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J. Biol. Chem., Vol. 276, Issue 31, 28933-28938, August 3, 2001
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,,,¶, and
From the National Laboratory of Medical Molecular
Biology, Institute of Basic Medical Sciences, Chinese Academy of
Medical Sciences, and Peking Union Medical College, Chinese National
Human Genome Center, Beijing 100005, China and the
§ Institute of Molecular Biology, The University of Hong
Kong, Pokfulam Road, Hong Kong SAR, China
Received for publication, May 1, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Host cell factor 1 (HCF-1) is a
cellular transcriptional coactivator which coordinates the assembly of
enhancer complex through direct interactions with viral and cellular
trans-activators such as VP16, Oct-1, LZIP, and GA-binding protein.
These interactions are mediated by the Transcriptional regulation in eukaryotes involves a complicated
interplay between activators, repressors, basal transcription factors,
and chromatin (1). To coordinate the orderly assembly of
multicomponent enhancer complexes onto DNA targets, cells have evolved
specialized groups of regulators that interact with multiple transcription factors. Host cell factor 1 (HCF-1)1 represents one of
these multipartner transcriptional regulators and it functions as a
coordinator in the assembly of enhanceosome on viral and cellular DNAs
(2-5).
HCF-1 was first identified through its association with herpes simplex
virus transactivator VP16, a key regulator of lytic infection (6, 7).
The formation of the VP16-HCF-1 complex facilitates the nuclear
accumulation of VP16 (8) and the recruitment of the POU-homeodomain
transcription factor Oct-1 (9), leading to activation of viral
immediate early genes. In addition to VP16 and Oct-1, HCF-1 also
directly interacts with GA-binding protein, another cellular
transcription factor critically involved in the regulation of immediate
early gene expression (4). One notable mechanism for the regulation of
HCF-1 activity is through subcellular localization. Thus, HCF-1 is
ambiently found in sensory neurons and nuclear translocation leads to
reactivation of the virus from latency (10).
HCF-1 is expressed as a 230-kDa precursor, which is autocatalytically
cleaved into an array of tightly associated polypeptides ranging from
50 to 150 kDa (4, 6, 11-14). The interaction of HCF-1 with VP16 is
mediated by the first 380 residues of HCF-1 (2, 15). This N-terminal
domain, which consists of six kelch repeats, sufficiently stabilizes
VP16-induced complex with Oct-1 and activates transcription (8, 15). In
addition, this domain has an essential role in cell proliferation (16).
The kelch repeats represent an evolutionarily conserved module for
protein-protein interaction and they form a tertiary structure called
One additional cellular target of the A second human HCF-like protein termed HCF-2 has also been identified
(26). Although HCF-1 and HCF-2 share extensive sequence homologies
(~65% identical residues) throughout the In this study, we describe the identification of a novel ubiquitously
expressed human HCF-like protein, designated HCLP-1. HCLP-1 is smaller
in size than both HCF-1 and HCF-2, and it consists almost entirely of
one Plasmid Construction--
Plasmid pBDHCLP-1 expressing Gal4
DNA-binding domain (BD)-HCLP-1 was constructed by in-frame insertion
into plasmid pAS2-1 (CLONTECH) of a cDNA
(GenBankTM AF113131) encoding the full-length 406 amino
acids of HCLP-1. Gal4BD-HCLPN and Gal4BD-HCLPC express truncated
Gal4BD-HCLP-1 corresponding to amino acids 1-331 and 298-406,
respectively. pADLZIP was obtained by cloning the complete LZIP coding
sequence into Gal4 activation domain (AD) plasmid pGADGH
(CLONTECH). pADCREB, pADATF4, pADATF6, pADcFOS,
pADcJUN, and pADCEBP contain the complete coding regions of human CREB,
ATF4, ATF6, c-Fos, c-Jun, and CEBP-
pBD-HCFN380 expresses the wild-type version of the HCF-1
Northern Blotting--
Northern blot analysis was performed with
a 32P-labeled 1005-base pair random-primed fragment
generated by polymerase chain reaction amplification of HCLP-1 cDNA
(corresponding to nucleotides 248-1252). Blots of poly(A)+
RNAs from human tissues and cancer cell lines were probed as recommended by CLONTECH.
