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Journal of Cell Science, Supplem ent 16, 123-127 (1992)Printed
in G reat Britain © The Company o f Biologists Limited 1992
123
The role of the transcriptional activator protein DBP in
circadian liver gene
expression
JÉRÔME WUARIN, EILEEN FALVEY, DAN LAVERY, DALE TALBOT, ED
SCHMIDT, VINCENT OSSIPOW,
PHILIPPE FONJALLAZ and UELI SCHIBLER*
Department o f M olecular Biology, Sciences II, 30 Quai Ernest
Ansermet, CH-1211 Geneva-4, Switzerland
^Corresponding author
Summary
DBP, a liver-enriched transcriptional activator protein of the
leucine zipper protein family, accumulates according to a very
strong circadian rhythm (amplitude approx. 1000-fold). In rat
parenchymal hepatocytes, the protein is barely detectable during
the morning hours. At about 2 p.m., DBP levels begin to rise, reach
maximal levels at 8 p.m. and decline sharply during the night. This
rhythm is free-running: it persists with regard to both its
amplitude and phase in the absence of external time cues, such as
daily dark/light switches. Also, fasting of rats for several days
influences neither the amplitude nor the phase of circadian DBP
expression. Since the levels of DBP mRNA and nascent transcripts
also oscillate with a strong amplitude, circadian DBP expression is
transcriptionally controlled. While DBP mRNA fluctuates with a
similar phase and amplitude in most tissues examined, DBP protein
accumulates to high concentrations only in liver nuclei. Hence, at
least in
nonhepatic tissues, cyclic DBP transcription is unlikely to be
controlled by a positive and/or negative feedback mechanism
involving DBP itself. More likely, the circadian DBP expression is
governed by hormones whose peripheral concentrations also oscillate
during the day. Several lines of evidence suggest a pivotal role of
glucocorticoid hormones in establishing the DBP cycle.
Two genes whose mRNAs and protein products accumulate according
to a strong circadian rhythm with a phase compatible with
regulation by DBP encode enzymes with key functions in cholesterol
metabolism: HMG-coA reductase is the rate-limiting enzyme in
cholesterol synthesis; cholesterol 7-a hydroxylase performs the
rate-limiting step in the conversion of cholesterol to bile acid.
DBP may thus be involved in regulating cholesterol homeostasis.
Key words: liver transcription factor, DBP, circadian
rhythms.
Introduction
Our laboratory has chosen the liver as a paradigm for studying
cell-type-specific gene expression. By identifying cis- acting
albumin promoter elements and the transcription factors interacting
with them, we hoped to gain access to key regulators of hepatic
gene expression. A detailed functional dissection of the albumin
promoter revealed six cognate sites (A, B, C, D, E and F) for
sequence-specific DNA- binding proteins (Lichtsteiner et al.,
1987). Two of these, sites B and D, proved to be particularly
important for efficient cell-type-specific transcription (Maire et
al., 1989; Lichtsteiner and Schibler, 1989). The proteins binding
to these elements have been identified, purified and their cDNA and
genes isolated by molecular cloning techniques (for review, see
Crabtree et al., 1992). In this paper we concentrate on our results
with a protein, DBP, which binds to the albumin D promoter element
(Mueller et al., 1990). Studies on DBP expression have led to the
surprising discovery that in adult rats, DBP accumulates according
to a
very strong circadian rhythm (Wuarin and Schibler, 1990). In the
next paragraphs we will focus on the regulation of circadian DBP
expression and speculate on its purpose.
DBP, a liver-enriched transcriptional activator protein
DBP cDNAs have been obtained by screening a lambda gt 11 liver
cDNA expression library with a radiolabeled DNA probe encompassing
the albumin promoter element D. Sequencing of close to full-length
cDNAs revealed an open reading frame corresponding to 325 amino
acids (Mueller et al., 1990; see corrected sequence, Cell 65, 1991,
page 915). DBP dimerizes via a carboxy-terminal leucine zipper and
binds DNA via an adjacent basic region (Vinson et al.,1989). The
three-dimensional structure of at least one leucine zipper, that of
the yeast transcription factor GCN4, has been resolved by X-ray
crystallography (O’Shea et al., 1991). Moreover, the amino acid
residues located at func
-
124 ./ . Wuarin and others
Fig. 1. Schematic representation (see O ’Shea et al., 1992) of
dimerized coiled coils (leucine zippers) within a DBP homodimer.
