DBP (albumin D-site-binding protein), HLF (hepatic leukemia factor), and TEF (thyrotroph embryonic factor) are the three members of the PAR bZip (proline and acidic amino acid-rich basic leucine zipper) transcription factor family. All three of these transcriptional regulatory proteins accumulate with robust circadian rhythms in tissues with high amplitudes of clock gene expression, such as the suprachiasmatic nucleus (SCN) and the liver. However, they are expressed at nearly invariable levels in most brain regions, in which clock gene expression only cycles with low amplitude. Here we show that mice deficient for all three PAR bZip proteins are highly susceptible to generalized spontaneous and audiogenic epilepsies that frequently are lethal. Transcriptome profiling revealed pyridoxal kinase (Pdxk) as a target gene of PAR bZip proteins in both liver and brain. Pyridoxal kinase converts vitamin B6 derivatives into pyridoxal phosphate (PLP), the coenzyme of many enzymes involved in amino acid and neurotransmitter metabolism. PAR bZip-deficient mice show decreased brain levels of PLP, serotonin, and dopamine, and such changes have previously been reported to cause epilepsies in other systems. Hence, the expression of some clock-controlled genes, such as Pdxk, may have to remain within narrow limits in the brain. This could explain why the circadian oscillator has evolved to generate only low-amplitude cycles in most brain regions.[Keywords: PAR bZip proteins; circadian transcription factors; epilepsy; pyridoxal kinase] Supplemental material is available at http://www.genesdev.org.

The two highly related PAR basic region leucine zipper proteins TEF and DBP accumulate according to a robust circadian rhythm in liver and kidney. In liver nuclei, the amplitude of daily oscillation has been estimated to be 50‐fold and 160‐fold for TEF and DBP, respectively. While DBP mRNA expression is the principal determinant of circadian DBP accumulation, the amplitude of TEF mRNA cycling is insufficient to explain circadian TEF fluctuation. Conceivably, daily variations in TEF degradation or nuclear translocation efficiency may explain the discrepancy between mRNA and protein accumulation. In vitro, TEF and DBP bind the same DNA sequences. Yet, in co‐transfection experiments, these two proteins exhibit different activation potentials for two reporter genes examined. While TEF stimulates transcription from the albumin promoter more potently than DBP, only DBP is capable of activating transcription efficiently from the cholesterol 7 alpha hydroxylase (C7alphaH) promoter. However, a TEF‐DBP fusion protein, carrying N‐terminal TEF sequences and the DNA binding/dimerization domain of DBP, enhances expression of the C7alphaH‐CAT reporter gene as strongly as wild‐type DBP. Our results suggest that the promoter environment, rather than the affinity with which PAR proteins recognize their cognate DNA sequences in vitro, determines the promoter preferences of TEF and DBP.

SummaryDBP, 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 maxi mal 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, fast ing of rats for several days influences neither the ampli tude 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 circa dian DBP expression is governed by hormones whose peripheral concentrations also oscillate during the day. Several lines of evidence suggest a pivotal role of glu cocorticoid hormones in establishing the DBP cycle.Two genes whose mRNAs and protein products accu mulate 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 cho lesterol 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 cho lesterol homeostasis.

Background-Long-term clinical studies are essential for monitoring the effectiveness and safety of a drug. Information provided by long-term clinical studies complements the results of short-term, randomized, controlled trials, which often form the basis of regulatory approval for a new drug application. However, with increasing study duration, the use of placebo becomes less ethical, forcing an open-label study design where a reference estimate for comparison, a placebo-treated cohort, is no longer available. Moreover, as the duration of a study increases and the number of patients continuing in the study declines, missing data become more of a problem: they may bias the results. Therefore, standard analytical strategies used in short-term randomized, controlled trials (intent-totreat, per-protocol) may not always be appropriate for data generated in long-term studies.

Transcription initiation of protein-encoding genes involves the assembly of RNA polymerase II and a number of general transcription factors at the promoter. A mammalian RNA polymerase II complex containing all of the components required for promoter-specific transcription initiation can be isolated by immunopurification with a monoclonal antibody directed against the cyclin-dependent kinase CDK7, a subunit of the general transcription factor TFIIH. In vitro transcription by this immunopurified RNA polymerase II complex is effectively stimulated by thyroid embryonic factor (TEF), a basic leucine zipper transcription factor. Thus, the RNA polymerase II complex must also contain components required for activated transcription that interact with the transactivation domain of TEF. This conjecture was verified by affinity selection experiments in which the TEF transcription activation domain was used as a bait. Indeed, an RNA polymerase II complex containing all of the accessory proteins required for transcription initiation can be enriched by its affinity to recombinant proteins containing the TEF transactivation domain. These results are compatible with a mechanism by which TEF can recruit an RNA polymerase II holoenzyme to the promoter in a single step.In prokaryotes and eukaryotes, transcription initiation can be divided into three basic steps: assembly of a closed initiation complex at the promoter, isomerization of the closed complex to the open complex, and promoter clearance (4,11,19,20,43). In principle, transcriptional regulators can affect any of these steps. For example, the Escherichia coli protein CAP (catabolite activator protein) has been shown to facilitate the binding of RNA polymerase to the promoter, isomerization, and promoter escape (4,10,30,41,43). In eukaryotes, the transactivation domain of the herpes simplex virus protein VP16 has been shown to stimulate transcription initiation, perhaps by interacting with TFIIB (18, 37), TFIIH (60), and TFIID (29). Therefore, VP16 may have a role in promoter assembly. Yankulov et al. (66) have demonstrated that the VP16 transactivation domain may also stimulate elongation, possibly by increasing the processivity of RNA polymerase II. Other activators, like the human immunodeficiency virus TAT protein, may affect still other steps (26).Careful order-of-addition experiments with purified components of the general transcription machinery have suggested a stepwise assembly of initiation complexes in vitro. According to this model, the TATA box (or another core promoter element) is first recognized by TBP, the TATA box-binding subunit of the TFIID complex. TFIIA and TFIIB then join promoter-bound TFIID. The resulting TFIID-TFIIA-TFIIB (DAB)-promoter complex subsequently recruits RNA polymerase II and TFIIF. Finally, TFIIE and TFIIH enter the initiation complex, and isomerization can occur (2,3,8,44,45,51,53). A somewhat different view of initiation complex assembly has emerged with the discovery of a large multisubunit RNA polymerase II complex in yeast cells; ...