The liver-enriched transcription factor CREB-H is
a growth suppressor protein underexpressed
in hepatocellular carcinoma
King-Tung Chin1, Hai-Jun Zhou1,3, Chun-Ming Wong2, Joyce Man-Fong Lee2,
Ching-Ping Chan1, Bo-Qin Qiang3, Jian-Gang Yuan3, Irene Oi-lin Ng2and Dong-Yan Jin1,*
1Department of Biochemistry and2Department of Pathology, Faculty of Medicine, University of Hong Kong,
Hong Kong, China and3National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
Received January 24, 2005; Revised March 5, 2005; Accepted March 14, 2005DDBJ/EMBL/GenBank accession no. AF392874
LZIP to be a growth suppressor targeted by hepatitis
C virus oncoprotein. In search of proteins closely
related to LZIP, we have identified a liver-enriched
transcription factor CREB-H. LZIP and CREB-H rep-
resent a new subfamily of bZIP factors. CREB-H
activates transcription by binding to cAMP respons-
ive element, box B, and ATF6-binding element.
Interestingly, CREB-H has a putative transmembrane
(TM) domain and it localizes ambiently to the endo-
plasmic reticulum. Proteolytic cleavage that removes
the TM domain leads to nuclear translocation and
of hepatic gluconeogenic enzyme phosphoenolpyr-
uvate carboxykinase. This activation can be further
stimulated by cAMP and protein kinase A. CREB-H
transcript is exclusively abundant in adult liver. In
contrast, the expression of CREB-H mRNA is aber-
rantly reduced in hepatoma tissues and cells. The
enforced expression of CREB-H suppresses the pro-
liferation of cultured hepatoma cells. Taken together,
scription factor CREB-H is a growth suppressor that
plays a role in hepatic physiology and pathology.
The establishment and maintenance of a differentiated pheno-
type in a given tissue requires specific gene expression pro-
gram. In liver, this is accomplished by the coordinated action
of a group of liver-enriched transcription factors (LETFs).
factors-1 (HNF1), HNF3, HNF4, HNF6, CAATT enhancer-
binding proteins (C/EBPs) and D-box binding protein. These
factors are activated in a precise temporal and spatial order
during liver development (1,2), and they form a regulatory
network that controls the expression of a repertoire of liver-
specific genes (3). Thus, HNF3 is required for inducing the
endoderm to adopt a hepatic fate (4). Once the pre-hepatic
cells are committed, HNF4a is essential for complete differ-
entiation of hepatocytes (5) and C/EBPa regulates a number
of genes required for energy metabolism in adult liver (6).
Dysregulation of liver-specific transcription is a hallmark
of hepatocellular carcinoma (HCC) (7). Indeed, most of the
dysregulated genes identified in an HCC expression profiling
study are known to be regulated by LETFs (8). In addition,
among the LETFs, HNF1, HNF3b, HNF4a and HNF4g have
been shown to be up-regulated in HCC samples whereas the
expression of C/EBPa is reduced (8). In this regard, C/EBPa
may serve as a growth suppressor through stabilization of p21
(9,10), inhibition of cyclin-dependent kinases 2 and 4 (11,12),
and repression of E2F-dependent transcription (13,14).
bZIP transcription factors are a large family of sequence-
specific DNA-binding proteins characterized by a basic
domain, which interacts with DNA, and a leucine zipper or
coiled coil region critical for dimerization (15,16). C/EBPa
and CREB are two prototypic bZIP proteins that have been
extensively studied (6,17). In the CREB family of bZIP tran-
scription factors, box B-binding factor 2 (BBF2)/dCREB-A
(18,19) has been identified in a screen for proteins that spe-
cifically bind to the box B element in the fat-body-specific
enhancer of the Drosophila alcohol dehydrogenase gene. The
fat body, a functional counterpart of the mammalian liver, is a
major organ for energy metabolism in Drosophila. It is note-
worthy that BBF2 also binds to and activates CRE and box
*To whom correspondence should be addressed at Department of Biochemistry, The University of Hong Kong, 3rd Floor, Laboratory Block, Faculty of Medicine
Building, 21 Sassoon Road, Hong Kong. Tel: +852 2819 9491; Fax: +852 2855 1254; Email: firstname.lastname@example.org
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Nucleic Acids Research, 2005, Vol. 33, No. 61859–1873
B-like element located in the promoter of human alcohol
dehydrogenase as well as rat tyrosine aminotransferase
genes (18). Thus, box B-like elements and their binding
proteins likely represent an important and evolutionarily
conserved component in the liver-specific regulatory circuit.
Interestingly, a human liver-specific bZIP protein called
CREB-H has also been described. CREB-H specifically binds
to box B and activates box B-dependent transcription (20),
suggesting that it may be functionally equivalent to BBF2.
A mammalian homolog of BBF2 termed LZIP/CREB3 has
previously been identified and characterized (21–23). LZIP
interacts with hostcellfactor-1 and host cellfactor-likeprotein
1 (21,23,24). The ubiquitously expressed LZIP protein binds
to canonical CRE and regulates cell proliferation. It is also a
binding partner and transformation cofactor of hepatitis C
virus core protein. Loss of LZIP function in NIH3T3 cells
independent growth (22). In searching for additional factors
closely related to LZIP, we identified a mouse bZIP protein
orthologous to human CREB-H. LZIP and CREB-H represent
a new subfamily of bZIP proteins with a unique putative
transmembrane (TM) domain lined to the bZIP region.
Removal of TM domain and the remaining C-terminal
sequences translocates CREB-H from endoplasmic reticulum
(ER) to the nucleus and renders it a potent transactivator.
CREB-H activates transcription by binding not only to CRE
and box B but also to ATF6 element. CREB-H transcript is
exclusively abundant in adult liver. In contrast, the expression
of CREB-H mRNA is aberrantly reduced in hepatoma tissues
and cells. Overexpression of the full-length CREB-H and its
active form suppress proliferation of HepG2 hepatoma cells.
Our findings indicate that CREB-H is a liver-enriched bZIP
transcription factor critically involved in hepatic physiology
MATERIALS AND METHODS
The mouse CREB-H cDNA clone (IMAGE 1887290) was
obtained from Invitrogen. CREB-H (amino acids 1–479)
and CREB-HDTC (amino acids 1–318) cDNA fragments
were PCR-amplified and subcloned into expression vector
pcDNA3.1/V5-His (Invitrogen) via restriction sites XhoI
and XbaI, into pCMV-FLAG (Kodak) via restriction sites
HindIII and SalI, and into pEGFP-C (Clontech) via restriction
sites HindIII and XhoI. Mammalian expression plasmids
containing the complete coding region of human CREB and
ATF4, and the active form of human ATF6 (amino acids 1–
373) were constructed by PCR amplification of the corres-
ponding cDNA fragments and inserted into pCMV-FLAG
via restriction sites HindIII and XhoI. Plasmids expressing
human C/EBPa (9) and ATF6 (25) were kindly provided
by Dr Gretchen Darlington and Dr Ron Prywes, respectively.