The N- to C-polarity of the dimerization helices is from left to
right. The right hand panel shows a vertical projection of the
coiled coils. The D and A heptad positions
containing the hydrophobic amino acids are shown in black and
shaded, respectively. Hypothetical salt bridges between glutamic
acids (E) and arginines (R) are indicated by arrows (four on each
side of the paired amphipathic helices).
tionally strategic positions of leucine zippers have been
determined by elegant genetic (Hu et al., 1990) and biochemical
work (eg. Landschulz et al., 1989, and references therein; O ’Shea
et al., 1992). Leucine zipper regions form coiled-coil structures,
consisting o f parallel am phipathic a - helices that interact via
hydrophobic side chains o f amino acids located at positions A and
D (Fig. IB). W hile no particular D position must be occupied by a
leucine, a total of at least two leucines appear to be required
within this heptad repeat (Hu et al., 1990). However, not all
leucine zippers can form stable interactions with each other. To a
large extent, the specificity o f dim erization is dictated by salt
bonds between amino acids of opposite charges located in positions
G and E (see O ’Shea et al., 1992 and references therein). The
leucine zipper o f DBP (Fig. 1A) is somewhat unusual in two
respects. Firstly, it contains only two leucines, the absolute
minimum tolerated for dimerization (see above). Secondly, the G and
E positions consist o f four charged amino acids which,
theoretically, could establish as many as eight salt bridges.
However, the intrahelical repulsion created by the runs of three
consecutive arginines and glutamic acids in the E and G heptad
repeat positions, respectively, may also result in a
destabilization o f the dimerization helices. Indeed, our
cross-linking experiments with purified recom binant proteins
produced in E. coli suggest a three-fold higher dimerization
dissociation constant for the DBP zipper as com pared to the zipper
o f LAP, a C/EBP (Landschulz et al., 1989) related protein that
also has affinity for the albumin D site (Descom bes and Schi-
bler, 1991). In keeping with this observation, replacement o f the
DBP zipper with the LAP zipper increases the affinity of the DBP
chimeric protein to its DNA cognate site by about three-fold.
Perhaps the dimerization properties of DBP have to be evaluated in
the light o f its role in diurnal transcription activation (see
below). The circadian accumulation (see below) and action of this
protein may call for a high off-rate o f DNA binding to ensure a
rapid equilibrium between specific and non-specific occupancy.
The N-terminal moiety o f DBP contains the transcription
activation domain(s). In cotransfection experiments with wild-type
DBP and DBP target genes, over-expression of the N-terminal two
thirds o f DBP results in a strong attenuation o f DBP-m ediated
transcription activation. This squelching is specific, since
transcription activation through LAP is not impaired by an excess o
f N-terminal DBP sequences (E. F., unpublished results). Thus, the
transcription activation domains of DBP and LAP appear to inter
act with different targets o f the general transcription
apparatus.
In liver, the majority of DBP is highly phosphorylated (V. O.,
F. Fleury and U. S., unpublished data). Dephosphorylation o f DBP
with acidic potato phosphatase does not appear to influence the
equilibrium DNA-binding constant o f DBP. W e cannot exclude,
however, that the dynamics o f DNA binding (Kon and A"0ff) are m
odulated by phosphorylation, as has recently been suggested for the
serum response factor SRF (M arais et al., 1992). Alternatively,
phosphate groups may be involved in regulating the transcription
activation potential o f DBP.
The DBP gene
The rat gene specifying DBP has recently been isolated and
characterized in our laboratory. It contains four exons and three
introns and encom passes about 5.6 kb from c a p - to
polyadenylation sites (J. W. and E. F., unpublished data). The prom
oter is located within a CpG island and is devoid o f a canonical
TATA box. As has been observed for many prom oters o f this type,
transcription initiation occurs at multiple start sites spread over
about 50 nucleotides. The 5 ' untranslated region of DBP m RNA is
extraordinarily long (about 400 nucleotides) and contains three
upstream AUGs. W e remain uncertain whether these sequences are
relevant for controlling translation initiation in the animal, but
deletion o f the sequences containing the three upstream AUGs
increases the accum ulation of DBP both in vivo (transient
transfection) and in vitro (reticulocyte lysate) (S. Rufino and U.
