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Vol. 54, Issue 1, 129-158, March 2002
Viszeral und Transplantationschirurgie, Medizinische Hochschule Hannover (H.S., J.K.); and Center of Drug Research and Medical Biotechnology, Fraunhofer Institut für Toxikologie und Aerosolforschung, Hannover, Germany (J.B.)
Abstract
I. Transcription Factors and Gene Regulation
A. Principal Mechanisms
B. Chromatin Higher Order Structure and Transcription Factor Function
1. ATP-Utilizing Chromatin Remodeling Complexes: Switch/Sucrose Nonfermenting and Relatives.
a. Switch/Sucrose Nonfermenting Subunits and Their Interaction with DNA.
b. Switch/Sucrose Nonfermenting Complex and Cell Cycle Control: Impact on Liver Regeneration?
c. Components of the Switch/Sucrose Nonfermenting Complex as Cofactors for Nuclear Receptors.
d. Further Multiprotein Complexes with Homology to the Switch/Sucrose Nonfermenting ATPase.
2. Chromatin Modification: Reversible Acetylation of Histone Lysines.
3. Chromatin Modification: Reversible Phosphorylation of Histone Serines and Threonines.
4. Chromatin Modification: Reversible Ubiquitination of Histone Lysines.
5. Chromatin Modification: Reversible DNA Methylation.
C. Epigenetics
D. Position-Effect Variegation
E. Formation of the Multiprotein Complex
II. Classification of Liver-Enriched Transcription Factors
A. DNA-Binding Domain of Hepatocyte Nuclear Factor-1
B. DNA-Binding Domain of Hepatocyte Nuclear Factor-3
C. DNA-Binding Domain of Hepatocyte Nuclear Factor-4
D. DNA-Binding Domain of Hepatocyte Nuclear Factor-6
E. DNA-Binding Domain of CCAAT/Enhancer-Binding Proteins
III. Molecular Regulation of Liver Function
A. Liver-Specific Gene Expression
B. Liver-Enriched Transcription Factors
IV. Hepatocyte Nuclear Factors
A. The Hepatocyte Nuclear Factor-1 Family
1. Dimerization Cofactor of Hepatocyte Nuclear Factor-1and Liver-Specific Gene Expression.
B. The Hepatocyte Nuclear Factor-3 Subfamily
C. The Hepatocyte Nuclear Factor-4 Subfamily
1. The Structure and Domains of Hepatocyte Nuclear Factor-4.
2. The Relevance of Hepatocyte Nuclear Factor-4 Splice Variants.
3. Homo- and Heterodimerization of Hepatocyte Nuclear Factor-4 Proteins.
4. Regulation of Hepatocyte Nuclear Factor-4 Function by Phosphorylation.
5. Agonistic and Antagonistic Ligands for the Nuclear Receptor Hepatocyte Nuclear Factor-4
6. Acetylation of Nucleosomal Histones and Hepatocyte Nuclear Factor-4 by cAMP Response Element-Binding Protein.
7. Chicken Ovalbumin Upstream Promoter-Transcription Factors and Hepatocyte Nuclear Factor-4: Cooperation and Competition.
D. Hepatocyte Nuclear Factor-6
1. Splice Variants of Hepatocyte Nuclear Factor-6.
2. Hepatocyte Nuclear Factor-6 in Development.
3. Regulation of Hepatocyte Nuclear Factor-6 Expression by Growth Hormone.
4. Inhibitory Protein-Protein Interaction between Hepatocyte Nuclear Factor-6 and a Nuclear Receptor.
E. Coactivators for Hepatocyte Nuclear Factor-1 and Hepatocyte Nuclear Factor-4
F. The Hepatocyte Nuclear Factor Network and Tissue-Specific Gene Expression
1. Hepatocyte Nuclear Factor-1 Regulates Hepatocyte Nuclear Factor-4
2. Hepatocyte Nuclear Factor-1
3. Hepatocyte Nuclear Factor-6, OC-2, Hepatocyte Nuclear Factor-3, and CCAAT/Enhancer-Binding Proteins Regulate Hepatocyte Nuclear Factor-3
4. Hepatocyte Nuclear Factor-1in the Liver.
5. Competition and Cooperation ("Coopetition") between Hepatocyte Nuclear Factor-3
G. Human Disease Due to Mutations in Hepatocyte Nuclear Factors
H. Evidence from Knockout Experiments
I. Lack of Confirmation for Existence of Hepatocyte Nuclear Factor-5
V. Challenges for the Future
Acknowledgments
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Numerous studies have established the pivotal role of liver-enriched transcription factors in organ development and cellular function, and there is conclusive evidence for transcription factors to act in concert in liver-specific gene expression. During organ development and in progenitor cells the timely expression of certain transcription factors is necessary for cellular differentiation, and there is overwhelming evidence for hierarchical and cooperative principles in a networked environment of transcription factors. The search for molecular switches that control stem cell imprinting and liver-specific functions has lead to the discovery of many interactions between such different molecules as transcription factors, coactivators, corepressors, enzymes, DNA, and RNA. Many of these interactions either repress or activate liver-specific gene expression. It thus can be demonstrated that specific mutational changes in liver-enriched transcription factors lead to altered intermolecular interactions with the consequence of human disease. This review provides an overview of our current knowledge about liver-enriched transcription factors and their role in liver function and development. We review the basic principles of gene transcription, the role of liver-enriched transcription factors in liver gene regulation, and the classification of transcription factors by their DNA-binding domains.
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I. Transcription Factors and Gene Regulation |
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A. Principal Mechanisms
Transcription factors are trans-acting
DNA-binding proteins that bind to a specific cis-acting DNA
sequence within the regulatory element of a gene. Usually, control
regions can be found upstream of the start site of transcription,
although in some cases binding occurs within the coding region.
Transcription factors bound to their cognate cis-acting DNA
sequence interact with the transcriptional machinery and enable
selective gene expression and regulation. Frequently, this process
is governed by the binding of many different proteins to cognate
DNA-binding sites, which enables combinatorial control of gene
expression. An additional level of complexity is provided by
protein-protein interactions between transcription factors and
coactivators or corepressors. Together with the transcriptional machinery, these proteins form a multiprotein complex that enables regulated mRNA synthesis (for review see Pabo and Sauer, 1992
; Giordano
and Avantaggiati, 1999; Klug, 1999; Wolberger, 1999; Goodman and
Smolik, 2000).
Efficient gene transcription requires a permissive chromatin
environment for successful interaction between the
trans-acting transcription factors of the multiprotein
complex and the respective cis-acting target DNA template of
the nucleosome core particle. Therefore, the modulation of chromatin
structure with its effects on gene transcription represents a key
mechanism for transcriptional repression, derepression, and
transcriptional activation. In the following sections we briefly
summarize some of the fundamental mechanisms in the formation of the
multiprotein complex to provide newfound knowledge on gene
transcription and liver-enriched transcription factors, and we
deliberately exclude aspects of DNA repair and DNA miscoding, which
have been reviewed elsewhere (Krokan et al., 2000; Thompson and Schild,
2001).
B. Chromatin Higher Order Structure and Transcription Factor Function
Chromatin is composed of a histone octamer, the DNA of the
nucleosome core particle, and the linker DNA. The nucleosome core particle is formed by about 160 bp1 of DNA wrapped
around an octamer composed of two copies of each of the four histones
H2A, H2B, H3, and H4. Within the nucleosome core particle an
(H3)2(H4)2 tetramer, as
well as an H2A-H2B dimer, could be distinguished. These histone
oligomers could be recombined with DNA in vitro to generate the
characteristic X-ray diffraction pattern of chromatin (Kornberg and
Thomas, 1974; Kornberg and Lorch, 1999). Figure
1 shows the basic entities that form
chromatin, and Fig. 2 shows a schematic
transection of the nucleosome core particle based on X-ray findings by
Luger et al. (1997). X-Ray and electron crystallography revealed the
coiling of DNA in left-handed superhelical turns around the histones
(Finch et al., 1977). Crystallographic analysis of the nucleosome
showed that the histones form a left-handed protein superhelix matching
that of the DNA in the nucleosome core particle (Klug et al., 1980;
Arents and Moudrianakis, 1995; Kornberg and Lorch, 1999).
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The human genome consists of 2.91 billion base pairs, which would be
theoretically about 1.8 m long if stretched out as one long chain
(Venter et al., 2001). DNA is organized into chromatin to achieve the
required high level of compaction to pack this DNA into a nucleus with
a diameter less than 6 nm (Lewin, 1994). The orderly packaging of DNA
in the nucleus plays an important role in the functional aspects of
gene regulation. Only a small percentage of chromatin is made available
to transcription factors and the transcriptional machinery, whereas the
remainder of the genome is in a state that is essentially inaccessible
to the RNA polymerases. ATP-dependent chromatin remodeling as well as
chromatin modifications by acetylation of lysines, DNA methylation,
phosphorylation of serines and threonines, and ubiquitination of
lysines play key roles in altering chromatin higher order structure and
function (Bradbury, 1992; Shilatifard, 1998; Giordano and Avantaggiati, 1999; Spencer and Davie, 1999; Stein et al., 1999). Acetylations and
phosphorylations markedly affect the charge densities of well defined,
very basic N- and C-terminal domains of histones (for details see also
Fig. 2), whereas ubiquitination adds a bulky globular protein,
ubiquitin, to lysines in the C-terminal tails of H2A and H2B (for
review see Bradbury, 1992; Bird and Wolffe, 1999; Kornberg and Lorch,
1999).
New findings on ATP-dependent chromatin remodeling as well as chromatin modifications by covalent acetylation, phosphorylation, ubiquitination, and DNA methylation demonstrate the importance of these alterations for the regulation of many genes, although their precise role in liver gene expression remains largely unknown. The following sections provide an overview of recent data and hopefully stimulate further investigations on the role of chromatin remodeling and chromatin modifications in liver function, liver regeneration, and liver development. Additionally, the concepts of epigenetics and position-effect variegation (PEV), and their possible impact on gene transcription and expression, are discussed, because these concepts are of fundamental importance in gene transcription but are widely neglected in molecular investigations of liver-specific gene expression.
1. ATP-Utilizing Chromatin Remodeling Complexes: Switch/Sucrose
Nonfermenting and Relatives.
The activation of a gene requires
accessibility for transcription factors, activators, coactivators, and
transcription machinery to the various regulatory regions. Several
ATP-consuming chromatin remodeling complexes have been identified that
enable gene activation by altering the stable structure of nucleosomes
(for review see Devine et al., 1999; Muchardt and Yaniv, 1999; Wade and
Wolffe, 1999; Tyler and Kadonga, 1999; Sudarsanam and Winston, 2000).
; Peterson and
Herskowitz, 1992). The altered nucleosome structure can be
distinguished from normal nucleosomes by their slow electrophoretic
mobility in nondenaturing gels, whereas the protein content in normal
and altered nucleosomes remains unchanged (Schnitzler et al., 1998). It
could be demonstrated that the SWI/SNF complex binds directly to
nucleosome cores and uses the energy of ATP hydrolysis to disrupt
DNA/histone interactions and to create an altered nucleosome core
conformation that is also stable in the absence of SWI/SNF (Coté
et al., 1998). It has been postulated that the alteration of the
nucleosome structure by chromatin remodeling complexes affects the
preferred bending of the DNA as it coils around the histone octamer,
leading to facilitated binding of transcription factors to their DNA
template. The reverse transition from altered to normal nucleosomes is
also catalyzed by the same SWI/SNF complex using ATP hydrolysis
(Coté et al., 1998; Schnitzler et al., 1998).
It could be demonstrated that the SWI/SNF complex contacts the DNA
strand at two points creating a loop and that only nucleosomes within
this loop are being altered (Bazett-Jones et al., 1999). In yeast only
about 6% (329 of 5460) of the genes tested were affected 2-fold or
more by the inactivation of SWI2/SNF2. Of these 329 genes, 203 genes
were elevated 2-fold or more in the absence of SWI2/SNF2, indicating
that chromatin remodeling can favor activation as well as repression of
transcriptional activity (Holstege et al., 1998). These results
prompted the hypothesis that the limited pool of SWI/SNF complexes is
recruited to a small number of specific promoters, which in turn will
bind either transcriptional activators or repressors (Holstege et al.,
1998; Muchardt and Yaniv, 1999).