Yeast Two-hybrid Analysis--
Gal4-based yeast two-hybrid
assays were performed in strain SFY526 as previously described (30,
31).
Western Blotting and Immunofluorescence Microscopy--
Western
blot analysis and confocal laser-scanning immunofluorescence microscopy
were carried out as detailed elsewhere (31, 32).
Cell Transfection and Luciferase Assays--
HeLa cells in
12-well plates were transfected using 3 µl of LipofectAMINE
2000TM (Life Technologies) per well. Cell extracts were
prepared in a commercial lysis buffer (Promega). Luciferase reporter
assays were performed according to the manufacturer's instructions
(Promega) and luciferase activity was measured with a LB9507
luminometer (EG&G).
Protein Affinity Binding--
Recombinant proteins GST,
GST-HCLP-1, His-taged TRX, and His-tagged TRX-LZIP were expressed from
Escherichia coli and purified as per the manufacturers'
protocols (Amersham Pharmacia Biotech and Novagen). Equal amounts of
TRX and TRX-LZIP were loaded onto either GST- or GST-HCLP-1-bound
glutathione-Sepharose (Amersham Pharmacia Biotech). The resins were
washed with three changes of 1 × phosphate-buffered saline. Bound
proteins were solubilized in SDS-gel loading buffer (50 mM
Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.001%
bromphenol blue, and 10% glycerol).
Gel Mobility Shift Assay--
Probe labeling and gel
mobility shift assays were performed as previously described (30, 32,
33). Oligonucleotides used to produce the CRE motif are 5'-AGCTT
GGTGACGCGGATCCGGTGACGCA-3' and 5'-AGCTTGCGTCACCGGATCCGCGTCACCA-3'.
Identification of HCLP-1 as a Novel HCF-like Protein--
To mine
for novel human kelch repeat proteins related to HCF, we searched the
current nucleotide data bases via the tblastn server
(www.ncbi.nlm.nih.gov) for expressed sequence tags homologous to the
coding sequences of the
HCLP-1 is a 406-amino acid protein composed almost entirely of six
internally repeated sequences of 45~71 residues (Fig.
1A). These kelch repeats are
expected to form four-stranded, anti-parallel
To determine the expression patterns of HCLP-1 mRNA, we analyzed
poly(A)+ RNA from various human tissues and cancer cell
lines for the presence of transcripts that hybridize to an HCLP-1
cDNA probe. HCLP-1-specific transcripts of ~2 kilobases and/or
1.8 kilobases in size were detected in all tested human tissues and
cancer cell lines (Fig. 2). Thus, HCLP-1
is a novel and ubiquitously expressed HCLP-1 Associates with LZIP but Not with VP16--
HCF-1 has
been shown to associate with VP16 (6, 7) and LZIP (18, 19). These
interactions are mediated by the
We addressed this issue by using a Gal4-based yeast two-hybrid assay
(Fig. 3A). In this experiment,
we used two irrelevant pairs of proteins (columns 1 and
6) as positive controls. As previously described, the Tax
oncoprotein of human T-cell leukemia virus type 1 can self-associate
(30). Therefore, Tax fused to Gal4BD interacts potently with Tax fused
to Gal4AD (Fig. 3A, column 1). Likewise, Tax
binds to a cellular partner called Int-6 (34), albeit with lower
affinity (column 6). Next we verified that the
To define the specificity of the interaction between HCLP-1 and LZIP,
we tested whether HCLP-1 could distinguish LZIP from other cellular
bZIP transcription factors. Seven bZIP proteins fused to Gal4AD were
queried in parallel for binding to Gal4BD-HCLP-1 (Fig. 3A,
columns 4 and 8-13). Notably, HCLP-1 interacts
with LZIP only but not with any of the other six bZIP factors, namely CREB, ATF4, ATF6, c-Jun, c-Fos, and CEBP-
The observed interaction between HCLP-1 and LZIP in yeast cells does
not exclude that it could be mediated through another yeast protein. To
challenge this possibility, we performed in vitro pull-down
assays with GST-HCLP-1 and His-tagged TRX-LZIP purified from E. coli. In agreement with a direct physical contact between HCLP-1
and LZIP, Fig. 3B verified that TRX-LZIP bound to GST-HCLP-1
(lanes 3 and 5; also compare lane 3 to
lane 2), but not to GST alone (lanes 1 and
4).