S., unpublished data).
Circadian and developmental DBP expression
DBP accum ulates in parenchym al hepatocytes according to a very
robust circadian rhythm. The DBP level measured at the time of
maximal accum ulation (8 p.m. in Lewis rats) exceeds the one
determined at the time of minimal accumulation (8 a.m.) by about
three orders o f magnitude. This rhythm is transcriptionally
controlled, free-running and independent o f food or water uptake
(W uarin and Schibler,1990). W hile DBP mRNA cycles with the same
phase and a similarly strong am plitude in a variety o f
non-hepatic tissues (lung, spleen, kidney), the DBP protein accum
ulates to much lower levels in these cell types as com pared to
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Circadian liver gene expression 125
hepatocytes. As DBP mRNA appears to be associated with polysom
es in all tissues (E. S., unpublished results), it is conceivable
that protein stability rather than translation efficiency accounts
for the differential DBP accumulation in various cell types.
In rodents, circadian rhythmicity com mences shortly after
weaning (for reviews on circadian rhythms, see Edmunds, 1988). As a
consequence, DBP oscillation is only observed in rats older than
three weeks (M ueller et al., 1990; Yano et al., 1992). In the
liver of younger animals (birth to three weeks), DBP accumulates to
very low levels and does not cycle during the day (D. L. and E. S.,
unpublished data).
How is the oscillation o f DBP transcription regulated? As
circadian mRNA expression is sim ilar in tissues that do or do not
accum ulate the protein, DBP itself is unlikely to control its own
transcription by positive and/or negative feedback loops. M ore
likely, DBP transcription is governed by hormones whose secretion
is also rhythmic. Several lines o f evidence are com patible with
glucocorticoid hormones participating in the “downphase” of
circadian DBP expression. In rats, corticosterone shows highest
peripheral levels at the dark/light switch (6 p.m. in our animal
facility; for circadian glucocorticoid secretion, see Meier, 1975
and references therein), several hours after DBP transcription
rates have clim axed (between 2 p.m. to 4 p.m.). Injection of
dexamethasone around noon results in a strong attenuation of DBP
mRNA accum ulation during the afternoon. M oreover, in homozygous
fa/fa Zucker rats, which exhibit higher corticosterone levels than
their w ild-type counterparts (Guillaume-Gentil et al., 1990), the
DBP cycle is phase-shifted towards earlier hours and is reduced in
am plitude. For the two following reasons, we consider it likely
that DBP transcription is directly repressed by glucocorticoid
hormones. (1) In hepatoma cells, dexamethasone addition leads to a
rapid extinction of DBP mRNA accumulation (E. F., unpublished
data). (2) The DBP prom oter contains a GRE, as judged from DNase 1
footprinting studies with recom binant glucocorticoid receptor.
In an alternative attem pt to localize cis-acting DBP promoter
elements, DNase 1 hypersensitive sites in rat liver nuclei were
mapped (D. T., unpublished data). Such sites have been observed
exclusively within the 1 kb 5 ' flanking region of the DBP gene.
Some of these appear to be more efficiently cleaved in nuclei
isolated during the afternoon, when the DBP gene is most active,
than in nuclei isolated in the morning, when the DBP gene is not or
very weakly transcribed. The importance o f these elements for
circadian DBP expression is currently being tested in transgenic
mice.
Purpose of circadian DBP expression
In order to decipher the physiological significance of circadian
DBP expression, one needs to identify bona fide DBP target genes.
As m entioned earlier, DBP has been recognized as a transcription
factor w hich binds to an albumin prom oter element in vitro.
