Hepatocyte Nuclear Factor 4? Is Implicated in
Endoplasmic Reticulum Stress–Induced Acute Phase
Response by Regulating Expression of Cyclic
Adenosine Monophosphate Responsive Element
Binding Protein H
Jennifer Luebke-Wheeler,1Kezhong Zhang,2Michele Battle,1Karim Si-Tayeb,1Wendy Garrison,1Sodhi Chhinder,1
Jixuan Li,1Randal J. Kaufman,2-4and Stephen A. Duncan1
Loss of the nuclear hormone receptor hepatocyte nuclear factor 4? (HNF4?) in hepatocytes
results in a complex pleiotropic phenotype that includes a block in hepatocyte differentia-
tion and a severe disruption to liver function. Recent analyses have shown that hepatic gene
expression is severely affected by the absence of HNF4?, with expression of 567 genes
reduced by >2.5-fold (P < 0.05) in Hnf4??/?fetal livers. Although many of these genes are
factors, and this raises the possibility that the dependence on HNF4? for normal expression
of some genes may be indirect. We postulated that the identification of transcription factors
whose expression is regulated by HNF4? might reveal roles for HNF4? in controlling
hepatic functions that were not previously appreciated. Here we identify cyclic adenosine
monophosphate responsive element binding protein H (CrebH) as a transcription factor
whose messenger RNA can be identified in both the embryonic mouse liver and adult mouse
liver and whose expression is dependent on HNF4?. Analyses of genomic DNA revealed an
HNF4? binding site upstream of the CrebH coding sequence that was occupied by HNF4?
in fetal livers and facilitated transcriptional activation of a reporter gene in transient trans-
fection analyses. Although CrebH is highly expressed during hepatogenesis, CrebH?/?mice
were viable and healthy and displayed no overt defects in liver formation. However, upon
CrebH?/?mice displayed reduced expression of acute phase response proteins. Conclusion:
These data implicate HNF4? in having a role in controlling the acute phase response of the
liver induced by ER stress by regulating expression of CrebH. (HEPATOLOGY 2008;48:
Abbreviations: Alb1, albumin 1; Apoc3, apolipoprotein c3; ATF, activating transcription factor; ChIP, chromatin immunoprecipitation; Creb, cyclic adenosine
monophosphate responsive element binding protein; CRP, C-reactive protein; DE, definitive endoderm; EMSA, electrophoretic mobility shift analysis; ER, endoplasmic
chain reaction; RT-PCR, reverse-transcription polymerase chain reaction; SAA3, serum amyloid A3; SAP, serum amyloid P-component; Ttr, transthyretin; VE, visceral
From the1Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI; and2Department of Biological Chemistry,
3Department of Internal Medicine, and4Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, MI.
Received April 9, 2008; accepted May 14, 2008.
Funding for this project was provided by an American Heart Association fellowship to W.G., by a Scientist Development Grant to K.Z., by National Institutes of Health
grants DK66226 and DK55743 to S.A.D., by National Institutes of Health grants DK042394, HL052173, and HL057346 to R.J.K., and by gifts from the Marcus
R.J.K. is an investigator of the Howard Hughes Medical Institute.
Copyright © 2008 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of this article.
studies have shown that the nuclear hormone receptor
hepatocyte nuclear factor 4? (HNF4?), as part of this
network, is essential for initiating and maintaining hepa-
tocyte differentiation and liver function.3-6Loss of
HNF4? in the fetal hepatoblasts results in reduced ex-
pression of over 500 genes encoding factors that contrib-
ute to all aspects of hepatic function.6HNF4? has also
been shown to regulate expression of other transcription
factors, including HNF1?7,8and peroxisome prolifera-
of hepatocyte gene expression by HNF4? may in some
cases be indirect. We therefore sought to uncover novel
transcription factors that were expressed during hepatic
development that could potentially be regulated by
HNF4?. We proposed that identification of such factors
would implicate HNF4? in regulating aspects of liver
function that heretofore were not recognized. In the cur-
rent study, we report the identification of a cyclic adeno-
sine monophosphate responsive element binding protein
of HNF4? regulation that is expressed in differentiating
for the expression of acute phase response proteins in-
duced by endoplasmic reticulum (ER) stress.
epatocyte gene expression is controlled by a
complex network of transcription factors that is
established during hepatogenesis.1,2Recent
Materials and Methods
Oligonucleotide Array Analysis. RNA was isolated
from tissues with the RNeasy kit (Qiagen), and comple-
mentary RNA probes were prepared according to the di-
rections described in the Affymetrix expression analysis
RT-PCR. RT-PCR3and quantitative real-time RT-
PCR20were performed as described previously. Primer
sequences are available upon request.