In addition to the in vivo yeast two-hybrid analysis and
in vitro protein affinity binding assays, confocal
laser-scanning immunofluorescence microscopy was also performed to
further probe the interaction between HCLP-1 and LZIP within human
cells. We generated anti-HCLP-1 serum (
We carried out additional yeast two-hybrid interactive assays to define
the functional domains of LZIP and HCLP-1 (Fig.
4). Various truncated mutants of LZIP
were constructed and their HCLP-1 binding activities were individually
assessed. We observed that the mutant LZIP-M4 induced LacZ reporter
expression as potently as the wild type LZIP (Fig. 4A). It
is noteworthy that this mutant with amino acids 109-315 contains an
intact bZIP region. By contrast, neither the LZIP-M2 mutant comprising
amino acids 1-109 nor the LZIP-M5 mutant comprising amino acids
235-371 can interact with HCLP-1. One interpretation for these data is
that the bZIP region (amino acids 152-220) is required for interaction
with HCLP-1. We noted that mutants LZIP-M3 and LZIP-M4, both lacking
the HBM motif, still interact with HCLP-1. These results implicate that the HBM region, which mediates the association between HCF1 and LZIP,
might be dispensable for the interaction between HCLP-1 and LZIP.
On the other hand, we were unable to narrow down a minimal LZIP-binding
domain in HCLP-1. Neither the N-terminal (amino acids 1-331) nor the
C-terminal (amino acids 298-406) portion of HCLP-1 is sufficient to
bind LZIP (Fig. 4B). Thus, the entire HCLP-1 Inhibits LZIP-dependent Transcription in
Mammalian Cells--
Previously, we and others have shown that LZIP is
a nuclear CRE-activating factor (19, 24). The finding that HCLP-1
interacts with LZIP predicts a functional impact of the former on the
transcriptional activity of the latter. To explore this, we expressed
epitope-tagged HCLP-1 protein in HeLa cells and assessed the effects of
HCLP-1 on Gal4BD-LZIP activation of luciferase expression driven by
Gal4-binding enhancer elements. The expression of HCLP-1 tagged with a
V5 epitope was verified by Western blot analysis, and an ~50-kDa
species recognized by the anti-V5 antibody was specifically detected
from the pcHCLP-1-transfected cells (Fig.
5A, compare lane 2 with lane 1). When we increased the expression of HCLP-1,
the Gal4BD-LZIP-dependent transcription of luciferase
reporter decreased progressively (Fig. 5B; GAL4-LZIP,
To further characterize this inhibitory effect, the HCLP-1-expressing
plasmid (pcHCLP-1) paired with either an LZIP-expressing plasmid or an
empty vector was transfected into HeLa cells, and the activation of
CRE-dependent reporter expression was assayed. Again, we
observed that increased expression of HCLP-1 reproducibly repressed
cell-endogenous (Fig. 5C;
Above, we showed a direct interaction of HCLP-1 with LZIP (Fig. 4) and
the functional consequence of this interaction (Fig. 5). One salient
point of our findings is that the HCLP-1-binding domain in LZIP
contains an intact bZIP DNA-binding region. This prompts us to ask
whether HCLP-1 would inhibit the DNA binding activity of LZIP. We and
others have shown that LZIP binds canonical CRE sites in
vitro and in vivo (19, 24). We therefore assessed the
influence of HCLP-1 on LZIP binding to CREs in an electrophoretic mobility shift assay (Fig. 6). Various
combinations of recombinant proteins produced from E. coli
were incubated with 32P-labeled CRE oligonucleotides and
the protein-DNA complex was analyzed on a nondenaturing PAGE gel. An
LZIP-specific retarded band (Fig. 6; highlighted by an
arrow) was observed when TRX-LZIP was mixed with labeled
probe containing CRE sites (Fig. 6, lane 2). When TRX was
added, this band was not seen (Fig. 6, lane 1). The addition
of a 50-fold excess of unlabeled CRE oligonucleotides depleted the
activity to bind with the labeled probe (Fig. 6, lane 3),
lending further support to the specificity of the band. This
LZIP-specific signal diminished progressively when increased amounts of
GST-HCLP-1 were added to the reaction (Fig. 6, lanes 7-9).
