A plasmid, pME-CREB-H, expressing the CREB-H protein was isolated
from full-length-enriched cDNA libraries described previously (15
and contained 2586 bp of the full-length CREB-H cDNA in an SRα-driven expression vector, pME18S (GenBank
accession no. AB009864). A plasmid, pME-CREB-HdelTM, that expresses
CREB-H lacking the putative transmembrane domain was constructed
by insertion of a PCR product (amino acids 1–320) between
RI and Xba
I sites of pME18S.
A PCR product of CREB-H was produced by amplifying with two primers:
and TM-XBE, AAggatccgaattcTCTAGATCATGTCTGGGCTGACTTGCTGGTGGACTGC,
TURBO (Stratagene). The nucleotide sequence
of the construct was validated by a 377 autosequencer (PE Biosystems).
To confirm the nucleotide sequence of the CREB-H ORF, a fragment
containing the ORF was amplified by PCR from cDNA libraries of HepG2
cells and human liver tissue with KOD dash DNA polymerase (Toyobo)
and subcloned into a TA cloning vector, pT7blue (Novagen). Five
clones containing the ORF were validated by sequencing.
A plasmid, pGST–CREB-H, expressing the GST–CREB-H fusion
protein was constructed by an in-frame insertion of a fragment containing
the CREB-H lacking the transmembrane domain between the BamHI
and EcoRI sites of a GST-expressing plasmid, pGEX-3X
(Pharmacia Biotech). A fragment containing the CREB-H lacking the
transmembrane domain was amplified with the following primers: Bam5′-2, 5′-TTGGATCCCCATGAATACGGATTTAGCTGCTGG-3′; and TM-XBE with pfu TURBO.
The nucleotide sequence of the constructs was checked by the 377
Plasmids expressing fusion proteins of the GAL4 DNA-binding
domain and CREB-H deletion mutants were constructed by an in-frame
insertion of DNA fragments encoding the full-length and the truncated
CREB-H proteins corresponding to amino acids shown in Figure . The CREB-H products were inserted between
dIII and Bam
HI sites of a
plasmid pEF/Gal4-VP16 based on pSG5 (Promega) containing
of the human elongation factor 1α cDNA
promoter derived from pEF-BOS (17
and an Eco
encoding a fusion protein of the DNA-binding domain of Gal4 (amino
acids 1–147) and the acidic transactivator domain of VP16
(amino acids 413–490) linked by the AAGCTTAGATCT (Hin
II, encoding Lys-Leu-Arg-Ser) sequence.
Figure 6 CREB-H activates transcription
of luciferase through the box-B element. The luciferase-reporter
construct was transfected into COS7 cells with a CREB-H-expressing
plasmid pME-CREB-H, or pME-CREB-HdelTM as well as a control vector
plasmid, pME18S. (more ...)
Plasmids expressing GFP fusion proteins with the full-length CREB-H
and a deletion mutant of the transmembrane domain were constructed
by replacing an EcoRI–HindIII
fragment encoding GFP with the GAL4 DNA binding domain of pEF/Gal4
plasmids containing CREB-H.
A luciferase reporter plasmid, pbox-B-Luc, was constructed by
an insertion of oligonucleotides of the box-B element into an Xho
I site of pRBGP-luc derived from pGL2-basic (Promega)
and containing the rabbit β-globin TATA
). The oligonucleotide
sequences for the box-B element of the Drosophila mulleri
) were as follows: box-B-sense,
5′-TCGAGCTCGGATGTACACGTAATCGTATTACTC-3′; and box-b-anti-sense, 5′-CGAGAGTAATACGATTACGTGTACATCCGAGCT-3′.
The plasmids were purified with a Qiagen Plasmid Kit (Qiagen)
according to the manufacturer’s instruction and their nucleotide
sequences were certified by the ABI 377 autosequencer.
Northern blot analysis
Northern blot analyses were performed with Multiple Tissue Northern
Blots I and II (Clontech). A 708 bp probe was produced by PCR with
primers P7 (5′-TGGGCCACCAGCTTGGAGCAGAGAC-3′) and Bam3 (5′-AAGGATCCTCACTCCTGACAGTGCCCAGCCCCAGGTC-3′). PCR products were purified by a
QIAquick gel extraction kit (Qiagen) and labeled with [α-32P]dCTP by a
Random Primer DNA Labeling Kit v.2 (Takara). Blots were prehybridized
for 1 h and then hybridized for 18 h at 42°C
in a hybridization buffer of 50% formamide, 5× SSC,
5× Denhardt’s solution, 0.5% SDS
and 500 µg/ml denatured salmon
sperm DNA. Hybridized blots were washed with 2× SSC/0.1% SDS
at room temperature for 30 min and then with 0.1× SSC/0.1% SDS
at 55°C for 60 min. The washed membranes
were analyzed by a BAS-2500 bio-image analyzer (Fuji film).
