Abstract To better understanding of DNA replicating-coupled chromatin assembly and transcription regulation, we cloned and sequenced cDNA encoding the chicken p46 polypeptide, chp46, homologous to the p48 subunit of chicken chromatin assembly factor-1, chCAF-1p48. The cDNA encoding a protein consists of 424 amino acids including a putative initiation Met, is a member of the WD protein family, with seven WD repeat motifs, and exhibits 90.3% identity to chCAF-1p48, and 94.3% identity to the human and mouse p46 polypeptides. The p46 polypeptide fusion protein were synthesized by in vitro translation system and expressed in Escherichia coli under induction by 50 µM IPTG and single step purified with glutathione-agarose beads, showed that GST-tagged protein of approximately 72 kDa, were dramatically synthesis in Escherichia coli BL-21 cells. The in vitro experiment established that chp46 interacts with chicken histones, chHDAC-1, and chHAT-1. The in vitro immunoprecipitation experiment, involving truncated mutants of chp46, revealed not only that two regions comprising amino acids 33-179 and 375-404 are necessary for its binding to H2B, but also that two regions comprising amino acids 1-32 and 405-424 are necessary for its binding to H4. Furthermore, the GST pulldown affinity assay, involving truncated mutants of chp46, revealed that a region comprising amino acids 359-404 binds to chHAT-1 in vitro. Taken together, these results indicate not only that chp46 should participate differentially in a number of DNA-utilizing processes through interactions of its distinct regions with histones and chHAT-1, but also that the proper propeller structure of chp46 is not necessary for its interaction with chHAT-1

Abdul Rauf Patong

Ahyar Ahmad

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A. Ahmad & A.R. Patong 
 
Proceeding of The  International Seminar on Chemistry 2008 (pp. 611-615) 
Jatinangor, 30-31 October 2008  
 
 611 
Functional interaction of chicken p46 polypeptide with histones, histone 
deacetylase-1 and histone acethyl transferase-1 
 
Ahyar Ahmad*, Abdul Rauf Patong 
 
Department of Chemistry, Hasanuddin University, Makassar, 90245, Indonesia,  
*e-mail: ahyar1@yahoo.com 
 
Abstract 
 
To better understanding of DNA replicating-coupled chromatin assembly and transcription 
regulation, we cloned and sequenced cDNA encoding the chicken p46 polypeptide, chp46, 
homologous to the p48 subunit of chicken chromatin assembly factor-1, chCAF-1p48. The cDNA 
encoding a protein consists of 424 amino acids including a putative initiation Met, is a member of the 
WD protein family, with seven WD repeat motifs, and exhibits 90.3% identity to chCAF-1p48, and 
94.3% identity to the human and mouse p46 polypeptides. The p46 polypeptide fusion protein were 
synthesized by in vitro translation system and expressed in Escherichia coli under induction by 50 
µM IPTG and single step purified with glutathione-agarose beads, showed that GST-tagged protein of 
approximately 72 kDa, were dramatically synthesis in Escherichia coli BL-21 cells. The in vitro 
experiment established that chp46 interacts with chicken histones, chHDAC-1, and chHAT-1. The in 
vitro immunoprecipitation experiment, involving truncated mutants of chp46, revealed not only that 
two regions comprising amino acids 33-179 and 375-404 are necessary for its binding to H2B, but 
also that two regions comprising amino acids 1-32 and 405-424 are necessary for its binding to H4. 
Furthermore, the GST pulldown affinity assay, involving truncated mutants of chp46, revealed that a 
region comprising amino acids 359-404 binds to chHAT-1 in vitro. Taken together, these results 
indicate not only that chp46 should participate differentially in a number of DNA-utilizing processes 
through interactions of its distinct regions with histones and chHAT-1, but also that the proper 
propeller structure of chp46 is not necessary for its interaction with chHAT-1.  
 