The diminution was caused by HCLP-1 but not by GST, since the binding
activity was unaffected by the addition of GST (Fig. 6, lanes
4-6). Notably, GST-HCLP-1 alone was unable to bind the CRE
oligonucleotides (Fig. 6, lane 10). Thus, HCLP-1 inhibits LZIP-mediated transcription through the modulation of its DNA binding
activity.
Here we report on the identification and characterization of human
HCLP-1, a novel and ubiquitously expressed HCF-1 is a unique transcriptional coactivator which functions as a
coordinator in the assembly of multicomponent enhanceosomes onto DNA
targets (2-5). HCF-1 has no histone acetyltransferase activity and a
major mechanism for its regulation of transcription is through
protein-protein interaction with viral and cellular transactivators.
Thus, HCF-1 associates with VP16, Oct-1, LZIP, and GABP. In one
perspective, the kelch In this context, it is not surprising that the HCF-like HCLP-1 protein
identified in this study interacts selectively with LZIP but not with
VP16. Although LZIP and VP16 share a common tetrapeptide HBM recognized
by HCF-1, they exhibit dramatically different sensitivities to HCF-1
point mutants in the HCLP-1 is smaller in size and the entire molecule consists of a
six-bladed Both HCF-1 and LZIP have been implicated in cell proliferation. A
proline-to-serine point mutant at position 134 in the 
-propeller domain comprising
the first 380 residues of HCF-1 with six kelch repeats. Here we
describe the identification and characterization of a novel HCF-like
kelch repeat protein, designated HCLP-1. HCLP-1 is a ubiquitously
expressed nuclear protein which is composed almost entirely of a
six-bladed -propeller. HCLP-1 selectively interacts with LZIP but
not with VP16. The physical interaction between HCLP-1 and LZIP leads
to the repression of the LZIP-dependent transcription. The
HCLP-1-binding domain of LZIP maps to residues 109-315, which contain
the bZIP DNA-binding motif. Electrophoretic mobility shift assay
demonstrates that HCLP-1 indeed interferes with the binding of LZIP to
its DNA target. Thus, HCLP-1 serves a transcriptional co-repressor function mediated through its inhibitory interaction with the LZIP
transcription factor. Our findings suggest a new mechanism for
transcriptional regulation by HCF-like proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-propeller, which has been found in many different polypeptide
contexts (17). In HCF-1, gross alterations in the six-bladed
-propeller structure disrupt both the VP16-binding and the
cell-cycle progression activities (9, 15, 16).
-propeller domain of HCF-1 is
a bZIP transcription factor known as LZIP or Luman (18, 19). LZIP is
the human ortholog of the fruit fly BBF2/dCREB-A protein, which has
been shown to activate Drosophila fat body- and mammalian
liver-specific transcription (20, 21). The subportion of LZIP that
contains its bZIP domain is highly homologous to counterparts in other
members of the bZIP family of transcription factors including CREB,
CREM, and ATF6 (18, 19, 22). LZIP binds to a canonical cAMP-responsive
element (CRE) and activates CRE-dependent transcription
(19, 22-24). While the cellular targets of LZIP are poorly understood,
LZIP has been implicated in cell proliferation and it acts as a binding
partner and transforming cofactor of the hepatitis C virus core
protein, a viral protein with oncogenic potential (24). HCF-1
recognizes a tetrapeptide HCF-binding motif (HBM) shared by LZIP, VP16,
and another HCF-1 partner termed Zhangfei (18, 23, 25). However, VP16
and LZIP show drastically different sensitivities to individual HCF-1 point mutants, suggesting that other structural motifs in the -propeller may also contribute to the specificity of the binding (9). HCF-1 functions as a coactivator in the context of LZIP, and the
physical interaction with HCF-1 is necessary for coordinated assembly
of enhanceosome and optimal transcriptional activation by LZIP
(5).
-propeller and
self-association domains, HCF-2 is bound poorly by VP16 and LZIP,
indicating the fine specificity of transcriptional control. Experiments
with HCF-1 and HCF-2 chimeras have localized determinants for this
selectivity to the fifth and sixth blades of the -propeller (26).