In Situ Hybridization and Immunohistochemistry.
In situ hybridization was performed as described else-
where.23A probe used to detect CrebH was generated by
in vitro transcription of a fragment from CrebH com-
plementary DNA, which was generated by PCR using
primers atcgaattcccatcagccctttcaactcc and atcggatccag-
gccagcctggtctacaag and subsequently cloned into the
EcoR1/BamH1 sites of pBSII-KS. Immunohistochem-
istry was performed with previously described proce-
dures5with antibodies that detect HNF4? (Santa Cruz
Chromatin Immunoprecipitation and EMSA. Im-
munoprecipitation of chromatin was performed with the
Upstate ChIP assay kit (Upstate #17-295) as described
previously,6and EMSA was performed as described else-
Analysis of Luciferase Expression. DNA was intro-
duced into 293T cells with Lipofectamine Plus reagent
(Invitrogen). Cell extracts were collected 48 hours after
transfection and processed with a luciferase assay dual
reporter system (Promega). Each experiment was per-
formed in triplicate, the entire experiment was repeated
on five separate occasions, and data were combined. Sta-
tistical significance was determined with the Student t
test, with P ? 0.05 considered significant.
Animals and Embryonic Stem Cells. The Medical
ical Center Institutional Animal Care and Use Commit-
tee approved all animal experiments and procedures. The
generation of Hnf4??/?(Hnf4?tm1Dnl), Hnf4?loxP/loxP
(Hnf4?tm1Sad), Foxa3Cre [Tg(Foxa3-cre)1Khk], AlfpCre
[Tg(Alb1-cre)1Khk], and VillinCre [Tg(Vil-cre)997Gum] mice
the appearance of a vaginal plug was considered to be 0.5
days post coitum, and the genotype of all embryos was de-
serum gonadotropin, used in superovulation, was obtained
from A.F. Parlow at the National Hormone and Peptide
CrebH Messenger RNA (mRNA) Is Highly Enriched
in the Fetal Liver. Using Affymetrix oligonucleotide
array analyses, we attempted to identify novel tran-
scription factors whose expression was enriched in fetal
livers. RNA was isolated from pools of livers, hearts,
and heads that were dissected from E10.5 mouse em-
bryos and used to generate probes that were hybridized
to Affymetrix oligonucleotide GeneChip Murine Ge-
nome U74v2 A and B arrays. A comparison of liver,
heart, and head arrays revealed 1208 genes whose ex-
pression was predicted to be increased ?3.0-fold in the
fetal liver samples compared to the heart and head
samples. Of these genes, 78 encoded potential fetal
liver–enriched transcription factors.10This list in-
cluded Hnf4?, CCAAT/enhancer binding protein al-
pha, Forkhead box A1 (Foxa1), and Foxa3 (see
Supplementary Table 1), all which have previously
been shown to be expressed in hepatoblasts during em-
bryonic development; however, other hepatoblast tran-
scription factors, including HNF1?, HNF1?, and
HNF6, were not identified, which suggested that the
screen was not saturated.
