Functional Investigation of the Hepatocyte Nuclear Factor 4 (HNF4) Binding Sites in the
Promoter of the Liver Glycogen Synthase (GYS-2) gene
Komsan Anyamaneeratch1,*, Pinnara Rojvirat2, Sarawut Jitrapakdee1,#
1
Department of Biochemistry, Faculty of Science, Mahidol University
2
Division of Multidiscriplinary, Kanjanaburi Campus, Mahidol University
*e-mail: new42531@hotmail.com, #e-mail: sarawut.jit@mahidol.ac.th
Abstract
Glycogen synthesis or glycogenesis plays an essential role in storing excess glucose as a
fuel during fasting period. The liver glycogen synthase (GYS-2) is the rate-limiting enzyme
which regulates glycogen synthesis in liver. The activity of glycogen synthase is allosterically
regulated by glucose-6-phosphate while is inactivated by a reversible phosphorylation. However,
recent reports have shown that the regulation of GYS-2 is also subjected to transcriptional
control. To understand transcriptional regulation of the GYS-2 gene, the 1.5 kb 5’-flanking
region of the mouse GYS-2 gene was cloned and sequenced. The GYS-2 promoter contains no
TATA or CCAAT box locating within the first 100 nucleotides upstream of the transcription
start site. To examine whether the GYS2 gene promoter (-1424/+21) contains full functional
regulatory elements, it was ligated to upstream of the luciferase reporter gene. Transient
transfections of the GYS-2-luciferase reporter construct into HepG2 cells showed that it can drive
expression of the luciferase reporter gene 2.5-fold higher than the promoterless construct,
indicating that this promoter contains full basal regulatory sequences. Truncation of the GYS-2
promoter fragment from -643 to -198 resulted in a significant decrease in the reporter gene
activity, suggesting that the activator element(s) is located between these regions. Furthermore
deletion of the 5’-flanking region to -38/+21 resulted in a marked increase of luciferase activity,
suggesting that the minimal promoter is located in 38 nucleotides proximal to the transcription
start site. Bioinformatics analysis identified two potential binding sites of the liver-enriched
transcription factor, HNF4. One of which resembled the classical DR-1 locating at -29/-17
while the other resembled the HNF4-specific binding motif (H4-SBM). Gel retardation assays of
these two binding sites with purified HNF4 have demonstrated that HNF4 binds to DR-1 with
higher affinity than the H4-SBM. Mutation of DR-1 but not H4-SBM resulted in a marked
reduction of HNF4-transactivation of the reporter construct, suggesting that DR-1 rather than
H4-SBM is required for an HNF4-mediated transcriptional induction of the GYS-2 gene.
Keywords: Glucose homeostasis, liver, glycogenesis, glycogen, and transcriptional regulation
Introduction
Liver is a vital organ which regulates central glucose homeostasis by storing and
releasing glucose during feeding and starvation periods, respectively. During postprandial
period, excess glucose is stored as glycogen by glycogenesis in liver and skeletal muscle or as
lipid by de novo fatty acid synthesis in liver and adipose tissue. Conversely, during short term
starvation period, glycogen is converted to glucose as a fuel especially for brain and red blood
cells (RBCs) by glycogenolysis. Therefore, glycogenesis and glycogenolysis constitute important
mechanism to control glucose disposal, protecting the body against hypoglycemia and
hyperglycemia. The imbalance of these two pathways can lead to the development of type 2
diabetes. Glyogenesis in skeletal muscle and liver are regulated by two glycogen synthase
isozymes, namely, the glycogen synthase-1 (GYS-1) and the glycogen synthase-2 (GYS-2),
respectively. In skeletal muscle, glycogen is used locally as the source of energy during fasted
state because muscle lacks glucose-6-phosphatase (G6Pase) while in liver, glycogen can be
converted to glucose which is subsequently released to the circulation to increase levels of
plasma glucose during fasting period. Therefore, liver glycogen appears to regulate plasma
glucose levels during feeding and fasting periods.