Two models have been proposed to explain why some promoters are
SWI/SNF-dependent, whereas others are not. The first model describes
the SWI/SNF complex as primarily regulating the DNA binding of
transcriptional modulators. In this model SWI/SNF-dependent promoters
are thought to have weak activator-binding sites covered by
nucleosomes, whereas SWI/SNF-independent promoters either have high-affinity activator-binding sites or are located in a
nucleosome-free region (Muchardt and Yaniv, 1999). In this context it
is interesting to note that nucleosome-free linker DNA can be bound to
linker histones (H1, H1°, H5, etc.) and that there is evidence for
the involvement of linker histones in transcriptional regulation. A
scenario has been proposed in which the reversible and controllable binding/displacement of linker histones to the nucleosomal entry/exit point determine the accessibility of nucleosomal DNA to the
transcriptional machinery (Zlatanova et al., 2000). The second proposed
model on SWI/SNF-dependent and SWI/SNF-independent promoters suggests that the SWI/SNF complex exerts its major effect in transcriptional activation at a step subsequent to transcriptional activator-promoter recognition. The recruitment of the SWI/SNF complex by the DNA-binding protein may then allow the binding of secondary transcriptional regulators that in turn either facilitate or prevent the recruitment of
the TATA-binding protein (TBP) (Ryan et al., 1998).
In some cases it could be found that the SWI/SNF complex is associated
with the polymerase II holoenzyme, which lead to the hypothesis that
the SWI/SNF complex is involved in the assembly of the preinitiation
complex (Wilson et al., 1996). Furthermore, the SWI/SNF complex was
shown to be able to facilitate the binding of TBP on nucleosomes in
vitro (Imbalzano et al., 1994).
Drosophila and human cells contain complexes related to
yeast SWI/SNF. These complexes contain about 10 subunits, and each contains a homolog of the yeast SWI2/SNF2 helicase-like subunit as well
as one or two homologs of yeast SNF5 (SNF5 = sucrose nonfermenting 5), SWI3 (switch 3), and SWP73 (an associated protein) (Muchardt and
Yaniv, 1999).
The composition of the mammalian SWI/SNF complex appears to be highly
variable in contrast to the respective complex in yeast and drosophila.
In human and mouse cells at least two homologs of the SWI2/SNF2 ATPase
subunit exist, known as brm (also called Brahma or
SNF2a) and brahma-related protein-1 (also known as BRG-1 or SNF2b). The
brm and the BRG-1 protein are 75% identical.
Purification experiments revealed that different mixtures of
brm and BRG-1-associated complexes can be found in
mammalian cells (Muchardt and Yaniv, 1999; Sudarsanam and Winston,
2000).
BRG-1 is capable of remodeling mononucleosomes and nucleosomal arrays
as a purified protein in vitro. The addition of further subunits of the
human SWI/SNF complex (hSNF5/INI1, BAF155, BAF170) to BRG-1 increases
the remodeling activity to a level comparable with that of the whole
human SWI/SNF complex. On this basis it was postulated that these
proteins define the functional core of the human SWI/SNF complex
(Phelan et al., 1999; Phelan et al., 2000).
a. Switch/Sucrose Nonfermenting Subunits and Their Interaction with
DNA.
Studies with brm deletion mutants revealed a
region with homology to the AT-hook present in high-mobility-group
protein I/Y (HMGI/Y). This region was shown to be required for the
tethering of brm to chromatin. In vitro this domain is able
to mediate binding to the minor groove of DNA with a preference for
A+T-rich sequences. Deletion of this sequence in brm leads
to increased extractability of the protein (Muchardt and Yaniv, 1999).
b. Switch/Sucrose Nonfermenting Complex and Cell Cycle Control:
Impact on Liver Regeneration?
Several observations suggest that
the SWI/SNF complex is also involved in cell cycle control (Muchardt
and Yaniv, 1999; Sudarsanam and Winston, 2000). It has been observed
that growth arrest or differentiation leads to increased accumulation
of brm protein, whereas rapidly dividing cells contain
mainly BRG-1 (Muchardt et al., 1998). The levels of brm
and BRG-1 are also regulated during the cell cycle. At the
G2/M transition the two proteins are phosphorylated. This
phosphorylation leads to proteolytic degradation of the
brm protein, whereas BRG-1 remains stable through mitosis (Muchardt et al., 1996; Sif et al., 1998; Muchardt and Yaniv,
1999). From these observations it is likely that the ratio between
brm and BRG-1-associated complexes is dependent on the phase of the cell cycle, the stage of development, and the specific tissue, and it is likely that each form has a specific function (Muchardt and Yaniv, 1999). The phosphorylation of brm
and BRG-1 during mitosis prevents the SWI/SNF complex from remodeling
chromatin in vitro (Sif et al., 1998). Furthermore, it was proposed
that mini-cycles of phosphorylation and dephosphorylation of the
brm and BRG-1 proteins regulate the attachment of these
proteins to nuclear structures during interphase (Muchardt and Yaniv,
1999).
It could be demonstrated that the retinoblastoma protein and BRG-1 form
a complex and cooperate to induce cell cycle arrest (Dunaief et al.,
1994). In the yeast two-hybrid system an interaction between
BRG-1/brm protein family members and retinoblastoma protein family members including pRB, p107, and p130 was observed. These interactions influence cellular proliferation because both BRG-1 and
brm, but not mutants of these proteins, which are unable to bind pRB family members, inhibit the formation of drug-resistant colonies when transfected into the SW13 human adenocarcinoma cell line,
which lacks endogenous BRG-1 or brm (Strober et al., 1996). Mouse brm null mutants (
/) showed increased hepatocyte
proliferation in the adult. In addition, embryonic fibroblasts isolated
from the brm / mice showed defects in
G1-checkpoint controls. In culture these cells
fail to arrest at confluency. This lack of contact inhibition can be
correlated with a lack of induction of the CDK inhibitor p27 at
confluency (Reyes et al., 1998). One consequence of the interaction
between brm/BRG-1 and the p105Rb retinoblastoma tumor
suppressor could be demonstrated in transient transfection experiments
as a synergistic repression of transcription factor E2F1, a protein
known to regulate cell cycle progression (Trouche et al., 1997).
Cyclin E, another cell cycle protein, was found to associate with both
BRG-1 and BAF155, a human homolog of SWI3. The interaction with cyclin
E, which is independent of p105Rb, leads to phosphorylation of the
SWI/SNF subunits by cyclin E-associated kinase activity, and cyclin E
and cyclin D1 can partially rescue BRG-1-induced growth arrest
(Shanahan et al., 1999).
After liver resection mammalian liver regeneration leads to the
controlled induction of a proliferative response in hepatocytes that
terminates as soon as the hepatic mass has been restored. Among other
proteins the retinoblastoma protein family members as well as the
cyclins E and D1 have been associated with different roles in
hepatocyte cell cycle control after partial hepatectomy in mice and
rats (Trautwein et al., 1999). The role of brm and BRG-1 in
mammalian liver regeneration remains to be determined. Since several
lines of evidence suggest that brm and BRG-1 play important
roles in cell cycle control, and since brm and BRG-1 are
known to influence the transcription of several genes through chromatin
remodeling, it seems likely that these mammalian SWI/SNF subunits play
an important role in liver regeneration. It would be valuable to
investigate the role of brm and BRG-1 during liver regeneration. Furthermore, studies on protein-protein interactions of
brm and BRG-1 with the retinoblastoma protein family and
cyclin E would be interesting, because it is likely that liver
regeneration may influence the phosphorylation status of brm
and BRG-1 and thus hepatocyte proliferation and/or senescence.
c. Components of the Switch/Sucrose Nonfermenting Complex as
Cofactors for Nuclear Receptors.
Components of the SWI/SNF
complex can function as coactivators for several nuclear receptors
including the glucocorticoid receptor, the retinoic acid receptor, and
the estrogen receptor (Muchardt and Yaniv, 1993; Chiba et al., 1994). A
ligand-dependent interaction of the estrogen receptor, the
glucocorticoid receptor (GR) or the progesterone receptor with the
BRG-1 protein has been demonstrated (Ichinose et al., 1997; Fryer and
Archer, 1998). Prebinding of GR to a nucleosomal template in vitro
facilitates nucleosome disruption by the SWI/SNF complex
(Östlund-Farrants et al., 1997). On the other hand, it could be
shown that GR-induced chromatin remodeling requires the SWI/SNF complex
(Fryer and Archer, 1998).
GR as well as the liver-enriched hepatocyte nuclear factor-4 (HNF-4)
belong both to the superfamily of nuclear receptors that share several
structural similarities (Hadzopoulou-Cladaras et al., 1997). Whether
components of the SWI/SNF complex also interact with HNF-4 as
coactivators or corepressors would be an interesting field of research
in view of the known impact of HNF-4 on the regulation of liver function.
d. Further Multiprotein Complexes with Homology to the
Switch/Sucrose Nonfermenting ATPase.
In the last few years
several multiprotein complexes with homology to the SWI/SNF ATPase
subunit have been identified [e.g., NURF (nucleosome remodeling
factor), CHRAC (chromatin-accessibility complex), ACF (ATP-utilizing
chromatin assembly and remodeling factor), RSF (remodeling and spacing
factor), NuRD (nucleosome remodeling histone deacetylase complex), and
RSC (remodel the structure of chromatin)] (Zhang et al., 1998, 1999;
Muchardt and Yaniv, 1999; Stein et al., 1999; Ahringer, 2000). This
diversity suggests that chromatin remodeling complexes are numerous and may each be involved in specific cellular pathways.
Molecular analysis of the NuRD subunits revealed that this
ATP-utilizing chromatin remodeling complex contains the human
dermatomyositis-specific autoantigen Mi-2 and a histone deacetylase
core complex. Furthermore, the NuRD complex has been involved in DNA
methylation (Zhang et al., 1998, 1999). Therefore, the NuRD complex
represents an example of a protein complex that is able to influence
transcriptional activity by several different mechanisms: ATP-dependent
chromatin remodeling, chromatin deacetylation, and DNA methylation
(Wade and Wolffe, 1999; Ahringer, 2000; Guschin et al., 2000). The NuRD complex (also known as the Mi-2 complex) has been associated with transcriptional silencing (Wade and Wolffe, 1999). The histone deacetylases HDAC1 and HDAC2, as well as the two histone-binding proteins RbAp46 and RbAp48, belong to this complex and to the SIN3
complex. The NuRD and the SIN3 complex represent the two major HDAC
complexes that have specific functions in development rather than being
required for general cellular processes (Ahringer, 2000).
2. Chromatin Modification: Reversible Acetylation of Histone
Lysines.
Expressed genes are located in highly acetylated
chromatin. The acetylation status of nucleosomes is regulated by a
group of enzymes, histone acetyltransferases (HATs) and HDACs. Examples of acetylation sites of histones H3 and H4 are shown in Fig. 2. Both
groups of enzymes contain numerous family members, most of which have
been highly conserved during evolution. The noncatalytic components of
these complexes can either target the catalytic unit to specific sites
of the genome or regulate its enzymatic specificity. DNA methylation
and histone acetylation have also been linked together, whereby
methylation is used to direct gene repression through a histone
deacetylase complex (Gray et al., 1999) (see also Fig.
3).
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; Wade and Wolffe, 1997; Workman and Kingston, 1998). In
particular, a number of transcriptional regulatory proteins, including
GCN5, PCAF, p300/CBP, TFII250, and the nuclear hormone receptor
coactivators ACTR and SRC-1, have been found to possess intrinsic HAT
activity (Kuo et al., 1998; Wang et al., 1998a,b; Chen et al., 1999).
Mutational analyses of yeast GCN5 indicated a direct role for the HAT
activity in histone acetylation and transcriptional activation of
target genes in vivo (Kuo et al., 1998, Wang et al., 1998a,b). These
findings suggest a mechanism whereby the activators recruit HAT
complexes to the promoters of target genes, allowing for acetylation of
histones to increase the accessibility of transcription factors. In
addition, it has recently been shown that p300/CBP and
p300/CBP-associated factor (p/CAF) are able to acetylate nonhistone
proteins, including some transcription factors such as p53 and
components of the general transcription machinery such as TFIIE (Gu and
Roeder, 1997; Imhof et al., 1997).
