E4BP4: a vertebrate protein related to Vrille

A novel member of the bZIP family of DNA-binding proteins has been described and designated E4BP4. It displays an unusual DNA-binding specificity that overlaps that of the activating transcription factor family of factors (ATF/CREB). When expressed in a transient transfection assay with a suitable reporter plasmid, E4BP4 strongly represses transcription in a DNA-binding-site-dependent manner. Examination of a series of deletion mutants reveals that sequences responsible for the repressing potential of E4BP4 lie within the carboxyl-terminal region of the protein. No similarity has been found between this region and the repressing domains of other known eukaryotic transcriptional repressors (Cowell, 1992).

The bZIP factor E4BP4 overlaps in DNA binding site specificity with the transcriptional activator CREB and members of the ATF family of transcription factors, but is an active transcriptional repressor. The repressing activity of E4BP4 maps to a small 'domain' of 65 amino acids that retains its ability to repress transcription when transferred to the heterologous DNA binding domain of the yeast transcriptional activator GAL4. This segment of the E4BP4 polypeptide contains a high proportion of charged amino acids and does not resemble the repression domains that have been characterized so far from other active transcriptional repressors such as the Drosophila Kruppel, Engrailed and Even-skipped proteins. A mutation that changes the charge configuration of this repression module results in a complete loss of repressor activity. The E4BP4-GAL4 fusion protein is able to repress the residual transcription from minimal promoters containing the adenovirus E4 or E1b TATA box. This is consistent with a mechanism of action whereby E4BP4 interacts with some component of the general transcription machinery to cause repression of basal and activated transcription. Although a number of nuclear proteins are able to interact with the E4BP4 repression domain in vitro, these proteins do not appear to include the general transcription factors TFIIB or TBP (Cowell, 1994).

A repression domain from the active transcriptional repressor E4BP4 maps to a 65 amino acid segment near the C-terminus of the polypeptide. The E4BP4 repression domain interacts specifically with the TBP binding repressor protein Dr1. Mutants that affect the ability of E4BP4 to bring about transcriptional repression are also deficient in their binding of Dr1. The results are discussed in the light of evidence for squelching of a 'global' repressor by a DNA binding defective E4BP4 mutant (Cowell, 1996).

The transcription factor E4BP4 has been isolated by lambda gt11 expression cloning using a probe containing the CRE/ATF-like sequence located between -2764 bp and -2753 bp in the upstream regulatory region for the human IL-1 beta gene. DNaseI protection, gel mobility shift analysis, and cotransfection studies were performed to investigate the binding and functional properties of E4BP4 using IL-1 beta promoter sequences. By DNaseI footprinting, a protection pattern was generated over the CRE/ATF-like site and the flanking sequences by bacterially produced E4BP4. Competition experiment by gel shift assay indicates that E4BP4 binds specifically to a CRE/ATF-like site, not an NF kappa B-like site. In cotransfection studies, E4BP4 represses promoter activity and this repression is mediated through the CRE/ATF-like site. Mutational analysis of E4BP4 suggests that the DNA binding as well as repression activities require the leucine heptad repeat domain. Analysis of E4BP4 produced in Escherichia coli and Sf9 cells infected with recombinant baculovirus indicates that baculovirus produced protein shows enhanced binding to the CRE/ATF-like site compared to the E. coli-produced protein. Analysis of posttranslational modifications indicates that E4BP4 produced in Sf9 cells is phosphorylated and this phosphorylation is important for the DNA binding activity of E4BP4 (Chen, 1995).