Interestingly, a Caenorhabditis elegans HCF, which is structurally similar to human HCF-2 and is more distantly related to
human HCF-1, conserves the abilities to stabilize the VP16-induced enhancer complex and to promote cell proliferation (27).
-propeller domain. In contrast to HCF-1 and HCF-2, HCLP-1
associates with LZIP but not with VP16. More importantly, the
interaction with HCLP-1 leads to inhibition rather than activation of
the LZIP-mediated transcription. Mechanistically, HCLP-1 modulates the
CRE binding activity of LZIP by targeting residues 109-315, which
contain the bZIP domain. Our findings suggest that HCLP-1 is a specific
transcriptional corepressor for LZIP.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, respectively. The ATF6 (28) and
CEBP- (29) cDNAs were kindly provided by Dr. Ron Prywes and Dr.
Gretchen Darlington, respectively.
-propeller domain (residues 1-380) fused to Gal4BD (a gift from Dr.
Tom Kristie). pADVP16 contains the full-length VP16 derived from herpes
simplex virus type 1 (kindly provided by Dr. Gary Hayward). pM
(CLONTECH), pGalLZ, and pGalVP16 are SV40
promoter-driven vectors expressing Gal4BD, Gal4BD-LZIP, and
Gal4BD-VP16AD, respectively. pLZm4VP16 is derived from pVP16
(CLONTECH) by inserting sequences coding for
residues 109-315 of LZIP. Prokaryotic expression vectors pGSTHCLP-1
and pET32aHisLZ for glutathione S-transferase (GST)-HCLP-1 and polyhistine-tagged thioredoxin (TRX)-LZIP were derived from pGEX-4T-1 (Amersham Pharmacia Biotech) and pET32a+ (Novagen). pcHCLP-1
and pcLZ were constructed by in-frame insertion of the full-length
HCLP-1 and LZIP, respectively, into plasmid pcDNA3.1/V5/HisB (Invitrogen). pGal-LUC (a gift from Dr. Karen Kibler) is a luciferase reporter plasmid derived from pGL3-basic (Promega). pCRE-LUC was from Stratagene.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-propeller domain of HCF-1. Further analysis
of the HCF-1-related sequences identified from this search revealed
that a group of human expressed sequence tags including AA488367,
AA167356, and AW992905 encode a single non-HCF-1 and non-HCF-2 kelch
repeat protein. A fetal brain cDNA library was then screened for
clones that match the above ESTs and a full-length human cDNA for a
novel HCF-like protein designated HCLP-1 was obtained.
-sheets that fold into
propeller-like barrel structures (17). Thus, the whole HCLP-1 molecule
will form a six-bladed -propeller. A kelch repeat is characterized
by a pair of glycines immediately preceded by four hydrophobic residues
and followed by a tryptophan (17). In HCLP-1, phenylalanine is found at
position 
1 relative to the glycine pair in five of the six repeats,
and valine or leucine is present at position 2. However, the third
and the sixth kelch repeats of HCLP-1 are relatively long. HCLP-1
shares significant homology with the HCF-1 and HCF-2 kelch domains
(Fig. 1B).
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Fig. 1.
HCLP-1 is a novel HCF-like protein.
A, alignment of kelch repeats in HCLP-1. Conserved residues
are shaded. B, alignment of HCLP-1 with
corresponding sequences in HCF-1 (SWISS-PROT accession number p51610)
and HCF-2 (GenBankTM identification number 7019405).
Compatible residues are highlighted by shading. HCLP-1
shares ~30% identical and 45% similar amino acid residues with
HCF-1 and/or HCF-2.
-propeller protein related to
HCF-1 and HCF-2.
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Fig. 2.
Expression of HCLP-1 mRNA in human
tissues and cells. The 32P-labeled HCLP-1 cDNA
probe was hybridized to poly(A)+ RNA from human tissues
(panel A) and cancer cell lines (panel B). The
same blots were stripped and hybridized subsequently with the -actin
probe (bottom).
-propeller domain of HCF-1 and a
tetrapeptide HBM motif present in both VP16 and LZIP (18, 23). Studies
with point mutants suggest that VP16 might recognize HCF-1 and HCF-2
through similar mechanisms, albeit the interaction with HCF-2 is weaker
(26). Because HCLP-1 forms a structurally related -propeller, we
asked whether HCLP-1 would also interact with LZIP and/or VP16.
