Gene name - PAR-domain protein 1
Cytological map position - 66A12-66A17
Function - transcription factor
Symbol - Pdp1
FlyBase ID: FBgn0016694
Genetic map position - 3-
Classification - PAR-domain bZip protein
Cellular location - nuclear
In vertebrates, transcriptional control of skeletal muscle genes during differentiation is regulated by enhancers that direct the combinatorial binding and/or interaction of MEF2 and the bHLH MyoD family of myogenic factors (Drosophila homolog: Nautilus). Drosophila Mef2 plays a role similar to its vertebrate counterpart in the regulation of the Tropomyosin I (TmI) gene in the development of Drosophila somatic muscles. However, unlike vertebrates, Drosophila Mef2 interacts with a muscle activator region that does not have binding sites for myogenic bHLH-like factors, such as Nautilus, or any other known Drosophila transcription factors. A novel protein regulator of the muscle activator region has been identified and named Pdp1 (for PAR domain protein 1). Pdp1 is a novel transcription factor, highly homologous to the PAR subfamily of mammalian bZIP transcription factors HLF, DBP and VBP/TEF. This is the first member of the PAR subfamily of bZIP transcription factors to be identified in Drosophila. Pdp1 may function as part of a larger protein/DNA complex that interacts with Mef2 to regulate transcription of Drosophila muscle genes (S. C. Lin, 1997).
For some perspective on the function of Pdp1, consider a brief look at mef2 regulation of TmI, and something termed the 'mut1' region within an intronic enhancer. Previous studies have shown that Drosophila TmI gene is a target gene for mef2 regulation. The TmI gene contains two different muscle enhancers within the first intron of the gene. Both enhancers contain a Mef2 binding site: mutation in either site reduces expression in somatic body wall muscles. For one enhancer, the proximal muscle enhancer (PME), a high level of gene expression in somatic muscles requires the cooperative activity of Mef2 and a cis-acting 60 base pair muscle activator (MA) region located within the enhancer (Lin, 1996). Further analysis of the MA region, of the PME reveals that multiple protein complexes bind to this region. Four complexes in particular, known as 4, 5, 6 and 8, fail to bind to a mutant fragment lacking a site called mut1, suggesting that formation of the complex requires sequences in the mut1 region. Deletion of the mut1 region, and the adjacent mut6 and mut2 regions, eliminates all enhancer directed muscle expression in larvae and adults (M.-H. Lin, 1997).
In order to identify regulatory factors that bind to the mut6, mut1 and mut2 regions and regulate MA function, radioactive oligonucleotides containing the sequence spanning this region were used to screen a lambda gt11 embryonic cDNA expression library (Singh, 1989). In this screening process, library sequences that code for a protein that binds the radioactive sequence were identified and subsequently characterized. One such library sequence identified the protein Pdp1, a PAR domain bZip transcription factor related to known vertebrate transcription factors. Subsequent experiments have shown that Pdp1 binds to a 10 base pair sequence that matches the consensus binding sequence for vertebrate PAR proteins. An additional weak Pdp1 binding site was identified adjacent to the strong site, but significant filling of both sites could not be demonstrated. The weak binding site differs from the consensus binding site for PAR bZIP proteins. Pdp1 functions as a transcriptional activator simulating reporter expression approximately 90 fold over background (S. C. Lin, 1997). The region spanning the two Pdp1-binding sites is hypersensitive to DNase I digestion, suggesting that the conformation of the DNA/chromatin in this region can be altered (M.-H. Lin, 1997). It is likely that Pdp1 binds DNA as a homodimer since the vertebrate PAR domain and other bZIP proteins bind DNA as homodimers or heterodimers.
How does Pdp1 function to regulate TmI expression? It has been previously shown that the MA can interact directly with Mef2 to mimic the activity of the PME (Lin, 1996). The identifed Pdp1 strong and weak binding sites within the MA are necessary but not sufficient for function of the MA enhancer alone or for function of the MA enhancer plus the Mef2 site (the entire proximal muscle enhancer). Thus other cis-acting elements must be present in the MA region that act in concert with Pdp1 to regulate its function. This is consistent with previous mutation analysis of the MA indicating that other cis-elements in the MA region spanning the mut3 to mut5 region also contribute to MA activity. This is also consistent with previous electrophoretic mobility shift assay (EMSA) results using nuclear extracts in which eight protein/DNA complexes could be identified that form in the MA region (M.-H. Lin, 1997). Four of these can be unambiguously assigned to the Pdp1 strong binding site and an additional complex can be assigned to the mut3 region. Thus, it is suggested that a minimal transcriptional activation complex might consist of Pdp1 homodimers or heterodimers bound to adjacent sites, or more likely Pdp1 binds to the strong Pdp1-binding site and interacts with another factor(s) bound at the Pdp1 weak site. It has been proposed that additional factors bind within the 60 bp region to perhaps form a complex to promote transcriptional activation (S. C. Lin, 1997).