The E2A-HLF (hepatic leukemia factor) oncoprotein, generated in pro-B lymphocytes by fusion of the trans-activation domain of E2A to the basic region/leucine zipper (bZIP) domain of HLF, functions as an anti-apoptotic transcription factor in leukemic cell transformation. When introduced into interleukin 3 (IL-3)-dependent mouse pro-B lymphocytes, E2A-HLF prevents apoptosis induced by growth factor deprivation, suggesting that IL-3 mediates cell survival through activation of a transcription factor whose activity can be constitutively replaced by the chimeric oncoprotein. Four bZIP transcription factors have been considered as candidates for this putative IL-3-regulated factor, each of which binds avidly to the DNA consensus sequence recognized by E2A-HLF and is related to the Caenorhabditis elegans CES-2 (cell death specification protein) neuron-specific mediator of cell death. The expression and binding activity of the Nfil3 protein (also called E4bp4), but not of Hlf, Dbp, or Tef, was found to be regulated by IL-3 in mouse pro-B cell lines (Baf-3 and FL5.12). Northern blot analysis shows that Nfil3/E4bp4 is regulated as a 'delayed-early' IL-3-responsive gene, requiring de novo protein synthesis. In the absence of IL-3, enforced expression of the human NFIL3/E4BP4 cDNA promotes the survival but not the growth of IL-3-dependent pro-B cells. These results implicate NFIL3/E4BP4 (nuclear factor regulated by IL-3/adenovirus E4 promoter binding protein) in a distinct growth factor-regulated signaling pathway that is responsible for the survival of early B-cell progenitors, and whose alteration by E2A-HLF leads to childhood B lineage leukemia (Ikushima, 1997).

Hematopoietic cells require cytokine-initiated signals for survival as well as proliferation. The pathways that transduce these signals, ensuring timely regulation of cell fate genes, remain largely undefined. The NFIL3 (E4BP4) transcription factor, Bcl-xL, and constitutively active mutants of components in Ras signal transduction pathways have been identified as key regulation proteins affecting murine interleukin-3 (IL-3)-dependent cell survival. Expression of NFIL3 is regulated by oncogenic Ras mutants through both the Raf-mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. NFIL3 inhibits apoptosis without affecting Bcl-xL expression. By contrast, Bcl-xL levels are regulated through the membrane proximal portion in the cytoplasmic domain of the receptor (betac chain), that is shared by IL-3 and granulocyte-macrophage colony-stimulating factor. Activation of either pathway alone is insufficient to ensure cell survival, indicating that multiple independent signal transduction pathways mediate the survival of developing B-lymphoid cells (Kuribara, 1999).

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. Chicken pineal genes were sought that are induced by light in a time-of-day-dependent manner. Isolated was the chick homolog of bZIP transcription factor E4bp4 (cE4bp4), which shows high similarity to vrille, one of the Drosophila clock genes. cE4bp4 is expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase is nearly opposite that of the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays show that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5'-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies show that the prolongation of the light period to early subjective night maintains the high level expression of the pineal cE4bp4, and presumably as a consequence delays the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 are followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene (Doi, 2001).

PAR domain family: Cloning and cyclic expression

The treatment of cultured rat-1 fibroblasts or H35 hepatoma cells with high concentrations of serum induces the circadian expression of various genes whose transcription also oscillates in living animals. Oscillating genes include rper1 and rper2 (rat homologs of the Drosophila clock gene period), and the genes encoding the transcription factors Rev-Erb alpha, DBP, and TEF. In rat-1 fibroblasts, up to three consecutive daily oscillations with an average period length of 22.5 hr have been recorded. The temporal sequence of the various mRNA accumulation cycles is the same in cultured cells and in vivo. The serum shock of rat-1 fibroblasts also results in a transient stimulation of c-fos and rper expression and thus mimics light-induced immediate-early gene expression in the suprachiasmatic nucleus (Balsalobre, 1998).

The expression of dbp, a putative clock-controlled transcription factor, was investigated in the mouse brain by in situ hybridization using antisense cRNA probe. Positive dbp mRNA signals were detected in various parts of the brain, with the highest expression in the suprachiasmatic nucleus (SCN). The circadian expression profile was investigated in SCN and extra-SCN areas. In the SCN, dbp mRNA signals show a peak at early daytime (ZT/CT4) and a trough at early nighttime (ZT/CT16) in both light-dark and constant dark conditions. In the cerebral cortex and the caudate-putamen, dbp mRNA is also expressed in a circadian manner, but the phase of dbp mRNA expression in these structures shows a 4-8 hr delay compared to that in the SCN. These findings indicate that the circadian expression profile of dbp in the extra-SCN brain areas is different from that in the SCN (Yan, 2000).