Although, GYS-2 (E.C. 2.4.1.11) is post-translationally regulated via a reversible
phosphorylation and allosteric regulation by glycogen synthase kinase 3 (GSK3) and glucose-6phosphate (G-6-P), respectively(1), recent studies have shown that this isozyme is also
transcriptionally regulated. In mouse, GYS-2 has previously been identified as a direct target of
transcriptional regulation in adipose tissue by the peroxisome proliferator-activated receptor
gamma (PPAR-. Interestingly, HNF4, a liver-specific transcription factor which shares the
similar binding site to PPAR-, can also bind the GYS-2 promoter through the direct repeat-1
(DR-1) and enhances the expression of GYS-2 gene in this tissue. In support of this evidence, the
liver-specific hnf4-null mice also show marked reduction of GYS-2 mRNA expression
compared to the wild-type mice. Interestingly, mutation of the DR-1 in mouse GYS-2 promoter
reduces HNF4-dependent transactivation by only 50%, suggesting the presence of an alternate
HNF4-binding site elsewhere. Also although HNF4 and PPAR have been shown to regulate
transcription of GYS-2 gene, the full characterization of this gene promoter has not been well
studied.
Here we have mapped the important regions and identified the minimal sequences of
GYS-2 promoter required for basal transcriptions of this gene in HepG2 cells. We have also
shown the presence of an alternative binding site of HNF4, namely the H4-SBM motif, in the
GYS-2 gene promoter and presented the functional relevance of this site with respect to the
previously identified DR-1 in the regulation of basal and HNF4-mediated transcriptional
activation of GYS-2 gene in a context of luciferase reporter gene assay.
Methodology
Cloning of mouse GYS-2 promoter from mouse genomic DNA. Full-length GYS-2
promoter (-1424 to +21) was isolated from mouse genomic DNA by PCR with forward [5’-GGT
ACC ATG TCA CTG AGT CCC AGA AGA C-3’; underline represents a KpnI restriction site
introduced] and reverse primers, [5’-CTC GAG AGC GTG CCC AGC TCC TTG-3’; underline
indicates an XhoI restriction site]. PCR was performed in a 50 µl-total volume containing
genomic DNA, 1x PCR buffer [20 mM Tris-HCl pH 8.4, 50 mM KCl], 1.5 mM MgCl2, 0.2 mM
dNTPs, 0.5 µM of each primer and 1 U of Taq DNA polymerase (Invitrogen). The amplification
profiles consisted of an initial denaturation at 94ºC for 5 min followed by 30 cycles of
denaturation at 94ºC for 45 sec, annealing at 60ºC for 45 sec and extension at 72ºC for 2 min,
followed by a final extension at 72ºC for 10 min in a mini-thermal cycler (BioRad). The PCR
product was cloned into pGEM-T Easy vector (Promega) before verified by automated DNA
sequencing. GYS-2 promoter was then subcloned into the above restriction sites in luciferase
repoter vector, pGL4.12-basic (Promega).
Generation of 5’-truncated promoter constructs. Five deletion constructs containing
various 5’-lengths of GYS-2 promoter were generated by PCR using 5’-nested deletion primers
(Table 1) and a common 3’-primer (5’-CTC GAG AGC GTG CCC AGC TCC TTG-3’;
underline indicates an XhoI site ). PCR was performed described above. Furthermore, two
shorter fragments were obtained by double digestion of the full-length GYS-2 promoter (XbaI
and XhoI, -197/+21; EcoRI and XhoI, -813/+21) (New England Biolabs) and were subcloned into
luciferase reporter vector. For constructing the mutHNF4DR or mutH4SBM mutants lacking the
DR-1 or H4-SBM, the pGL4-del200R2 (+38/+21) was used as template to produce the above
two mutants using site-directed mutagenesis. Mutagenesis was performed in a 50 μl-reaction
mixture containing 1x Pfu polymerase buffer (100 mM KCl, 100 mM (NH4)2SO4, 200 mM TrisHCl pH 8.8, 20 mM MgSO4, 1% Triton® X-100 and 1 mg/ml BSA), 0.2 mM dNTPs, 125 ng of
each of primer, 100 ng of template and 2.5 units of Pfu Turbo polymerase. PCR profiles
consisted of an initial denaturation at 95 °C for 1 min, followed by 20 cycles of denaturation at
95 °C for 30 sec, annealing at 50 °C for 3 min and extension at 68 °C for 7 min, followed by a
final extension at 68 °C for 8 min. The PCR mixture was digested with DpnI at 37oC overnight.