Drosophila Mef2 and the MA can function together like a mini-enhancer, driving high-level muscle-specific expression as do myogenic factors and MEF2 in vertebrates. It is not known how Drosophila Mef2 interacts with the MA region. This may involve a direct interaction between the two proteins to direct promoter activity or each could interact independently with promoter elements. There is a repressor-like activity located in the 1B(b) region that (in the context of a MA and an adjacent sequence) can repress MA activity (Gremke, 1993). Thus it is possible that Mef2 may have at least two roles in regulating TmI expression -- one role to overcome this repressor activity, possibly through a direct interaction with a repressor and a second role in cooperatively interacting with Pdp1 and other MA factors to stimulate high levels of transcription (S. C. Lin, 1997).
The Drosophila circadian clock consists of two interlocked transcriptional feedback loops. In one loop, Clock/Cycle activates period expression, and Period protein then inhibits Clock/Cycle activity. Clock is also rhythmically transcribed, but its regulators are unknown. vrille (vri) and Par Domain Protein 1 (Pdp1) encode related PAR family bZIP transcription factors whose expression is directly activated by Clock/Cycle. Vri and Pdp1 proteins are shown to feed back and directly regulate Clock expression. Repression of Clock by Vri is separated from activation by Pdp1 since Vri levels peak 3-6 hours before Pdp1. Rhythmic vri transcription is required for molecular rhythms, and the clock stops in a Pdp1 null mutant, identifying Pdp1 as an essential clock gene. Thus, Vri and Pdp1, together with Clock itself, comprise a second feedback loop in the Drosophila clock that gives rhythmic expression of Clock, and probably of other genes, to generate accurate circadian rhythms (Cyran, 2003).
vri and Pdp1 encode basic zipper transcription factors with highly conserved basic DNA binding domains, suggesting they bind the same set of target genes. vri and Pdp1 are both direct targets of Clk/Cyc. First, a test was performed to see which Pdp1 isoform(s) are clock-controlled since four alternative promoters and alternative splicing generate six Pdp1 isoforms in vivo. RNase protection probes specific for the different isoforms revealed that only Pdp1 RNA levels oscillate in adult fly heads (Cyran, 2003).
Taking time points every three hours during a light-dark (LD) cycle revealed that vri and Pdp1epsilon RNA levels oscillate with similar phases to one another, but peak levels of Pdp1epsilon are not reached until 3-6 hr after the peak of vri RNA levels. Oscillating Pdp1epsilon RNA levels are also seen in constant darkness. Pdp1epsilon RNA levels were high at both ZT2 and ZT14 in per0 and tim01 mutants. Pdp1epsilon RNA is low at both ZT2 and ZT14 in ClkJrk and cyc0 mutants at levels close to the Pdp1epsilon RNA levels at ZT2 in wild-type flies. The phase of Pdp1epsilon RNA expression in wild-type flies, and the loss of rhythms in clock mutants, are both consistent with Pdp1epsilon transcription being regulated in a similar manner to per, tim, and vri transcription. Indeed, analysis of the first 4 kb of sequence upstream of the start site of Pdp1epsilon transcription reveals six perfect CACGTG E boxes, which are potential Clk/Cyc binding sites. This is similar to the vri promoter, which has 4 E boxes in 2.4 kb. Thus, Pdp1epsilon is the clock-regulated Pdp1 transcript (Cyran, 2003).
The different phases of vri and Pdp1epsilon RNAs may reflect subtly different transcriptional activities of their promoters and/or different mRNA half-lives. Thus, the vri promoter could be stronger than the Pdp1epsilon promoter, and vri RNA may have a shorter half-life than Pdp1epsilon RNA. Indeed, the vri 3' UTR contains seven copies of an AATAA element, likely to be associated with mRNA instability (Cyran, 2003).