-propeller
domain of HCF-1 is able to interact with both LZIP and VP16
(columns 2 and 3). In contrast, HCLP-1 can
associate only with LZIP (compare column 4 to column
7), but not with VP16 (compare column 5 to column
7). Hence, HCLP-1 can discriminate between LZIP and VP16.
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Fig. 3.
Interaction of HCLP-1 with LZIP.
A, yeast two-hybrid assay. The indicated Gal4BD and Gal4AD
fusion pairs were assayed for -galactosidase activity in the yeast
strain SFY526. Relative -galactosidase activity was expressed as
arbitrary units. pGAD424 is the expression vector for Gal4AD alone.
B, LZIP binds to immobilized GST-HCLP-1 but not to GST.
Equal amounts of bacterially expressed TRX and TRX-LZIP proteins were
loaded onto glutathione-Sepharose preincubated with 2 µg of GST or 2 µg of GST-HCLP-1. The resins were washed three times with
phosphate-buffered saline. Bound proteins were solubilized in SDS gel
loading buffer, resolved by 12% SDS-PAGE, transferred to
polyvinylidene difluoride membrane, and probed with an anti-His tag
monoclonal antibody from CLONTECH (lanes
1-3). GST and GST-HCLP-1 were stained with Coomassie Blue
(lanes 4 and 5). C, nuclear
localization of HCLP-1 and LZIP in HeLa cells. Exponentially growing
HeLa cells were fixed and stained with rabbit anti-HCLP serum -HCN
directed against the N-terminal 331 residues of HCLP-1 (panel
1), or with rabbit anti-LZIP antibody -ZN (panel 2),
or with -HCN preincubated with 1 µg of His-tagged HCLP-1
(His-HCLP-1). The -HCN and -ZN antibodies were used,
respectively, at 1:500 and 1:100 dilutions. Bar, 20 µm.
Specificity of -ZN has been verified elsewhere (24).
. Thus, HCLP-1 is a specific partner of LZIP.
-HCN) directed against
bacterially produced HCLP-1 protein containing the N-terminal 331 residues. The specificity of this antiserum was verified by
immunofluorescent staining. HeLa cells were probed with -HCN, or
preimmune serum, or -HCN neutralized with excess amount of purified
His-tagged HCLP-1 protein (His-HCLP-1). HCLP-1-specific staining was
found in the nucleus of interphase HeLa cells probed with -HCN (Fig. 3C, panel 1), but not in cells stained with
preimmune (data not shown) or neutralized (Fig. 3C,
panel 3) sera. Because both -HCN and the anti-LZIP
antibody -ZN were from rabbits, a direct co-localization of HCLP-1
and LZIP cannot be demonstrated with indirect immunofluorescence microscopy. However, when considered together with the previously documented (24) nuclear localization of LZIP (Fig. 3C,
panel 2), our data indicate that endogenous HCLP-1 and LZIP
proteins reside in the same intranuclear compartment within human
cells, consistent with an intracellular protein-protein contact between the two entities.
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Fig. 4.
Mapping of functional domains in LZIP
(A) and HCLP-1 (B). Schematic
representation indicates the fragments of LZIP or HCLP-1. The truncated
proteins were individually tested for binding to HCLP-1 or LZIP in the
yeast two-hybrid interactive assay. Minus ( ) and
plus (++) indicate the relative levels of -galactosidase
activity. HBM, bZIP, and the kelch repeats were highlighted.
-propeller structure of HCLP-1 might be necessary for interaction with LZIP.
).
By sharp contrast, the reporter expression dependent on a Gal4BD
version of VP16, which does not interact with HCLP-1 (see Fig.
3A for reference), was totally unaffected by HCLP-1 (Fig.
5B; GAL4-VP16, 
). These results correlate the protein-protein interaction with an inhibitory effect on
transcription.
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Fig. 5.
HCLP-1 protein inhibits
LZIP-dependent transcription. A, expression
of HCLP-1 in HeLa cells. Cells were mock transfected (lane
1) or transfected with pcHCLP-1 (0.1 µg; lane 2).
