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.

Our laboratory has chosen the liver as a paradigm for studying cell-type-specific gene expression. By identifying exacting 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, Cell65, 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 functionally 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 of parallel amphipathic a-helices that interact via hydrophobic side chains of amino acids located at positions A and D (Fig. IB). While 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 of dimerization 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 of 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 of 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 of the dimerization helices. Indeed, our cross-linking experiments with purified recombinant proteins produced in E. coli suggest a three-fold higher dimerization dissociation constant for the DBP zipper as compared to the zipper of LAP, a C/EBP (Landschulz et al., 1989) related protein that also has affinity for the albumin D site (Descombes and Schi-bler, 1991). In keeping with this observation, replacement of 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 of 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 of DNA binding to ensure a rapid equilibrium between specific and non-specific occupancy.

The N-terminal moiety of 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 of DBP results in a strong attenuation of DBP-mediated transcription activation. This squelching is specific, since transcription activation through LAP is not impaired by an excess of N-terminal DBP sequences (E. F., unpublished results). Thus, the transcription activation domains of DBP and LAP appear to interact with different targets of the general transcription apparatus.

In liver, the majority of DBP is highly phosphorylated (V. O., F. Fleury and U. S., unpublished data). Déphosphorylation of DBP with acidic potato phosphatase does not appear to influence the equilibrium DNA-binding constant of DBP. We cannot exclude, however, that the dynamics of DNA binding (W>n and Á”off) are modulated by phosphorylation, as has recently been suggested for the serum response factor SRF (Marais et al., 1992). Alternatively, phosphate groups may be involved in regulating the transcription activation potential of 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 encompasses about 5.6 kb from cap-to polyadenylation sites (J. W. and E. F., unpublished data). The promoter is located within a CpG island and is devoid of a canonical TATA box. As has been observed for many promoters of this type, transcription initiation occurs at multiple start sites spread over about 50 nucleotides. The 5’ untranslated region of DBP mRNA is extraordinarily long (about 400 nucleotides) and contains three upstream AUGs. We remain uncertain whether these sequences are relevant for controlling translation initiation in the animal, but deletion of the sequences containing the three upstream AUGs increases the accumulation 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 accumulates in parenchymal hepatocytes according to a very robust circadian rhythm. The DBP level measured at the time of maximal accumulation (8 p.m. in Lewis rats) exceeds the one determined at the time of minimal accumulation (8 a.m.) by about three orders of magnitude. This rhythm is transcriptionally controlled, free-running and independent of food or water uptake (Wuarin and Schibler, 1990). While DBP mRNA cycles with the same phase and a similarly strong amplitude in a variety of non-hepatic tissues (lung, spleen, kidney), the DBP protein accumulates to much lower levels in these cell types as compared to hepatocytes. As DBP mRNA appears to be associated with polysomes 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 commences 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 (Mueller 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 of DBP transcription regulated? As circadian mRNA expression is similar in tissues that do or do not accumulate the protein, DBP itself is unlikely to control its own transcription by positive and/or negative feedback loops. More likely, DBP transcription is governed by hormones whose secretion is also rhythmic. Several lines of evidence are compatible 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 climaxed (between 2 p.m. to 4 p.m.). Injection of dexamethasone around noon results in a strong attenuation of DBP mRNA accumulation during the afternoon. Moreover, in homozygous fa/fa Zucker rats, which exhibit higher corticosterone levels than their wild-type counterparts (Guillaume-Gentil et al., 1990), the DBP cycle is phase-shifted towards earlier hours and is reduced in amplitude. 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 promoter contains a GRE, as judged from DNase 1 foot-printing studies with recombinant glucocorticoid receptor.

In an alternative attempt to localize civ-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 of 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 mentioned earlier, DBP has been recognized as a transcription factor which binds to an albumin promoter element in vitro. Indeed, in adult rats, albumin transcription appears to fluctuate with a phase compatible with that of DBP accumulation (Wuarin and Schibler, 1990; J. W., unpublished results). However, this circadian transcription can hardly be physiologically relevant, since due to their long half-lives, neither albumin mRNA nor its protein product accumulate in a cyclic fashion. In order for a gene product to oscillate during the day, it must be unstable. The impact of the half-life on the accumulation of diur-nally synthesized mRNAs is illustrated by the mathematical simulations shown in Figs 2 and 3. If one assumes that the transcription rates of DBP target genes parallel the circadian accumulation of DBP (shaded areas in Fig. 2) one can derive the accumulation profiles for the corresponding mRNAs of any given half-life (shown for T1/2 between 1 hour and 20 hours). Even if the transcriptional amplitude of a gene were infinite, the level of 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 HMG Co A reductase and cholesterol 7-a hydroxylase, which catalyze the rate-limiting steps in cholesterol synthesis and cholesterol conversion to bile acids, respectively. Enzyme activities as well as mRNAs from these genes accumulate according to a robust circadian rhythm with amplitudes of around 10-fold and a phase compatible with that of DBP accumulation (Clarke et al., 1984; Noshiro et al., 1990; D. L. and J. W., unpublished results). Furthermore, and in keeping with DBP accumulation, circadian expression of these enzymes is not detected until after weaning. Genomic clones encompassing the promoter regions of both of these genes have been obtained and are currently being examined for the presence of DBP-binding sites. Thus far, such elements have been discerned within the cholesterol 7-a hydroxylase promoter. Furthermore, in cotransfection experiments, a CAT reporter gene driven by this promoter is strongly activated by DBP. While none of the data thus far obtained prove that the circadian expression of cholesterol 7-a hydroxylase and HMG CoA reductase is governed by DBP, this transcription factor appears to be a likely player in the control of cholesterol homeostasis. In the model we are currently testing, DBP would simultaneously activate the diurnal production of bile acid and cholesterol. Such a coordination may be pivotal, since the massive cholesterol utilization during the activity period (evening and night hours for rats and mice) may call for a large, simultaneous increase in its synthesis.

DBP, a liver-enriched transcriptional activator of the bZip protein family, accumulates 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, corticosterone 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 time 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 pacemaker SCN see Kornhauser et al., 1992; Meijer and Rietveld, 1989). In the case of adrenal hormone secretion, the SCN is believed to set the pace of CRF secretion in the hypothalamus. This in turn may dictate the rhythmic release of ACTH from the anterior pituitary gland, eventually resulting in a cyclic secretion of corticosterone in the adrenal gland (Meijer and Rietveld, 1989).

Deciphering of the physiological purpose of DBP oscillation will require the unequivocal identification of downstream genes. At present we are testing the hypothesis that one of the DBP functions is related to cholesterol homeostasis, because the rate-limiting enzymes in cholesterol synthesis (HMG coA reductase) and utilization (cholesterol 7-a hydroxylase) both fluctuate with a circadian phase compatible with that of 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 become available. In the case of DBP, both gain-of-function (constitutive expression of DBP throughout the day) and loss-of-function (DBP gene knockout by homologous recombination) experiments should now be technically feasible.

We were surprised to observe that, while transcription of 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. coil-derived recombinant DBP with a 17-fold lower Kd 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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