Plasmids expressing the fusion protein GAL4BD-CREB-H
and its deletion mutants were constructed by in-frame inser-
SalI and XbaI for BD-FL and BD-(1–318); EcoRI and SalI for
for BD-(1–62) and BD-(1–280); SalI and HindIII for BD-
(1–120), BD-(1–180) and BD-(1–240). Plasmid expressing
the bZIP region of CREB-H (CREB-HZ; amino acids 206–
318 plus C-terminal polyhistidine tag) was constructed by
PCR amplification and in-frame insertion to the bacterial
expression plasmid pET32c (Novagen) via restriction sites
NdeI and XhoI.
Reporter plasmid pCRE-Luc and expression plasmid for the
catalytic subunit of protein kinase A (PKA) were from Strata-
gene. pATF6-Luc was derived from pGL3-basic (Promega)
and contains six copies of canonical ATF6-binding sites (26).
pPEPCK-Luc containing ?490 to +73 of rat phosphoenol-
pyruvate carboxykinase (PEPCK) promoter (27) was a kind
gift from Dr Marc Montminy. pGAL4-Luc was a gift from
Dr Karen Kibler. pSV-bGal was purchased from Promega.
Lentiviral expression vector for CREB-H was derived from
Polyclonal antiserum a-CH8 and a-CH9 were raised in two
rabbits, respectively, against a purified GST-CREB-H (1–84)
fusion protein, which contains the N-terminal 84 amino acids
of mouse CREB-H. The crude antiserum was further purified
by pre-adsorbing the anti-glutathione S-transferase (GST)
antibodies onto a GST–Sepharose column. Rabbit polyclonal
anti-V5 and mouse monoclonal anti-FLAG were from Sigma.
Mouse monoclonal anti-BrdU conjugated to fluorescein was
from Roche. Mouse monoclonal antibodies against green
fluorescent protein (GFP), V5, calnexin and GM130 were
from Santa-Cruz, Invitrogen, Affinity Bioreagents and BD
random-primed fragment generated by restriction digestion
of mouse CREB-H cDNA (corresponding to nucleotides
258–918) using EcoRI and PstI. Blots of poly(A)+RNAs
from mouse tissues and cancer cell lines were purchased
from Clontech and were probed as recommended by the
Electrophoretic mobility shift assay (EMSA)
Recombinant polyhistidine-tagged protein of CREB-H bZIP
region (CREB-HZ; amino acids 206–318) was expressed
and purified from Escherichia coli according to Novagen’s
instructions. Probe labeling and EMSA were performed as
described previously (28). Canonical sequences for CRE,
AP1, Sp1 and kB motifs have also been described previously
(22). Sequences of other sense-strand oligonucleotides used
are as follows: box B in fruitfly alcohol dehydrogenase-1 gene
(29), 50-GATCTTGTACACGTAATCGTAGGATCC-30; C/
EBP in human albumin gene (30), 50-GATCTTGATTTTG-
TAATGGGGGGATCC-30; CRE in rat PEPCK gene (31),
element in human GRP78 (an ER chaperone) gene (32),
ERSE-II element in human Herp (a TM protein of ER)
gene (33), 50-ATTGGGCCACG-30.
Harvested cells were lysed either by repeated freezing and
thawing or in RIPA buffer (50 mM Tris–HCl, pH 7.4,
1860 Nucleic Acids Research, 2005, Vol. 33, No. 6
150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium
deoxycholate) supplemented with 2 mM phenylmethyl-
sulfonyl fluoride, 2 mg/ml pepstatin A, 2 mg/ml aprotinin,
2 mg/ml leupeptin and 2 mg/ml soybean trypsin inhibitor. Sam-
ples containing equal amounts of protein were separated by
SDS–PAGE and electroblotted onto Immobilon-P membranes
(Millipore) using Hoefer SemiPhor semi-dry blotting appar-
atus (Amersham). Blots were blocked with 5% skim milk,
followed by incubation with primary antibodies. Blots were
then incubated with goat anti-rabbit or anti-mouse secondary
antibody conjugated to horseradish peroxidase (Amersham)
and visualized by enhanced chemiluminescence (Amersham).
Cell cultures, transient transfection, lentiviral
transduction and luciferase assays
Human hepatoma cell lines HepG2 and Hep3B, human cer-
vical carcinoma cell line HeLa and mouse hepatoma cell line
Hepa1-6 were grown in DMEM containing 10% fetal bovine
serum, 100 U/ml penicillin-G and 100 mg/ml streptomycin.
Cells were maintained at 37?C in a humidified atmosphere at
5% CO2. For luciferase reporter assays, cells were cultured in
fected using LipofectAMINE 2000TMreagent (Invitrogen).
HepG2 cells were transfected by calcium phosphate co-
precipitation method. All cells were transfected for at least
16 h before harvest. For forskolin (Fsk) treatment, cells were
incubatedinthepresence of10mMFskfor12 hbeforeharvest.
Reporter assays were performed using the Dual-LuciferaseTM
kit (Promega). Luminescence was measured with a LB9570
Preparation of lentiviral stock and lentiviral transduction
of HepG2 cells were performed using the manufacturer’s
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed as detailed elsewhere (34),
except that the cross-linking step was performed by adding
1% formaldehyde to HepG2 or Hepa1-6 cells for 5 min. The
primers used for PCR amplification of the endogenous human
PEPCK promoters are forward primer 1, 50-GACTGTG
ACCTTTGACTATGGGGTGACATC-30(?454 to ?424);
GTTATGC-30(?150 to ?123); forward primer 2, 50-GTTGA-
GGGCTCGAAGTCTCCCAGCATTC-30(?339 to ?311);
reverse primer 2, 50-GGCACGAATGTGAGGTCACCTGA
AATAG-30(+206 to +234). The primers for PCR amplifica-
tion of rat PEPCK promoter are forward primer, 50-GTG-
primer, 50-CTTGGTAGCTAGCCCTCCTC-30(?21 to ?2).
DNA was PCR-amplified from the immunoprecipitated
chromatin (number of cycles: 30; annealing temperature:
65?C; extension time: 25 s). Amplification of human MAD1
promoter was performed as described previously (34). The
number of cycles for all PCR was optimized to ensure that
the amplification is within a quantitative linear range.
MCF-7 and HepG2 were cultured on cover-slips that are
situated inside the wells of a 6-well plate. Transfected or
lentivirus-transduced cells were washed with phosphate-
buffered saline (PBS) and then fixed in ice-cold 50% acet-
one/50% methanol for 10 min, followed by three washes with
PBS. Cells were co-stained for CREB-H with calnexin/
GM130. The cover-slips containing cells were mounted on
glass slide using VECTORSHIELD agent (Vector). Confocal
immunofluorescence microscopy was then performed on
BioRad MRC1024 system and the images were captured at
63· magnification with the help of the LaserSharp software.
Patients and tumor samples
Sampling of tumor tissues was performed as detailed else-
where (35). In short, 26 Chinese patients (18 males and 8
females) who had had surgical resection of the tumors at
the University of Hong Kong Medical Center were randomly
selected for study. Paired samples of their HCCs and corres-
ponding non-tumorous liver tissues were used. The patients’
ages ranged from 29 to 74 years (mean: 56.85 years). The
tumor size ranged from 2.3 to 27 cm (mean: 8.86 cm), with
20 (76.9%)of thembeing >5cm indiameter.Eight(30.8%)of
the tumors were at earlier stage (tumor, node, and metastasis
stages I and II), and the remaining 69.2% (18 cases) were at
more advanced stages (stages III and IV). The sera were pos-
itive for hepatitis B surface antigen in 21 (80.77%) patients.