10 μl of the digest were transformed into Escherichia coli DH5α. The correct mutagenesis was
verified by nucleotide sequencing before replacing the equivalent fragments in the parental
plasmid.
Table 1 Oligonucleotides used for generating 5’-deletion mutagenesis and site-specific mutants
Construct
Oligonucleotides Sequence (5’-3’)
-38/+21
GGT ACC TTG GTC TAA AGG CCA AAG GCC
-134/+21
GGT ACC CCA CCG TGC AGT CTG TGG
-334/+21
GGT ACC GCT ATG CAG AAA GAC ACC GTG
-436/+21
GGT ACC GAC ATT TAA AAT CCA TGA ACG GAA C
del800F
-643/+21
GGT ACC GGT GGT GTT TGT CTG TAT GGG
mutHNF4DR
mutHNF4DR-F
AGG AGT TTG GTC TAA GAA TTC AAG GCC AAA GGA CTG
mutHNF4DR-R
CAG TCC TTT GGC CTT GAA TTC TTA GAC CAA ACT CCT
mutH4SBM-F
CAA AGG CCA AAG GAC GAA TTC TCC GTC ATG CTC AGT CTG
mutH4SBM-R
CAG ACT GAG CAT GAC GGA GAA TTC GTC CTT TGG CCT TTG
del200F
del300F
del500F
del600F
mutH4SBM
Cell culture and transient transfection. HepG2 cells (ATCC No. HB-8065) were cultured
in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine
serum (FBS), 1x penicillin and streptomycin and maintained at 37oC under 5% CO2. The day
before transfection, 1.5 × 105 cells were seeded in 24-well plate. Transfections were carried out
using LipofectAMINETM 2000 reagent (Invitrogen). Each transfection was performed with 0.07
pmole of luciferase reporter construct, 250 ng pRSV--gal with or without 0.07 pmole of
plasmid encoding human HNF4 (pcDNA-HNF4) (3). The transfected cells were further
maintained at 37ºC with 5% CO2 for 48 h. Luciferase activity was measured using the luciferase
reporter assay system (Promega). -Galactosidase activity was measured using 2-nitrophenyl-D-galactopyranoside (ONPG) as a substrate. The relative luciferase activity was normalized with
-Galactosidase activity and expressed as “relative luciferase” expression.
Electrophoretic mobility shift assay (EMSA). Synthesized oligonucleotides, which are
labeled with biotin at 3’-end, contain DR-1 site (5’- TCT AAA GGC CAA AGG CCA AAG
GA -3’) or H4-SBM site (5’- AGG ACT GGA CTC TTG TCA TGC TC -3’) of GYS-2 gene.
Two complementary oligonucleotides were annealed in 1x annealing buffer (10 mM Tris pH7.4,
100 mM NaCl and 1 mM EDTA) by heating at 95oC for 5 min before slowly cool down. The
nuclear proteins of HepG2 cells were prepared as described previously (4). Binding reactions
were performed in a 20 μl-reaction mixture containing 1x binding buffer (10 mM Tris, pH 7.4,
50 mM KCl, 1 mM DTT, 5% (v/v) glycerol), 1 μg of poly (dI–dC), 1% (v/v) NP-40, 120 pmole
annealed probe, and 15 μg of nuclear extracts or 0.1 μg purified 6×His hHNF4α, at 4°C for 30
min. The protein- DNA complexes were analyzed by 5% non-denaturing acrylamide gel
electrophoresis in 0.5% TBE (44.5 mM Tris–HCl, pH 8.0, 44.5 mM boric acid and 1.25 mM
EDTA). The specificity of each site was examined by including 5-, 10- and 50-fold excess
unlabeled oligonucleotide probes, respectively. Supershift assay was performed by incubating 1
g of anti- HNF4polyclonal antibody SantaCruz Biotech)in the binding
reactionThe DNA and protein complexes were visualized using the Non-radioactive Nucleic
Acid Detection Kit (Pierce).