PAR domain family: Transcriptional targets

A chicken liver cDNA expression library was screened with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter and clones for a transcription factor have been isolated and named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and is found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search reveals that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control (Iyer, 1991).

Hepatic cytochrome P450 cholesterol 7 alpha-hydroxylase, CYP7, is regulated in vivo at the protein and the mRNA level in response to multiple physiological factors, including liver cholesterol synthesis, bile acid feedback inhibition, and diurnal rhythm. An investigation was carried out to see whether the liver transcription factor DBP (albumin promoter D-site binding protein), which undergoes a striking diurnal rhythm in rat liver (DBP levels during evening/morning approximately 100:1), contributes to the diurnal regulation of CYP7 gene expression. DNase I footprinting analysis using bacterially expressed DBP and a cloned 5'-flanking DNA segment of the rat CYP7 gene revealed five distinct DBP-binding sites, designated A-E, distributed between nucleotides (nts) -41 and -295 relative to the CYP7 transcription start site. CYP7-directed gene transcription in HepG2 cells transfected with a 5'-CYP7 promoter-chloramphenicol acetyl-transferase reporter is activated up to 12-fold upon cotransfection of a DBP expression vector. 5'-Deletion analyses and site-specific mutagenesis reveals that this stimulating effect of DBP can in part be ascribed to its functional interaction with three different DBP binding sites. C/EBP beta (LAP), another liver-enriched basic-leucine zipper transcription factor, binds to these same sites but effects a more modest increase in CYP7-directed gene transcription (up to 3-4-fold) when expressed in HepG2 cells. Competition for CYP7 promoter-binding sites between C/EBP, which undergoes an approximately 2-fold diurnal change in rat liver, and the diurnally regulated DBP, is proposed to determine the relative rates of basal versus diurnally regulated CYP7 gene transcription and thus may be a primary mechanism for setting the 3-6-fold amplitude that characterizes the circadian rhythm of liver CYP7 expression. Moreover, since DBP is first expressed in rat liver 3-4 weeks after birth, these findings may account for both the enhanced expression and the onset of the diurnal pattern of CYP7 enzyme levels at this stage of development (Lee, 1994).

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 the 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. These 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 (Fonjallaz, 1996).

To study the molecular mechanisms of circadian gene expression, attempts were made to identify genes whose expression in mouse liver is regulated by the transcription factor DBP (albumin D-site-binding protein). This PAR basic leucine zipper protein accumulates according to a robust circadian rhythm in nuclei of hepatocytes and other cell types. The Cyp2a4 gene, encoding the cytochrome P450 steroid 15alpha-hydroxylase, is a novel circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone and estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displays circadian kinetics indistinguishable from those of the highly related CYP2A5 gene. Proteins encoded by both the Cyp2a4 and Cyp2a5 genes also display daily variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprinting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeed regulated by DBP. These conclusions are corroborated by genetic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expression in the liver are significantly impaired in a mutant mouse strain homozygous for a dbp null allele. These experiments strongly suggest that DBP is a major factor controlling circadian expression of the Cyp2a4 and Cyp2a5 genes in the mouse liver (Lavery, 1999).

PAR domain family proteins: Regulation of behaviorial circadian rhythms

DBP, a PAR leucine zipper transcription factor, accumulates according to a robust circadian rhythm in liver and several other tissues of mouse and rat. DBP mRNA levels also oscillate strongly in the suprachiasmatic nucleus (SCN) of the hypothalamus, believed to harbor the central mammalian pacemaker. However, peak and minimum levels of DBP mRNA are reached about 4 h earlier in the SCN than in liver, suggesting that circadian DBP expression is controlled by different mechanisms in SCN and in peripheral tissues. Mice homozygous for a DBP-null allele display less locomotor activity and free-run with a shorter period than otherwise isogenic wild-type animals. The altered locomotor activity in DBP mutant mice and the highly rhythmic expression of the DBP gene in SCN neurons suggest that DBP is involved in controlling circadian behavior. However, since DBP-/- mice are still rhythmic and since DBP protein is not required for the circadian expression of its own gene, dbp is more likely to be a component of the circadian output pathway than a master gene of the clock (Lopez-Molina, 1997).