All specimens were obtained immediately after surgical
resection, snap-frozen in liquid nitrogen and kept at ?70?C.
Frozen sections were cut from the tumor and non-tumorous
liver blocks separately and stained for histological examina-
tion to ensure homogeneous cell populations of tissues.
RNA extraction and RT–PCR analysis
Total RNA was extracted from human HCC and their corres-
ponding non-tumorous livers with TRIzol reagent as described
by the manufacturer (Invitrogen).cDNA was synthesized from
1 mg of total RNA by using oligo(dT)16as primer and with
Gene-Amp RNA PCR kit (Applied Biosystems). PCR ampli-
fication using a set of primers (forward: 50-AGTGTTCTCCA-
GAACTTTGC-30; reverse: 50-TGCACGTCCTGAGCCAGT-
30) was performed to give a product of 286 bp (from nucleotide
1187–1472). A fragment of b-actin was amplified as a control.
The PCR was performed in a 9700 thermocycler (Perkin-
Elmer). PCR was stopped at the exponential phases, 28 cycles
for CREB-H and 28 cycles for b-actin, with one cycle of hot-
start at 95?C for 12 min, followed by amplification at 94?C for
30 s, 53?C for 30 s, 72?C for 30 s and a final elongation at 72?C
for 5 min. The PCR products were analyzed by electrophoresis
on 1.5% agarose gels, and their signal intensities were meas-
ured using GelWorks ED Intermediate Software (UVP).
Growth suppression assays
For b-galactosidase (b-gal) staining experiments, 5 · 105
HepG2 cells were cultured in each well of a 6-well plate,
and co-transfected at day 0 with pSV-bgal (2 mg) and a
CREB-H-expressing plasmid (0.2 mg; either pcDNA3.1/
V5-CREB-H or pcDNA3.1/V5-CREB-HDTC) at a ratio of
10:1. On day 1 and day 5 post-transfection, cells were har-
vested and stained with X-gal for b-gal activity. Briefly, the
cells were washed three times with PBS and then fixed by
adding 0.2% glutaraldehyde in PBS for 5 min at room
Nucleic Acids Research, 2005, Vol. 33, No. 61861
temperature. Cells were washed again three times with PBS
and then X-gal/PBS was added. b-gal staining was performed
at 37?C in a humidified atmosphere at 5% CO2for at least 2 h.
Cell growth was calculated by counting the number of blue
cellclusters(more than four cells)in five different fieldsat10·
and 40· magnification under microscope.
For BrdU staining experiments, 1 · 105HepG2 cells were
cultured on cover-slips in 6-well plate. Cells were transfected
with either a control plasmid pcDNA3.1/V5-b-gal or a CREB-
H-expressing plasmid for 16 h. Cells were then incorporated
with BrdU (10 mM) for 6 h, washed three times with PBS
and fixed with ice-cold methanol/acetone (1/1, v/v). Cells
were subsequently stained with rabbit anti-V5 and fluorescein-
conjugated mouse anti-BrdU. A Texas red-conjugated second-
ary antibody against rabbit IgG (Cappel) was used to visualize
the V5 signals. For quantitative analysis, 200 transfected cells
from each transfection were examined. Cells were counted as
BrdU-positive if the intensity of BrdU staining was increased
more than 5-fold compared with adjacent untransfected cells
on the same focal plane. Three independent transfections were
analyzed for each group of cells. The percentage of blue cells
reported represents the average of three transfections.
CREB-H belongs to a new subfamily of bZIP
In search of proteins closely related to LZIP, we identified
several mouse expressed sequence tag clones that might
encode novel bZIP proteins homologous to LZIP. DNA
sequencing of one clone (IMAGE 1887290) confirmed that
this cDNA codes for a protein of 479 amino acids harboring
a characteristic bZIP domain (GenBank accession no.
AF392874). The coding sequence is reasoned to be complete
in light of the existence of an in-frame termination codon
immediately upstream of the putative initiating ATG. More
than 75% of the residues in this novel protein are similar to
those in LZIP.
At the time of initial identification, this mouse LZIP-like
protein had no existing counterparts in the databases, except
for a paralogous protein termed OASIS/CREB3L1 (36). Sub-
sequently, a highly homologous human protein was identified
in a search for liver-specific transcription factor through ran-
dom sequencing of cDNAs in libraries derived from human
liver. This protein designated CREB-H is specifically
expressed in hepatocytes (20). In light of the high homology
between the two proteins (67% identity and 73% similarity),
the LZIP-related protein we identified likely represents the
mouse ortholog of human CREB-H. Notably, the official
nameforCREB-H asapprovedbyMouse GenomeInformatics
is cAMP responsive element binding protein 3-like 3 protein
(CREB3L3; http://www.informatics.jax.org). For simplicity
and consistency, hereafter we will call it mouse CREB-H.
The mouse CREB-H locus was found to locate in chromosome
10. The gene is 8.5 kb in length and contains 12 exons.
Interestingly, several additional mammalian bZIP proteins
significantly homologous to LZIP and CREB-H have been
identified more recently. These include AIBZIP/Atce1/
CREB4/CREB3L4 (37,38) and human BBF2H7/CREB3L2
(39). These proteins contain a short stretch of hydrophobic
amino acids, which was predicted to be a TM domain by two
programs HMMTOP (Hungarian Academy of Sciences; http://
www.enzim.hu/hmmtop) and PredictProtein (Columbia Uni-
versity; http://cubic.bioc.columbia.edu/predictprotein). The
C-terminal TM domain immediately adjacent to the bZIP
region is highly conserved in this unique group of bZIP
proteins. Indeed, phylogenetic analysis based on protein
sequences segregates these bZIP proteins into a new subfamily
distinct from CREB and ATF6.
CREB-H preferentially recognizes CRE, ATF6 and
box B elements
Human CREB-H binds to CRE and box B (20). However,
mouse Atce1 closely related to CREB-H has been shown to
recognize kB motif instead of CRE (38). To clarify this issue,
recombinant polyhistidine-tagged mouse CREB-HZ protein,
which contains the bZIP region (amino acids 206–318), was
expressed and purified from E.coli. This protein was then
tested for its ability to bind various32P-labeled DNA elements
using the EMSA assay. In addition to CRE, box B and kB site,
canonical ATF6-binding element (26) was also examined
because ATF6 has a TM domain (40) and shares some sim-
ilarity to CREB-H subfamily proteins. ERSE-I and ERSE-II
are two different ATF6-responsive elements found in the
promoter of GRP78 (32) and Herp (33) genes, respectively.
CREB-H was found to bind strongly to canonical CRE, ATF6
and box B elements (Figure 1A, lanes 1, 5 and 6), but not to
AP-1 or kB motif (lanes 2 and 4). CREB-H also bound less
potently to CRE in the rat PEPCK gene, ERSE-I and ERSE-II
(lanes 8–10), but it hardly recognized Sp1 and C/EBPa motifs
(lanes 3 and 7). Results from the competition EMSA assay
using 10- and 50-fold cold oligonucleotides verified the spe-
cific interaction of CREB-H with CRE, ATF6 and box B
elements (Figure 1B).
Proteolytic processing of CREB-H in cultured cells
We raised two polyclonal antisera (a-CH8 and a-CH9) in
rabbits against the N-terminal 84 amino acids of CREB-H.