It could be demonstrated that 17
-estradiol treatment of the human
breast cancer cell line MCF-7 causes a rapid and dramatic increase of
acetylation of histones at the promoter of 17-estradiol receptor
target genes including pS2, cathepsin D, c-Myc, and EB1. Surprisingly
this acetylation seems to be a transient phenomenon despite the
continuous presence of hormone. It was found that the p160 coactivators
such as the acetylase SRC-3 can be acetylated by p300/CBP and that such
acetylation disrupts hormone receptor-coactivator interaction. These
findings show the possible role of histone acetylation in gene
activation and the possible role of acetylase protein acetylation in
transcriptional attenuation (Chen et al., 1999). However, the precise
role of histone acetylation and nonhistone protein acetylation in the
process of transcriptional activation in vivo remains largely unclear.
The role of reversible acetylations of histone lysines and
transcription factors for the regulation of liver-specific
genes is becoming increasingly evident, as could be shown for several
coactivators of the liver-enriched transcription factors HNF-1 and
HNF-4. In the second part of this review (see section "Molecular
Regulation of Liver Function"), the HNF-1 and HNF-4 coactivators will
be discussed in some detail.
3. Chromatin Modification: Reversible Phosphorylation of Histone
Serines and Threonines.
Histone H1 and H3 phosphorylations
correlate with the process of chromosome condensation. The subunits of
histone H1 kinase have now been shown to be cyclins and the p34CDC2
kinase product of the cell cycle control gene CDC2. It is probable that
all of the processes that control chromosome structure and function
relationships are also involved in the control of the cell cycle
(Bradbury, 1992; Spencer and Davie, 1999).
).
4. Chromatin Modification: Reversible Ubiquitination of Histone
Lysines.
Ubiquitin-dependent proteolytic pathways are largely
responsible for selective protein turnover in the cytosol of
eukaryotes. Although ubiquitinated histones are present in substantial
levels in vertebrate cells, the roles they play in specific biological processes and the cellular factors that regulate this modification are
not well characterized. Ubiquitinated H2B (uH2B) has been identified in
the yeast S. cerevisiae, and mutation of the conserved ubiquitination site could confer defects in mitotic cell growth and
meiosis. uH2B was not detected in rad6 mutants, which are defective for
the ubiquitin-conjugating enzyme Ubc2, thus identifying Rad6 as the
major cellular activity that ubiquitinates H2B in yeast (Robzyk et al.,
2000).
5. Chromatin Modification: Reversible DNA
Methylation.
Cytosine residues in the sequence 5'CpG
(cytosine-guanine) are often postsynthetically methylated in animal
genomes. The methyl-CpG-binding proteins MeCP1 and MeCP2 interact
specifically with methylated DNA and mediate transcriptional
repression. MeCP2 is an abundant nuclear protein that is essential for
mouse embryogenesis (Nan et al., 1998). MeCP2 binds tightly to
chromosomes in a methylation-dependent manner. It contains a
transcriptional-repression domain that can function at a distance in
vitro and in vivo. A region of MeCP2 that localizes with the
transcriptional-repression domain associates with a corepressor complex
containing the transcriptional repressor mSin3A and histone
deacetylases (see Fig. 3). Transcriptional repression in vivo is
relieved by the deacetylase inhibitor trichostatin A, indicating that
deacetylation of histones (and/or of other proteins) is an essential
component of this repression mechanism. Two global mechanisms of gene
regulation, DNA methylation and histone deacetylation, can be linked by
MeCP2 (Nan et al., 1998).
; Clark et al.,
1997). In addition, methylation-dependent, sequence-specific
DNA-binding proteins such as MDBP may act as transcriptional repressors
(Asiedu et al., 1994).
There are several situations in which 5'CpG islands in the promoter
region of genes become de novo methylated in normal development, thereby silencing the expression of the associated gene (Feil and
Khosla, 1999; Jones and Laird, 1999). Examples of genes silenced by 5'CpG island methylation include genes that are transcriptionally repressed by parental-specific imprinting and genes on the inactive X
chromosome in female mammals (Issa et al., 1994, 1996; Jaenisch, 1997).
During aging, CpG islands associated with nonimprinted autosomal genes
can show gradual increases in methylation (Issa et al., 1994 and 1996).
DNA methylation may also contribute to immobilization of mammalian
transposons, suppression of transcriptional noise, and the control of
tissue-specific gene expression, but decisive evidence on these points
is lacking (Bird and Wolffe, 1999). The methylation of tumor suppressor
gene promoters (e.g., RB1, VHL, CDKN2, CDKN2B, MLH1, and APC) is
regarded as one potential hit paving the way to carcinogenesis together
with loss of heterozygosity or mutational inactivation in such tumors
as retinoblastoma, renal cell carcinoma, melanoma, and colorectal
cancer (Jones and Laird, 1999). CpG methylation is involved in the
repression of viral genomes, while the methylation of exogenous DNA
introduced into cells compromises efforts at gene therapy (Garrick et
al., 1998). A striking and widespread de novo methylation of CpG
islands occurs as a consequence of in vitro cell culture of immortal
cell lines (Jones and Laird, 1999).
Figure 4 shows a model proposed by Bird
and Wolffe (1999) on the effect of DNA methylation on the range of
transcriptional regulation beyond that which could be achieved by
chromatin modification alone. It is assumed that DNA methylation is
able to contribute a significant additional level of gene repression.
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C. Epigenetics
The inheritance of information during cell replication on the
basis of gene expression levels is known as epigenetics, as opposed to
genetics, which refers to information inherited on the basis of gene
sequence. Enzymatic methylation of the C-5 position of cytosine
residues can effect epigenetic inheritance by altering the expression
of genes and by transmission of DNA methylation patterns through cell
division (Bird and Wolffe, 1999; Jones and Laird, 1999; Wolffe and
Matzke, 1999). Epigenetic control of gene expression can be considered
from the standpoint of normal development, which requires stable
repression of genes not required in specific cell types (Wolffe and
Matzke, 1999). Interactions between repeated DNA sequences can trigger
the formation and the transmission of inactive genetic states and DNA
modifications. Methylation induced by DNA repeats can template
chromatin modifications and transcriptional repression by MeCP2 binding
to methylated CpG with subsequent recruitment of histone deacetylase
(Nan et al., 1998; Jones and Laird, 1999) (see also Fig. 3).
D. Position-Effect Variegation
The chromosomes of most higher eukaryotes consist of distinct
regions that are cytologically distinguishable owing to differences in
condensation. In a typical chromosome, heterochromatin differs from
euchromatin in sequence composition, function, and cytological appearance and is predominantly located in the pericentric region. The
DNA of heterochromatin consists almost entirely of repetitive sequences
and encodes relatively few genes. In Drosophila, genes juxtaposed to heterochromatin are frequently inactivated, a phenomenon known as PEV. Inactivation is believed to result from the spreading of
the heterochromatin state along the chromosome (Dorer and Henikoff 1994, 1997). The extent of PEV spreading may vary from cell to cell,
producing mosaic expression of nearby genes. In contrast with the
growing understanding of transacting factors, little is known of
cis-acting requirements for heterochromatin formation and
PEV. Experiments with Drosophila using a
mini-white reporter gene, a commonly used eye color marker
in Drosophila P transposons, and PEV to explore
the requirements for heterochromatin formation revealed that variegated
expression of mini-white occurs when it is present in repeat
arrays. Variegation was particularly strong for repeated transposons at
a euchromatic site near heterochromatin, but also resulted from repeats
at a site distant from heterochromatin (Dorer and Henikoff, 1994).
Inactivation strengthened with increasing copy number, a phenomenon
that can also be observed for the transgene in numerous transgenic
animals and plants (Dorer and Henikoff, 1997; Garrick et al., 1998).
Experiments using the lox/Cre system of site-specific recombination to
generate transgenic mouse lines showed that the reduction in copy
number results in a methylation at the transgene locus (Garrick et al.,
1998).
E. Formation of the Multiprotein Complex
The expression of any gene is accomplished primarily through the
interaction of protein transcription factors with characteristic nucleotide sequences located in the control regions of the gene, which
are most commonly located near to, or upstream from, the actual coding
region. The binding of a set of such factors, or regulatory proteins,
acts as a molecular switch for the activation of the RNA polymerase II
(RNA pol II) and other components of the transcriptional machinery,
which are common to all genes. The supply of a particular combination
of such transcription factors ensures that a gene is switched on in the
right cell or tissue and at the right time (Duncan et al., 1998; Klug,
1999).
Transcription initiation by RNA pol II requires interaction between
cis-acting promoter elements and trans-acting
factors. The eukaryotic promoter consists of core elements, which
include the TATA and CAAT box and other DNA sequences that define
transcription start sites, and regulatory elements, which either
enhance or repress transcription in a gene-specific manner. The core
promoter is the site for assembly of the transcription preinitiation
complex, which includes RNA pol II and the general transcription
factors TBP, transcription factor IIB (TFIIB), TFIIE, TFIIF, and TFIIH (for review see Roeder, 1996; Hampsey, 1998; Shilatifard, 1998).
Regulatory elements bind gene-specific factors, which affect the rate
of transcription by interacting, either directly or indirectly, with
components of the general transcriptional machinery. A third class of
transcription factors, termed coactivators, is not required for basal
transcription in vitro but often mediates activation by a broad
spectrum of activators. Accordingly, coactivators are neither
gene-specific nor general transcription factors, although gene-specific
coactivators have been described in metazoan systems including humans.
Transcriptional repressors include both gene-specific and general
factors. Similar to coactivators, general transcriptional repressors
affect the expression of a broad spectrum of genes yet do not repress
all genes. General repressors either act through the core
transcriptional machinery or are histone-related and presumably affect
chromatin function, thus preventing RNA transcription ( Chang and
Jaehning, 1997; Hampsey, 1998; Yamaguchi et al., 1998).
Figure 5 depicts a schematic model on the
formation of the multiprotein complex within the promoter region of a
gene. In this model acetylated chromatin is made accessible for
transcription factors (DNA-binding transactivators) in the control
region (promoter regions and enhancer-binding sites) of the respective
gene by ATP-dependent chromatin remodeling complexes. After
transcription factor binding, the RNA polymerase II, general initiation
factors, and mediators bind at the promoter region. Then RNA polymerase II elongation factors bind additionally to the multiprotein complex to
enable mRNA transcription. In this process extensive protein-protein interactions occur that enable the fine tuning of an orchestrated regulation of gene transcription (for review see Roeder, 1996; Hampsey,
1998; Shilatifard, 1998; Wolberger, 1999).
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II. Classification of Liver-Enriched Transcription Factors |
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Transcription factors achieve recognition of the DNA-binding site
through protein-DNA and protein-protein interactions via discrete
substructures or protein domains that serve binding to DNA. The
DNA-binding motifs of transcription factors contain characteristic amino acid sequences and form characteristic three-dimensional structures that allow the classification of different types of transcription factors. The three-dimensional structure of these DNA-binding motifs leads to DNA sequence-specific DNA binding through
the formation of hydrogen bonds and Van der Waals contacts (Pabo and
Sauer 1992; Klug, 1999).
A. DNA-Binding Domain of Hepatocyte Nuclear Factor-1
The first DNA-binding motif identified in X-ray crystallographic
studies is the helix-turn-helix (HTH) motif (Wintjens and Rooman,
1996). POU domain transcription factors have two separate helix-turn-helix DNA-binding subdomains, the POU homeodomain (POUhd) and the POU-specific domain (POUs). Each subdomain recognizes a
specific subsite of 4 or 5 bp in the octamer recognition sequence (Van
Leeuwen et al., 1997). The POU domain family of transcription factors
was defined after the observation that the products of three mammalian
genes, Pit-1, Oct-1, and Oct-2, and the protein encoded by the
Caenorhabditis elegans gene unc-86, shared a region of
homology, known as the POU domain (Schonemann et al., 1998).
Molecular characterization of the genes whose sequence alterations
cause impressive phenotypes in the fruit fly, Drosophila melanogaster, has led to the identification of the human homeobox genes, also referred to as the HOX genes and defined as "master genes" for their crucial role in embryogenesis (McGinnis and
Krumlauf, 1992). They all share a homeobox region, known as a 180-bp
highly conserved sequence encoding a 60-amino acid DNA-binding domain, also called the "homeodomain", conferring to the resulting proteins the ability to act as transcription factors (Gehring et al., 1994; Chariot et al., 1999). The 39 human HOX genes are organized in four
distinct clusters (loci A, B, C, and D) and can be aligned on the basis
of homology within the homeobox to define paralogs (Acampora et al.,
1989; Scott, 1992). Besides a critical involvement in cell phenotype
determination along the anterior-posterior axis during embryonic
development, the HOX genes also play a key role in differentiation and
tumoral development (Chariot et al., 1999; Cillo et al., 1999; Morata
and Sanchez-Herrero, 1999).