Indeed, in adult rats, albumin transcription appears to fluctuate
with a phase com patible with that o f DBP accum ulation (W uarin
and Schibler, 1990; J. W., unpublished results). However, this
circadian tran-
Fig. 2. Circadian accumulation of mRNAs from putative DBP target
genes as a function of their half-lives. As a first approximation,
it is assumed that the transcription rates of DBP target genes
(shaded areas) follow the accumulation profile of DBP. As
demonstrated in Figs 2 and 4, the precise circadian transcription
patterns o f DBP target genes depends on the affinity o f their DBP
cognate sites and on the contribution of other, perhaps
constitutively expressed transcription factors participating in the
regulation of these genes. Accumulation profiles are shown for mRNA
half-lives of one to twenty hours.
scription can hardly be physiologically relevant, since due to
their long half-lives, neither albumin m RNA nor its protein
product accum ulate in a cyclic fashion. In order for a gene
product to oscillate during the day, it must be unstable. The
impact o f the half-life on the accum ulation of diurnally
synthesized mRNAs is illustrated by the m athem atical simulations
shown in Figs 2 and 3. If one assumes that the transcription rates
o f DBP target genes parallel the circadian accum ulation of DBP
(shaded areas in Fig. 2) one can derive the accum ulation profiles
for the corresponding mRNAs of any given half-life (shown for T 1/2
between 1 hour and 20 hours). Even if the transcriptional am
plitude of a gene were infinite, the level o f mRNAs with
half-lives of more than ten hours would fluctuate by two-fold or
less (see Fig. 3, solid circles). However, the transcriptional
fluctuation of most DBP target genes may only be five- to tenfold,
since constitutively expressed activators in addition to DBP may be
involved in controlling their expression. For mRNAs of such genes
to cycle, their half-lives would have to be very short (see Fig. 3,
open circles). Clearly then, DBP oscillation can be physiologically
relevant only for those target genes whose mRNA and protein
products are unstable.
Two particularly attractive candidates for DBP target genes are
those encoding the liver-enriched enzymes HM G
-
126 J. Wuarin and others
a (m a x ) /a (m in )
10
O S(max)/S(min)= 5
• S(max)/S(min)= infinite
'O - .O -o.>-0 - 0-0
5 10 15
T1/2 (h ou rs )
2 0
Fig. 3. Amplitudes of mRNA accumulation as a function of
halflives and amplitude of synthesis. Solid circles: Amplitudes of
the accumulation (a) of mRNAs whose amplitude of synthesis (S) is
infinite, such as shown in Fig. 2. Open circles: amplitudes of the
accumulation (a) of mRNA whose amplitude of synthesis (S) is 5fold.
The amplitudes (maximal/minimal accumulation) were determined
graphically from accumulation profiles such as shown in Fig. 2.
Co A reductase and cholesterol 7 -a hydroxylase, which catalyze
the rate-lim iting steps in cholesterol synthesis and cholesterol
conversion to bile acids, respectively. Enzym e activities as well
as mRNAs from these genes accum ulate according to a robust
circadian rhythm with am plitudes of around 10-fold and a phase com
patible with that o f DBP accum ulation (Clarke et a l , 1984;
Noshiro et al., 1990; D. L. and J. W., unpublished results).
Furtherm ore, and in keeping with DBP accum ulation, circadian
expression of these enzymes is not detected until after weaning.
Genomic clones encom passing the prom oter regions o f both o f
these genes have been obtained and are currently being examined for
the presence of DBP-binding sites. Thus far, such elem ents have
been discerned within the cholesterol 7 -a hydroxylase promoter.
Furthermore, in cotransfection experiments, a CAT reporter gene
driven by this prom oter is strongly activated by DBP. W hile none
o f the data thus far obtained prove that the circadian expression
of cholesterol 7 -a hydroxylase and HM G CoA reductase is governed
by DBP, this transcription factor appears to be a likely player in
the control o f cholesterol homeostasis. In the model we are
currently testing, DBP would sim ultaneously activate the diurnal
production of bile acid and cholesterol. Such a coordination may be
pivotal, since the m assive cholesterol utilization during the
activity period (evening and night hours for rats and mice) may
call for a large, sim ultaneous increase in its synthesis.