Of the mRNAs identified, we chose to focus our anal-
yses on CrebH because it was highly enriched in the fetal
HEPATOLOGY, Vol. 48, No. 4, 2008LUEBKE-WHEELER ET AL.1243
liver and was a member of the ATF/CREB family, other
members of which are known to have important roles in
controlling liver gene expression, and analyses of the hu-
being exclusively expressed in the adult liver and unde-
tectable in other tissues.11Using reverse-transcription
polymerase chain reaction (RT-PCR), we first confirmed
compared to the heart and head. Figure 1A shows that,
like Albumin 1 (Alb1) mRNA, CrebH mRNA was en-
riched in three independent E10.5 liver samples com-
pared to heart and head samples. Real-time quantitative
RT-PCR (not shown) demonstrated that CrebH mRNA
levels were 45 times greater in the liver compared to the
Hepatic Expression of CrebH Initiates During
Liver Bud Formation and Continues Throughout
Hepatogenesis. As a first step toward determining
whether CrebH had the potential to act downstream of
Fig. 1. Fetal expression of CrebH initiates in the primary liver bud and
continues throughout hepatogenesis. (A) RT-PCR analyses revealed the
presence of CrebH and Alb1 mRNAs in livers isolated from E10.5
embryos. Amplification of Rna pol2 (Pol2) was used as a loading control,
whereas reactions lacking reverse transcriptase (?RT) and a DNA tem-
plate (0DNA) confirmed the absence of contaminating DNA. (B) RT-PCR
analyses uncovered CrebH and Hnf4? mRNA in livers isolated from
mouse embryos at daily intervals ranging from E10.5 to E18.5 and in
adult livers. Amplification of hypoxanthine guanine phosphoribosyl trans-
ferase (Hprt) was used as a loading control, whereas reactions lacking
reverse transcriptase (?RT) and a DNA template (0DNA) confirmed the
absence of contaminating DNA. (C-H) Radioactive in situ hybridization
analyses revealed the presence of CrebH mRNA (arrows; silver grains)
during early development. (C,F) Sagittal sections through an E8.5 (6-8
somite stage) embryo identified CrebH mRNA in the extraembryonic
visceral endoderm (VE) but not in the definitive endoderm (DE); the
extraembryonic/embryonic boundary is indicated by arrowheads. CrebH
mRNA was also found to be present (D,G) in the primary liver bud (Lb;
outlined with dashes) in transverse sections through an E9.5 embryo and
(E,H) in the expanding clusters of hepatoblasts (outlined with dashes) in
transverse sections through an E10.5 embryo. (C,D,E) H&E-stained
bright-field images and (F,G,H) corresponding dark-field images are
Fig. 2. CrebH is expressed in the adult liver and gastrointestinal tract.
(A) RT-PCR analyses of CrebH and Hnf4? were performed on mRNA
extracts from the adrenal gland (Adr), brain (Brn), colon (Col), duodenum
(Ddn), heart (Hrt) ileum (Ile), kidney (Kid), liver (Liv), lung (Lng), ovary
(Ovr), stomach (Sto), testis (Tes), and uterus (Utr). (B-G) The distribution
of CrebH mRNA in (B,E) the liver, (C,F) small intestine, and (D,G)
stomach was identified (white arrows, silver grains) with radioactive in
situ hybridization analysis. CrebH mRNA was present in the hepatocytes
(h, arrow) of the liver, in the epithelial cells of the villi (v, arrow) but not
the crypts (c, white arrows; a yellow dashed line demarcates the villi/
crypt border) of the small intestine, and in the surface lining cells (sl) of
the stomach. (B-D) H&E-stained bright-field images and (E-G) corre-
sponding dark-field images are presented.
1244LUEBKE-WHEELER ET AL. HEPATOLOGY, October 2008
pared expression of CrebH to that of Hnf4? during he-
patic development. Livers were isolated from embryos at
E10.5 through E18.5 and from adults, and mRNAs en-
coding CrebH and HNF4? were measured by RT-PCR.
levels early in development, begins to increase around
is reached in the adult liver. This dynamic pattern of
seen to increase over this developmental time course. In
situ hybridization analyses were next performed to assess
expression of CrebH mRNA during the onset of hepatic
development. Figure 1C,F shows that during specifica-
tion of the hepatic lineage from the ventral endoderm at
E8.0 (6-8 somites), CrebH mRNA is restricted to the
extraembryonic visceral endoderm, and it was not de-
tected in the definitive endoderm, as has previously been
described for Hnf4?.12,13Approximately a day later in
development at E9.5, like Hnf4?,12,13CrebH mRNA was
detected in the definitive endoderm forming the primary
liver bud (Fig. 2D,G), and expression continued in the
hepatoblasts as they delaminated from the bud and in-
CrebH Is Expressed in the Epithelial Cells of the
Adult Liver, Pyloric Stomach, and Small Intestine.
The expression profile of CrebH mRNA in adult mouse tis-
was performed with complementary DNA derived from a
spectrum of adult mouse tissues (Fig. 2A). Hnf4? mRNA
viously.13-15Like Hnf4?, CrebH mRNA was identified in
the liver, pyloric stomach, duodenum, and ileum; however,
in contrast to Hnf4?, CrebH mRNA was not identified in
also expressed Hnf4? mRNA, although not all Hnf4?-pos-
itive tissues expressed CrebH.