The liver-enriched transcription factor hepatocyte nuclear
factor-1 (HNF-1) contains a variant homeodomain and shares homeodomain, as well as short acidic and basic sequences, with the POU family of
transcriptional activators (Baumhueter et al., 1990). HNF-1 is composed
of HNF-1
or HNF-1 homo- or heterodimers (Song et al., 1998).
B. DNA-Binding Domain of Hepatocyte Nuclear Factor-3
The hepatocyte nuclear factor-3 (HNF-3)/fork head (fkh)
family contains a large number of transcription factors and folds into
a winged helix motif. Despite having almost invariable amino acid
sequences in their principal DNA-binding helices, HNF-3/fkh proteins
show a wide diversity of sequence-specific binding. Previous studies of
chimeric HNF-3/fkh proteins demonstrated that the binding specificity
is primarily influenced by a region directly adjacent to the binding
helix (Marsden et al., 1998; Jin et al., 1999). In NMR and X-ray
crystallographic studies it is found that in comparison with HNF-3, the
HNF-3/fork head (fkh) family member Genesis contains an extra small
helix directly prior to the N terminus of the primary DNA contact
helix. Due to the insertion of this helix, a shorter and slightly
repositioned primary DNA contact helix is observed, which is believed
to lead to the DNA-binding specificity differences among various family
members (Marsden et al., 1998).
C. DNA-Binding Domain of Hepatocyte Nuclear Factor-4
The liver-enriched transcription factor hepatocyte nuclear
factor-4 (HNF-4) belongs to the group of zinc finger proteins and is
frequently seen as a member of the nuclear receptor superfamily with
unknown ligand (Taraviras et al., 1994). Zinc-fingers are small
DNA-binding peptide motifs. These motifs can be used as modular
building blocks for the construction of larger protein domains that
recognize and bind to specific DNA sequences (Klug, 1999). Steroids and
thyroid hormones, as well as vitamin D, retinoids, and some nutrient
metabolites (fatty acids, prostaglandins, farnesol metabolites) act
through binding to members of the zinc-finger containing superfamily of
nuclear hormone receptors. These receptor proteins bind directly to
specific DNA recognition sequences (hormone response elements) in the
promoter region of target genes to facilitate transcription. The
formation of several sets of heterodimers among family members as well
as cross-talk with other signaling systems results in an intricate
regulatory network with distinct particularities for each receptor type
(Meier, 1997).
D. DNA-Binding Domain of Hepatocyte Nuclear Factor-6
HNF-6 is a liver-enriched transcription factor that contains a
single-cut domain and a novel type of homeodomain. Comparative trees of
mammalian, Drosophila, and C. elegans proteins
showed that HNF-6 defines a new class of homeodomain proteins called onecut class. It could be demonstrated that C. elegans
proteins of this class bind to HNF-6 DNA targets. Thus, depending on
their sequence, these targets determine for HNF-6 at least two modes of
DNA binding, which hinge on the homeodomain and on the linker that
separates it from the cut domain, and two modes of transcriptional stimulation, which hinge on the homeodomain (Lannoy et al., 1998).
E. DNA-Binding Domain of CCAAT/Enhancer-Binding Proteins
Many transcription factors bind DNA to form dimeric (2:1)
protein-DNA complexes. Examples include basic region leucine zipper (bZIP) proteins and basic region helix-loop-helix zipper (bHLHZIP) proteins. These two families of transcription factors follow an assembly pathway in which two protein monomers bind DNA sequentially and form their dimerization interface while bound to DNA (Kohler et
al., 1999). Dimerization of these transcription factors stabilizes the
protein-DNA complexes and can lead either to homodimers with the same
transcription factor or to heterodimers with other members of the same
family of transcription factors (Horiuchi et al., 1997).
The bZIP family of proteins is one of the largest and most conserved
groups of eukaryotic transcription factors/repressors (Niu et al.,
1999). These transcription factors use an atypically simple motif for
DNA recognition called the basic region, yet family members
discriminate differentially between target sites that differ only in
half-site spacing. Two such sites are the cAMP-response element (CRE)
and the AP-1 target site (Metallo and Schepartz, 1994). The DNA-binding
motif of transcription factors belonging to the bZIP family is
bipartite, consisting of a dimerization interface termed "leucine
zipper" and a DNA contact surface termed the "basic region".
Specificity of DNA binding has been shown to be imparted by the basic
region (Agre et al., 1989).
The CCAAT/enhancer-binding proteins (C/EBP, C/EBP, C/EBP
, and
C/EBP
) form a subfamily of bZIP transcription factors that display
sequence homology within the bZIP domain. The conserved basic region in
this subfamily contains two motifs that exhibit significant homology to
the bipartite nuclear localization signal promoting nuclear transport
of a bZIP transcription factor (Williams et al., 1997).
Further important members of the bZIP family of transcription factors
are c-jun, c-fos (AP-1), and CREB. The molecular chaperone bZIP
enhancing factor (BEF) has been shown to increase DNA binding of
transcription factors that contain a basic region leucine zipper (bZIP)
DNA-binding domain. BEF stimulates DNA binding by recognizing the
unfolded leucine zipper and promoting the folding of bZIP monomers to
dimers. Antisense experiments indicate that BEF is required for
efficient transcriptional activation by bZIP proteins in vivo
(Virbasius et al., 1999).
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III. Molecular Regulation of Liver Function |
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A. Liver-Specific Gene Expression
The transcription rate of genes encoding liver-specific proteins
is distinctly higher in hepatocytes as compared with other cell types
(Powell et al., 1984). The transcription of several hepatic genes is
activated during liver development and later modulated depending on
extracellular stimulation (Schmid and Schulz, 1990; Cascio and Zaret,
1991; Shiojori et al., 1991). Experiments using a cDNA library from
mouse liver poly(A)+ RNA that was then differentially screened with
poly(A)+ RNA from liver and nonliver cells provided strong evidence
that the predominant control of liver-specific gene expression resides
at the level of transcription (Derman et al., 1981; Aran et al., 1995).
Clones proven to be liver-specific were picked and used as templates
for hybridization with radioactive RNA newly transcribed in vitro in
nuclei isolated from liver and nonliver tissues. The hybridization
signals obtained with RNA synthesized with liver nuclei were at least
10 times more intense than those obtained with nuclei from other
tissues. Because the cDNA clones represented an unbiased population of transcripts, the findings led to the conclusion that liver-specific gene expression is primarily a consequence of transcriptional regulation (Derman et al., 1981).
Transient transfection assays in which the introduced gene does not
integrate into the genome have been instrumental in identifying the
regulatory sequences in DNA that confer liver-specific gene expression.
Analyses performed on a wide variety of genes that code for entirely
different proteins show shared regulatory sequences. Moreover,
characterization of the regulatory sequences of a number of genes has
shown that each gene contains a combination of some or all of the
liver-specific shared motifs (Benvenisty and Reshef, 1991; Aran et al.,
1995). It is this combination of cis-regulatory elements
rather than a single element that appears to be required for
liver-specific gene expression. Finally, these shared motifs bind
distinct cognate liver-enriched transcription factors and have aided in
isolating and characterizing these factors (for review see De Simone
and Cortese, 1991; Lai and Darnell, 1991; Aran et al., 1995).
B. Liver-Enriched Transcription Factors
Six families of liver-enriched transcription factors have been
characterized so far: HNF-1, HNF-3, HNF-4, HNF-6, C/EBP, and D-binding
protein (DBP). The analysis of the tissue distribution of these
factors and the determination of their hierarchical relations have led
to the hypothesis that the cooperation of liver-enriched transcription
factors with the ubiquitous transactivating factors is necessary, and
possibly even sufficient, for the maintenance of liver-specific gene
transcription (Hayashi et al., 1999).
HNFs are a heterogeneous class of evolutionarily conserved
transcription factors that contain several families of liver-enriched transcription factors that are required for cellular differentiation and metabolism (Duncan et al., 1998). The liver-enriched transcription factor family containing the C/EBPs was formerly called HNF-2 and will
be reviewed separately along with the D-binding protein (DBP).
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IV. Hepatocyte Nuclear Factors |
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The HNF-1, HNF-3, HNF-4, and HNF-6 families of transcription
factors contain several members. It should be noted that liver-enriched transcription factors are not exclusively expressed in the liver. For
example, HNF-1, -3, -3, -3, -4, and -6 are also
expressed in pancreatic -cells (Vaisse et al., 1997). HNF-1 and
HNF-4 play there a critical role in normal pancreatic -cell
function. Mutations in these liver-enriched transcription factors
result in two forms of maturity-onset diabetes of the young (MODY),
MODY3 and MODY1, respectively (Yamagata et al., 1996; Vaisse et al., 1997; Chevre et al., 1998). There are many more examples of relevant extrahepatic functions of liver-enriched transcription factors, but it
is beyond the scope of this review to provide a complete summary of
those extrahepatic functions.
A. The Hepatocyte Nuclear Factor-1 Family
HNF-1 is a transcriptional regulator composed of HNF-1 and
HNF-1 hetero- and homodimers. These homeoproteins share identical DNA-binding domains but have different transcriptional activation properties (Kuo et al., 1991; Song et al., 1998).
The HNF-1 gene was assigned by somatic cell hybrids and recombinant
inbred strain mapping to mouse chromosome 5 near Bcd-1 and to human
chromosome 12 region q22-qter, revealing a different chromosomal region
for these two species (Kuo et al., 1990). The HNF-1 gene was
assigned to human chromosome 17 and murine chromosome 11. These
chromosomal localizations differ from that of the HNF-1 gene,
indicating that both genes are not clustered on the genome (Bach et
al., 1991).
HNF-1 is one of the most important transactivators of liver-specific
albumin transcription (Maire et al., 1989). HNF-1 acts as an accessory
factor to enhance the inhibitory action of insulin on mouse
glucose-6-phosphatase gene transcription (Streeper et al., 1998).
HNF-1 is also an accessory factor required for activation of
glucose-6-phosphatase gene transcription by glucocorticoids (Lin et
al., 1998). Several lines of evidence point to a direct transactivation
of the mouse ferrochelatase promoter by HNF-1 in the liver (Muppala
et al., 2000).
Plasma lipoprotein(a) concentrations are highly heritable and
predominantly determined by the liver-specific apolipoprotein(a) [apo(a)] gene. Elevated levels of lipoprotein(a) in the plasma are a
risk factor for coronary artery disease and stroke. Positive regulation
of transcription of the apo(a) gene is dependent on the binding of
HNF-1 to a regulatory element located downstream of the mRNA start
site (Wade et al., 1994).
HNF-1 is able to repress the transcription of liver-specific genes
as demonstrated for the sucrose-isomaltase gene. Glucose represses
transcription of this gene in cooperation with three HNF-1-binding
sites in the sucrose-isomaltase promoter. Mutagenesis of the
HNF-1-binding sites showed that the two distal HNF-1-binding sites are
crucial for the glucose regulation of the sucrose-isomaltase gene
(Rodolosse et al., 1998).
A number of genes that are predominantly expressed in the liver are
positively regulated by HNF-1 interacting with the respective cis-acting HNF-1-binding elements in the promoters of these
genes (see also Table 1).
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Serum colloid osmotic pressure is believed to control hepatic output of
plasma proteins. Many plasma proteins that are secreted from the liver,
including albumin, have a HNF-1-binding site in their promoter. The
activity of HNF-1 in highly differentiated hepatoma cells was shown
to be modulated by a fluctuation in the level of oncotically active
macromolecules like dextran or albumin in the surrounding cell culture
medium. Higher oncotic pressures lead to a decrease in HNF-1 mRNA
levels (Pietrangelo and Shafritz, 1994).
1. Dimerization Cofactor of Hepatocyte Nuclear Factor-1 and
Liver-Specific Gene Expression.