Albumin D-binding protein (DBP) is a PAR leucine zipper transcription factor that is expressed according to a robust circadian rhythm in the suprachiasmatic nuclei, harboring the circadian master clock, and in most peripheral tissues. Mice lacking DBP display a shorter circadian period in locomotor activity and are less active. Thus, although DBP is not essential for circadian rhythm generation, it does modulate important clock outputs. The role of DBP in the circadian and homeostatic aspects of sleep regulation have been studied by comparing DBP deficient mice (dbp-/-) with their isogenic controls (dbp+/+) under light-dark (LD) and constant-dark (DD) baseline conditions, as well as after sleep loss. Whereas total sleep duration was similar in both genotypes, the amplitude of the circadian modulation of sleep time, as well as the consolidation of sleep episodes, was reduced in dbp-/- under both LD and DD conditions. Quantitative EEG analysis demonstrates a marked reduction in the amplitude of the sleep-wake-dependent changes in slow-wave sleep delta power and an increase in hippocampal theta peak frequency in dbp-/- mice. The sleep deprivation-induced compensatory rebound of EEG delta power is similar in both genotypes. In contrast, the rebound in paradoxical sleep is significant in dbp+/+ mice only. It is concluded that the transcriptional regulatory protein DBP modulates circadian and homeostatic aspects of sleep regulation (Franken, 2000).

E4BP4, a basic leucine zipper transcription factor structurally related to Vrille, contains a DNA-binding domain closely related to DBP, HLF, and TEF, which are PAR proteins. The phase of e4bp4 mRNA rhythm is opposite that of the dbp, hlf, and tef rhythms in the suprachiasmatic nucleus (SCN), the mammalian circadian center, and the liver. The protein levels of E4BP4 and DBP also fluctuate in almost the opposite phase. Moreover, all PAR proteins activate, whereas E4BP4 suppresses, the transcriptional activity of the reporter gene containing a common binding sequence in transcriptional assays in vitro. An electrophoretic mobility shift assay has demonstrated that E4BP4 is not able to dimerize with the PAR proteins, but is able to compete for the same binding sites with them. Furthermore, sustained low e4bp4 and high dbp mRNA levels are found in mCry-deficient mice. These results indicate that the E4BP4 and PAR proteins are paired components of a reciprocating mechanism wherein E4BP4 suppresses the transcription of target genes during the time of day when E4BP4 is abundant, and the PAR proteins activate them at another time of day. E4BP4 and the PAR proteins may switch back and forth between the on-off conditions of the target genes (Mitsui, 2001).

On the basis of these results, two working models are proposed that can explain the relationship between the e4bp4 gene and the putative core feedback loop including CLOCK/BMAL1, mPERs, and mCRYs. It is hypothesized that the e4bp4 gene is regulated by an unidentified transcriptional repressor (X) that is regulated by CLOCK/BMAL1 and the negative elements, mPERs and mCRYs, as in the case of the dbp, hlf, and tef genes. When X mRNA is translated rapidly and the produced X protein accumulates in the nuclei of the SCN cells with little delay and depresses expression of e4bp4, the phase of the e4bp4 rhythm thus is expected to be opposite that of the dbp, hlf, and tef rhythms. Rapidly translated E4BP4 suppresses the transcriptions of target genes during the time of day when E4BP4 is abundant, and the PAR proteins activate them at another time of day. Thereby, the E4BP4 and PAR proteins increase the amplitude of the rhythmically expressed transcript levels of the target genes. In a second model, the e4bp4 gene is regulated by an unidentified repressor (Y) that is indirectly regulated by CLOCK/BMAL1, mPERs, and mCRYs via DBP-, HLF-, or TEF-mediated regulation. In addition to these two models, it also is conceivable that the rhythmic expression of the e4bp4 gene is controlled by the cycling presence of an unidentified positive element that drives the rhythmic expression of the bmal1 gene, depending on mPER2. It is noteworthy that in all three models, the existence of an unidentified activator or repressor is indicated (Mitsui, 2001).