Fig. 3. CrebH is a direct target of HNF4? transcriptional activity. (A) Schematic showing the genomic location and sequence of the identified
HNF4? binding site with respect to CrebH (exons are shown as boxes). (B) The ability of the HNF4? protein to bind the putative HNF4?-binding site
was confirmed by EMSA. Radiolabeled oligonucleotides representing binding sites were incubated with liver nuclear extracts in the presence of
anti-HNF4? antibody, and this resulted in a retarded migration of HNF4?-bound complexes (arrows) or anti-Pes1 antibody (negative control).
Alternatively, EMSAs were performed with nuclear extracts from COS-7 cells or COS-7 cells expressing HNF4?. A previously described HNF4? binding
site in the Apoc3 promoter served as a positive control, and a Foxa (Hnf3) binding site within the Ttr promoter served as a negative control. (C) 293T
cells were transfected with plasmids in which expression of luciferase was driven by the HIV basal promoter (pZLHIVS) or, in addition, a 207-bp
fragment from the CrebH gene that contained the HNF4? binding site (pCrebH-Luc1) in the presence or absence of exogenously expressed HNF4?.
Luciferase levels from five independent experiments are presented as fold differences with respect to cells transfected with pZLHIVS alone.
Significance was determined by the Student t test (P ? 0.05). (D) ChIP analyses were performed on chromatin extracted from two independent E18.5
livers (L1 and L2) or brains (B1 and B2), which acted as negative control tissue that did not express HNF4?. Chromatin was precipitated with
anti-HNF4? or anti-Pes1 (negative control), and specific primers were used to amplify input chromatin or chromatin precipitated from the Pol2
promoter (negative control), Apoc3 promoter (positive control), or CrebH.
HEPATOLOGY, Vol. 48, No. 4, 2008LUEBKE-WHEELER ET AL.1245
specific cell types that expressed CrebH mRNA. Tissues
lacking CrebH mRNA, such as the kidney (based on RT-
PCR analysis), showed no hybridization above back-
ground and therefore acted as convenient negative
the hepatocytes of the adult liver (Fig. 2B,E) and in the
epithelium of the villi, but not the crypts, of the small
intestine (Fig. 2C,F). CrebH transcripts were also identi-
fied in the surface epithelial cells of the pyloric stomach
(Fig. 2D,G) but not in the glands.
HNF4? Regulates Transcription Through an
HNF4?-Binding Site Within the Putative Transcrip-
tional Regulatory Region of the CrebH Gene. The
is continuously expressed in the hepatic cells throughout
liver development in a manner indistinguishable from
HNF4?, raising the possibility that CrebH is a direct
transcriptional target of HNF4?. To identify potential
DNA sequences that could facilitate HNF4?-mediated
expression of CrebH, we therefore examined 27.5 kb of
the CrebH genomic DNA sequence, including sequences
extending 10 kb upstream of exon 1, for the presence of
any of 215 known HNF4? binding sites, using an
HNF4? motif finder generated by Sladek and colleagues
(http://www.sladeklab.ucr.edu/links.html). This analysis
lying 3.7 kb upstream of CrebH exon 1 (Fig. 3A). The
ability of HNF4? to bind the aforementioned sequence
was confirmed with electrophoretic mobility shift analy-
ses (EMSAs) on nuclear extracts from adult liver. Figure
3B shows that HNF4? protein could be detected in a
complex with a well-characterized binding site (H4.21;
HNF4? antibody. A complex with a similar migration
pattern was identified when the same extracts were incu-
bated with the H4.77 HNF4?-binding site from the
CrebH gene, but not when extracts were incubated with a
FoxA transcription factor binding site from the Transthy-
retin (Ttr) gene, which acted as a control for binding
specificity. Similar results were obtained when extracts
from COS-7 cells expressing exogenous HNF4? were
used (Fig. 3B). Other protein-DNA complexes were also
detected in the extracts and likely reflect the binding of
ing sites, such as retinoid A receptor, retinoid X receptor,
and chicken ovalbumin upstream promoter transcription
The ability of exogenous HNF4? to activate tran-
scription via the HNF4? binding site within the puta-
tive CrebH promoter region was studied by transient
transfection analyses in 293T cells, which do not ex-
press endogenous HNF4?. Figure 3C shows that lucif-
erase levels measured in 293T cells transfected with a
reporter plasmid containing only the human immuno-
deficiency virus (HIV) basal promoter to regulate tran-
scription of the luciferase reporter gene (pHIV-Luc)
were not affected by the additional introduction of
exogenously expressed HNF4?. Similar results were
obtained when 293T cells were transfected with the
same reporter plasmid containing an additional 123-bp
element from CrebH that included the HNF4?-binding
site (pCrebH-Luc). However, the introduction of exog-
enously expressed HNF4? to pCrebH-Luc–transfected
293T cells now resulted in an approximately 2.5-fold in-
duction (Student t test, P ? 0.05) of luciferase activity in
comparison with controls, and this demonstrated that
HNF4? can activate transcription through this element
of the CrebH gene.