Direct regulation of Pdp1epsilon expression by Clk/Cyc make it likely that Pdp1epsilon protein would be found in clock cells as shown for vri. Pdp1 protein is detected at night (ZT21) but not by day (ZT10) in larval pacemaker cells, marked by the neuropeptide pigment dispersing factor (PDF). Oscillation of Pdp1 protein continues in constant darkness in wild-type pacemaker cells but is blocked by null or dominant-negative mutations in the per, tim, Clk, and cyc clock genes (Cyran, 2003).
A robust oscillation in Pdp1 levels is also visible in photoreceptor cells of the adult eye, which contain functional clocks. Low Pdp1 levels are seen during the day at ZT9, and high levels in the middle of the night at ZT18. The oscillation is especially clear in the outer photoreceptor cell nuclei. Pdp1 at ZT18 colocalizes with ELAV, which marks the nuclei of neurons. Although antibodies to Pdp1 do not distinguish between the different Pdp1 isoforms, RNase protection data and Western blots detect rhythmic expression of only Pdp1epsilon in fly heads. Pdp1 protein is thus rhythmically detectable in both central and peripheral clock cells and it is a nuclear protein, as predicted by its ability to activate transcription (Cyran, 2003).
Current models of the Drosophila circadian oscillator are based upon rhythmic activation of per/tim transcription by cycling levels of Clk/Cyc, and rhythmic repression of per/tim transcription by cycling levels of Per/Tim. While these models explain Per and Tim oscillations, the molecular mechanisms underlying cycling of Clk/Cyc have been unknown. This study identifies Vri as a rhythmically expressed Clk repressor and Pdp1epsilon as a rhythmically expressed Clk activator. Vri and Pdp1epsilon are shown to directly regulate Clk transcription by binding the same site in the Clk promoter. Pdp1 is required for circadian clock oscillation and for Clk expression, thus establishing it as a novel and essential clock gene. Vri and Pdp1epsilon proteins accumulate with a phase delay that presumably underlies sequential repression and activation of Clk transcription. Thus, Vri, Pdp1epsilon, and Clk form a second feedback loop in the circadian oscillator responsible for regulating rhythms in Clk/Cyc levels (Cyran, 2003).
A second feedback loop in the Drosophila clock, interlocked to the first feedback loop, has been predicted to explain antiphase rhythms of Clk and per expression. Direct regulation of vri and Pdp1epsilon transcription by Clk/Cyc, and direct regulation of Clk expression by Vri and Pdp1epsilon proteins establishes the existence of this second loop and identifies its components (Cyran, 2003).
The first loop of this model starts with activation of per and tim expression by Clk/Cyc at about noon. Per/Tim then feeds back to inhibit Clk/Cyc activity during the second half of the night. In the second loop, Clk/Cyc also activates vri and Pdp1epsilon transcription at about noon. vri and Pdp1epsilon RNAs and proteins accumulate with different kinetics such that Vri protein accumulates first and represses Clk expression. Pdp1epsilon protein then accumulates and activates Clk transcription after Vri-mediated repression ends in the middle of the night. However, newly produced Clk protein is inactive due to the presence of Per repressor. Once Per is degraded, Clk/Cyc reactivates per/tim and vri/Pdp1epsilon transcription to start a new cycle. The two loops are linked together by Clk/Cyc and restart simultaneously (Cyran, 2003).
Conceptually, a molecular clock must separate the phases of clock gene transcription and repression otherwise clock components reach a stable steady state. The delay separating active transcription and repression of per/tim is controlled by the Double-time and Shaggy/GSK3 protein kinases that regulate Per/Tim accumulation and nuclear transport. The phases of Clk transcription and repression are separated by two mechanisms: (1) accumulation of Vri protein before Pdp1epsilon, which ensures that repression of Clk precedes activation; and (2) Per inhibition of Clk/Cyc activity in the early morning which prevents reactivation of vri and Pdp1epsilon transcription even when Clk levels are high (Cyran, 2003).
Does the model fit the data? The model explains the observation that Vri represses Clk independently of nuclear Per/Tim. It also suggests that in a per0 background, Clk expression is repressed because of high Vri protein levels. High levels of Vri must therefore dominate over high Pdp1epsilon levels and suppress Clk expression in per0 flies. Indeed, overexpression of vri is dominant and stops the clock in an otherwise wild-type background with constantly low Clk expression (Cyran, 2003).