Keywords: Chromatin, immunoprecipitation experiment, histones, propeller structure  
 
 
Introduction 
 
Alterations in chromatin structure are preferentially 
involved in the regulation of cell functions, including 
gene expression in eukariotes. In recent years, 
knowledge concerning the characteristics of  the p48 
subunit of CAF-1 (CAF-1p48), which has seven WD 
repeat motifs and is a member of the WD protein 
family, in numerous DNA-utilizing processes has been 
accumulated (1). CAF-1p48 was identified as a 
polypeptide that is tightly associated with the catalytic 
subunit of human histone deacetylase-1 and 2 
(HDAC-1 and -2) (2). On the other hand, the p46 
polypeptide (p46), with seven WD (Trp-Asp) repeat 
motifs, is a member of the WD protein family and is 
originally characterized as an Rb-binding protein 
(RbAp46). We have found that chicken CAF-1p48, 
chCAF-1p48, interacts with chHDAC-1 and 2 in vivo, 
in an immunoprecipitation experiment followed by 
Western blotting (3). Furthermore, the GST pulldown 
affinity assay, involving deletion mutants of both 
chCAF-1p48 and chHDAC-2, revealed not only that 
chCAF-1p48 binds to two regions of chHDAC-2 
comprising amino acids 82-180 and 245-314, 
respectively, but also 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 in 
vitro interaction. On the other hand, the p46 
polypeptide (p46), which is a CAF-1p48 homolog, 
together with the latter, is contained in repressor 
complexes with HDAC-1 and -2, and mSin3 (the 
mammalian homolog of Sin3), Rb (the retinoblastoma 
protein), or Mi2 plus MeCP2  to repress transcription 
(4).  
In this study we cloned cDNA encoding the 
chicken p46, chp46, comprising 424 amino acids 
including a putative initiation Met, exhibiting 90.3% 
homology to chCAF-1p48, and carrying seven WD 
repeat motifs. The p46 polypeptide fusion protein 
were synthesized in Escherichia coli with induction 
by 50 µM IPTG and single step purified with 
glutathione-agarose, showed that protein of 
approximately 72 kDa, were dramatically synthesis in 
Escherichia coli BL-21 cells. The in vitro experiment 
established that chp46 interacts with chicken histones, 
chHDAC-1, and chHAT-1.  
The in vitro immunoprecipitation experiment, 
involving truncated mutants of chp46, revealed not 
only that two regions comprising amino acids 33-179 
and 375-404 are necessary for its binding to H2B, but 
also that two regions comprising amino acids 1-32 and 
405-424 are necessary for its binding to H4. The GST 
pulldown affinity assay revealed not only that chp46, 
as well as chCAF-1p48, associates with chHAT-1, but 
ISBN 978-979-18962-0-7 
 
A. Ahmad & A.R. Patong 
 
Proceeding of The  International Seminar on Chemistry 2008 (pp. 611-615) 
Jatinangor, 30-31 October 2008  
 
 612 
also that a C-terminal region, comprising amino acids 
359-404 is essential for this interaction. These results 
suggest not only that the proper propeller structure of 
chp46, probably due to its WD repeat motifs, should 
not be necessary for its in vitro interaction with 
chHAT-1, but also that chp46 should be involved in a 
number of DNA-utilizing processes in distinct 
manners.  
 
Materials and Methods 
 
Materials  
 
pBluescript II SK(-) and pCite4a(-) were purchased 
from Stratagene and Novagen, respectively. pGEX-
2TK plasmid and glutathione-agarose beads were a 
product of Amersham Pharmacia Biotech. A single 
Tube Protein
TM
 System 3 were purchased from 
Novagen. The anti-HA and anti-FLAG M2 beads 
were purchased from Santa Cruz, Biotech. Inc.  and 
Eastman Kodak Co., respectively. 
 