Using chromatin immunoprecipitation (ChIP) analy-
ses, we next addressed whether HNF4? could occupy the
in fetal livers. Figure 3D shows that, in contrast to se-
Fig. 4. HNF4? is essential for expression of CrebH in the liver but is
dispensable for expression in the small intestine. RT-PCR analyses of Hnf4?
and CrebH mRNA were performed on RNA isolated from liver (lanes 1-4),
colon (lanes 5-8), or small intestine (lanes 9-13) that had been collected
from Hnf4?loxP/?(con) or Hnf4?loxP/loxP(mut) mice that expressed Cre
recombinase in the hepatocytes (Alfp.Cre; lanes 1-4), colonic epithelial cells
(Foxa3.Cre; lanes 5-8), or small intestine epithelial cells (Villin.Cre; lanes
9-13), respectively. Amplification of hypoxanthine guanine phosphoribosyl
transferase (Hprt) was used as a loading control, and omitting DNA template
from the reaction (0DNA) served as a negative control.
1246LUEBKE-WHEELER ET AL.HEPATOLOGY, October 2008
quences from the Pol2 gene that do not contain HNF4?
binding sites, a known HNF4? binding site within the
Apoc3 promoter (H4.21) as well as the HNF4? binding
site within the CrebH gene could be precipitated from
chromatin isolated from fetal livers with an antibody that
specifically recognizes HNF4?. Importantly, the levels of
products identified when precipitations were performed
on brain extracts, which lack HNF4?, or when liver ex-
tracts were precipitated with an unrelated antibody (anti-
Pes) were low to undetectable. Cumulatively, these data
demonstrate that the CrebH gene contains an HNF4?
recognition element that is occupied by HNF4? in fetal
HNF4? Is Essential for Expression of CrebH in the
Fetal Liver but Dispensable for Expression in the
Small Intestine. To definitively determine whether
E18.5 embryos in which HNF4? was specifically deleted
in differentiating hepatocytes (Hnf4?loxP/loxPAlfp.cre),5,16
colonic epithelial cells (Hnf4?loxP/loxPFoxA3.cre),17or
small intestine epithelial cells (Hnf4?loxP/loxPVillin.cre),
and we measured CrebH levels by RT-PCR. Figure 4
shows that although CrebH expression was detected in
two different control livers, it was not identified in
HNF4?-null livers under identical conditions. As ex-
pected, CrebH was also undetectable in colons regardless
ingly, and in contrast to the liver, CrebH mRNA was
detected in both control and HNF4?-null small intes-
tines. We therefore conclude that HNF4? is dispensable
for CrebH expression in the gastrointestinal tract but is
essential for CrebH expression in the liver.
CrebH Is Dispensable for Hepatogenesis and He-
patocyte Differentiation. The finding that fetal liver ex-
pression of CrebH is dependent on the presence of
HNF4? raised the question of whether the absence of
CrebH could account for any aspect of the phenotype
associated with Hnf4??/?fetal livers. To test this, we
Fig. 5. Generation of mice harboring conditional and null alleles of CrebH. (A) Diagram showing the targeting strategy used to generate a
CrebHloxPneoallele as well as the alleles CrebH?(in response to Cre recombinase activity) and CrebHloxP(in response to Flpe recombinase activity).
The position of HindIII restriction endonuclease recognition sequences (H), a neomycin phosphotransferase cassette (Neo), loxP (solid circles) and
Frt (solid rectangles) sites, oligonucleotide PCR primers, and Southern blot probes are shown with respect to exons (numbered open rectangles). (B,C)
Autoradiograph of a Southern blot of HindIII-digested embryonic stem cell genomic DNA hybridized to (B) 5? and (C) 3? probes, which identify a
27.7-kb wild-type CrebH (wt) fragment and 9.7-kb and 11.3-kb CrebHloxPneo (loxPneo) fragments, respectively. (D,E) PCR analyses of ear-punch
genomic DNA from CrebH?/?, CrebH?/?, CrebH?/?, CrebH?/loxP, and CrebHloxP/loxPmice using primers shown in panel A. The sizes of CrebH?(wt),
CrebH?(null), and CrebHloxP(loxP) amplicons are indicated in base pairs. (F) RT-PCR analyses of RNA extracted from the individual livers of
CrebH?/?(?/?), CrebH?/?(?/?), and CrebH?/?(?/?) mice using primers that identify CrebH, Pol2 (input RNA control), and Hnf4? (hepatocyte
control) mRNA. The omission of reverse transcriptase (?RT) and a template (0 DNA) ensured the absence of contaminating DNA.