Interestingly, HNF-1, but not
HNF-1, is expressed in the liver. Under physiologic conditions as
well as in transfection experiments with HNF-1 and HNF-1, stable
homodimer formation can be found in the liver, whereas in other organs,
heterodimers also are detected. From these data it was assumed that the
extent of heterodimerization may be regulated in a tissue-specific
manner. Furthermore, it could be shown that exclusive expression of
HNF-1 is associated with repression of a subset of
hepatocyte-specific genes in the dedifferentiated hepatocyte cell line
C2, in differentiated F9 cells, in somatic hybrids between hepatocytes
and fibroblasts, and in the lung (Mendel et al., 1991a).
is unique among the vertebrate homeodomain-containing proteins
in that it dimerizes in the absence of its DNA recognition sequence
(Mendel et al., 1991b). A dimerization cofactor of HNF-1 (DCoH)
could be identified that displays a restricted tissue distribution and
does not bind to DNA, but, rather, selectively stabilizes HNF-1
homodimers. The formation of a stable tetrameric DCoH-HNF-1 complex
requires the dimerization domain of HNF-1 and does not change the
DNA-binding characteristics of HNF-1, but enhances its
transcriptional activity. DCoH regulates the formation of transcriptionally active tetrameric complexes and thus may contribute to the developmental and tissue specificity of the complex (Mendel et
al., 1991b). DCoH plays an important role in liver development and
liver-specific gene expression, because HNF-1 is regarded as an
important regulator of the transcriptional network in liver development
and liver-specific gene expression.
The chromosomal localization of the genes for DCoH was assigned to
chromosomes 10 in both humans and mice by Southern blot analyses of
somatic cell hybrids (Milatovich et al., 1993). DCoH functions as both
a transcriptional coactivator and a pterin dehydratase (Cronk et al.,
1996). The human DCoH (also named pterin-4 -carbinolamine dehydratase) is a bifunctional protein proposed to be involved in
entirely different biochemical functions. The protein coding region of
the gene is about 5 kb long and contains 4 exons. Within the
5'-flanking sequence, potential regulatory regions include consensus
binding sites for transcription factor Sp1, an AP-1, and several
AP-2-binding sites; however, the 5' upstream region lacks both a
proximal TATA and CAAT box promoter element (Thony et al., 1995).
B. The Hepatocyte Nuclear Factor-3 Subfamily
The mammalian HNF-3/fkh family consists of at least 30 distinct
members and is expressed in a variety of different cellular lineages
(Qian and Costa, 1995). The HNF-3 gene subfamily is composed of three
proteins (, , and ) that mediate hepatocyte-enriched transcription of numerous genes whose expression is necessary for organ
function (Samadani and Costa, 1996). All three transcription factors
share strong homology in the winged-helix/fork head DNA-binding domain
(region I) that overlaps with the nuclear localization signal (Qian and
Costa, 1995). HNF-3, -, and - are able to recognize the same
DNA sequence (Samadani and Costa, 1996; Pani et al., 1992a,b). They
also possess two similar stretches of amino acids at the carboxyl
terminus (regions II and III) and a fourth segment of homology at the
amino terminus (region IV) (Pani et al., 1992a,b).
The HNF-3 proteins demonstrate homology with the Drosophila
homeotic gene fork head in regions I, II, and III, suggesting that
HNF-3 may be its mammalian homolog (Pani et al., 1992a). Experiments
using site-directed mutagenesis within regions II and III (amino acids
361-458) of HNF-3 demonstrated their importance for
transactivation. In cotransfection assays with expression vectors that
produced different truncated HNF-3 proteins, amino-terminal sequences defined by conserved region IV also contributed to
transactivation, but region IV activity required the participation of
the region II-III domain (Pani et al., 1992a).
HNF-3 and HNF-3 regulate gene expression in endoderm-derived
hepatocytes, and intestinal, pancreatic, and bronchiolar epithelium (Rausa et al., 1997; Clevidence et al., 1998). HNF-3 may also play
an important role in development and maintenance of urogenital tract
epithelial cells (Clevidence et al., 1998; Kopachik et al., 1998).
HNF-3 and HNF-3 are members of a large family of developmentally regulated transcription factors that participate in embryonic pattern
formation (Rausa et al., 1997; Clevidence et al., 1998).
Stimulation of HNF-3 gene transcription upon retinoic acid-induced
differentiation of mouse F9 embryonal carcinoma cells can give rise to
three distinct differentiated cell types; visceral endoderm, parietal
endoderm, and primitive endoderm, which indicates that HNF-3 may
play an important role in differentiation during primitive endoderm
formation, an extremely early event during murine embryogenesis (Jacob
et al., 1994).
A number of liver-specific genes that are predominantly expressed in
the liver are positively regulated by HNF-3, -, or - through
interaction with the respective cis-acting HNF-3-binding elements in the promoters of these genes (see also Table
2). In contrast, HNF-3 bound to the
HNF-3-binding site of the human aldolase B promoter completely
antagonizes transactivation of the liver-specific aldolase B gene by
HNF-1 and DBP (Gregori et al., 1993).
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Partial hepatectomy produced minimal fluctuation in HNF-3 (, ,
and ) and transthyretin expression, suggesting that HNF-3, -,
and - expression is not influenced by proliferative signals induced
during liver regeneration. In acute-phase livers a dramatic reduction
in HNF-3 expression was observed, which correlates with a decrease
in the expression of target genes, such as the transthyretin gene (Qian
et al., 1995).
C. The Hepatocyte Nuclear Factor-4 Subfamily
The HNF-4 subfamily belongs to the nuclear receptor superfamily,
which contains more than 150 proteins that represent nuclear receptors
for steroids, retinoids, thyroid hormone, and vitamin D, as well as
many related proteins (Mangelsdorf et al., 1995). HNF-4 subfamily
members include HNF-4, HNF-4, and HNF-4 and many splice
variants. HNF-4 was formerly classified as an orphan member of the
steroid/thyroid nuclear receptor superfamily, because HNF-4 had no
defined ligand. Hertz et al. (1998) reported that fatty acyl-CoA
thioesters are ligands of HNF-4. Therefore it seems no longer
justified to think of the HNF-4 subfamily members as orphan members of
the larger nuclear receptor superfamily.
HNF-4 participates in the regulation of several genes involved in
diverse metabolic pathways (e.g., glucose, cholesterol, and fatty acid
metabolism), in the synthesis of blood coagulation factors, and in
developmental processes determining the hepatic phenotype (see also
Table 3) (Sladek et al., 1990; Jiang et
al., 1995; Yamagata et al., 1996; Hadzopoulou-Cladaras et al., 1997).
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HNF-4 (gene symbol, TCF14) is an upstream regulator of HNF-1
expression (Yamagata et al., 1996) and is expressed in the mammalian
liver, kidney, and digestive tract (Sladek et al., 1990; Holewa et al.,
1997). The human HNF-4 gene was mapped to chromosome 20q in a region
syngenic with mouse chromosome 2, to which the HNF-4 ortholog has been
assigned (Argyrokastritis et al., 1997; Chevre et al., 1998).
HNF-4 was first identified in Xenopus and showed distinct
activation and expression profiles in oogenesis and embryogenesis of
Xenopus laevis (Holewa et al., 1997).
A novel HNF-4 subtype called HNF-4 could be located on human
chromosome 8. Northern blot analysis revealed that HNF-4 is expressed in the kidney, pancreas, small intestine, testis, and colon
but not in the liver, whereas HNF-4 RNA was found in all of these
tissues (Drewes et al., 1996).
An example of negative HNF-4 regulation is the mitochondrial HMG-CoA
synthase gene. HNF-4 binds to the mitochondrial HMG-CoA synthase
nuclear receptor response element and represses peroxisome proliferator-activated receptor (PPAR)-dependent activation of reporter
gene linked to the mitochondrial HMG-CoA synthase gene promoter
(Rodriguez et al., 1998). Another example of negative regulation by
HNF-4 is the acyl-CoA oxidase gene. Both PPAR and HNF-4 efficiently
bind to the acyl-CoA oxidase gene enhancer element, but PPAR
exhibits much stronger transactivation than HNF-4. As a result, HNF-4
suppressed the gene-activating function of PPAR, when they were
expressed together, due to competition for a common binding site
(Nishiyama et al., 1998). An example of repression by HNF-4 could be
found in studies of the rat arginase promoter activity that is
stimulated by C/EBPs and DBP (Chowdhury et al., 1996).
1. The Structure and Domains of Hepatocyte Nuclear
Factor-4.
HNF-4 contains two transactivation domains, designated
AF-1 and AF-2, which activate transcription in a cell type-independent manner. Deletion of AF-1 results in 40% reduction of the
HNF-4-mediated activation. AF-1 consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of
transcription. This short transactivator belongs to the class of acidic
activators, and it is predicted to adopt an amphipathic -helical
structure. In contrast, the AF-2 transactivator is complex, spanning
the 128-366 region of HNF-4, and it cannot be further dissected
without impairing activity (Hadzopoulou-Cladaras et al., 1997). AF-1
shares common structural motifs and molecular targets with the
activation domains of p53, NF-
B-p65, and VP-16 (a herpes simplex
virus-1 virion protein), implying that these activators may function
through common mechanisms (Green et al., 1998). Remarkably, AF-1
interacts with multiple proteins that act at distinct steps during
transcription (including TBP; the TBP-associated factors
TAFII31 and TAFII80; TFIIB;
TFIIH-p62; and the coactivators CBP, ADA2, and PC4) providing a
possible mechanism for the functional synergy exhibited by this activator in vivo (Green et al., 1998).
). The 360-366 region of HNF-4 contains a motif that is highly conserved among transcriptionally active nuclear receptors, and it is
essential for AF-2 activity, but it is not necessary for dimerization
and DNA binding of HNF-4. Thus, HNF-4 deletion mutants lacking the
361-465 region bind efficiently to DNA as homo- and heterodimers and
behave as dominant negative mutants (Hadzopoulou-Cladaras et al.,
1997). Remarkably, the full transactivation potential of AF-2 is
inhibited by the region spanning residues 371-465 (region F). The
inhibitory effect of region F on the HNF-4 AF-2 activity is a unique
feature among members of the nuclear receptor superfamily, and it has
been proposed that it defines a distinct regulatory mechanism of
transcriptional activation by HNF-4 (Hadzopoulou-Cladaras et al.,
1997). In later studies a repressor domain has been localized to
residues 428-441 in region F of HNF-4 that is sufficient by itself to
repress the activity of the AF-2 domain. Multiple mutations within this
repressor domain enhance activity (Iyemere et al., 1998).
2. The Relevance of Hepatocyte Nuclear Factor-4 Splice
Variants.
Further complexity of gene control by HNF-4
transcription factors can be anticipated by the differential splicing
of the 10 initially identified exons of the HNF-4 gene (Nakhei et
al., 1998). Thus, so far, seven distinct splice variants have been identified in human and murine cDNA samples. HNF-41 represents the
initially identified transcript, whereas HNF-42 through HNF-47 are the splice variants identified subsequently (Sladek et al., 1990;
Hata et al., 1992, 1995; Chartier et al., 1994; Drewes et al., 1996;
Kritis et al., 1996; Furuta et al., 1997; Nakhei et al., 1998).
HNF-41, HNF-42, and HNF-43 were initially referred to as
HNF-4A, HNF-4B, and HNF-4C, respectively (Hata et al., 1992, 1995;
Kritis et al., 1996). In all HNF-4 splice variants the DNA-binding
domain remains unchanged (Viollet et al., 1997; Nakhei et al., 1998).
The impact of these different splice variants on the regulation of
downstream target gene regulation remains largely to be determined. The
consequences of the existence of different splice variants on the
regulation of gene transcription are still not fully understood.
, the two splice variants
HNF42 and HNF43 with additional exons were detected. Both
HNF-4 splice variants share HNF-4-binding sites with HNF-4 but
have lower DNA-binding activities and weaker transactivation potential
than HNF-4 (Holewa et al., 1997).
In cotransfection experiments evidence was obtained that HNF-4 is
significantly less active than HNF-42 and that the HNF-4 splice
variant HNF-44 has no detectable transactivation potential. Therefore, the differential expression of distinct HNF-4 proteins may
play a key role in the differential transcriptional regulation of
HNF-4-dependent genes (Drewes et al., 1996).
3. Homo- and Heterodimerization of Hepatocyte Nuclear Factor-4
Proteins.
Studies with in vitro translated HNF-4 protein show
that it binds to its recognition site as a dimer, and cotransfection
assays indicate that it activates transcription in a sequence-specific fashion in nonhepatic (HeLa) cells (Sladek et al., 1990). It has been proposed that HNF-4 forms homodimers in contrast to other members
of the nuclear receptor superfamily that also form heterodimers with
other members of the nuclear receptor superfamily like retinoid X
receptor (RXR-) (Jiang et al., 1995). Later, it could be demonstrated that another orphan member of the nuclear hormone receptor
superfamily called SHP (short heterodimer partner), which contains the
dimerization and ligand-binding domain found in other family members
but lacks the conserved DNA-binding domain (Seol et al., 1996),
specifically inhibits transactivation by HNF-4 and other hormone
receptor superfamily members with which it interacts (Seol et al.,
1996; Lee et al., 2000). Therefore, it has been suggested that SHP
functions as a negative regulator of receptor-dependent signaling
pathways (Seol et al., 1996; Lee et al., 2000). SHP represses nuclear
hormone receptor-mediated transactivation via two separate steps: first
by competition with coactivators and second by direct effects of its
transcriptional repressor function (Lee et al., 2000).