The genes albumin, cholesterol 7alpha hydroxylase, and cytochrome P450 (Cyp2c6, Cyp2a4, and Cyp2a5) are thought to be candidates for the target genes of the PAR proteins in the liver. Conversely, the interleukin 3 gene is thought to be a target of E4BP4 in T lymphocytes. The consensus-binding site for E4BP4 and the PAR proteins is different from the CLOCK/BMAL1 E-box binding site. Therefore, E4BP4 and the PAR proteins potentially could regulate a set of output genes that do not also possess an E-box. Moreover E4BP4 and the PAR proteins regulate the transcriptional activity of the mPer1 promoter in a transcriptional assay in vitro. Thus, the cyclic activities of E4BP4 and the PAR proteins may feed back onto the central clock mechanism (Mitsui, 2001).

E4BP4 has been shown to behave as an active transcriptional repressor that directly suppresses the transcriptional activities of genes whose promoters it binds. This active repression is mainly because of a small transferable repression domain of 65 amino acids in the C-terminal half of the protein. However, in the case of DBP the PAR (proline and acidic amino acid-rich) domain, which resides amino-terminal to the basic region, has been shown to act as an activation domain. Some other examples of the combination of activator and active repressor that bind the same site are found in genes controlling segmentation during early Drosophila development (e.g., Fushi tarazu and Engrailed). It has been proposed that competition between an activator and repressor is not only required, but also is sufficient to establish all-or-none switches in gene expression. E4BP4 and the PAR proteins may switch back and forth between the on-off conditions (Mitsui, 2001).

The Drosophila transcription factor Vrille contains a DNA-binding domain closely related to mammalian E4BP4, but lacks a PAR domain. It therefore is comparable to E4BP4. Recently, Vri was shown to be required for a functional Drosophila clock: reducing vri gene dosage caused period shortening and elimination of the normal vri cycle generated long-period rhythms or arrhythmicity. Therefore, this family of transcription factors may have an important role in circadian clocks in both Drosophila and mammals. However the phase of the e4bp4 oscillation is opposite that of the mPer1, mPer2, and mPer3 rhythms, which are regulated directly by CLOCK/BMAL1. In contrast, the cycling of vri mRNA in Drosophila is regulated directly by dCLOCK/dBMAL1, and vri mRNA thereby oscillates with the same phase as per mRNA in adult heads. Thus, there may be a difference in the way E4BP4 and Vri are utilized in the mammalian/Drosophila clocks (Mitsui, 2001 and references therein).

Negative control of circadian clock regulator E4BP4 by Casein kinase I-mediated phosphorylation

Light-dependent transcriptional regulation of clock genes is a crucial step in the entrainment of the circadian clock. E4bp4, a vertebrate ortholog of Drosophila Vrille, is a light-inducible gene in the chick pineal gland, and it encodes a bZIP protein that represses transcription of cPer2, a chick pineal clock gene. Prolonged light period-dependent accumulation of E4BP4 protein is temporally coordinated with a delay of the rising phase of cPer2 in the morning. E4BP4 is phosphorylated progressively and then disappears in parallel with induced cPer2 expression. Characterization of E4BP4 revealed Ser182, a phosphoacceptor site located at the amino-terminal border of the Ser/Thr cluster, which forms the phosphorylation motifs for casein kinase 1 (CK1). This serine/threonine cluster is evolutionarily conserved from vertebrate E4BP4 to Drosophila Vrille. CK1 physically associates with E4BP4 and phosphorylates it. CK1-catalyzed phosphorylation of E4BP4 results in proteasomal proteolysis-dependent decrease of E4BP4 levels, while E4BP4 nuclear accumulation is attenuated by CK1 in a kinase activity-independent manner. CK1-mediated posttranslational regulation is accompanied by reduction of the transcriptional repression executed by E4BP4. These results not only demonstrate a phosphorylation-dependent regulatory mechanism for E4BP4 function but also highlight the role of CK1 as a negative regulator for E4BP4-mediated repression of cPer2 (Doi, 2004).