HEPATOLOGY, Vol. 48, No. 4, 2008 LUEBKE-WHEELER ET AL.1247
generated two strains of mice, one of which harbored a
null allele of CrebH (CrebH?/?) and the other of which
harbored a CrebH allele that could be conditionally dis-
rupted by expression of Cre recombinase (CrebHloxP/loxP).
Details of the targeting strategy are shown in Fig. 5A.
ated that contained a single loxP site between exons 11
and 12 of CrebH and a cassette, which was flanked by frt
rect targeting of the CrebH locus was confirmed by
the altered CrebH allele was successfully transmitted
through the germ line to generate CrebH?/loxPNeomice.
transgenic mouse [B6.FVB-Tg(EIIa-cre)C5379Lmgd/J],
and this resulted in Cre-mediated recombination between
loxP sites in the germ line18(Fig. 5A,D). We also produced
mice containing a conditionally null CrebH allele (CrebH?/
recombinase from the human ?-actin gene promoter [B6;
If CrebH were essential for a central aspect of hepato-
genesis, we predicted that CrebH?/?embryos would die
during late gestation stages. However, crosses of
CrebH?/?mice yielded CrebH?/?offspring at the ex-
pected Mendelian ratio as determined with polymerase
chain reaction (PCR) analyses of genomic DNA isolated
from 162 weanlings. RT-PCR analyses identified CrebH
mRNA in control livers but not in CrebH?/?livers (Fig.
5F), and this is consistent with a loss of CrebH activity in
the mutant mice. CrebH?/?mice were found to be long-
lived, fecund, and apparently healthy. Examination of
E18.5 embryos revealed no obvious difference between
control and mutant embryos (Fig. 6A), and the overall
anatomy of the liver and gastrointestinal tract of
eosin (H&E) histological staining and HNF4? immuno-
histochemistry performed on sections through E18.5
CrebH?/?livers found them to be indistinguishable from
cleotide array analyses revealed that mRNA levels were
comparable between control and mutant embryos (not
shown). Cumulatively, these data demonstrate that
CrebH is dispensable for hepatogenesis and hepatocyte
differentiation in the mouse.
Loss of CrebH Results in Reduced Expression of
Acute Phase Response Proteins Induced by Tunicamy-
cin. CrebH is a member of the CREB/ATF family of
bZip transcription factors that contains a transmembrane
domain that directs its localization to the ER.11,20Recent
age of CrebH, allowing the N-terminus to translocate to
the nucleus, where it can activate transcription of target
genes including those involved in the acute phase re-
sponse.20,21We therefore examined whether the expres-
sion of acute phase genes in response to treatment with
tunicamycin, which induces ER stress by blocking glyco-
sylation and protein folding, was affected in CrebH?/?
mice. Figure 7 shows that the expression of mRNAs en-
coding the acute phase response proteins C-reactive pro-
tein (CRP), serum amyloid P-component (SAP), and
serum amyloid A3 (SAA3) was robustly induced in
CrebH?/?mice in response to treatment with tunicamy-
cin as expected. However, in contrast to control animals,
expression of each of these acute phase mRNAs was se-
verely reduced. Similar results were obtained for both
Fig. 6. CrebH is not essential for development of the liver. (A)
Micrographs of viscera (lower panels) showing livers (*) and gastroin-
testinal tracts (?) dissected from E18.5 CrebH?/?, CrebH?/?, and
CrebH?/?embryos (upper panels). (B) Micrographs of sections through
CrebH?/?and CrebH?/?E18.5 livers stained with H&E or for the
presence of HNF4? with immunohistochemistry. Scale bar ? 100?M.
1248LUEBKE-WHEELER ET AL.HEPATOLOGY, October 2008
liver and serum SAP protein levels (see Supplementary
component of the systemic inflammatory response to ER
stress. Moreover, they imply an indirect role for HNF4?
in this response by mediating CrebH expression.