4. Regulation of Hepatocyte Nuclear Factor-4 Function by
Phosphorylation.
HNF-4 DNA-binding activity is modulated
post-translationally by phosphorylation (Ktistaki et al., 1995; Viollet
et al., 1997). Phosphorylated HNF-4 is concentrated in distinct nuclear
compartments within the cell, as evidenced by in situ
immunofluorescence and electron microscopy. Inhibition of HNF-4
phosphorylation with genistein results in a loss of the nuclear
compartmentalization of HNF-4 associated with a significantly decreased
ability to activate endogenous target genes (Ktistaki et al., 1995).
). Further experiments demonstrated that phosphorylation of HNF-4
by cAMP-dependent protein kinase A at serine residues leads to a
reduced DNA-binding affinity of HNF-4 in vitro (Viollet et al., 1997).
It could be demonstrated that in vivo phosphorylation of HNF-4 depends
on the diet; it is decreased by a carbohydrate-rich diet and is
increased by fasting or in refed animals given glucagon or
isoproterenol and phosphodiesterase inhibitors (Viollet et al., 1997).
Phosphorylation of HNF-4 by cAMP-dependent protein kinase A at serine
residues might be involved in the transcriptional inhibition of liver
genes by cAMP inducers (Viollet et al., 1997).
5. Agonistic and Antagonistic Ligands for the Nuclear Receptor
Hepatocyte Nuclear Factor-4.
In 1998 Hertz and coworkers
published the discovery of several ligands for HNF-4 with agonistic and
antagonistic effects on HNF-4 transcriptional activity (see also
Tables 4 and
5). It could be demonstrated that
long-chain fatty acids directly modulate the transcriptional activity
of HNF-4 by binding as their acyl-CoA thioesters to the
ligand-binding domain of HNF-4. This binding shifts the
oligomeric-dimeric equilibrium of HNF-4, because it could be shown
that the binding of saturated (C14:0)-CoA to the ligand-binding domain
of HNF-4 leads to increased HNF-4 dimerization and activates
binding of the HNF-4 dimer to its cognate enhancer element, whereas
saturated (C16:0)-CoA only activates binding of the HNF-4 dimer to
its cis-acting element. In contrast, the antagonistic
ligands 
-3 and -6 polyunsaturated fatty acyl-CoAs, (C18:3,
w-3)-CoA, and saturated (C18:0)-CoA decrease the transcriptional activity of HNF-4. (C18:3, w-3)-CoA and saturated (C18:0)-CoA were
shown to lower the affinity of HNF-4 to its cognate enhancer element. Furthermore, it could be demonstrated that saturated (C18:0)-CoA leads to decreased HNF-4 dimerization (Hertz et al., 1998).
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6. Acetylation of Nucleosomal Histones and Hepatocyte Nuclear
Factor-4 by cAMP Response Element-Binding Protein.
CBP possesses
an intrinsic acetyltransferase activity capable of acetylating
nucleosomal histones as well as several nonhistone proteins. It could
be demonstrated that CBP can acetylate HNF-4 at lysine residues within
the nuclear localization sequence. CBP-mediated acetylation is crucial
for the proper nuclear retention of HNF-4, which is otherwise
transported out to the cytoplasm via the CRM1 pathway. Acetylation also
increases HNF-4 DNA-binding activity and its affinity of interaction
with CBP itself, and is required for target gene activation.
Acetylation is a key post-translational modification that may affect
several properties of a transcription factor critical for the execution
of its biological functions (Soutoglou et al., 2000a).
7. Chicken Ovalbumin Upstream Promoter-Transcription Factors and
Hepatocyte Nuclear Factor-4: Cooperation and Competition.
Chicken
ovalbumin upstream promoter-transcription factor (COUP-TF) and HNF-4
were both frequently called orphan members of the steroid/thyroid
receptor superfamily and exhibit ubiquitous and liver-enriched tissue
distribution, respectively (Kimura et al., 1993). COUP-TFs strongly
inhibit transcriptional activation mediated by nuclear hormone
receptors, including HNF-4. COUP-TFs repress HNF-4-dependent gene
expression by competition with HNF-4 for common binding sites found in
several regulatory regions (Kimura et al., 1993; Ktistaki and
Talianidis, 1997b). In contrast, promoters, such as the HNF-1 promoter,
which are recognized by HNF-4 but not by COUP-TFs, are activated by
COUP-TFI and COUP-TFII in conjunction with HNF-4 more than 100-fold
above basal levels, as opposed to about 8-fold activation by HNF-4
alone (Ktistaki and Talianidis, 1997b). This enhancement was strictly
dependent on an intact HNF-4 E domain. In vitro and in vivo evidence
suggests that COUP-TFs enhance HNF-4 activity by a mechanism that
involves their physical interaction with the amino acid 227-271 region
of HNF-4 (see also Fig. 6) (Ktistaki and
Talianidis, 1997b). Therefore, in certain promoters, COUP-TFs act as
auxiliary cofactors for HNF-4, orienting the HNF-4 activation domain in
a more efficient configuration to achieve enhanced transcriptional
activity (Kimura et al., 1993; Ktistaki and Talianidis, 1997b). An
example of COUP-TF- associated repression of a liver-specific gene
provides the gene for rat ornithine transcarbamylase, an ornithine
cycle enzyme (Kimura et al., 1993). Therefore, COUP-TF plays a dual
regulatory role depending on the promoter context. Repression of a
tissue-specific promoter by a ubiquitous transactivator and
derepression by a related tissue-enriched transactivator is potentially
an important mechanism for tissue-specific activation of a gene (Kimura
et al., 1993; Ktistaki and Talianidis, 1997b).
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D. Hepatocyte Nuclear Factor-6
In contrast to HNF-5 there is plenty of evidence for the existence
of HNF-6 (see Table 6). The human
gene for HNF-6 has been mapped to chromosome bands 15q21.1-21.2
and the rat gene to chromosome 8q24-q31 by Southern blotting of DNA
from somatic cell hybrids and by fluorescence in situ hybridization
(Vaisse et al., 1997; Rastegar et al., 1998). Interspecific backcross
analysis determined that the murine HNF-6 gene is located in the middle
of mouse chromosome 9 (Rausa et al., 1997).
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Transcription factors of the onecut class, whose prototype is HNF-6,
are characterized by the presence of a single-cut domain and by a
peculiar homeodomain. Human OC-2, the second mammalian member of this
class, is located on human chromosome 18. The distribution of OC-2 mRNA
in humans is tissue-restricted, the strongest expression being detected
in the liver and skin. The amino acid sequence of OC-2 contains several
regions of high similarity to HNF-6. The recognition properties of OC-2
for binding sites present in regulatory regions of liver-expressed
genes differ from, but overlap with, those of HNF-6 (Jacquemin et al.,
1999). It might be that in the future, HNF-6 and OC-2 will be regarded
as two members of a bigger family.
1. Splice Variants of Hepatocyte Nuclear Factor-6.
Two rat
cDNA species coding for two isoforms, HNF-6 (465 residues) and
HNF-6 (491 residues) could be identified, which differ only by the
length of the spacer between the two DNA-binding domains. The two HNF-6
isoforms are generated by alternative splicing of three exons that are
more than 10 kb apart from each other. Exon 1 codes for the N-terminal
part and the cut domain, exon 2 codes for the 26 HNF-6-specific
amino acids, and exon 3 codes for the homeodomain and the C-terminal
amino acids (Rastegar et al., 1998). Both isoforms stimulate
transcription. The affinity of HNF-6 and HNF-6 for DNA depends on
the target sequence. Binding of HNF-6 to DNA involves the cut domain
and the homeodomain, but the latter is not required for binding to a
subset of sites (Lannoy et al., 1998).
2. Hepatocyte Nuclear Factor-6 in Development.
Observations
that HNF-6 contributes to the control of the expression of
transcription factors and is expressed at early stages of liver,
pancreas, and neuronal differentiation suggest that HNF-6 participates
in several developmental programs (Landry et al., 1997). HNF-6
recognizes the 138 to 126 region of the HNF-3 promoter.
Site-directed mutagenesis of this HNF-6 site diminishes reporter gene
expression, suggesting that HNF-6 activates transcription of this
promoter and may thus play a role in epithelial cell differentiation of
gut endoderm via regulation of HNF-3 (Samadani and Costa, 1996).
Later, it was recognized that HNF-6 is required for HNF-3 promoter
activity and that HNF-6 also recognizes the regulatory region of
numerous liver-specific genes (Rausa et al., 1997). In situ
hybridization studies of staged specific embryos demonstrate that HNF-6
and its potential target gene, HNF-3, are coexpressed in the
pancreatic and hepatic diverticulum. More detailed analysis of the
developmental expression patterns of HNF-6 and HNF-3 provides evidence of colocalization in hepatocytes, intestinal epithelial, and
pancreatic ductal epithelial and exocrine acinar cells. The expression
patterns of these two transcription factors do not overlap in other
endoderm-derived tissues or the neurotube (Rausa et al., 1997).
3. Regulation of Hepatocyte Nuclear Factor-6 Expression by Growth
Hormone.
HNF-6 expression can be regulated and modulated by
growth hormone (GH) (Lahuna et al., 1997, 2000). In hypophysectomized
rats, HNF-6 mRNAs increase within 1 h after a single injection of
GH. The same GH-dependent induction could be reproduced on isolated hepatocytes. DNA binding experiments showed that the transcription factors STAT5 (signal transducer and activator of transcription 5) and
HNF-4 bind to sites located around 110 and 650 of the hnf-6 gene,
respectively. Furthermore, it could be demonstrated that STAT5 binding
is induced and HNF-4-binding affinity is increased in the liver within
1 h after GH injection to hypophysectomized rats (Rastegar et al.,
2000). Using transfection experiments and site-directed mutagenesis, it
could be found that STAT5 and HNF-4 stimulated transcription of an
hnf-6 gene promoter-reporter construct. Consistent with earlier
findings that HNF-6 stimulates the hnf-4 and hnf-3 gene promoters,
GH treatment of hypophysectomized rats increased the liver
concentration of HNF-4 and HNF-3 mRNAs. Together, these data
demonstrate that GH stimulates transcription of the hnf-6 gene by a
mechanism involving STAT5 and HNF-4. They show that HNF-6 participates
not only as an effector, but also as a target, to the regulatory
network of liver transcription factors, and that several members of
this network are GH-regulated (Lahuna et al., 2000). In protein-DNA
interaction studies and in transfection experiments, it could be found
that the liver-enriched transcription factor C/EBP binds to the
hnf-6 gene and inhibits its expression. This inhibitory effect involved
an N-terminal subdomain of C/EBP and two sites in the hnf-6 gene
promoter. Using liver nuclear extracts from GH-treated
hypophysectomized rats, it was found that GH induces a rapid, transient
decrease in the amount of C/EBP protein. This GH-induced change is
concomitant with the transient stimulatory effect of GH on the hnf-6
gene. Stimulation of the hnf-6 gene by GH therefore involves lifting of
the repression exerted by C/EBP in addition to the GH-induced
stimulatory effects of STAT5 and HNF-4 on that gene (Pierreux et al.,
1999).
4. Inhibitory Protein-Protein Interaction between Hepatocyte
Nuclear Factor-6 and a Nuclear Receptor.
HNF-6 inhibits the
glucocorticoid-induced stimulation of two genes coding for enzymes of
liver glucose metabolism, 6-phosphofructo-2-kinase and
phosphoenolpyruvate carboxykinase. Binding of HNF-6 to DNA is required
for inhibition of glucocorticoid receptor activity. In vitro and in
vivo experiments suggest that this inhibition is mediated by a direct
HNF-6/glucocorticoid receptor interaction involving the amino-terminal
domain of HNF-6 and the DNA-binding domain of the receptor (Pierreux et
al., 1999).