Transfection and luciferase assay
COS7 cells were grown in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal calf serum and seeded in six-well
plates. At 48 h after seeding, cells were washed with OPTI-MEM I
Reduced Serum Medium (Life Technologies) and transfected with Lipofectamine
2000 (Life Technologies) in OPTI-MEM I Reduced Serum Medium. In
each transfection, 0.4 µg of the luciferase-reporter
plasmid, 1 µg of the CREB-H-expressing
plasmid, 1 ng of the pRL-CMV plasmid (Promega) and 10 µl
of Lipofectamine 2000 were used. At 24 h after transfection, cells
were lysed and assayed for the Firefly and the Renilla luciferase
activities by a Dual-Luciferase Reporter Assay System (Promega)
according to the manufacturer’s instructions. The results
were normalized against the Renilla luciferase
activities obtained from the pRL-TK plasmid as an internal control.
Gel mobility shift assay
The plasmids were transformed into Escherichia coli
Transformed bacteria were pre-cultured overnight, transferred to
fresh medium and grown for 2 h. After induction with 1 mM IPTG for
2 h, bacteria were sonicated in a sonication buffer containing 50
mM Tris–HCl pH 8.0, 50 mM NaCl, 5 mM EDTA and 2 mM DDT.
The GST–CREB-H fusion protein was purified by glutathione–Sepharose
4B in the sonication buffer, washed with the sonication buffer,
and eluted by an elution buffer containing 50 mM Tris–HCl
pH 8.0, 50 mM NaCl, 5 mM EDTA, 2 mM DDT and 20 mM glutathione. For each
binding reaction, 0.1 µg of GST–CREB-H
was used. Annealed oligonucleotides were labeled with [γ-32
P]ATP with a
Megalabel DNA 5′-end-labeling kit (Takara).
GST–CREB-H and labeled oligonucleotides were incubated
for 30 min at room temperature in 10 µl
of a binding buffer containing 50 mM Tris–HCl
pH 8.0, 5 mM EDTA, 1 mM DTT, 50 mM KCl, 0.1 µg/µl poly(dI–dC) and 20% glycerol.
Electrophoresis was performed for 2 h at 10 mA with a mini-gel system,
a 4.5% acrylamide gel and a 1× electrode
buffer of a GelShift Assay Kit (Stratagene). The gels were dried
down, and the autoradiogram was analyzed by the BAS-2500 bio-image
analyzer (Fuji film). The nucleotide sequences of the probes were
as follows: 5′-TCGAGCTCGGATGGCTGACGTCAGAGATTACTC-3′ and 5′-CGAGAGTAATCTCTGACGTCAGCCATCCGAGCT-3′ for CRE of the somatostatin promoter
); 5′-TCGAGCTCGGATGATTTTGTAATGGGGTTACTC-3′ and 5′-CGAGAGTAACCCCATTACAAAATCATCCGAGCT-3′ for the C/EBP element of
the albumin promoter (21
); 5′-TCGAGCTCGGATCAAAGTTTAGTCAATTACTC-3′ and 5′-CGAGAGTAATTGACTAAACTTTGATCCGAGCT-3′ for the AP-1 element of the c-jun
); 5′-TCGAGCTCGGAGGGGAATCTCCCGGGTTACTC-3′ and 5′-CGAGAGTAACCCGGGAGATTCCCCTCCGAGCT-3′ for the NF-κB
of the IL-2 receptor-α promoter (23
For the box-B element, the same sequences for the luciferase construct,
pbox-B-luc, were used.
Transfected cells were seeded on a chamber slide (Lab-Tek). After
incubation for 24 h, the cells were washed with phosphate-buffered
saline (PBS), fixed with 3.7% formaldehyde in PBS for 5
min, washed with PBS and permeabilized with 0.1% Triton
X-100 in PBS for 5 min. The cells were incubated with 0.5 µg/ml
of 4′,6-diamidino-2-phenylindole (DAPI;
Polysciences, Inc.) for 30 min. The chamber slide was washed three times
with PBS and placed with a drop of 90% glycerol in PBS.
Microscopic examination was carried out under fluorescent light.