Western blotting was performed to verify expression of the V5-tagged
HCLP-1. The mouse monoclonal anti-V5 antibody (Invitrogen) was used at
1:1000 dilution. B, effects of HCLP-1 expression on
Gal4BD-LZIP transactivation. HeLa cells were transiently transfected
with pGalLUC (0.1 µg) plus either the Gal4-VP16-expressing plasmid
pGalVP16 (0.1 µg, ) or the Gal4-LZIP-expressing plasmid pGalLZ
(0.1 µg, ) plus the indicated amounts of pcHCLP-1. C,
effects of HCLP-1 expression on LZIP activation of CRE. HeLa cells were
transiently transfected with pCRELUC (0.1 µg) plus the indicated
amounts of pcHCLP-1 plus 0.1 µg of empty vector (0.1 µg, 
) or
expression plasmids for the indicated proteins (LZIP, 
; LZM4-VP16,
). All values represent the means of three independent
transfections, and error bars indicate S.D. from the
mean.
) and LZIP-stimulated (Fig. 5C; ) CRE-dependent luciferase activity. One
interpretation of this experiment is that HCLP-1 protein physically
binds endogenous LZIP in HeLa cells in a manner similar to its
interaction with exogenously overexpressed LZIP, leading to repression
of transcription. To assess whether the HCLP-1-binding domain of LZIP
can confer HCLP-1 responsiveness to transcription factors which do not
bind HCLP-1, we constructed a LZM4-VP16 chimera, which contains the HCLP-1-binding domain (amino acid 109-315) of LZIP and the activation domain of VP16. Interestingly, LZM4-VP16 also stimulated
CRE-dependent activity and again was repressed by HCLP-1 in
a dose-dependent manner (Fig. 5C, ). All of
this supports the notion that the interaction between HCLP-1 and LZIP
leads to the inhibition of LZIP-mediated transcription.
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Fig. 6.
HCLP-1 inhibits the DNA binding activity of
LZIP. Reactions contain bacterially expressed TRX (2 µg,
lane 1), TRX-LZIP (2 µg, lanes 2-9),
GST-HCLP-1 (0.1 nmol, lane 10). Purified GST (0, 0.1 and 0.5 nmol, lanes 4-6) and GST-HCLP-1 (0, 0.1, and 0.5 nmol,
lanes 7-9) were added individually to the reactions as
indicated on the top of the gel. A 50-fold excess of
unlabeled CRE oligonucleotides (lane 3) was used to compete
for binding. Relative intensity of bands was quantitated with the help
of a densitometer (Molecular Dynamics). Results are representative of
three independent experiments. P, free probe.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-propeller protein
related to HCF-1 and HCF-2 (Figs. 1 and 2). The direct interaction of
HCLP-1 with the cellular transcription factor LZIP was demonstrated in
two independent assays for protein-protein binding: yeast two-hybrid
analysis (Fig. 3A) and in vitro affinity chromatography (Fig. 3B). Confocal immunofluorescence
microscopy further supports that both proteins localize to the same
intranuclear compartment (Fig. 3C). Interestingly, HCLP-1
interacts specifically with LZIP but not with herpes simplex virus VP16
(Fig. 3A). As a consequence of the direct interaction,
HCLP-1 modulates the DNA binding activity of LZIP (Fig. 6), leading to
transcriptional repression (Fig. 5). Several lines of evidence are
consistent with the model that HCLP-1 inhibits
LZIP-dependent transcription through interference with DNA
binding. First, both LZIP and HCLP-1 localize predominantly to the
nucleus (Fig. 3C). Second, the HCLP-1-interacting domain in
LZIP coincides with the bZIP DNA-binding region (Fig. 4). Finally
and most importantly, electrophoretic mobility shift assay demonstrates
that HCLP-1 suppresses the binding of LZIP to CRE oligonucleotides in a
dose-dependent manner (Fig. 6). Taken together, our
findings point to a specific transcriptional corepressor function of
HCLP-1 mediated through physical interaction with a cellular transactivator.