E. Coactivators for Hepatocyte Nuclear Factor-1 and Hepatocyte Nuclear Factor-4
Multiple coactivators of HNF-1 and HNF-4 could be identified,
including CBP, p300, p/CAF, and a series of factors that have been
identified biochemically and by expression cloning (Kamei et al., 1996;
Torchia et al., 1997; Yoshida et al., 1997; Dell and
Hadzopoulou-Cladaras, 1999; Rachez et al., 2000; Soutoglou et al.,
2000a,b). These factors, with a molecular mass around 160 kDa, are
members of the p160 protein family and have been shown to exhibit an
intrinsic HAT (Bannister and Kouzarides, 1996; Ogryzko et al., 1996;
Chen et al., 1997; Glass et al., 1997; Imhof et al., 1997; Spencer et
al., 1997). Furthermore, a nuclear receptor coactivator (NCoA) gene
family within the p160 protein family has been proposed that includes
the homologous factors SRC-1 (also called NCoA-1), SRC-2 (also called
NCoA-2, TIF2, GRIP1) and SRC-3 (also called NCoA-3, ACTR, AIB1, p/CIP,
TRAM-1, RAC3) (Torchia et al., 1997; Rachez et al., 2000). The NcoA
family members SRC-1, SRC-2, and SRC-3 share a conserved N-terminal
bHLH, PAS A domain, a serine/threonine-rich region, and a C-terminal
glutamine-rich region (Torchia et al., 1997).
SRC-1, SRC-3, and CBP all contain several related leucine-rich, charged
helical interaction motifs (also termed LCDs) with a consensus core
LXXLL sequence motif that is required for the assembly of coactivator
complex, which provides receptor-specific mechanisms of gene activation
and allows the selective inhibition of distinct signal-transduction
pathways. Mutation of this consensus core motif leads to abolished
interaction with nuclear receptors (Torchia et al., 1997). This leads
to the inevitable question whether mutations in these LCD domains may
lead to disturbances in liver development or liver function due to
reduced HNF-1 and HNF-4 transactivation potential.
Possibly, conformational changes in the CBP holoprotein, perhaps in
part contributed by SRC-3 by forming the coactivator complex, modulate
interactions with transcription factors and associated regulatory
proteins, including protein kinases and histone acetylases (Bannister
and Kouzarides, 1996; Ogryzko et al., 1996; Torchia et al., 1997).
SRC-1 contains a histone acetylase domain between amino acid residues
1107 and 1216 with intrinsic HAT activity specific for histones H3 and
H4 (Spencer et al., 1997). Furthermore, SRC-1 also contains two
p/CAF-interacting domains between amino acid residues 1207 and 1250 that bind p/CAF, another factor, with intrinsic histone acetylase
activity (Yang et al., 1996; Spencer et al., 1997). SRC-1 interacts
also with CBP/p300 through a conserved C-terminal domain of CBP/p300
and probably gets involved in a three-way interaction with CBP/p300 and
an interacting nuclear receptor or transcription factor (Kamei et al.,
1996; Yao et al., 1996).
SRC-3 and CBP are a functional complex, necessary for the activity of
several CBP-dependent transcription factors as well as nuclear
receptors (Torchia et al., 1997). Whether SRC-3 is required for
transactivation by HNF-1 or HNF-4 remains to be determined. SRC-3 forms
complexes with significant portions of CBP in the cell and is
required for transcriptional activity of nuclear receptors and other
CBP/p300-dependent transcription factors (Torchia et al., 1997). The
major CBP interaction domain of SRC-3 could be mapped to amino acid
residues 758-1115, with an internal 200-amino acid domain that could
still interact, whereas a minimal nuclear receptor interaction domain
could be mapped N-terminal of the CBP interaction domain to amino acid
residues 680-740, which were sufficient for binding of a liganded
nuclear receptor (Torchia et al., 1997).
It could be demonstrated that HNF-1 can physically interact with CBP,
p/CAF, SRC-1, and SRC-3 and that these interactions lead to increased
HNF-1-dependent transcription in functional assays using a
genome-integrated promoter. The transcriptional activation potential of
HNF-1 was strictly dependent on the synergistic action of CBP and
p/CAF. It could be shown that CBP and p/CAF can independently interact
with the N-terminal and the C-terminal domain of HNF-1, respectively
(see also Fig. 6) (Soutoglou et al., 2000b).
CBP binds to the HNF-4 AF-1 and AF-2 domains with the N terminus and
the N and C termini, respectively (see also Fig. 6) (Dell and
Hadzopoulou-Cladaras, 1999). Interestingly, in contrast to the other
nuclear hormone receptors the interaction between HNF-4 and CBP is
ligand-independent and leads to enhanced HNF-4 transcriptional activity
for liver-specific apolipoprotein CIII gene expression (Dell and
Hadzopoulou-Cladaras, 1999). Recruitment of CBP by HNF-4 results in an
enhancement of the transcriptional activity of the latter (Yoshida et
al., 1997; Dell and Hadzopoulou-Cladaras, 1999). CBP does not activate
gene expression in the absence of HNF-4, and dominant negative forms of
HNF-4 prevent transcriptional activation by CBP, suggesting that the
mere recruitment of CBP by HNF-4 is not sufficient for enhancement of
gene expression (Dell and Hadzopoulou-Cladaras, 1999).
As expected, it could be demonstrated, that p300 acts as a HNF-4
coactivator in a manner similar to that of CBP and that p300 and SRC-1
together are able to enhance the transcriptional activity of HNF-4 more
than SRC-1 or p300 alone (Wang et al., 1998a,b).
The acidic AF-1 domain of the activator HNF-4 interacts specifically
with the coactivators CBP, PC4, and ADA2 (see also Fig. 6). It was
speculated that AF-1 could affect the preinitiation step through
interaction with CBP and/or the ADA2-GCN5 complex by increasing
acetylation of histones and rendering the chromatin more accessible to
the transcription machinery (Green et al., 1998). Furthermore, it was
hypothesized that AF-1 could act also at a postinitiation step,
promoting the opening of the DNA double helix through its interaction
with PC4 (Brandsen et al., 1997; Green et al., 1998). PC4 and ADA2 are
general coactivators that function cooperatively with TBP-associated
factors (TAFs) and mediate functional interactions between upstream
activators and the general transcriptional machinery (Ge and Roeder,
1994; Barlev et al., 1995). PC4 was shown to possess two ssDNA-binding
domains that might be implicated in the opening of the DNA double helix during gene transcription (Brandsen et al., 1997). It could be demonstrated that affinity-purified PC2, which lacks independent activity, acts in synergy with the upstream stimulatory activity (USA)-derived coactivator PC4 to mediate the effects of HNF-4 (Malik et
al., 2000). ADA2 was shown to display specific interactions with acidic
domains of activators such as the HNF-4 AF-1 domain and with the TBP
(Barlev et al., 1995).
Table 4 provides an overview of the HNF-4 coactivators and agonistic
ligands, while Fig. 6 provides a model of protein-protein and
protein-DNA interactions including the players HNF-1, HNF-4, and their
cofactors at the HNF-1 promoter.
F. The Hepatocyte Nuclear Factor Network and Tissue-Specific Gene Expression
The presence of HNF-4 protein has been correlated with the
expression of the liver phenotype in vitro: intertypic rat
hepatoma-human fibroblast hybrids that show extinction of
liver-specific gene expression are deficient for the expression of
HNF-4 and HNF-1, and reexpression of liver-specific genes in revertants
(or hybrid cell segregants) correlates with the reexpression of both
genes (Griffo et al., 1993). Because HNF-4 is an upstream regulator of
HNF-1 expression, it was proposed that the HNF-4 gene is the primary
target of the pleiotropic extinguisher (Griffo et al., 1993).
Dedifferentiated H5 variant cells of a rat hepatoma cell line that show
a pleiotropic loss of hepatic functions and fail to express both HNF-1
and HNF-4 (Descharette and Weiss, 1974; Faust et al., 1994) could be
directed toward redifferentiation by stable transfection of
epitope-tagged HNF-4 cDNA (Späth and Weiss, 1997). The forced
expression of only HNF-4 in these H5 variant cells lead to the
activation of a subset of liver-specific genes including
1-antitrypsin, -fibrinogen, and transthyretin, but not of the
endogenous HNF-4 gene. Treatment of the HNF-4tag-expressing cells with
dexamethasone induced expression of the transgene by 10-fold, resulting
in enhanced expression of target genes of both glucocorticoid hormones
and HNF-4 (Späth and Weiss, 1997). The set of activated hepatic
genes was extended by treatment of cells with the demethylating agent
5-azacytidine followed by selection in dexamethasone-containing
glucose-free medium. Some of the colonies that developed reexpressed
the entire set of hepatic functions tested (Späth and Weiss,
1997). In dedifferentiated rat hepatoma H5 cells, the effects of HNF-4
expression extend to the reestablishment of differentiated epithelial
cell morphology and simple epithelial polarity. The acquisition of
epithelial morphology occurs in two steps. First, expression of HNF-4
results in reexpression of cytokeratin proteins and partial
reestablishment of E-cadherin production. Only the transfectants are
competent to respond to the synthetic glucocorticoid dexamethasone,
which induces the second step of morphogenesis, including formation of
the junctional complex and expression of a polarized cell phenotype
(Späth and Weiss, 1998).
1. Hepatocyte Nuclear Factor-1 Regulates Hepatocyte Nuclear
Factor-4 Expression.
Liver-specific expression of the mouse
HNF-4 gene was studied by analyzing the promoter region for required
DNA elements. Experiments with reporter constructs in transient
transfection assays and in transgenic mice revealed distal enhancer
elements at kb 5.5 and 6.5 that were sufficient to drive
liver-specific expression of the mouse HNF-4 gene in animals (Zhong
et al., 1994). A HNF-1-binding site between bp 98 and 68 played an
important role in the hepatoma-specific promoter activity of HNF-4 in
transient transfection assays but was not sufficient to drive
liver-specific expression of a reporter gene in transgenic mice (Zhong
et al., 1994).
2. Hepatocyte Nuclear Factor-1 and Hepatocyte Nuclear Factor-4
Regulate Hepatocyte Nuclear Factor-1 Expression.
The HNF-1
gene contains a relatively short promoter segment located between
positions 82 and 40 to direct cell type-specific HNF-1
transcription. This region contains a single site for HNF-4 (Tian
and Schibler, 1991). Transfection experiments revealed that a short
region between 118 and 8 is crucial for cell type-specific expression of the HNF-1 gene in HepG2 cells. This region contains two positive cis-elements called site A, a HNF-4-binding
site, and site B, a HNF-1-binding site. Mutational analyses of these sites and cotransfection assays showed that HNF-4 and HNF-1 can transactivate the HNF-1 gene (Miura and Tanaka, 1993).
negatively regulates its own
expression in transient transfection experiments as well as the
expression of HNF-4-dependent genes (ApoCIII and Apo AI) that lack
HNF-1-binding sites in their promoter region. DNA binding and
cell-free transcription experiments failed to demonstrate any direct or
indirect interaction of HNF-1 with the regulatory regions of ApoCIII
or ApoAI. From these observations it was assumed that HNF-1 is able
to impede HNF-4 binding or activity. An indirect negative
autoregulatory mechanism for HNF-1 expression was described, which
in turn may affect HNF-4-dependent transcription of other liver-specific genes (Kritis et al., 1993). Later, it could be found
that this repression is exerted by a direct interaction of HNF-1
with AF-2, the main activation domain of HNF-4. The dual functions of
gene activation and repression suggest that HNF-1 is a global
regulator of the transcriptional network involved in the maintenance of
the hepatocyte-specific phenotype (Ktistaki and Talianidis, 1997a).
Figure 6 shows a model of the complex molecular interactions that are
involved in the regulation of the HNF-1 gene. Numerous coactivators
as well as the positive HNF-4 ligands appear to be necessary for
optimal HNF-1 expression. In this context it is interesting to note
that the HNF-4 coactivators p300/CBP as well as SRC-1 and SRC-3 bind to
the activation domain AF-2 of HNF-4. It may well be that HNF-1
competes with coactivator binding at the activation domain AF-2 of
HNF-4 and thus exerts its indirect negative autoregulation.
Additionally, it might be that this hypothetical competition is further
modulated by tissue-specific coactivator availability.
3. Hepatocyte Nuclear Factor-6, OC-2, Hepatocyte Nuclear
Factor-3, and CCAAT/Enhancer-Binding Proteins Regulate Hepatocyte
Nuclear Factor-3 Expression.