In the current analyses, we have identified CrebH as a
direct target of HNF4?. Although our data support the
view that CrebH expression is strictly dependent on
HNF4? in the liver, we have also found that CrebH
continues to be robustly expressed in HNF4?-null small
intestinal CrebH expression, ChIP analyses have revealed
that, like the liver, HNF4? occupies its binding site
within the CrebH promoter in intestinal tissue (data not
shown). These data suggest that the regulation of CrebH
by HNF4? is tissue-dependent and importantly that oc-
cupancy of a promoter by HNF4? in vivo does not nec-
essarily correlate with transcriptional regulation.1It is
intriguing that CrebH expression is independent of
HNF4? in the small intestine yet is strictly dependent
on HNF4? in the liver. One possible explanation
could be that an intestinal regulatory element exists
within the CrebH promoter that is controlled specifi-
cally by intestinal transcription factors, thereby obvi-
ating any requirement for HNF4?. Alternatively,
HNF4?, which in contrast to the liver is robustly ex-
pressed in the gut, could potentially compensate for
loss of HNF4? in Hnf4?loxP/loxPVillin.cre mice. Efforts
to address possible redundancy between these two
HNF4 family members through the generation of
Hnf4g?/?animals are currently underway.
To examine the role of CrebH in liver function, we
generated CrebH?/?mice that were viable and healthy.
Although at the onset of the project very little was known
tion factors, a number of reports have recently emerged
that raise a potential role for this factor in controlling
hepatic function.11,20-22Most relevant is the finding by
Zhang et al.20that proinflammatory cytokines or ER
stress could activate CrebH through proteolytic cleavage
and that depletion of CrebH with short hairpin RNA
inhibited expression of acute phase proteins.20In addi-
tion, CrebH was shown to transactivate expression of a
luciferase reporter gene through the CRP and SAP pro-
moters, and this suggested that CrebH directly regulates
ined the expression of mRNAs encoding acute phase re-
sponse proteins in CrebH?/?mice following a treatment
with tunicamycin that induced ER stress. We found that
expression of CRP, SAP, and SAA3 mRNAs following
treatment was severely diminished in CrebH?/?animals
compared to controls. Although our data confirm an im-
portant role for CrebH in the liver, the contribution of
CrebH to gut function, particularly in response to ER-
induced stress, remains to be determined. However, with
to address this issue in future studies.
important role in a pathway that controls expression of
acute phase proteins in response to ER stress. Moreover,
because hepatic expression of CrebH is dependent on
HNF4?, these results demonstrate that HNF4? makes
an indirect yet crucial contribution toward the induction
of a systemic inflammatory response by ER stress.
for providing COS-7 cell extracts and helping with the
identification of HNF4?-binding sites.
The authors thank Frances Sladek
1. Odom DT, Zizlsperger N, Gordon DB, Bell GW, Rinaldi NJ, Murray
tion factors. Science 2004;303:1378-1381.
2. Kyrmizi I, Hatzis P, Katrakili N, Tronche F, Gonzalez FJ, Talianidis I.
Plasticity and expanding complexity of the hepatic transcription factor
network during liver development. Genes Dev 2006;20:2293-2305.
3. Li J, Ning G, Duncan SA. Mammalian hepatocyte differentiation requires
the transcription factor HNF-4alpha. Genes Dev 2000;14:464-474.
4. Hayhurst GP, Lee YH, Lambert G, Ward JM, Gonzalez FJ. Hepatocyte
hepatic gene expression and lipid homeostasis. Mol Cell Biol 2001;21:
Fig. 7. The response to tunicamycin-in-
duced ER stress is reduced by the loss of
CrebH. CrebH?/?(gray bars) and CrebH?/?
(white bars) mice were given tunicamycin (2
?g/g of body weight) by intraperitoneal injec-
tion. Livers were isolated at 24 hours and
processed for quantitative RT-PCR of mRNAs
encoding the acute phase proteins (A) CRP, (B)
SAA3, and (C) SAP.
HEPATOLOGY, Vol. 48, No. 4, 2008LUEBKE-WHEELER ET AL. 1249
5. Parviz F, Matullo C, Garrison WD, Savatski L, Adamson JW, Ning G, et
al. Hepatocyte nuclear factor 4alpha controls the development of a hepatic
epithelium and liver morphogenesis. Nat Genet 2003;34:292-296.