The loss of circadian PAR bZip transcription factors results in epilepsy

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. 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 (Gachon, 2004).

To elucidate the molecular link between PAR bZip transcription factors and epileptic seizures, PAR bZip-regulated genes were sought that could provide insight into the control of brain electrical activity. To this end brain mRNA populations from double-knockout mice, which are not prone to epileptic attacks, and triple-knockout animals, which are susceptible to audiogenic and spontaneous seizures, were compared by Affymetrix high-density oligonucleotide microarray hybridization. Three pools of brain RNA, composed of equivalent amounts of RNA from five males and five females each, were prepared for the two genotypes and compared by hybridization with Affymetrix oligonucleotide microarrays containing probe features for ~12,000 genes (representing 30%-50% of all genes). This resulted in a total of nine comparisons (Gachon, 2004).

Animals homozygous for the disrupted Tef allele contained considerably less Tef transcripts than animals homozygous for a Tef wild-type allele. Interestingly, NFIL3/E4BP4, a putative antagonist of PAR bZip transcription factors, is also down-regulated in TEF-deficient animals, suggesting that TEF stimulates the production of its own competitive inhibitor. In the brain, TEF also appears to stimulate the expression of mPer2 and Dec1, two transcriptional regulators that had been implicated in the negative limb of the circadian oscillator (Gachon, 2004).

Among the genes positively regulated by TEF was Pdxk. Pdxk mRNA levels scored about twofold less in Tef-deficient mice in nine out of nine comparisons. PDXK performs the last step in the conversion of B6 vitamers into pyridoxal phosphate (PLP), a coenzyme of numerous decarboxylases and transaminases involved in amino acid and neurotransmitter metabolism. Even moderate reductions in PLP levels have previously been associated with a susceptibility to epileptic seizures and an increase in delta EEG activity (Sharma, 1994), and thus the transcription of Pdxk was examined in greater detail to evaluate whether this gene might be a direct target gene of TEF. As a first step, the transcriptional start sites within the promoter of the murine Pdxk gene were determined using RACE technology. The sequencing of 13 RACE products yielded two major and several minor cap sites, located between 65 bp and 91 bp upstream of the translation initiation codon. A short conserved sequence block encompassing a PAR bZip recognition sequence (PARRE) could be discerned in the first intron, 12-20 bp downstream of the exon-intron splice junction. A double-stranded oligonucleotide containing this putative binding site was used in electromobility shift assays (EMSA) with liver and brain nuclear extracts harvested at 4-h intervals around the clock to investigate whether this sequence element indeed binds PAR bZip proteins. This element forms a prominent protein:DNA complex with liver nuclear proteins prepared from wild-type mice at times when DBP and TEF reach zenith levels (ZT10). A protein:DNA complex reflecting a liver nuclear protein with circadian accumulation is still discerned in double-knockout mice but not in triple-knockout mice, strongly suggesting that this complex contains TEF. Similar complexes were observed in EMSA experiments with brain nuclear extracts prepared from animals of the three genotypes. As expected on the basis of the temporal mRNA accumulation profiles, the PAR bZip proteins revealed by these EMSA studies accumulate at high levels throughout the day in the brain of wild-type mice. Likewise, TEF levels do not appear to follow a circadian rhythm in the brain of double-knockout animals (Gachon, 2004).