We then performed western blotting with these antibodies to
probe CREB-H protein in Hepa1-6 hepatoma cells that were
either mock-transfected (Figure 2B, lanes 1 and 5), transfected
with an untagged CREB-H-expressing plasmid (Figure 2B,
lanes 2–4) or transfected with a FLAG-tagged CREB-H
expressing plasmid (Figure 2B, lanes 6–8). Consistent with
our results from northern blot analysis (see below in Figure 6),
the amount of endogenous CREB-H protein in Hepa1-6 cells
was very low (Figure 2B, lanes 1 and 5). In contrast, several
additional a-CH8/9-reactive protein bands were observed in
CREB-H-overexpressing Hepa1-6 cells (Figure 2B, lanes 2
To verify the identity of these reactive bands, we tested the
pre-immune sera (Figure 2B, lanes 4 and 8) and performed
protein blocking assay by pre-incubating a-CH8/9 with an
excess amount of recombinant CREB-H protein (Figure 2B,
lane 3 and 7). Because neither pre-immune nor depleted a-
CH8/9 recognized the bands of ?80 and 50 kDa in size
(Figure 2B, lanes 3, 4, 7 and 9), these bands should represent
specific CREB-H-derived species. While the ?80 kDa pro-
teins (Figure 2B, lanes 2 and 6, bands with an arrow) plausibly
represent the full-length CREB-H and FLAG-CREB-H, the
1862Nucleic Acids Research, 2005, Vol. 33, No. 6
50 kDa species (bands with an arrowhead) might be a cleaved
product (Figure 2B, compare lane 2 to 3, and lane 6 to 7). Both
forms of CREB-H might have extensive post-translational
modifications because their sizes are larger than predicted.
Since a-CH8/9 recognizes the N-terminus of CREB-H, the
appearance of 50 kDa a-CH-reactive species suggests site-
specific proteolysis of CREB-H at the C-terminus. To verify
the specificity of a-CH8/9 and to further characterize the
proteolytic processing of CREB-H, we expressed two versions
of CREB-H proteins (Figure 2A, full-length CREB-H and
truncated CREB-HDTC) fused to GFP. In CREB-HDTC, a
C-terminal part including the TM domain was removed
(Figure 2A). Comparison of this artificially truncated protein
with the proteolytic fragments of CREB-H might shed light
on the cleavage site in CREB-H. To this end, Hepa1-6 cells
were transfected with GFP-CREB-H and GFP-CREB-HDTC
plasmids and the cell extracts were examined by western
blotting (Figure 2C).
With increasing amounts of protein loaded, a-CH9 was
able to detect a band of ?110 kDa in size that corresponds
to GFP-CREB-H (Figure 2C, upper panel, lanes 2–4). The blot
was re-probed with a monoclonal anti-GFP antibody (a-GFP;
Figure 2C, lower panel, lanes 3 and 4). The reaction of the
110 kDa band with both a-CH9 and a-GFP verified the iden-
tity of GFP-CREB-H and it also lent further support to the
specificity of a-CH9.
Likewise, both a-CH9 and a-GFP recognized the GFP-
CREB-HDTC protein of 75 kDa (Figure 2C, lane 5). Interest-
ingly, in cells that had been transfected with a GFP-CREB-H
plasmid but not a GFP-CREB-HDTC construct, an a-CH9-
and a-GFP-reactive band of about the same size was also
observed (Figure 2C, compare lane 4 with lane 5, bands
with arrows). This species plausibly represents a processed
form of CREB-H that had been cleaved at a specific site.
Because the size of this cleavage product is almost identical
to that of GFP-CREB-HDTC, the actual cleavage site was
predicted to be very close to the junction between bZIP and
TM regions (Figure 2A). It is noteworthy that this cleaved
species of 77 kDa shown in Figure 2C corresponds to the
50 kDa band in Figure 2B. The difference in molecular
mass (?27 kDa) was accounted for by GFP. In addition,
when we performed immunoprecipitation with a-CH8 and
extracts of cells transfected with either FLAG-CREB-H or
GFP-CREB-H plasmid, two major protein species correspond-
ing to the full-length and cleaved forms of CREB-H, respect-
ively, were found in the precipitates (Figure 2D and E).
Next, we asked whether proteolysis also occurs in HepG2
cells stably transduced with a lentivirus expressing CREB-H.
In keeping with results obtained from transfected cells, a
strong ?80 kDa band and a weaker ?50 kDa species reactive
to a-CH9 were observed in extracts of lentivirus-transduced
cells (cf. Figure 2F, lane 2 with Figure 2B, lane 5). These two
protein bands were not seen when a-CH9 was depleted with
recombinant CREB-H (Figure 2F, lane 4). As a size reference
for the ?50 kDa band observed in lentivirus-transduced cells,
protein extract of HepG2 cells expressing CREB-HDTC,
which did not react to depleted a-CH9 (Figure 2F, lane 5),
was also examined (cf. lane 3 with lane 2).
CREB-H activates CRE- and ATF6 element-dependent
Human CREB-H has been shown to activate box B-dependent
transcription (20). Box B is a fat-body-specific enhancer in
Figure 1. CREB-H preferentially recognizes CRE, ATF6 and box B elements. (A) EMSA assay. EMSA was performed using 0.2 mg of purified CREB-HZ protein
Nucleic Acids Research, 2005, Vol. 33, No. 61863
Drosophila and box B-like sequences in mammalian cells
have not been characterized (18). In light of the strong binding
of CREB-H to CRE and ATF6 element (Figure 1), we next
asked whether CREB-H was able to drive transcription from
CRE or ATF6 element. To address this, we co-transfected a
CREB-H expressing plasmid and either pCRE-Luc or pATF6-
Luc reporter plasmid into HepG2 hepatoma cells and assayed
for luciferase activity. For comparison, we also tested the
activities of ATF4, ATF6, CREB and C/EBPa on CRE and
ATF6 enhancers (Figure 3A and B). While the full-length
CREB-H weakly activated CRE-dependent transcription
(Figure 3A, 1.6-fold activation), it was capable of stimulating
the ATF6 enhancer to 4.9-fold (Figure 3B). This potency is
comparable with that of ATF4, CREB and C/EBPa (11.3-,
3.9- and 6.9-fold activation, respectively).
Itisgenerally accepted that removal of the TM domainfrom
ATF6 and LZIP leads to the activation of these transcription
factors (40,41). The proteolytic cleavage of ATF6 can be
induced in vivo in response to ER stress (40), but the physio-
logical stimuli for the activation of LZIP remain elusive.