Results
Localization of minimal cis-regulatory elements in the GYS-2 promoter. To determine the
promoter activity and localize the essential cis-acting elements within the GYS-2 gene promoter
required for basal transcriptional activity, a series of 5’-truncated promoter constructs was
generated either by PCR or digesting with restriction enzymes. Promoter fragments were cloned
into the pGL4.12-basic vector, a promoterless reporter plasmid. Following transient transfections
of these reporter constructs into HepG2 cells, the relative luciferase activity was determined. As
shown in Figure 1, the 1.5 kb GYS-2 promoter fragment (pGL4-G22) was capable of driving the
expression of luciferase approximately 2.5-fold higher than the empty vector while further
deletions to nucleotides -813 and to -643 increased the luciferase activity to 5-fold and 8-fold,
respectively, suggesting the presence of negative regulatory elements locating between
Figure 1 Localization of minimal promoter elements required for basal transcription of the mouse GYS-2 promoter. The
1424-bp flanking regions were progressively deleted from its 5’-end, fused to the pGL4-basic vector and transfected into
HepG2 cells. The luciferase activity of each construct was normalized to the β-galactosidase activity and shown as
relative luciferase activity. The relative luciferase activities shown are the means + the standard deviations for triplicate
determinations. Relative luciferase activities are also shown as fold increase of the activity of pGL4-basic construct,
which was arbitrarily set as 1. *, p < 0.005; **, p < 0.001
Figure 2 Putative cis-regulatory elements of GYS-2 promoter identified by PROMO database
nucleotides -1424 and -643. However, further deletion to nucleotides -436 resulted in a dramatic
decrease of the luciferase activity to 2.5-fold, suggesting that the positive regulatory elements are
located between nucleotide positions -644 and -236. Further deletions to nucleotides -334 and –
198 did not significantly affect the promoter activity. However progressive deletions to
nucleotides -134 resulted in a significant increase of promoter activity to 5.5-fold suggesting the
presence of the second negative regulatory elements between nucleotides -197 and -135. Further
truncation to nucleotide -38 did not affect promoter activity, indicating that the first 38
nucleotides proximal to the transcription start site are sufficient to drive transcription of GYS-2.
As shown in Figure 2, nucleotide sequence analysis of mouse GYS-2 promoter by PROMO
database (5, 6) revealed that GYS-2 promoter lacked both TATA and CAAT boxes but contained
several binding sites of potential transcription factors including general transcription factors;
USF-1, USF-2 and Sp1; transcription factors associated with energy homeostasis, CCAATenhancer binding prteoin (C/EBPs); liver-specific transcription factors, HNF1, HNF3 and
HNF4; and hormone-responsive transcription factor, SREBP-1c. Interestingly, in the first 38
Figure 3 EMSA of DR-1 and H4-SBM sequences of GYS-2 promoter. Gel shift and super shift assay were
performed using biotin-labeled probes containing DR-1 (A) or H4-SBM (B) sequence. Lane 1, free probes; lane
2, probes incubated with nuclear proteins from HepG2 cells; lane 3-5, the excess unlabeled DR-1 (A) or H4SBM (B) nucleotides (5x, 10x and 50x) were incubated with nuclear proteins and probes; lane 6-8, the excess
unlabeled H4-SBM (A) or DR-1 (B) nucleotides (5x, 10x and 50x) were incubated with nuclear proteins and
probes; lane 9, probes incubated with nuclear protein and antibodies against HNF4; lane 10, HNF4 site 1 and
3 of mouse PC promoter was used as a positive control of DR-1 and H4-SBM elements, respectively.
nucleotides proximal to the transcription start site, this region contained two HNF4 binding
sites which contains the classical direct repeat-1 (DR-1) (-29/-17) (5’-AGGCCAAAGGCCA-3’)
and the HNF4-specific binding motif (H4-SBM) (-10/+2) (5’-TGGACTCTTNNNN-3’) (7).