-propeller domain of HCF-1 serves as a
specialized platform for interactions with multiple partners. It is of
great interest to understand better the modular structure and the
molecular mechanism through which the kelch domain recognizes the
target transcription factors. In this regard, the comparison with other
HCF-like proteins provides novel opportunities to study the structure
and functions of HCF-1. The findings that a closely related HCF-2
interacts poorly with VP16 and hardly with LZIP (26) strongly suggest
that the recognition of targets by the -propeller has specificity.
-propeller domain (9), implicating that HCF-1
binds VP16 and LZIP through different mechanisms. Our findings that
HCLP-1 interaction with LZIP is independent of the HBM (Fig. 4)
revealed that the -propeller domain of HCLP-1 can recognize a
distinct motif in LZIP. Further analysis will shed light on the
identity of this recognition motif.
-propeller (Fig. 1). Structurally, HCLP-1 is most similar
to a newly identified and naturally occurring 50-kDa fragment of HCF-1,
which has been found in the cytoplasm of primary G0 cells
as a proteolytic product of the full-length protein (14). This
HCFp50 fragment contains the intact -propeller domain
and retains the ability to interact with VP16. HCFp50 has
been suggested to mediate the suppression of viral immediate early gene
expression through sequestration of VP16 in the cytoplasm (14). This
resembles the action of the hepatitis C virus core protein, which
retains LZIP in the cytoplasm (24). We note that endogenous HCLP-1
protein localizes to the nucleus of interphase HeLa cells (Fig.
3C). However, we do not rule out the possibility that HCLP-1
could be a cytoplasmic protein during particular phases of the cell
cycle. Taking the similarity between HCLP-1 and HCFp50 into
account, we postulate that HCLP-1 might also inhibit LZIP activity by
sequestering it in the cytoplasm under certain circumstances. On the
other hand, the HCLP-1 modulation of the LZIP-dependent DNA
binding activity demonstrated in this study (Fig. 6) provides novel
mechanistic insights into how HCF-like -propeller proteins regulate
transcription. It is of great interest to see whether similar forms of
HCF-like proteins such as HCFp50 might act through a
similar mechanism when targeted to the nucleus.
-propeller domain of HCF-1 leads to G0/G1 cell cycle
arrest (15, 16). In contrast, loss of LZIP function induced by the
hepatitis C virus core protein correlates with oncogenic transformation
(24). We have previously proposed a model in which LZIP serves a tumor suppressor function in the development of hepatocellular carcinoma (24). Intriguingly, HCLP-1 has recently been identified independently as a specific tumor antigen for hepatocellular carcinoma
(GenBankTM AF244137). This raises the possibility that
HCLP-1 may have a role in hepatocarcinogenesis by targeting the LZIP
tumor suppressor. Further investigations are required to test this hypothesis.
| |
ACKNOWLEDGEMENTS |
|---|
We thank H.-f. Kung for encouragement and support; Y. P. Ching for helpful discussions; Y. Zhou, A. C. S. Chun, and R. W. M. Ng for technical assistance; J. Vogel, T. Kristie, K. V. Kibler, K.-T. Jeang, G. S. Hayward, D. Repka, G. J. Darlington, and R. Prywes for providing reagents; and Y. P. Ching, B. C. B. Ko, A. C. S. Chun, R. W. M. Ng, A. C. Y. Lo, O. S. W. Wong, and S. F. Chan for critical reading of manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by Hong Kong Research Grants Council Grant N-HKU015/00 under NSFC/RGC JRS, University of Hong Kong Grant 10300147.32993.43700.305.01, National Natural Science Foundation of China Grants 39830070 and 3001161945, National Program for Key Basic Research Project G1998051002, and National High Technology R&D Program.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) AF113131 for HCLP-1.
¶ To whom correspondence may be addressed. Tel.: 852-22990777; Fax: 852-28171006; E-mail: dyjin@hkucc.hku.hk or yuanjg@mail.east.net.cn.
Leukemia and Lymphoma Society Scholar.
Published, JBC Papers in Press, May 30, 2001, DOI 10.1074/jbc.M103893200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: HCF, host cell factor; HCLP-1, HCF-like protein 1; CREB, cAMP-responsive element-binding protein; CRE, cAMP-responsive element; HBM, HCF-binding motif; BD, DNA-binding domain; AD, activation domain; GST, glutathione S-transferase; TRX, thioredoxin.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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