CREB-H also contains a TM domain, which is probably
removed through site-specific proteolytic cleavage (Figure 2)
as exemplified in the cases of ATF6 (42) and SREBPs
(43,44). To test the hypothesis that CREB-H could also be
Figure 2. Proteolytic processing of CREB-H in hepatoma cells. (A) Schematic representation of the structure of CREB-H and CREB-HDTC. (B) Western blot
line Hepa1-6 for 24 h. Cells were then harvested and lysed. The whole cell extracts thus obtained were analyzed by 10% SDS–PAGE followed by western blotting
H. The full-length CREB-H protein and the 50 kDa cleaved product of CREB-H are indicated with arrow and arrowhead, respectively. The 110- and 72 kDa cross-
reactive bands are highlighted with asterisk. Trace amount of a 45 kDa band (marked with ‘#’) was seen in mock- and pFLAG-CREB-H-transfected Hepa1-6 cells
cross-reactive protein (C) Western blot analysis was performed as in B, except that GFP-CREB-H (GFP-CH) and GFP-CREB-HDTC (GFP-DTC) constructs were
used. Lanes 2–4 received increasing amounts of protein (protein amt.). The blot in the upper panel was probed with a-CH9 (lanes 5–6) or a-CH9 depleted with an
H. CREB-H protein was immunoprecipitated (IP) with a-CH8 antibody and the precipitates were immunoblotted (IB) with a-FLAG and a-GFP antibodies,
respectively. (F) Western blot analysis was performed with extracts of HepG2 cells transduced with a lentivirus expressing CREB-H (LV-CH). Protein extract of
HepG2 cells transfected with CREB-HDTC (CHDTC) plasmid (lanes 3 and 5) was used as a size reference for the proteolytic fragment.
1864Nucleic Acids Research, 2005, Vol. 33, No. 6
activated through regulated proteolysis, we asked whether the
truncated CREB-HDTC devoid of the TM and C-terminal
domains (Figure 2A) might have a higher transcriptional
activity. Indeed, CREB-HDTC acted as a transcriptional
activator on both CRE and ATF6 element, resulting in
?100- and 30-fold increase of luciferase activity (Figure 3A
and B). In this setting, CREB-HDTC is more potent than
CREB, ATF4, ATF6 and C/EBPa. This raised the possibility
that the truncated CREB-HDTC might be functionally
equivalent to the physiologically active form of CREB-H
generated through site-specific proteolysis in vivo (see
CREB-H is activated in response to cAMP stimulation
cAMP is a ubiquitous second messenger and regulator of gene
transcription (45,46). In one well-characterized pathway,
cAMP activates PKA, which phosphorylates and activates
CREB (17). In addition, C/EBPs are also responsive to
cAMP stimulation (6). This prompted us to investigate the
regulation of CREB-H by cAMP. We measured the activities
of CREB-H and CREB-HDTC on the pCRE-Luc and pATF6-
Luc reporters with orwithout the addition of Fsk, which stimu-
lates adenylate cyclase leading to increased cAMP levels
(Figure 3A and B). Fsk did not activate ATF6 element-
dependent transcription significantly with the exception of
Figure 3. Transcriptional activity of CREB-H and potentiation by cAMP. Empty vector (VEC) and plasmids (0.3 mg of each) expressing CREB-H, CREB-
HDTC (CHDTC), ATF4, ATF6, CREB and C/EBPa were individually co-transfected with pCRE-Luc (A), pATF6-Luc (B) or pPEPCK-Luc (C) plasmid (0.2 mg)
into HepG2 (A and B) and Hep3B (C) cells with or without the addition of Fsk or catalytic subunit of PKA (PKAcs). An SV40 promoter-driven Renilla
luciferase plasmid was also transfected into the cells. The cell lysates were measured for Firefly luciferase luminescence and the readings were normalized by
the luminescence of Renilla luciferase. (D) ChIP assay was performed with a-CH8 and extracts of HepG2 cells transfected with pcDNA-CREB-H. One control
the proximal promoter of human PEPCK gene (panels 1 and 2). Experiments were also carried out with primers for amplifying human MAD1 promoter (panel 3).
Similar ChIP assays were performed in mock-transfected (panel 4) and CREB-H-expressing (panel 5) Hepa1-6 cells carrying a rat PEPCK promoter. Enforced
expression of CREB-H was achieved by co-transfection with pcDNA-CREB-H. No CREB-H-bound DNA could be detected when input chromatin was omitted
(data not down).
Nucleic Acids Research, 2005, Vol. 33, No. 6 1865
CREB-H, which was stimulated mildly by Fsk (Figure 3B,
?80% increase in luciferase activity). As to CRE-dependent
transcription, both CREB-H and CREB-HDTC were modestly
responsive to Fsk stimulation, resulting in up to 100% increase
in reporter activity (Figure 3A, 3.7- and 227.8-fold activation,
respectively). However, in the control experiment in which the
empty vector alone was co-transfected, Fsk also stimulated
CRE-dependent transcription (Figure 3A, 3.7-fold activation).
Thus, while it remains unclear as to whether Fsk specifically
stimulates CREB-H, Fsk activation of endogenous trans-
cription factors alone unlikely accounted for the increase of
CRE-dependent luciferase activity observed in the presence of
CREB-HDTC and Fsk. Thus, cAMP might be directly or
indirectly involved in regulating CREB-H and CREB-HDTC.
CREB-H activates PEPCK promoter
While the DNA-binding and gene-activating abilities of LZIP,
CREB-H and other transcription factors in the same subfamily
have been well documented (20,22,23,37,38,47,48), their
physiological targets remain to be identified. As a first step
toward identifying the gene targets of CREB-H (20), we asked
whether it might bind and activate CRE-dependent transcrip-
tion in liver. In this regard, the gene encoding gluconeogenic
enzyme PEKCK has been extensively used as a model for
studying the regulation of CREB- and C/EBP-dependent tran-
scription in liver (49–51). Based on this reasoning, we have
demonstrated that CREB-H binds modestly to the CRE in rat
PEPCK promoter (Figure1A, lane 8). Because CREB-H binds
moderately to the rat PEPCK CRE, here we set out to invest-
igate whether it might activate the PEPCK promoter. Indeed,
CREB-HDTC but not the full-length CREB-H stimulated the
PEPCK promoter activity when overexpressed in hepatoma
cell line Hep3B (Figure 3C, lane 3). PEPCK is abundantly
expressed in liver and is an important metabolic enzyme that
controls gluconeogenesis in both liver and adipose tissues. It is
well known that cAMP/PKA can activate the PEPCK gene
transcription through the cAMP response unit in the PEPCK
promoter (6,50). As such, when the cAMP pathway is stimu-
lated by either treatment of Fsk or the expression of PKA
catalytic subunit in Hep3B cells, the PEPCK promoter is
stimulated (Figure 3C, lanes 4 and 7) (52). Consistent with
our finding that Fsk was able to further enhance CREB-HDTC
activation of CRE (Figure 3A), we observed that the expres-
sion of CREB-HDTC cooperated with either PKA or Fsk in the
activation of PEPCK promoter (Figure 3C, lanes 6 and 8).
Because the reporter assays were performed in cells trans-
fected with PEPCK-Luc plasmid, we next investigated the
binding of CREB-H to genomic PEPCK promoter in
CREB-H-expressing cells using ChIP assay (Figure 3D).
We observed that a-CH8 but not pre-immune IgG precipitated
a CREB-H–DNA complex containing endogenous PEPCK
promoter (Figure 3D, panels 1 and 2, lane 3 compared with
lane 2). In a control experiment, a-CH8 did not pull down an
irrelevant genomic sequence in human MAD1 promoter
(Figure 3D, panel 3, lane 3). In further support of the speci-
ficity of the interaction between CREB-H and the PEPCK
promoter, ChIP assaysperformed
expressing Hepa1-6 cells (see Figure 5) yielded no CREB-
H–DNA complex (Figure 3D, panel 4), whereas the PEPCK
promoter was amplified from the CREB-H-containing
Hepa1-6 cells. Thus, our results consistently revealed that
Subcellular localization of CREB-H
We expressed CREB-H and CREB-HDTC proteins in MCF-7
cells and determined their subcellular localization. CREB-H
localized predominantlyinthe cytoplasm(Figure4A,panel1),
whereas CREB-HDTC was exclusively in the nucleus (panel
5). Because ATF6 with a TM domain localizes to ER mem-
brane and is activated by intramembrane proteolysis (40),
we investigated the association of CREB-H with ER by
co-staining cells for CREB-H and calnexin, an ER marker.