Cloning and sequencing of cDNA encoding chp46  
 
A PCR product of 435 bp, corresponding to a part of 
the coding region of hup46, was first prepared from 
chicken DT40 cDNA, using sense and antisense 
oligonucleotide primers containing sequences 5'-
CTGATGATCAGAAACTTATGATATGGG-3' and 
5'-AGCAAATGACCCAAG  
GTTCATTGGG-3', respectively, which were 
constructed based on the amino acid sequences 
(DDQKLMIWD and PNEPWVICS) in hup46 
deduced from its cDNA (5). To obtain full-length 
chp46 cDNA, using the resultant PCR product as a 
probe, we screened the DT40 lambda ZAP II cDNA 
library (3) as described (6). The entire nucleotide 
sequences of both strands of the largest cDNA insert 
were sequenced by the dye terminator method 
(Applied Biosystems Division, Perkin Elmer).  
 
Plasmid construction  
 
We constructed the pCitep46 and/or pCiteFLAGp46 
plasmid carrying the gene encoding chp46 as 
described (3,7). First a sense primer containing NcoI 
(5') with the sequence 5'-CCATGGCGAGTA 
AGGAAGTGCTGGAGG-3' and an antisense primer 
containing SalI (3') with the sequence 5'-
CTCGAGTTACTGTCCTTGACCTTCCAG-3' were 
constructed. Next we prepared the DNA fragment 
encoding the full-length coding region of chp46 by 
PCR using the parental plasmid (pB(II)SKp46) 
carrying the full-length chp46 cDNA as a template 
with these primers, followed by digestion with NcoI 
plus SalI. We replaced the resultant NcoI/SalI 
fragment with the NcoI/SalI fragment of the 
pCiteFLAGp48 plasmid carrying the gene encoding 
chCAF-1p48 (8). The deletion mutants of chp46 were 
obtained as described (8). We also use pCiteHAH2A, 
pCiteHAH2B, pCiteHAH3, pCiteHAH4, 
pCiteHAHDAC-1 and pCiteHAp60 were obtained as 
described (3, 8). Expression of the chp46 in E. coli 
and by in vitro translation system E. coli BL-21 cells 
were transformed with pGEX-2TKchp46 and pGEX-
2TKchHAT-1, respectively, harboring the full-length 
chp46 and chHAT-1 cDNAs, and grown to A600nm = 
~0.2 in 400 ml of LB medium supplemented with 200 
µg/ml ampicillin. After induction by the addition of 50 
µM isopropyl ß-D-thiogalactopyranoside (IPTG) 
overnight at 20oC, the cells were collected by 
centrifugation and purification by glutathione-agarose 
beads, essentially as described (3).  
 
Production of [
35
S]Met-labeled proteins 
 
To produce [
35
S]Met-labeled FLAG-chp46 fusion 
protein, chp46 polypeptide, CAF-1p48, CAF-1p60,  
HDAC-1, HAT-1, and histone H4 a Single Tube 
ProteinTM System 3 (Novagen) was used with a 
following modification. The reaction mixture was set 
up at room temperature: 1 µl DNA template (free 
from RNase, Mg
+2
 and salt) plus 1 µl nuclease-free 
water and 8 µl STP3 transcription kit. It was mixed 
by stirring with the pipet tip and incubated at 30
o
C for 
30 minute. Next, 5 µl  (15 mCi/ml) 
35
S-Met 
(Amersham Pharmacia Biotech.) and 30 µl in vitro 
STP3 translation kit were added to the transcription 
reaction products as mentioned above. Reactions 
were typically performed in 50 µl volumes by adding 
5 µl nuclease-free water and translation reactions 
were incubate at 30
o
C about 1 hr. After the reaction 
was completed, the final products were saved as 
[
35
S]Met-labeled protein and stored at –20
o
C until 
used.  
 