The liver-enriched
transcription factor HNF-6 recognizes the 138 to 126 region of the
HNF-3 promoter and is required for HNF-3 promoter activity
(Samadani and Costa, 1996). Similar to HNF-6, another member of the
onecut class of transcription factors called OC-2, with
tissue-restricted expression in liver and skin, stimulates
transcription of the HNF-3 gene in transient transfection experiments, suggesting that OC-2 participates in the network of
transcription factors required for liver differentiation and metabolism
(Jacquemin et al., 1999).
requires
134 bp of HNF-3 proximal sequences and binds four nuclear proteins, including two ubiquitous factors. One of these promoter sites
interacts with a cell-specific factor, LF-H3 , whose binding activity correlates with the HNF-3 tissue expression pattern. Furthermore, there is a binding site for the HNF-3 protein within its own promoter, suggesting that an autoactivation mechanism is
involved in the establishment of HNF-3 expression. It has been
proposed that both the LF-H3 and HNF-3 sites play an important role
in the cell type-specific expression of the HNF-3 transcription factor (Pani et al., 1992b).
Later studies demonstrated that members of the C/EBP and proline and
acidic amino acid-rich subfamilies of basic region leucine zipper
transcription factors bind to the LF-H3 site, and cotransfection of
HepG2 cells showed that these factors are able to activate a HNF-3
promoter reporter construct. The LF-H3 -C/EBP binding sequence also
confers HNF-3 promoter stimulation in response to interleukin (IL)-1
and IL-6. Upstream of this HNF-3 proximal promoter region, an
IFN-stimulated response element core sequence (231 to 210) was
found that mediates transcriptional induction by IFN- but not
IFN-. Gel mobility supershift assays demonstrated that an
IFN--induced protein-DNA complex is disrupted by an antibody specific for interferon-regulatory-factor-1/interferon-stimulated gene
factor-2. Surprisingly, the effect of the three cytokines (IL-1, IL-6,
and IFN-) in combination, as assayed by the same model, is not
synergistic. HNF-3 joins the C/EBP family on the list of
liver-enriched transcription factors, the expression of which is
modulated by cytokines (Samadani et al., 1995).
4. Hepatocyte Nuclear Factor-1 Regulates Hepatocyte Nuclear
Factor-3 in the Liver.
HNF-3 is an important regulator of
liver-specific genes, and the expression of this factor is reduced in
the liver injured by the toxin carbon tetrachloride [CCl(4)]
(Nakamura et al., 1999). HNF-3 is thought to be involved in
anterior-posterior regionalization of the primitive gut. In the
HNF-3 locus, 170 kb contain all elements important in the regulation
of HNF-3. A 3'-enhancer could be identified that contains a HNF-1
and --binding site that was shown to be crucial for enhancer
function in vitro (Hiemisch et al., 1997).
5. Competition and Cooperation ("Coopetition") between
Hepatocyte Nuclear Factor-3 and Hepatocyte Nuclear
Factor-3.
Studies using embryoid bodies in which one or both
HNF-3 or HNF-3 genes were inactivated showed that HNF-3 was
necessary for expression of HNF-3. HNF-3 positively regulated the
expression of HNF-4/HNF-1 and their downstream targets,
implicating a role in diabetes. In these studies HNF-3 acted as a
negative regulator of HNF-4/HNF-1, demonstrating that HNF-3
and HNF-3 have antagonistic transcriptional regulatory functions in
vivo. HNF-3 did not appear to act as a classic biochemical repressor
but, rather, exerted its negative effect by competing for HNF-3-binding
sites with the more efficient activator HNF-3. In addition, the
HNF-3/HNF-3 ratio was modulated by the presence of insulin,
providing evidence that the HNF network may have important roles in
mediating the action of insulin (Duncan et al., 1998).
G. Human Disease Due to Mutations in Hepatocyte Nuclear Factors
Haploinsufficiency of HNF-4 due to a nonsense mutation (Q268X)
in exon 7 of the HNF-4 gene leads to an autosomal-dominant, early-onset form of noninsulin-dependent diabetes mellitus
(maturity-onset diabetes of the young; gene named MODY1) in humans
associated with an abnormal pancreatic -cell function (Yamagata et
al., 1996; Lindner et al., 1997; Stoffel and Duncan, 1997). This
mutation deletes 187 C-terminal amino acids of the HNF-4 protein. It
has been shown that the mutant HNF-4 protein has lost its
transcriptional transactivation activity, and fails to dimerize and
bind DNA, implying that the MODY1 phenotype is due to a loss of
HNF-4 function (Stoffel and Duncan, 1997). Several genes encoding
components of the glucose-dependent insulin secretion pathway (glucose
transporter 2, aldolase B, glyceraldehyde-3-phosphate dehydrogenase,
and liver pyruvate kinase) as well as fatty acid-binding proteins and
cellular retinol-binding protein are dependent upon functional HNF-4
and are down-regulated in embryonic stem cells induced to differentiate into visceral endoderm and lacking proper HNF-4 function (Stoffel and Duncan, 1997). Interestingly, individuals of a family with MODY1
(Dresden-11) and an inherited nonsense mutation, R154X, in the HNF-4
gene showed no abnormalities in lipid metabolism or coagulation except
for a paradoxical 3.3-fold increase in serum lipoprotein(a) levels
(Lindner et al., 1997).
Hemophilia B Leyden is an X chromosome-linked bleeding disorder
characterized by very low plasma levels of blood coagulation factor IX
during childhood. After puberty, plasma factor IX levels gradually rise
to a maximum of 60% of normal, probably under the influence of
testosterone. Single point mutations in the factor IX promoter region
of hemophilia B Leyden patients have been reported at 20, 6, 5,
+8, and +13. In addition, one promoter mutation (G
C at 26) has
been detected that abolishes factor IX expression throughout life. The
severity of the hemophilia phenotype appears to be directly related to
the degree of disruption of HNF-4 binding to the factor IX promoter and
transactivation (Reijnen et al., 1994).
It could be demonstrated that HNF-6 is a major determinant of protein C
gene activity. Individuals affected by protein C deficiency are at risk
for venous thrombosis. One such affected individual was shown earlier
to carry a 14 T 
C mutation in the promoter region of the protein
C gene. It could be shown that the 14 T C mutation reduces HNF-6
binding to the protein C promoter. In transient transfection
experiments, HNF-6 transactivated the wild-type protein C promoter, and
introduction of the mutation abolished transactivation by HNF-6 (Spek
et al., 1998). This was the first report describing the putative
involvement of HNF-6 and of a HNF-6-binding site in human pathology.
H. Evidence from Knockout Experiments
Mice lacking HNF-1 fail to thrive and die around weaning after
a progressive wasting syndrome with a marked liver enlargement. The
transcription rate of genes like albumin and
1-antitrypsin is reduced, whereas the gene
coding for phenylalanine hydroxylase is totally silent, giving rise to
phenylketonuria. Mutant mice also suffer from severe Fanconi syndrome
caused by renal proximal tubular dysfunction. The resulting massive
urinary glucose loss leads to energy and water wasting. HNF1-deficient
mice may provide a model for human renal Fanconi syndrome (Pontoglio et
al., 1996). Mice deficient in HNF-1 develop Laron dwarfism and
noninsulin-dependent diabetes mellitus (Lee et al., 1998).
Targeted disruption of the HNF-4 gene, expressed in visceral
endoderm, leads to early embryonic death due to malfunction of the yolk
sac and impaired gastrulation in HNF-4 / mouse embryos (Chen et
al., 1994; Stoffel and Duncan, 1997; Duncan et al., 1998).
I. Lack of Confirmation for Existence of Hepatocyte Nuclear Factor-5
Site III of the liver-type promoter of
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase contains a TGTTTGC
sequence. This TGTTTGC sequence has been called the hepatocyte nuclear
factor-5 motif or the HNF-5-TGTTTGC sequence motif, which can be found in several liver-specific genes (Grange et al., 1991; Lemaigre et al.,
1993). It has been postulated that a protein named HNF-5 binds to this
sequence (Grange et al., 1991, Rigaud et al., 1991). Later, it could be
demonstrated that HNF-3 can bind to the putative HNF-5-TGTTTGC sequence
motif of the rat tyrosine aminotransferase (TAT) gene promoter, whereas
the HNF-5-TGTTTGC sequence motifs from other promoters or enhancers do
not bind HNF-3 (Lemaigre et al., 1993). The HNF-5-TGTTTGC sequence
motif of the albumin enhancer binds eH-TF (Zaret et al., 1990; Liu et
al., 1991), and the HNF-5-TGTTTGC sequence motif of the A domain of the
transferrin gene enhancer binds EBP45 and EBP40 (Petropoulos et al.,
1991). Although eH-TF, EBP45, and EBP40 produce footprints with typical hypersensitive sites, they differ from HNF-3, HNF-3, and HNF-3 (Liu et al., 1991; Petropoulos et al., 1991). Unfortunately the postulated transcription factor HNF-5 that binds to the HNF-5-TGTTTGC sequence motif (Grange et al., 1991) could not be defined in greater detail yet. It is also interesting, that over several years, no further
publications on HNF-5 can be found. This is probably due to the lack of
confirmation regarding the identity of this postulated transcription factor.
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V. Challenges for the Future |
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TopPreviousNextReferences
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The expression of liver-specific genes requires a timely and
coordinated expression of different transcription factors from distinct
chromosomes. As an example, the 1-antitrypsin
gene contains binding sites for HNF-1, HNF-3, HNF-4, and HNF-6, that
have been shown to interact with the liver-enriched transcription
factors HNF-1, HNF-3, HNF-41, HNF-42, HNF-6, and
HNF-6 (Sladek et al., 1990; De Simone and Cortese, 1991; Samadani
and Costa, 1996). Liver-enriched transcription factors that bind to the
regulatory sequences of the 1-antitrypsin gene
have been assigned to human chromosome 12 region q22-qter, chromosome
20q, and chromosome 15 region q21.1-21-2. Furthermore, the
transcription factors that bind to the
1-antitrypsin-regulatory sequence also
influence the transcriptional activity of each other. Thus, a
considerable challenge for further investigations on the regulation of
transcriptional networks will be the understanding of the molecular
basis of the orchestration of transcriptional events that are
interdependent and at the same time separated on different chromosomes.
It can be expected that the chromatin remodeling complexes, as well as biochemical modifications of chromatin, play pivotal roles in liver
development and liver-specific gene expression. In the future the exact
role of chromatin higher order structure and function in liver
development and liver function will need to be determined. Protein-protein interactions between transcription factors and cofactors as well as between components of multiprotein complexes and
transcription factors are coming more into focus and illustrate the
true complexity of gene transcription. In the posthuman genome era and
with the availability of the human DNA sequence, we find ourselves
confronted with a plethora of new challenges ahead that will provide
newfound knowledge on the origin of life and the molecular basis of
disease. There is optimism that new platform technologies in functional
genomics will unveil the secrets of gene regulation and phenotype expression.
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Acknowledgments |
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This work was supported in part by research grants from Novartis Pharma GmbH Germany, BU Transplantation and Immunology to H.S. and J.K. and the Lower Saxony and Ministry of Science and Culture to J.B.
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Footnotes |
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Address correspondence to: Dr. Jürgen Borlak, Center for Drug Research and Medical Biotechnology, Fraunhofer Institut für Toxikologie und Aerosolforschung, Nicolai Fuchs Str. 1 30625 Hannover, Germany. E-mail: borlak{at}ita.fhg.de
This article is dedicated to Jemima Ann Schrem.
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Abbreviations |
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bp, base pair; PEV, position-effect variegation; SWI/SNF, switch/sucrose nonfermenting; TBP, TATA-binding protein; CDK, cyclin-dependent kinase; GR, glucocorticoid receptor; HNF, hepatocyte nuclear factor; IFN, interferon; IL, interleukin; NuRD, nucleosome-remodeling histone; HDAC, histone deacetylase; HAT, histone acetyltransferase; RNA pol II, RNA polymerase II; TFII, transcription factor II; bZIP, basic region leucine zipper; BEF, bZIP-enhancing factor; C/EBP, CCAAT/enhancer-binding protein; DBP, D-binding protein; PPAR, peroxisome proliferator-activated receptor; SHP, short heterodimer partner; CBP, cAMP response element-binding protein; COUP-TF, chicken ovalbumin upstream promoter-transcription factor; GH, growth hormone; STAT5, signal transducer and activator of transcription 5; p/CAF, p300/CBP-associated factor; NCoA, nuclear receptor coactivator.
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