As shown in Figure 4A, full-length CREB-H co-localized with
calnexin (panels 3 and 4 compared with panel 7). These results
suggest that CREB-H, similar to ATF6, localizes to the ER.
Next, we examined the localization of CREB-H protein in
HepG2 cells homogenously expressing V5-tagged CREB-H
from a lentiviral vector. Again, a typical cytoplasmic local-
ization pattern was observed when we stained the lentivirus-
transduced cells with anti-V5 (Figure 4B, panels 1 and 5). We
noticed a significant co-localization of CREB-H and calnexin
(Figure 4B, panels 1–4). While the staining patterns of CREB-
H and the Golgi marker GM130 were distinct, a fraction of
CREB-H was also found in the Golgi apparatus (Figure 4B,
panels 5–7). Thus, the full-length but inactive CREB-H
(Figure 3) is retained predominantly in the ER. On the con-
trary, CREB-HDTC acts as a potent transactivator plausibly
owing to constitutive nuclear localization.
CREB-H expression is abundant in adult liver but
reduced in hepatoma cells
We performed northern blot analysis of CREB-H mRNA in
mouse tissues and cells. Consistent with previous findings in
human (20), CREB-H mRNA was exclusively and abundantly
detected in adult mouse liver (Figure 5A, lane 5). On the other
hand, the CREB-H signal was not observed in a mouse hep-
atoma cell line Hepa1-6 (Figure 5B, lane 7). In addition, we
most hepatoma cell lines tested (data not shown). It is, how-
ever, noteworthy that mouse CREB-H transcript is highly
expressed in 3T3 fibroblasts immortalized by mammary sar-
coma virus (M-MSV-BALB/3T3), cancerous subcutaneous
adipocytes (L-M) and NB41A3 cancer cells derived from
neuroblastoma (Figure 5B, lanes 4, 5 and 12).
Consistent with results from northern blot analysis, CREB-
H protein was detected in mouse liver, but not in brain or
heart (Figure 5C, lanes 1–3). The identity of the endogenous
CREB-H protein in mouse liver was verified by comparing
with untagged recombinant CREB-H (Figure 5C, lane 4) and
by antibody depletion experiments (Figure 5C, lanes 5–8).
Notably, the truncated CREBDTC form was undetectable in
the blot, implicating that CREB-H is not constitutively active
in mouse liver.
Because CREB-H is abundantly expressed in normal
adult liver but underexpressed in liver cancer cell lines, we
speculated that CREB-H might be critically involved in
hepatocarcinogenesis. With this hypothesis in mind, we per-
formed RT–PCR screening of CREB-H transcripts in human
HCC tissues. The CREB-H mRNA was significantly
1866Nucleic Acids Research, 2005, Vol. 33, No. 6
underexpressed in HCC samples when compared with corres-
ponding non-tumorous liver (Figure 6, P < 0.001 by Wilcoxon
test). A more than 2-fold reduction in steady-state amounts
of CREB-H mRNA was observed in 11 of the 26 HCC tissues
(see Figure 6A for representative RT–PCR results and
Figure 6B for a scatterplot).
Growth suppressive activity of CREB-H in hepatoma
The underexpression of CREB-H in HCC cells and tissues
(Figures 5 and 6) suggests that it might play a role in hepato-
carcinogenesis. Considered together with the tumor suppress-
ive activities of other bZIP proteins, such as C/EBPa (11,12),
C/EBPb (53) and LZIP (22), we queried whether CREB-H
might also serve a growth suppressive function in the liver. We
carried out a growth assay in HepG2 cells using transient
co-transfection of pSV-bgal, a b-gal expression vector driven
by SV40 promoter, plus excess amount (10·) of CREB-H
expressing plasmids. This ensures that the blue cells are the
CREB-H transfected cells when staining with X-gal. This
method haspreviouslybeen usedtostudythe growthsuppress-
ive activity of various proteins, including C/EBPa and
On day 1 after co-transfection, slight differences were seen
in the number of blue cell clusters among the groups that had
received vector alone, full-length CREB-H and CREB-HDTC
(Figure 7A, panels 1–3). On average, four cell clusters (of >4
cells) were seen in vector-transfected cells, whereas two and
one clusters were evident in CREB-H- and CREB-HDTC-
overexpressing cells, respectively (Figure 7B, columns 1–3).
However, on day 5 after transfection, significant differences
were observed among vectors, CREB-H and CREB-HDTC
groups (Figure 7A, panels 4–9; Figure 7B, columns 4–6).
On average, four cell clusters increased to seven cell clusters
in vector-transfected cells, whereas only one or less than one
Figure 4. Subcellular localizations of CREB-H and CREB-HDTC. (A) pcDNA-CREB-H (panels 1–4) and pcDNA-CREB-HDTC (panels 5–7) plasmids were
separatelytransfected into MCF-7cells.Cells were fixed andco-stainedwith a-CH8(green,panels1 and 5)and a-calnexin(red,panels2and 6). Green(CREB-H)
and red (calnexin) fluorescent signals were overlaid by computer assistance (panels 3, 4 and 7). Panel 4 is a high magnification image of a portion of panel 3. Co-
localizations are shown in yellow. The same fields are shown in panels 1–3 and 5–7. Arrows indicate transfected cells. (B) Similar co-localization studies were
performed with HepG2cells transducedwith a lentivirus expressingV5-taggedCREB-H(LV-CH-V5). Green (CREB-H) and red(calnexin or GM130)fluorescent
Nucleic Acids Research, 2005, Vol. 33, No. 6 1867
cell cluster was found in CREB-H- and CREB-HDTC-
overexpressing cells (Figure 7B, columns 4–6). Western
blot analysis confirmed that the protein expression levels of
CREB-H and CREB-HDTC in HepG2 cells on day 1 and day
5 post-transfection were similar (data not shown). Thus,
CREB-H and CREB-HDTC inhibited the proliferation of
To further characterize the growth inhibitory activity of
CREB-H, we measured 5-bromo-20-deoxyuridine (BrdU)
incorporation in CREB-H- and CREB-HDTC-expressing
Figure 5. CREB-H is exclusively expressed in the liver of adult mouse and is differentially expressed in mouse cancer cell lines. Northern blot analysis was
performed on mouse tissues (A) and cancer cell lines (B). CREB-H transcripts are arrowed. Western blotting was also performed on protein extracts from mouse
tissues (C).Mousebrain,heart and livertissues were probed witha-CH9 (lanes 1–4) and depleteda-CH9 (lanes 5–8). Recombinant CREB-H(rCH)preparedfrom
transfected Hepa1-6 cells was also loaded as a positive control (lanes 4 and 8). CREB-H protein is highlighted with an arrowhead. The 72 kDa cross-reactive bands
are indicated with an asterisk.