Immunoprecipitation experiment and GST pulldown 
affinity assay  
 
The in vitro immunoprecipitation experiment was 
performed with 5 µl of  [
35
S]Met-labeled chp46 
polypeptide, CAF-1p48, CAF-1p60,  HDAC-1, HAT-
1, and histone H4, 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 standing for 60 min, 20 µl of 
the reaction mixture was removed as an input sample, 
and the remaining mixture was added to 20 µl of anti-
HA beads (Santa Cruz, Biotech. Inc.) or anti-FLAG 
M2 beads (Eastman Kodak Co.), followed by gentle 
rotation for 60 min at 4
o
C. The affinity beads were 
collected by centrifugation at 9,500 X g for 10 sec, 
and then washed with 1 ml of the bead-binding buffer 
containing 0.1% 4-(2-aminoethyl)-benzosulfonyl 
fluoride (AEBSF) three times.  The beads were 
suspended in 30 µl of 2X SDS sample buffer and then 
A. Ahmad & A.R. Patong 
 
Proceeding of The  International Seminar on Chemistry 2008 (pp. 611-615) 
Jatinangor, 30-31 October 2008  
 
 613 
boiled for 5 min.  Aliquots (15 µl) of the resultant 
eluates were analyzed by 10 or 12% SDS-PAGE, and 
then the gels were washed with a fluorographic 
reagent (Amersham Pharmacia Biotech), dried, and 
subjected to fluorography. The GST pulldown affinity 
assay essentially as described (3), in 200 µl of the 
bead-binding buffer.  
 
Results and Discussion 
 
Cloning of cDNA encoding chp46  
 
To study the characteristics of chp46, we cloned and 
sequenced cDNA encoding it. Based on conserved 
amino acid sequences in hup46 and mouse p46 
(mop46) (5), we prepared the 435 bp PCR fragment, 
corresponding to a part of cDNAs encoding hup46 
and mop46, by PCR using cDNAs from DT40 cells. 
Sequence analysis of the largest cDNA insert of 1799 
bp among them revealed that the clone contained both 
an initiation codon and a 3' poly(A) tail, and also 
appeared to contain the full-length chp46 cDNA 
sequence. The amino acid sequence deduced from the 
nucleotide sequence, comprising 424 residues 
including a putative initiation Met. This chicken 
polypeptide exhibits 94.3% identity in amino acid 
sequences to hup46 and mop46, and 90.4% identity to 
chCAF-1p48.  
 
Expression of recombinant chp46 by in vitro 
translation system and in E. coli  
 
First, to construct a chimeric plasmid, pGEX-
2TKchp46, expressing the GST-chp46 fusion protein, 
chp46 cDNA was subcloned into the pGEX-2TK 
plasmid in frame. GST fusion proteins were 
synthesized in E. coli, extracted, and purified 
essentially as described previously (3). As shown in 
Figure 1A, the electrophoretic patterns on SDS-PAGE 
of whole cell lysates before and after induction with 
IPTG revealed that GST-chp46 fusion proteins of 
approximately 72 kD were dramatically accumulated 
in E. coli BL-21 cells containing the pGEX-
2TKchp46 plasmid. In lane 1 and 2, whole cell lysate 
of BL-21 cells containing the pGEX-2TKchp46 and 
pGEX-2TKchHAT-1 plasmid without induction by 
IPTG, lane 3 and 4, lysate of BL-21 cells containing 
the pGEX-2TKchp46 and pGEX-2TKchHAT-1 
plasmid with induction by 50 µM IPTG, lane 5 and 6, 
complex beads containing chp46 and HAT-1, lane 7 
and 8, chp46 and chHAT-1 fraction purified with 
glutathione-agarose beads. In this data revealed, the 
GST-chp46 fusion proteins were purified to more than 
95% homogeneity, using glutathione-agarose beads 
(see lane 7 in Figure 1A). Furthermore, chp46 also 
have high and stable expression level by in vitro 
transcription and translation system (Figure 1B) using 
a single Tube Protein
TM
 kit (Novagen).   
 
 
Figure 1 Expression followed SDS gel 
electrophoretic patterns of GST-chp46 (A) 
and FLAG-chp46 fusion protein (B).  
 
