, and then
their binding activities as to the GST-chHDAC-2 fusion protein were
examined as in Fig. 4. The mutant proteins used are denoted by
appropriate abbreviations, i.e. p48(1-375) is the
abbreviation for
chCAF-1p48-(1-375), as an example.
GST-HD2, GST-chHDAC-2 fusion protein. B, the
results obtained for the in vitro interaction of HA-tagged
chCAF-1p48 and its truncated proteins with chHDAC-2 are schematically
presented.
chCAF-1p48-(55-425) and
chCAF-1p48-(139-425), were the same as that of the parental chCAF-1p48 protein. On the other hand, two other truncated proteins,
chCAF-1p48-(181-425) and
chCAF-1p48-(268-425), did not exhibit the ability. These results indicated that the deletion of the region
comprising amino acids 139-180, in addition to the N-terminal region
comprising amino acids 1-138, resulted in the loss of the binding ability.
chCAF-1p48-(55-375) exhibited the activity but
chCAF-1p48-(139-375) had lost it, whereas both mutants similarly
lacked the C-terminal region comprising amino acids 376-425.
chCAF-1p48-(1-375) exhibited the binding activity but
chCAF-1p48-(1-328) did not revealed that a lack or disruption of
two C-terminal WD repeats causes loss of the binding ability. Second,
the finding that
chCAF-1p48-(139-425) possessed the binding
activity but
chCAF-1p48-(181-425) had lost it revealed that a lack
or disruption of two N-terminal WD repeats results in loss of the
binding ability. Third, the finding that the two mutants,
chCAF-1p48-(139-425) and
chCAF-1p48-(55-375), exhibited the
binding activity but
chCAF-1p48-(139-375) did not revealed that the
simultaneous lack of an N-terminal WD repeat and a C-terminal one also
causes loss of the binding ability. Taken together our results indicate
that among the seven WD repeats of chCAF-1p48, two N-terminal, two
C-terminal, or one N-terminal and one C-terminal, one is necessary for
the in vitro interaction of the p48 subunit with
chHDAC-2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-propeller structure, wherein each of the seven "blades"
contains the amino acid residues of one WD repeat unit and probably
function as a common platform for the protein-protein interaction
involved in chromatin metabolism. At least two N-terminal WD repeats
and two C-terminal WD repeats, respectively, are predominantly involved
in maintaining the propeller structure. Deletion of the two N-terminal
WD repeats with the shorter region preceding the second FD dipeptide
motif or that of the two C-terminal WD repeats with the shorter region
preceding the sixth WD dipeptide motif causes destruction of the proper surface loops and turns. This structural change does not allow the
interaction of chCAF-1p48 with chHDAC-2, probably with BD1 and/or BD2.
Similarly, simultaneous deletion of the first N-terminal WD repeat with
the shorter region preceding the second FD dipeptide motif and of the
last C-terminal WN dipeptide motif with the shorter region preceding
the motif does not allow the propeller structure to be maintained
properly, resulting in loss of the binding activity.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. C. M. Tiree for the pAS.1 plasmid.
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FOOTNOTES |
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* This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF097750.
To whom correspondence should be addressed. Tel: 81-985-85-3127;
Fax: 81-985-85-6503; E-mail: tnakayam{at}post1.miyazaki-med.ac.jp.
2 Y. Takami, H. Kikuchi, and T. Nakayama, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
CAF-1, chromatin
assembly factor-1;
ch, chicken;
GST, glutathione
S-transferase;
HDAC, histone deacetylase;
PCR, polymerase
chain reaction;
HA, hemagglutinin;
PAGE, polyacrylamide gel
electrophoresis;
IPTG, isopropyl
-D-thiogalactopyranoside;
BSA, bovine serum
albumin.
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REFERENCES |
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