6. Battle MA, Konopka G, Parviz F, Gaggl AL, Yang C, Sladek FM, et al.
Hepatocyte nuclear factor 4alpha orchestrates expression of cell adhesion
proteins during the epithelial transformation of the developing liver. Proc
Natl Acad Sci U S A 2006;103:8419-8424.
7. Tian JM, Schibler U. Tissue-specific expression of the gene encoding he-
patocyte nuclear factor 1 may involve hepatocyte nuclear factor 4. Genes
HNF-1? and HNF-1? by retinoic acid in F9 teratocarcinoma cells.
EMBO J 1991;10:2231-2236.
9. Pineda Torra I, Jamshidi Y, Flavell DM, Fruchart JC, Staels B. Character-
ization of the human PPARalpha promoter: identification of a functional
nuclear receptor response element. Mol Endocrinol 2002;16:1013-1028.
10. Gray PA, Fu H, Luo P, Zhao Q, Yu J, Ferrari A, et al. Mouse brain
organization revealed through direct genome-scale TF expression analysis.
11. Omori Y, Imai J, Watanabe M, Komatsu T, Suzuki Y, Kataoka K, et al.
CREB-H: a novel mammalian transcription factor belonging to the
CREB/ATF family and functioning via the box-B element with a liver-
specific expression. Nucleic Acids Res 2001;29:2154-2162.
12. Duncan SA, Manova K, Chen WS, Hoodless P, Weinstein DC, Bach-
varova RF, et al. Expression of transcription factor HNF-4 in the extraem-
bryonic endoderm, gut, and nephrogenic tissue of the developing mouse
embryo: HNF-4 is a marker for primary endoderm in the implanting
blastocyst. Proc Natl Acad Sci U S A 1994;91:7598-7602.
13. Taraviras S, Monaghan AP, Schutz G, Kelsey G. Characterization of the
14. Sladek FM, Zhong W, Lai E, Darnell JE Jr. Liver-enriched transcription
factor HNF-4 is a novel member of the steroid hormone receptor super-
family. Genes Dev 1990;4:2353-2365.
15. Drewes T, Senkel S, Holewa B, Ryffel GU. Human hepatocyte nuclear
Mol Cell Biol 1996;16:925-931.
16. Parviz F, Li J, Kaestner KH, Duncan SA. Generation of a conditionally
null allele of hnf4alpha. Genesis 2002;32:130-133.
Hepatocyte nuclear factor 4alpha is essential for embryonic development
of the mouse colon. Gastroenterology 2006;130:1207-1220.
18. Lakso M, Pichel JG, Gorman JR, Sauer B, Okamoto Y, Lee E, et al.
Efficient in vivo manipulation of mouse genomic sequences at the zygote
stage. Proc Natl Acad Sci U S A 1996;93:5860-5865.
High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP.
Nat Genet 2000;25:139-140.
20. Zhang K, Shen X, Wu J, Sakaki K, Saunders T, Rutkowski DT, et al.
Endoplasmic reticulum stress activates cleavage of CREBH to induce a
systemic inflammatory response. Cell 2006;124:587-599.
21. Chin KT, Zhou HJ, Wong CM, Lee JM, Chan CP, Qiang BQ, et al. The
underexpressed in hepatocellular carcinoma. Nucleic Acids Res 2005;33:
22. Shen X, Ellis RE, Sakaki K, Kaufman RJ. Genetic interactions due to
constitutive and inducible gene regulation mediated by the unfolded pro-
tein response in C. elegans. PLoS Genet 2005;1:e37.
33P-labeled RNA probes for determination of cellular expression patterns
of liver transcription factors in mouse embryos [published correction ap-
pears in Methods 1998;16:359-360]. Methods 1998;16:29-41.
24. Madison BB, Dunbar L, Qiao XT, Braunstein K, Braunstein E, Gumucio
of the vertical (crypt) and horizontal (duodenum, cecum) axes of the in-
testine. J Biol Chem 2002;277:33275-33283.
25. Lee CS, Sund NJ, Behr R, Herrera PL, Kaestner KH. Foxa2 is required
for the differentiation of pancreatic alpha-cells. Dev Biol 2005;278:
26. Chen WS, Manova K, Weinstein DC, Duncan SA, Plump AS, Prezioso
VR, et al. Disruption of the HNF-4 gene, expressed in visceral endoderm,
leads to cell death in embryonic ectoderm and impaired gastrulation of
mouse embryos. Genes Dev 1994;8:2466-2477.
1250LUEBKE-WHEELER ET AL.HEPATOLOGY, October 2008