Interaction of chp46 with histones, HDAC-1, CAF-
1p60 and HAT-1  
 
We have revealed that chCAF-1p48 binds to 
chHDACs (3) and chHAT-1 (10) in vivo and in vitro. 
On the other hand, it has been reported that a 
heterodimer of hup46 and HAT-1 is involved in the 
chemical modification of core histones with acetyl 
groups, especially in the acetylation of Lys-5 and Lys-
12 of histone H4 (11). Therefore, to determine 
whether or not chp46 bound to these chromatin-
related proteins, such as histone H4, HDAC-1 and 
CAF-1p60, we carried out an in vitro 
immunoprecipitation experiment, using [
35
S]Met-
labeled FLAG-tagged chp46 and [
35
S]Met-labeled 
HA-tagged histones, HDAC-1 and CAF-1p60. As 
shown in Figure 3A and 3C, chp46 bound to histone 
H4 and HDAC-1 but not to CAF-1p60. To determine 
whether or not the chp46 polypeptide, together with 
chCAF-1p48 as a positive control, bound also to 
HAT-1, the GST pulldown affinity assay was carried 
out. The chp46 was translated in vitro in the presence 
of [
35
S]Met, and then assayed its ability to interact 
with HAT-1. As shown in Figure 3B and 3C, the 
chp46 bound to GST-HAT-1 fusion protein, as well as 
the binding activity of CAF-1p48 to HDAC-2 and 
HAT-1 as a positive control, whereas under the same 
conditions ß -galactosidase as a negative control,  did 
not bind to it.  
A. Ahmad & A.R. Patong 
 
Proceeding of The  International Seminar on Chemistry 2008 (pp. 611-615) 
Jatinangor, 30-31 October 2008  
 
 614 
 
 
Figure 2 In vitro interaction of chp46 with histone 
H4, HDAC-1, CAF-1p60 and HAT-1. 
 
Two regions, two other regions and one distinct 
region of chp46 are necessary for the interaction with 
H2B, H4 and HAT-1 Next, we determined the 
region(s) of chp46 necessary for its binding ability as 
to H2B, H4 and HAT-1, because the interaction of 
p46 with one or more of these proteins could be 
thought to play a key role in the DNA-utilizing 
processes. We constructed C-terminal and N-terminal 
truncated mutants of FLAG-tagged chp46, and studied 
their in vitro interaction with the H2B, H4 and HAT-1 
protein. An in vitro immunoprecipitation experiment, 
involving truncated mutants of chp46, revealed not 
only that two regions comprising amino acids 33-179 
and 375-404 are necessary for its binding to H2B, but 
also that two regions comprising amino acids 1-32 and 
405-424 are necessary for its binding to H4 (Figure 
3). Furthermore, a GST pull down affinity assay, 
involving truncated mutants of chp46, revealed that a 
region comprising amino acids 375-404 bind to HAT-
1 in vitro (Figure 3).  
These results indicate not only that chp46 should 
participate differentially in a number of DNA-utilizing 
processes through the interaction of its distinct regions 
with H2B, H4 and HAT-1, but also that the propeller 
structure of chp46, based on the WD repeat motifs, is 
not necessary for its interaction with HAT-1. Very 
recently, that sucrose gradient fractionation of semi-
purified tagged Asf1-complexes showed the presence 
of HAT-1, RbAp48 is holomog of chp46 and histones 
H3/H4 at 5-6S fractions in the complexes (12). These 
findings suggest the possible involvement of HAT-1 
in regulating cytosolic H3/H4 pool mediated by Asf1-
containing cytosolic H3/H4 pre-deposition complex. 
 
 
 
 
 
 
 
 
 
 
Figure 3 Essential regions of the chp46 polypeptide 
for interaction with histone H2B and H4, 
and HAT-1.  
 
Conclusions 
 
In summary, chp46 should be involved in numerous 
DNA-utilizing processes in different manners for its 
binding to H2B, H4 and HAT-1, and the proper 
propeller structure of chp46 is not necessary for the 
interaction with HAT-1. The overall picture 
concerning the in vitro and in vivo interaction of 
chp46 with chHDACs and chHAT-1 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 for a number of different 
genomic loci, including genes encoding core histones. 
 
References  
 
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Jatinangor, 30-31 October 2008  
 
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