HNF4 transcription factor binds to both DR-1 and H4-SBM sequences in vitro. As H4SBM sequence is an alternative binding site for HNF4, electrophoretic mobility shift assays
(EMSA) of probes containing DR-1 or H4-SBM sequences using HepG2 nuclear extracts were
performed to examine if this site was capable of binding to HNF4. As shown in Figure 3A,
incubation of DR-1 probe with HepG2 nuclear extract produced a strong DNA-protein complex
while this complex was decreased approximately by 90-100% when 5-fold and 10-fold excess
amounts of unlabeled DR-1 probe were included in the binding reaction, respectively. To
compare the binding affinity of this H4-SBM and the DR1 with HNF4, different amounts (5x,
10x, 50x) of unlabeled H4-SBM or DR-1 competitor were included in the binding reactions. As
shown in Figure 3A, 50x excess amount of unlabeled H4-SBM was capable of completely
competing the complex formation while this only occurred when 10x excess amount of
unlabeled DR1 were included. To confirm this result we performed a similar experiment but
using H4-SBM as a probe instead of DR-1, incubation of this probe with HepG2 nuclear extract
produced a prominent DNA-protein complex similar to that of the DR-1 probe, as shown in
Figure 3B. Addition of excess amount of unlabeled H4-SBM competitor up to 50-fold was
required to completely inhibit this complex formation while this inhibition requires only 10x
Figure 4 HNF4 binds to HNF4 binding sites with different affinities. Various amounts of biotinylated
oligonucleotides (60, 120, 240, 360 and 480 fmole ) containing DR-1 or H4-SBM were incubated with 100 ng of
purified HNF4
excess amount of DR-1 competitor. Likewise, incubation of anti-HNF4 antibody in the reaction
inhibited this complex formation, as shown in Figure 3A and 3B. The use of these two probes in
these assays suggested that DR-1 binds HNF4 presence in HepG2 nuclear extract better than
H4-SBM.
HNF4α binds both HNF4α binding sites with different affinities. Since EMSA of both probes
using HepG2 nuclear extract was not sensitive enough to measure the relative binding affinity of
HNF4 to each probe, we conducted more quantitative EMSA in which different amounts (60,
120, 240, 360 and 480 fmole) of each probe were titrated with a limited amount of purified
HNF4 (100 ng). As shown in Figure 4, HNF4 appears to bind to DR-1 probe approximately
3-fold higher than to the H4-SBM.
To examine whether these two HNF4 binding sites, DR-1 or H4-SBM contributed to
the transcriptional regulation the GYS-2 promoter under the basal and the HNF4-mediated
transactivation conditions, the GYS-2 promoter luciferase constructs containing mutation of DR1 or H4-SBM were generated. As shown in Figure 5, mutation of the H4-SBM binding site had
no effect on promoter activity while mutation of the DR-1 site resulted in a reduction of
promoter activity by 60%. To examine the functional significance of the interaction of HNF4
Figure 5 Effect of mutations of HNF4 binding sites (DR-1 and H4-SBM) on GYS-2 promoter activity under
basal condition. The single and double mutations of DR-1 and H4-SBM were introduced into pGL4-del200R2
reporter construct and transiently transfected into HepG2 cells. The luciferase activity was normalized by galactosidase activity and shown as relative luciferase activity. The activity obtained from the mutated
promoter was compared to that of the wild-type (WT) construct which was arbitrarily set as 100. *, p < 0.05
Figure 6 Transactivation of the wild-type and mutant GYS-2 promoter-luciferase reporter constructs by HNF4.
The first 38 nucleotides proximal to transcription start site of GYS-2 promoter were transiently co-transfected
with empty expression vector (pcDNA) or plasmid encoding human HNF4 into HepG2 cells. The luciferase
activity of each construct was normalized by -galactosidase activity and expressed as relative luciferase
activity. Cells transfected with pGL4-del200R2 and empty vector arbitrarily set as 1.
with its binding site located in GYS-2 promoter, the wild-type and mutant reporter constructs of
the 38 nucleotides upstream of the transcription start site were also transiently co-transfected
with plasmid overexpressing human HNF4into HepG2 cells. As shown in Figure 6,
overexpression of HNF4 resulted in 12-fold increase of the reporter activity of the wild-type
promoter reporter construct while mutation of DR-1 site caused a 50% reduction of HNF4mediated transcriptional induction of the reporter activity. Unlike promoter activity of mutated
DR-1 site, mutation of H4-SBM site showed the increase of promoter activity in the same level
of wild-type construct. Thus, HNF4 drives the transcription of GYS-2 gene through DR-1 site
but not H4-SBM site.