Figure 6. CREB-H is underexpressed in human HCC tissues. Surgically resected HCC (T) and adjacent non-cancerous liver tissues (NT) from 26 patients were
subjected to RNA extraction and subsequent RT–PCR using primers that amplify 286 bp cDNA fragment of human CREB-H, as well as a fragment of b-actin.
Representative RT–PCR results were shown in (A). The normalized values of the relative quantities of the CREB-H PCR products as compared with the respective
amount of b-actin were shown in a scatterplot (B). The lines indicate the median.
1868Nucleic Acids Research, 2005, Vol. 33, No. 6
HepG2 cells using confocal microscopy. BrdU incorporation
indicates the cellular activity of DNA synthesis that dictates
competency of cell proliferation. This method has been widely
used to study cell growth and cell cycle progression. In par-
ticular, it has been adopted to demonstrate the growth sup-
pressive role of transiently expressed C/EBPa in cultured cells
(12). In our control experiment, the expression of b-gal did
not affect BrdU incorporation (Figure 8, panels 1–3). In stark
contrast, BrdU staining was undetectable in either CREB-
H- or CREB-HDTC-expressing HepG2 cells (Figure 8, panels
4–9; compare transfected cells with arrowheads to surround-
ing untransfected cells with arrows). Quantitative analysis by
cell counting indicates that none of the CREB-H- or CREB-
H-positive cells was incorporating BrdU, whereas ?50%
(47.6 – 5.0%) of control cells expressing b-gal were positive
for BrdU. In another word, BrdU incorporation was signific-
antly reduced in CREB-H- or CREB-HDTC-expressing cells
compared with control cells (P < 0.0001 by t-test). Thus,
the activation of CREB-H can inhibit S-phase entry and
In this study, we characterized a newly identified liver-
enriched transcription factor called CREB-H. CREB-H con-
tains a TM domain and represents a new subfamily of bZIP
proteins that have not been extensively studied. CREB-H
binds to CRE, box B and ATF6 enhancer elements (Figure 1)
and activates transcription from CRE, ATF6 site and PEPCK
promoter in response to cAMP/PKA (Figure 3). CREB-H
associated with the ER in the cytoplasm (Figure 4) is
proteolytically processed in hepatoma cells to generate an
active form devoid of the repressive TM domain (Figure 2).
This active form of CREB-H constitutively localizes to the
nucleus (Figure 4). CREB-H is exclusively expressed in adult
liver and is significantly underexpressed in HCC tissues and
cells (Figures 5 and 6). Finally, CREB-H functions as a growth
Figure 7. Overexpression of CREB-H suppresses growth in cultured human cells. (A) pcDNA3.1/V5 vector, pcDNA3.1/V5-CREB-H and pcDNA3.1/V5-CREB-
on day 5. (B) Cell clusters containing more than four cells were counted in five different fields at 10· and 40· magnifications and scored in a bar graph. Results
represent the average of five independent experiments. Error bars indicate the SE. On day 1, the number of blue cells in the CREB-HDTC group was significantly
lower thanin the groupthathad received vectoralone (P < 0.01by t-test; markedwith *).On day 5, the numbersof bluecells in CREB-Hand CREB-HDTC groups
were also significantly lower than the group with vector alone (P < 0.002 and P < 0.0001, respectively; marked with ** and ***).
Figure 8. Overexpression of CREB-H inhibits S-phase entry. (A) pcDNA3.1/
fixed. Cells were subsequently stained with a-BrdU-fluorescein and a-V5 or
a-CH8 antibodies. Red (BrdU) and green (b-gal, CREB-H or CREB-HDTC)
were obtained from three transfections.
Nucleic Acids Research, 2005, Vol. 33, No. 61869
suppressor in cultured cells (Figures 7 and 8). Thus, CREB-H
serves regulatory roles in liver-specific transcription and the
growth of hepatocytes.
Collectively, our data derive mechanistic insight into the
proteolytic processing, nuclear translocation and regulated
activation of CREB-H. In addition, we also provide the first
evidence for the growth suppressive activity of CREB-H. Our
findings have implications in liver physiology and carcino-
A new subfamily of bZIP proteins
Compared with prototypic bZIP proteins, such as CREB and
C/EBPa, CREB-H is unique in bearing a hydrophobic TM
domain immediately adjacent to the dimerization and DNA-
binding domains situated in the middle of the protein
(Figure 2A). Additional bZIP proteins that share a domain
architecture and high homologies with CREB-H can be
classified into a new subfamily structurally and functionally
different from CREB and ATF6.
In this expanding subfamily, LZIP/CREB3, OASIS and
BBF2H7 exhibit broad expression profiles, while CREB-H
and AIBZIP are specifically expressed in liver and prostate/
testis, respectively. LZIP/CREB3 has been implicated in the
suppression of herpes simplex virus replication and in the
establishment of viral latency in trigeminal ganglia (55)
through association with HCF-1 (21,23). OASIS has initially
beenisolated fromastrocytesinlong-termculture,which serve
as an in vitro gliosis model (36), implicating a role in inflam-
mation and stress responses. Detailed developmental expres-
sion profiling has revealed that OASIS is involved in the late
phase of osteoblast differentiation (56). BBF2H7 has recently
been retrieved from database as a novel human protein whose
C-terminal part was fused to the FUS genes in low grade
fibromyxoid sarcoma as a result of chromosomal translocation
(39). Human AIBZIP has been found to be expressed abund-
antly and specifically in prostate tissues and tumors under the
control of androgens, suggesting a role in prostate devel-
opment and carcinogenesis (37). Interestingly, the mouse
ortholog of AIBZIP termed Atce1 has also been found in
reproductive tissues exhibiting a restricted post-meiotic
expression pattern in spermatids in mouse testis (38). Appar-
ently, bZIP proteins in this emerging subfamily serve both
general and tissue-specific regulatory functions in various
bZIP proteins form homo- and heterodimers through their
leucine zipper domains.Selective dimerizationis anadditional
determinant for the functional diversity and specificity of bZIP
factors. In this regard, one recent study based on protein arrays
has raised the possibility of heterodimerization between bZIP
proteins within the emerging LZIP subfamily (57). Hence, it
would be of great interest to investigate whether and how
CREB-H might interact with other members of this subfamily,
such as LZIP and OASIS.
TM domain and activation of CREB-H
Although CREB-H and ATF6 are in phylogenetically separate
groups, both proteins have a TM domain immediately down-
stream of the bZIP region. ATF6 ambiently localizes to the ER
and functions as a proximal sensor of ER stress (58,59). In
response to the accumulation of unfolded proteins in the ER,
ATF6 is activated by regulated intramembrane proteolysis to
release its active N-terminal fragment, which translocates
into the nucleus to activate the transcription of ER-resident
chaperones and folding enzymes (40). This active form of
ATF6 binds to the consensus ER stress elements (ERSEs)
constitutively pre-occupied by NF-Y (32,33,60). CREB-H
binds modestly to ERSEs (Figure 1); however, it neither
activates the genes coding for ER chaperones, such as
GRP78 and GRP94, nor responds to the ER stress-inducing
agents, such as tunicamycin (K.-T. Chin and D.-Y.Jin, unpub-
lished data). Thus, it remains to be understood whether CREB-
H might regulate gene transcription through the ERSEs.