However, this model does not immediately explain why Clk RNA levels are high in ClkJrk and cyc0 mutants. In the absence of Clk/Cyc function, vri RNA levels are low, and the consequently low levels of Vri protein would not be sufficient to repress Clk expression. But how can expression of Clk RNA be maximal with very low Pdp1epsilon levels in ClkJrk and cyc0 mutants? This question is especially relevant given the very low levels of Clk in Pdp1P205 homozygous mutant larvae in constant darkness, which makes the existence of additional factors that positively regulate Clk expression in constant conditions unlikely. The simplest explanation is that the very low levels of Pdp1epsilon RNA present in ClkJrk and cyc0 mutants are still sufficient to give enough Pdp1epsilon protein to activate Clk when competition from Vri is minimal due to very low Vri protein levels. Indeed, Clk RNA levels are close to their peak at ZT3 and ZT6 in wild-type flies when both Vri and Pdp1epsilon levels are very low. In contrast, Pdp1epsilon protein is totally absent in Pdp1P205 null mutants because the Pdp1 gene is deleted and thus, Clk is at low levels. However, further work is required to test this hypothesis (Cyran, 2003).
The model can also be used to explain how clock-controlled genes are expressed with different phases. Genes activated by Clk/Cyc will reach maximum RNA levels at ~ZT14 and these include per, tim, vri, and Pdp1epsilon. Genes regulated by Vri and Pdp1epsilon will peak at ~ZT2 and Clk is one example. Another candidate Vri/Pdp1epsilon regulated gene is cryptochrome (cry), whose RNA levels oscillate in phase with Clk RNA and follow the same pattern as Clk in clock mutants. Indeed, overexpression of Vri represses cry expression, and the cry promoter contains functional Vri (and therefore probably also Pdp1epsilon) binding sites (Cyran, 2003).
It is also conceivable that certain DNA sequences bind Vri with higher affinity than Pdp1epsilon or vice versa. One could then imagine two promoters, one with 5 optimal Vri and another with 5 optimal Pdp1epsilon binding sites, that would give RNA expression profiles differing by ~2-4 hr. Such a mechanism may help to explain the multiple peaks of rhythmically expressed genes found in Drosophila (Cyran, 2003).
Most clock genes are conserved between Drosophila and mammals, and they function in a broadly similar mechanism. For example, peak levels of Bmal1 and Clock RNAs are antiphase to mPer1 and mPer2 in mice just as Clk RNA peaks in antiphase to Drosophila per. A recent study identified the clock-controlled Bmal1 repressor, which parallels the Vri repression of Clk data presented in this study. The data extend the similarities of the Drosophila and mammalian clocks and suggest the existence of a rhythmically transcribed Bmal1 transcriptional activator that plays an analogous role to Drosophila Pdp1 in the second mammalian feedback loop (Cyran, 2003).
However, the Bmal1 repressor is REV-ERBα, an orphan nuclear receptor, which is unrelated to Vri. Perhaps even more surprising is that REV-ERBα is dispensable for rhythmicity in mice, although it adds robustness and precision to the circadian clock. Posttranscriptional regulation of clock proteins in the first loop presumably compensates for the loss of rhythmic Bmal1 expression in the second loop in rev-erbα-/- mice. Posttranscriptional regulation of Clk protein also plays an important part in the Clk protein cycle. However, the magnitude of the period alterations in vri and Pdp1 heterozygous flies are comparable to those seen in mice homozygous for a rev-erbα knockout. Therefore, the Drosophila clock may rely more heavily on transcriptional control than the mammalian clock, especially in the second loop (Cyran, 2003).
Homologs of Vri and Pdp1 do exist in mammals and are even expressed with a circadian rhythm in pacemaker cells. However, genetic loss-of-function experiments suggest that none of the three mammalian Pdp1 homologs, either alone or in combination, affects the period length of circadian locomotor activity by more than 30 min. Similar loss-of-function experiments have yet to be performed for E4BP4, the mammalian homolog of Vri. The mammalian homologs of vri and Pdp1 may thus play only an ancillary regulatory role in the mammalian central clock, with their primary role being the regulation of rhythmic clock outputs (Cyran, 2003).
Tightly regulated and interconnected feedback loops are conserved in the circadian clocks of all the model organisms so far studied. A second interconnected loop adds robustness to oscillators. Two transcription loops also provide the potential for multiple inputs to the clock such as light, temperature, membrane potential, and redox state. Additionally, a second transcriptional loop provides a mechanism to regulate a novel phase of rhythmic expression of clock output genes. Such downstream genes presumably allow an organism to anticipate a constantly changing, but relatively predictable, environmental cycle, and adjust its behavior and physiology accordingly. The identification of downstream genes that link the molecular ticking of a central clock to changes in whole animal behavior and physiology is clearly the next major challenge in circadian biology (Cyran, 2003).