TaqMan RT-PCR technology was used to investigate the differences in Pdxk expression in the liver and brain of double- and triple-knockout mice. In the liver of double-knockout mice, Pdxk mRNA accumulates in a circadian fashion, with a phase compatible with that of TEF accumulation. In triple-knockout mice, Pdxk transcript levels are nearly constant throughout the day and amount to about one-third of the zenith levels attained in double-knockout mice. The Pdxk mRNA levels are also lower in the brain of triple-knockout mice (about two-fold on average). While the Pdxk mRNA concentrations determined in double-knockout mice are similar at most times of the day, the value obtained at ZT8 appears to be somewhat lower. However, in contrast to the clearly circadian expression pattern of liver Pdxk mRNA, the temporal accumulation profile of brain Pdxk transcripts did not score as circadian by the ANOVA test. The daily accumulation of Pdxk mRNA in liver and brain of homozygous Tef single-knockout mice was determined and it was found to be intermediate between double- and triple-knockout mice. This is in keeping with the low but significant seizure activities observed in the EEGs of Tef single-knockout animals (Gachon, 2004).

Several brain regions show significantly lower in situ hybridization signals in triple-knockout mice as compared with double-knockout mice. These include structures that have previously been implicated in the development of spontaneous and/or audiogenic epilepsies, such as cerebral cortex, amygadala, hippocampal formation, inferior colliculus, and periaqueductal gray (Gachon, 2004).

PLP is synthesized in the liver via the phosphorylation of B6 vitamers by PDXK, and a portion of the hepatic PLP is then exported into the bloodstream and transported to other sites, such as the brain. Before crossing the blood-brain barrier and the plasma membrane of target cells, PLP has to be dephosphorylated to pyridoxal (PL) by plasma phosphatases. In the target cells, PL is then rephosphorylated to PLP, which is the active coenzyme form of vitamin B6 (Gachon, 2004).

Whether the reduced Pdxk expression in the liver and brain of PAR bZip-deficient mice indeed affected the levels of PLP was examined. Both brain and liver levels of PLP are significantly reduced. Whereas PLP accumulation appears to follow a circadian rhythm in the liver of Tef wild-type mice, it is nearly invariable in the liver of PAR bZip triple-knockout mice and in the brain of double- and triple-knockout mice. Because Pdxk expression is more strongly dependent on TEF in a subset of brain structures, it is thought that in these structures the difference in PLP levels between TEF-proficient and TEF-deficient animals is considerably higher than the difference measured in extracts from total brains (Gachon, 2004).

In the brain, PLP is the coenzyme of several enzymes participating in the synthesis of neurotransmitters, such as gamma-aminobutyric acid (GABA), serotonin, and dopamine, which had previously been associated with epileptic seizures. Moreover, changes in PLP levels can also influence the levels of glutamate (Glu) and histamines by determining the rate of conversion of these neurotransmitters to other compounds. Whether the reduced levels of PLP in the brains of PAR bZip triple-knockout mice affected the brain concentrations of GABA, serotonin, dopamine, and histamine, was examined. The results revealed significantly reduced levels for serotonin and dopamine and increased levels of histamine in triple-knockout as compared with double-knockout animals (Gachon, 2004).

Since all three PAR bZip genes show circadian expression in the SCN and in peripheral tissues, whether these transcription factors are required for sustained rhythmicity in constant darkness (DD) was examined. The relevance of this issue is reinforced by the recent finding that PAR domain protein 1 (Pdp1), the only Drosophila PAR bZip domain protein, is an essential clock component in fruit flies. The circadian locomotor activity of triple-knockout mice was recorded using wheel running assays. PAR bZip-deficient mice are rhythmic in DD, suggesting that they still harbor a functional circadian pacemaker. Moreover, the period length of triple-knockout mice measured in DD is nearly identical to that measured for wild-type mice. Interestingly, Dbp single-knockout mice free-run with a period ~30 min shorter than that of wild-type mice, while Tef and Hlf single knockout mice free-run with periods ~-25 min longer than wild-type. Apparently, these positive and negative period differences counteract each other in triple-knockout animals, yielding a period length that is close to that observed for wild-type mice. Although triple-knockout animals display a rhythmic behavior in constant darkness, it was noticed that a large proportion of these animals show a spate of wheel running activity several hours before the onset of the dark phase in LD or the onset of the subjective night in DD (Gachon, 2004).