The ATF6 site (50-TGACGTGG-30) is another ER stress
responsive element identified through binding site screening
experiments using the bZIP domain of ATF6 (26). While it
remains to be seen that ATF6 can directly bind to the ATF6
site (32), XBP1,anotherbZIPproteinwhich isthe downstream
target of ATF6 (60), has been demonstrated to recognize
the ATF6 element (61). Noteworthily, both OASIS (47) and
CREB-H (Figure 2) interacted strongly with the ATF6 ele-
ment. Taken together with the fact that both CREB-H and
ATF6 have similar domain structure (data not shown) and
localize to ER (Figure 4), CREB-H and ATF6 are likely
regulated through distinct but related mechanisms. In this
scenario, CREB-H might activate unidentified genes that
contain recognition sites similar to the ATF6 element but
distinct to the ERSEs. Because ATF6 and XBP1 have been
shown to affect only a subset of ER stress targets (62), CREB-
H might target a different subset of genes. Meanwhile, it might
also cooperate with ATF6 and XBP1 to regulate some
ATF6 is an archetype of TM domain-containing bZIP fac-
tors and it is cleaved by site-1 and site-2 proteases in response
to ER stress to release an active N-terminal fragment (42,63).
Interestingly, LZIP is also cleaved by the same proteases that
act on ATF6 and the activated N-terminal form of LZIP likely
translocates into the nucleus to activate transcription (41).
However, the physiological stimuli that trigger LZIP cleavage
and activation are not understood. In particular, unlike ATF6
or OASIS (64) but similar to CREB-H, LZIP does not respond
to tunicamycin stimulation (K.-T. Chin and D.-Y. Jin, unpub-
lished data) (41). Currently, we have no evidence of CREB-H
involvement in ER stress response.
In this study, we obtained several lines of data that strongly
supportthe regulated activation ofCREB-H. First,we detected
a proteolytic fragment of CREB-H in hepatoma cells, which is
almost identical to CREB-HDTC in size (Figure 2). Second,
we demonstrated the ER and nuclear localizations of CREB-
H and CREB-HDTC, respectively (Figure 4). Finally, we
documented that CREB-HDTC activates transcription more
potently than CREB-H (Figure 3). These findings strongly
support the notion that CREB-H could be activated through
intramembrane cleavage and subsequent nuclear translocation
of an active form equivalent to CREB-HDTC. In this regard,
it will be of interest to elucidate the upstream signals and
molecular events that trigger the cleavage of CREB-H.
CREB-H activation of PEPCK promoter
We showed that the promoter of hepatic gluconeogenic
enzymePEPCK isactivated byoverexpression of
1870Nucleic Acids Research, 2005, Vol. 33, No. 6
CREB-HDTC, but not the full-length CREB-H (Figure 3C).
The cAMP response unit, which mediates the activation of
to bind a number of bZIP transcription factors, including
CREB, CREM, C/EBPa, C/EBPb, ATF2, ATF3 and AP-1
(49,50,52,65,66). As CREB-H
in vitro (Figure 1) and to endogenous PEPCK promoter
in vivo (Figure 3D), CREB-H likely activates PEPCK tran-
scription through direct binding to the CRE element (?91 to
?84) in its promoter. Plausibly, the regulation of PEPCK by
the liver-specific transcription factor CREB-H would be
Our results indicate that CREB-HDTC and cAMP/PKA
synergistically activate the PEPCK promoter (Figure 3C).
This raises the possibility that post-cleavage activation of
CREB-H by cAMP/PKA-dependent phosphorylation might
be responsible for regulating PEPCK expression and hence
glucose homeostasis in the liver. However, we could not
identify potential PKA phosphorylation sites in CREB-
HDTC (data not shown). It remains to be elucidated as to
whether CREB-H is phosphorylated by PKA on a non-
consensus site. Alternatively, CREB-H might cooperate
with other cAMP-responsive CRE-binding factors or tran-
scriptional co-regulators to activate PEPCK expression. In
this regard, CREB has been shown to synergize with other
transcription factors and co-activators to mediate cAMP
responsiveness (67,68). Nevertheless, it is of interest to see
whether cAMP and PKA are required for CREB-H activation
of PEPCK promoter. PKA inhibitors, such as H89, could be
used to address whether the kinase is dispensable for the
activity of CREB-HDTC on the PEPCK promoter.
Growth suppressive function of CREB-H in liver
LETFs are known to be required for liver development and for
maintaining its differentiated functions (1,2,4). In this study,
we characterized CREB-H, a novel LETF whose expression is
exclusively abundant in adult liver (Figure 5A). Notably,
CREB-H is underexpressed in HCC tissues and cell lines
(Figures 5B and 6). Our findings suggest that CREB-H
might play a pivotal role in hepatocyte growth and differen-
tiation. In this context, the loss of CREB-H function in HCC
might contribute to the initiation and/or progression of cancer.
In support of this model, overexpression of CREB-H suffi-
With respect to the liver-specific and growth suppressor
functions, we noticed a strong resemblance of CREB-H to
C/EBPa, another LETF of the bZIP family. C/EBPa is
involved in regulating hepatic growth and differentiation
(69). C/EBPa is also a multifaceted growth suppressor that
interferes with various growth regulatory pathways (9–
12,14,70). The mechanisms through which CREB-H induces
growth arrest in liver remain to be elucidated. In this regard,
we noted that forced overexpression of CREB-H in hepatoma
cells might trigger apoptosis because the number of CREB-H
transfected cell clusters (blue cells in Figure 7A) decreased
during prolonged culture. In addition, CREB-HDTC could not
be stably expressed in HepG2 or Hepa1-6 cells transduced
with a lentivirus carrying a CREB-HDTC expression cassette.
In fact, the cells were all dead within the first day of lentiviral
transduction (K.-T. Chin and D.-Y. Jin, unpublished data).
Since hepatitis C virus core protein targets LZIP (22) which
is structurally related to the liver-specific CREB-H, we rea-
soned that CREB-H might be a more physiologically relevant
target of hepatitis C virus core protein. Our preliminary work
suggests that CREB-H does not interact directly with hepatitis
C virus core protein (data not shown). However, CREB-H
could also be targeted indirectly through an interaction with
LZIP. Additionally, LZIP is a binding partner of transcrip-
tional co-activator HCF1 (71,72) as well as its related protein
HCLP-1 (24),bothofwhich are implicatedincellproliferation
(71). It remains to be seen whether CREB-H might interact
LZIP and partners of LZIP merits further study.
The authors thank Y.P. Ching for helpful discussions;
M. Montminy, K. Mori, K.V. Kibler, G.J. Darlington and
R. Prywes for reagents; and Y.P. Ching, J.W.P.Yam,
C.M. Wong, A.C.S. Chun, Y.T. Siu and K.L. Siu for critical
reading of the manuscript. D.-Y.J. is a Leukemia and
Lymphoma Society Scholar. This work was supported by
grants to D.-Y.J. from the Hong Kong Research Grants
Council (Project HKU 7294/02M and Project N-HKU015/
00). The National Natural Science Foundation (Project
3001161945) and the Ministry of Science and Technology
of China (Projects 2001AA221041 and G1998051002 under
the National Program for Key Basic Research Projects) also
provided support to B.-Q.Q. and J.-G.Y. in Beijing. Funding to
pay the Open Access publication charges for this article was
provided by the Hong Kong Research Grants Council.
Conflict of interest statement. None declared.
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