Discussion and Conclusion
Glycogen synthase-2 (GYS-2) is a liver-specific isozyme and its expression is also
transcriptionally regulated. Although GYS-2 gene promoter lacks both TATA and CCAAT
boxes, it is capable of driving the luciferase reporter gene, indicating that it may use other
sequence(s) to drive the basal transcription in HepG2 cells. The lack of both TATA and CCAAT
boxes is usually found in many housekeeping genes (8, 9). Deletion analysis of its 5’-end
revealed the presence of both negative and positive regulatory elements locating throughout the
1.5 kb region. Nevertheless, the shortest construct containing only 38 nucleotides was still able
to drive the expression of the luciferase reporter gene, indicating that these 38 nucleotides must
contain sufficient elements to drive the transcription of the GYS-2 gene in HepG2 cells. The use
of relatively short segment of cis-acting element to drive transcription is not unusual since the
similar length of minimal promoter of the flavin-containing monooxyenase-1 (FMO-1) gene was
also reported to contain sufficient information to drive its transcription. In FMO-1 gene
promoter, binding site of YY1 transcription factor was found in the 41 nucleotides proximal to
transcription start site. YY1, locating in upstream of initiator element (Inr), participates in the
negative regulation of FMO-1 gene. In contrast, YY1 binding to the Inr element of adenoassociated virus type 2 P5 promoter is important for basal promoter activity (10). Therefore,
putative YY1 binding site in the GYS-2 promoter may participate in the basal transcription of this
gene.
Bioinformatic analysis of the 1.5 kb of GYS-2 promoter, identified several putative
binding sites of transcription factors implicating in both energy homeostasis and liver-specific
expression. The CCAAT/enhancer binding protein  (C/EBP) has been reported to be
associated with the maintenance of energy homeostasis and its expression is important for
positive regulation of GYS-2 transcription (11). As described by Lee et al, the GYS-2 mRNA
levels were significantly decreased in the hepatocytes of c/ebp-null mice. This is consistent
with the presence of several putative binding sites of C/EBPs in the positive regulatory element
(nucleotides from -643 to -437) of GYS-2 promoter. Because GYS-2 expression is liver-specific,
it is not surprised to see the presence of several putative binding sites of liver-enriched
transcription factors in GYS-2 promoter. HNF3 binding sites were found in the negative
regulatory element from -1424 to -644. As reported in the previous study, overexpression of
HNF3 in mice reduced the levels of GYS-2 mRNA (12). Therefore, HNF3 may participate in
transcriptional regulation of GYS-2 gene. Furthermore, Odom and his co-workers have reported
that the HNF4 ablated mice showed a reduction of GYS-2 mRNA level (13).
HNF4 is one of the non-steroid nuclear receptors of ligand-dependent transcription
factor (NR2A1) (14, 15). The development of liver and maintenance of its function are regulated
by HNF4 . HNF4 plays an important role in the central metabolism by regulating
glucose homeostasis. Mutation of this transcription factor causes the development of a
monogenic form of diabetes known as the Maturity Onset of the Young 1 (MODY-1) (17). In the
previous studies, the direct repeat-1 (DR-1) sequence was identified as a HNF4 binding site.
Mandard and his colleagues characterized the function of DR-1 site in the mouse GYS-2
promoterand showed that DR-1 site is a binding site of both HNF4 and PPAR in hepatocyte
and adipocyte, respectively (2). Consistent with the previous study, we found that mutation of
the DR-1 site showed 50% reduction of HNF4-transactivation of GYS-2 promoter activity,
suggesting that it contained another HNF4 binding site located in GYS-2 promoter. Fang et al.
has reported a novel binding site of HNF4, named the HNF4-specific binding motif (H4-SBM)
and this sequence is present in the 38 bp proximal to transcription start site of GYS-2 promoter
and is also located downstream from DR-1 site. Although EMSA revealed that HNF4 can bind
to H4-SBM, mutation of H4-SBM did not affect the promoter activity both under basal condition
and HNF4-mediated transcriptional activation, suggesting that this H4-SBM is not relevant to
HNF4-transactivation of GYS-2 promoter. The identification of an alternative HNF4-binding
site warrants further investigation.
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Acknowledgements: This work was supported by the Institutional Strengthening Scholarship
from Faculty of Science, Mahidol University.