Seven different-sized RNAs are detected with the Pdp1 probe. Band 7, the smallest, is approximately 3.2 kb and corresponds to the size of the Pdp1 cDNA; RNA 1, the largest, is approximately 7.3 kb. The larger RNAs may represent precursor RNAs, alternately spliced Pdp1 RNAs or RNAs encoded by closely related genes (S. C. Lin, 1997).
The Pdp1 gene spans approximately 45 kb of DNA. There are nine exons in the gene. Unlike the vertebrate PAR genes, the intron/exon splice sites of Pdp1 do not delineate the PAR, basic, and leucine zipper domains within the protein. The locations and arrangements of the exons indicate that there are at least two and possibly four transcriptional start sites. The Pdp1-epsilon and Pdp1-phi cDNAs are not full length and could conceivably splice to upstream exons and thus their 5' ends may not reflect their start sites of transcription. The Pdp1-alpha and -gamma cDNAs end in poly(A) sequences just downstream from consensus polyadenylation signals and are probably cleavage sites used in vivo. The other cDNAs end in short poly(A) sequences that occur in the 3' UTR. These cDNAs are not associated with a consensus polyadenylation signal and probably result from alternative priming of the reverse transcriptase in making the libraries (Reddy, 2000).
Three of the six Pdp1 isoforms (alpha, beta, and delta) have a common amino-terminal peptide while the Pdp1-gamma,-epsilon, and -phi proteins have unique amino-terminal peptides spliced to the common carboxy-terminal domains of the protein encoded by exons 5-8 in the gene. The common exons encode the putative transcriptional activation domain and the PAR and bZIP domains. The role of the alternately generated amino-terminal peptides of the vertebrate PAR domain proteins has not been investigated and the amino acid sequences of the unique alpha and gamma ends do not show any unusual features. In contrast, the amino-terminal peptides of the epsilon and phi isoforms are considerably larger than the others and have large clusters of asparagines and glutamines. Accordingly, this region is very hydrophilic and likely to reside on the surface of the protein and could be subject to protein modification. Also, some transcriptional activation domains have high asparagine and glutamine content. The location of the epsilon and phi exons adjacent to each other and the similarity of their sequences suggests that they may have arisen through duplication. The Pdp1-beta protein is produced by insertion of an additional 14 amino acids between the amino-terminal a peptide and the common portion of the protein (Reddy, 2000).
The Pdp1-delta isoform is produced by alternate splicing of exon 7 to an internal splice site of exon 8. This results in an in-frame removal of the last 28 amino acids of the 48- amino-acid PAR domain and the first 12 amino acids of the 38-amino-acid basic domain. The 12-amino-acid basic region that is spliced out does not contain the core DNA binding domain; however, it does contain the extended basic region amino acids KKSRK. The extended basic sequence is highly conserved in all PAR proteins and has been shown to influence DNA binding specificity, suggesting that Pdp1-delta may have altered DNA binding properties (Reddy, 2000).
Bases in 5' UTR - 187
Bases in 3' UTR - 2010
Several proteins have high homology to the C-terminal region of Pdp1. These proteins are all transcription factors of a subfamily of three vertebrate bZIP proteins called the PAR family. This subfamily includes HLF (human hepatic leukemia factor), VBP/TEF (chicken vitellogenin protein/rat thryotrophic factor), and DPB (rat albumin D-box-binding protein). This subfamily is characterized by a C-terminal leucine zipper region and by additional homology over an approximately 48 amino acid central region that is proline- and acidic amino acid-rich (PAR). There is 44-46% identity in the PAR domain of Pdp1 with the other PAR family members. Pdp1 also has 86-91% amino acid identity within the 22 amino acid basic DNA-binding and forked domains and 45-52% identity in the leucine zipper region with the other PAR family members. The 21 amino acid region separating the PAR and the basic DNA domain, called the extended basic region and shown to contribute to the DNA-binding specificity, has 81% identity with HLF. There is also 69% identity with HLF from amino acid 61 to 100 in Pdp1. This region in HLF has been shown to function as a transcriptional activation domain. The Drosophila gap segmentation protein Giant is highly homologous to Pdp1 in the bZIP region (Lin, 1997b).
date revised: 17 December 2003
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