Conclusions and perspectives
DBP, a liver-enriched transcriptional activator of the bZip
protein family, accum ulates according to an extraordinarily strong
circadian rhythm. Preliminary results suggest that circadian DBP
expression may be controlled, at least in part, by the cyclic
secretion of glucocorticoid hormones. Like most (if not all)
circadian activities in mammals, cor
ticosterone synthesis and secretion is governed by the
suprachiasmatic nucleus (SCN), a small brain structure located
above the optical chiasma. Circadian outputs are entrained in the
SCN by external tim e cues, such as light/dark phases. Once
entrained, such outputs are free- running and persist for extended
time periods, until the clock is reset by new time cues (for
references on the circadian pacem aker SCN see Kornhauser et al.,
1992; M eijer and Rietveld, 1989). In the case o f adrenal hormone
secretion, the SCN is believed to set the pace o f CRF secretion in
the hypothalam us. This in turn may dictate the rhythmic release o
f ACTH from the anterior pituitary gland, eventually resulting in a
cyclic secretion o f corticosterone in the adrenal gland (M eijer
and Rietveld, 1989).
Deciphering o f the physiological purpose o f DBP oscillation
will require the unequivocal identification of downstream genes. A
t present we are testing the hypothesis that one of the D BP
functions is related to cholesterol hom eostasis, because the
rate-limiting enzymes in cholesterol synthesis (HM G coA reductase)
and utilization (cholesterol 7 -a hydroxylase) both fluctuate with
a circadian phase com patible with that o f DBP accumulation.
Clearly, the possible role of DBP in cholesterol metabolism will
remain hypothetical until scrutinized by a thorough genetic
analysis. The tools required for this endeavor have recently becom
e available. In the case of DBP, both gain-of-func- tion
(constitutive expression of DBP throughout the day) and
loss-of-function (DBP gene knockout by homologous recombination)
experiments should now be technically feasible.
W e were surprised to observe that, while transcription of
Fig. 4. Circadian transcription of two hypothetical DBP target
genes containing DBP cognate sites with different affinities. The
circadian DBP accumulation profile is schematically shown as a
solid line. Target gene A is assumed to contain a 10-fold stronger
DBP cognate site (equilibrium dissociation constant A'd=X) than
gene B (equilibrium dissociation constant ATd=10X). As a
consequence, half-saturation of the DBP site in gene B would
require 10-fold higher DBP concentrations than that of its
counterpart in gene A. Therefore, maximal transcriptional activity
of gene A would be expected to persist for a longer circadian
period than that of gene B. The periods of maximal transcription
for genes A and B are depicted as black bars. The genes encoding
cytochrome P450 CYP2C6 and cholesterol 7 -a hydroxylase constitute
putative candidates for the gene classes A and B, respectively.
-
Circadian liver gene expression 127
certain genes, such as the one encoding serum albumin, cycles
during the day, the accumulation of their products remains
relatively constant. Apparently, diurnally expressed transcription
factors like DBP may be used for genes whose products oscillate,
such as HMG coA reductase and cholesterol 7 -a hydroxylase, and
genes whose products accumulate constitutively, depending on their
stability.
The relative affinity of the DBP sites for their cognate factor
may be another important parameter in determining the temporal
pattern of putative DBP target gene expression. As illustrated by
the cartoon shown in Fig. 4, DBP target genes with low affinity
sites may be maximally expressed during a short circadian time
window, since the critical DBP concentrations required for
efficient occupancy may only be reached during a few hours during
the day. In contrast, genes with high affinity sites are expected
to be expressed during much longer time periods, as even low DBP
levels would suffice to fill the respective recognition sequences.
For example, the cytochrome P450 gene CYP2C6 contains a DBP site
within its promoter that binds E. coli-derived recombinant DBP with
a 17-fold lower Kc\ than the albumin D promoter element (Yano et
al., 1992). Run-on transcription experiments with nuclei isolated
at four hour intervals around the clock yielded more or less
constant transcription rates for the CYP2C6 gene (D. L.,
unpublished data). While further experiments are required to
identify this gene as a bona fide DBP target gene, these
considerations exemplify the versatile potentials of oscillating
transcription factors: circadian DBP accumulation may result in
strong, weak or no oscillation of mRNAs and proteins issued from
downstream genes, depending on the relative affinities of their
DBP-binding sites and the stabilities of their products.
We would like to thank Prof. G. Wanner, Department of Applied
Mathematics, University of Geneva, for preparing the computer
program used in Fig. 2. This research was supported by the Swiss
National Science Foundation and the State of Geneva.
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