The circadian expression of core clock components in the livers of triple-knockout and wild-type mice were compared. The rhythmic expression of the four clock genes Bmal1, Per1, RevErbalpha, and Cry1 is nearly identical in animals of these two genotypes. It is thus concluded that PAR bZip proteins are dispensable for circadian rhythm generation, and hence that they are regulators of outputs of the mammalian circadian timing system rather than core components of the clock (Gachon, 2004).

By controlling Pdxk expression, PAR bZip transcription factors may play an important role in the fine-tuning of neurotransmitter homeostasis in the brain. PLP, the product of the PDXK reaction, serves as the coenzyme for a number of enzymes involved in the synthesis and degradation of neurotransmitters, and changes in PLP levels can result in severe neurotransmitter disequilibria and brain pathologies characterized by epilepsy. For example, the disruption of the gene encoding tissue-nonspecific alkaline phosphatase (TNAP) causes an approximately threefold reduction in brain PLP levels, and 100% of the Tnap mutant mice succumb to lethal epileptic seizures before 20 d of age. gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter of the central nervous system, probably plays the most important role in preventing the uncontrolled activation of epileptic foci and the propagation/amplification of seizure waves through downstream regions. The function of PLP in determining the concentration of GABA is complex, as PLP is the coenzyme of both glutamate decarboxylase (GAD), which converts glutamate to GABA, and GABA transaminase (GABA-T), which degrades GABA to succinic semialehyde. Moreover, two GAD isoforms, GAD65 and GAD67, with possibly different functions are expressed in most glutamatergic neurons. Whereas GAD65 is believed to be mostly responsible for the synthesis of GABA used for neurotransmission in nerve endings, GAD67 may produce GABA that is used to a large extent for metabolic purposes involving the tricarboxylic acid cycle. GAD67 has a higher affinity for PLP than GAD65, and exists primarily in the holoenzyme form at physiological PLP levels. In contrast, a large fraction of GAD65 is found in the apoenzyme form, and the activity of this GAD isoform is thus more dramatically affected by changes in PLP concentration. It appears that the bulk of brain GABA is synthesized by GAD67, because Gad65-/- mice (genetic backgound 129xC57BL/6) contain normal basal levels of brain GABA. Since these mice are susceptible to spontaneous epileptic attacks, local reductions in GABA concentrations that are not revealed by measuring total brain GABA may be sufficient to provoke seizures. Hence, in spite of the virtually identical levels of total brain GABA measured in double- and triple-knockout mice, it remains possible that the susceptibility of PAR bZip triple-knockout animals to seizure is enhanced by a local reduction of GABA levels in key brain structures (Gachon, 2004 and references therein).

PLP also serves as the coenzyme of aromatic amino acid decarboxylase (AADC), an enzyme involved in the synthesis of the monoamines serotonin and dopamine. Both of these neurotransmitters have been associated with epileptic attacks. For example, young adults of a mouse strain homozygous for a serotonin receptor 5-HT2C null allele develop audiogenic seizures and mice homozygous for a dopamine receptor 2 (D2R) null allele develop epilepsies after injection of kainic acid at doses that do not provoke seizures in D2R wild-type littermates. Given these observations, it is suspected that the significantly reduced serotonin and dopamine concentrations in the brains of PAR bZip triple-knockout mice contribute to the susceptibility of these mice to epileptic attacks (Gachon, 2004 and references therein).

Two PLP-dependent enzymes, histidine decarboxylase and histamine oxidase, are involved in histamine synthesis and catabolism, respectively. Interestingly, high levels of PLP have been shown to result in a decrease of histamine levels, probably by promoting histamine degradation. Therefore, it is speculated that the high level of histamine found in the brain of PAR bZip triple-knockout mice is caused by a decrease in histamine catabolism, due to diminished PLP levels. Whether or not histamine imbalances play a role in the generation of epilepsies is still somewhat controversial (Gachon, 2004).

vrille: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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