InteractiveFly: GeneBrief

PAR-domain protein 1 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

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).

vrille, Pdp1, and Clock form a second feedback loop in the Drosophila circadian clock

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 Clk^Jrk 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 Clk^Jrk 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 Clk^Jrk and cyc⁰ mutants? This question is especially relevant given the very low levels of Clk in Pdp1^P205 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 Clk^Jrk 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 Pdp1^P205 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).

Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila

Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, this study monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). The scrib mutation was shown to impact the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch dedicated transcription factor. The Notch-dependent neoplasms require however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1, and bZIP factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally this work highlights some Notch/scrib specificities, in particular the role of the PAR domain containing bZIP transcription factor and Notch direct target Pdp1 for neoplastic growth (Logeay, 2022).

In this study, using Notch-driven paradigms of epithelial overgrowth in Drosophila wing discs, the molecular mechanisms are described underlying the cooperation between Notch and polarity loss during neoplasia. It was shown that epithelial polarity alterations redirect the transcriptional outcome of the Notch signaling pathway, thus defining a specific set of new neoplastic Notch direct targets. It was further shown that this redirection occurs mainly on pre-existing Su(H)-bound regions rather than new ones. Finally, it was shown that, similar to what was previously described for Ras signaling, the AP-1/Stat/Yki/Ftz-f1 transcription factors are required for the cooperation between Notch signaling and polarity loss during neoplastic growth (Logeay, 2022).

Although cancer genomes exhibit multiple mutations in cancer cells, their functional interactions remain difficult to monitor and model. Neoplastic tissues, generated upon the combination of Notch pathway activation and polarity loss through scrib mutation, experience many cellular stresses: DNA-damage responses, but also endoplasmic reticulum and unfolded protein response, starvation or oxidative stresses. However, even though present, these different stresses, in particular oxidative stress and DNA damage, are not individually necessary in the context of polarity loss as blocking them or the cellular response they promote (by CAT/SOD overexpression, or inhibition of p53) did not significantly suppress the NS tumorous behaviors. These observations suggest that the different stress pathways activated during polarity loss are either not required for fueling growth (they are rather a consequence than a cause of neoplastic growth), or might act 'redundantly' to activate a common core response required for increased growth (Logeay, 2022).

Although Drosophila and mouse models have demonstrated that overactive signaling pathways cooperate with epithelial polarity impairment to generate neoplastic growth, the vast majority of studies seeking to understand the underlying mechanisms have focused primarily on the cooperation between activated RasV12 and scrib mutants, especially in Drosophila. Importantly, the current study, investigating the cooperation between Notch and polarity, shows that many observations made for Ras can be extended to Notch, suggesting that the paradigms used are not specific to Ras but might represent a more general tumor growth paradigm. But, because the main, if not only, Notch pathway outcome is transcriptional, the NICD/scrib^- model allowed the modes of cooperation to be studied in greater detail. The cooperation between Notch pathway activation and polarity loss led to a specific transcriptional program, and in particular the activation of new Notch direct targets. This was not the consequence of a general redeployment to new target gene loci of Su(H), the Notch pathway-dedicated transcription factor, ruling out one possible model for the oncogene/polarity cooperation. Thus, what could be the mechanisms controlling which genes were activated in the different conditions? All 'Notch'-activating transcriptional complexes comprise NICD, Mastermind and Su(H). Although no differences in overall levels of NICD and Su(H) could be detected by western blot (data not shown), they could be modified in different ways post-translationally leading to different Notch responses (e.g. core Notch response, N-only, NS-only). Indeed, recent reports point towards different post-translational modifications for Su(H). However, whether they lead to different transcriptional programs, and whether they occur in vivo in the N and NS models, remain to be studied. Through the use of iRegulon, we demonstrated that the genes of the NS transcriptome, and most importantly the NS Notch direct targets, were enriched in their regulatory regions for elements corresponding to specific transcription factors, and in particular Stat92E or bZIP factors. The fact that similar transcription factor families were found in the overall group of upregulated genes and in the more limited subset of Notch direct genes suggests that the Notch output was controlled, at least in part, by factors that act more broadly on the genome. These analyses support a model in which polarity loss redirects the output of the Notch transcriptional program by the action of cooperating transcription factors. However, further work, such as detailed comparative ChIP analyses of the different factors in the different conditions, is required to establish this model firmly (Logeay, 2022).

Although this study demonstrated the involvement of a similar 'oncogenic module' as identified for the RasV12/scrib^- neoplastic model, there are specifics that are likely oncogene specific. First, unlike what was reported for RasV12/scrib^- transcriptomes, Yki/Sd/TEAD modules were not found to be enriched in the different Notch and scrib^- transcriptomes. In the case of Ras, it has been shown that Yki activity can reprogram Ras by promoting the expression of the Ras pathway-specific regulators Capicua and Pointed to promote aggressive growth. Both genes were either unaffected (capicua) or downregulated (pointed) in the NS Notch-driven neoplastic paradigm, suggesting that, even though Yki is clearly required, changes in the expression of capicua and pointed are unlikely to be mediators here. These differing results in the enrichment of Yki/Sd/TEAD motifs between Notch and Ras transcriptomes in the context of polarity loss might reflect the inhibitory effect Notch has on Yki activity in the wing pouch, in part through the action of vestigial. Furthermore, in the NS transcriptome, a contribution was identified of the E(spl) bHLH transcriptional repressors, canonical Notch targets, which represents thus a Notch specificity. However, the fact that motifs for E(spl)-HLH repressors are found in the upregulated transcriptome of NS and not N could suggest that in NS the repressive ability of E(spl)-HLH factors is antagonized, further allowing higher expression of Notch targets. More precisely, the previous work identified many incoherent feed-forward loops in the N hyperplastic transcriptome, including through the action of E(spl) repressors, which might thus be resolved in NS. It would be interesting to explore further the link between NS and E(spl)-HLH-mediated repression, but due to the high redundancy between the seven E(spl)-HLH factors (Δ, &gamma', β, 3, 5, 7, 8) and Dpn, the requirement of E(spl)-HLH-mediated repression in Notch-driven neoplasia could not be formally tested (Logeay, 2022).

By performing functional assays to identify the genes and processes required for NS tumor growth, this study demonstrated that the Notch direct targets associated with 'de novo' NS-specific Su(H) peaks were unlikely to be major contributors. We did show, however, that the bZIP PAR domain-containing factor Pdp1 is required for NS tumor growth and invasiveness. Su(H) is bound in the vicinity of Pdp1 in all wing discs set-ups, and in particular in N and NS, and Pdp1 represents a 'core' Notch target activated in all overgrowth conditions, albeit at higher levels in polarity-deficient conditions. Pdp1 is not only a Notch target, but also a Jak/Stat target, at least in the developing eye, and canonical tandem Stat92E putative binding sites are found in its second intron, although not overlapping with Su(H) binding, which is found in its first intron. Interestingly, Pdp1 is required for Stat92E phosphorylation and efficient Jak/Stat signaling, suggesting that Notch might amplify Stat92E signaling during wing disc neoplastic growth, both through ligand expression (Upd ligands are Notch direct targets) and Pdp1 expression (Logeay, 2022).

Although Pdp1 downregulation could suppress NS neoplastic growth, it was not as efficient as JNK inhibition, or Yki downregulation, suggesting that other factors in parallel to Pdp1 might be involved, such as the previously identified Atf3, but also the other Notch direct target Ets21C. Indeed, RNAi-mediated knockdown of Atf3 or Ets21C partly suppressed Bx>NS tumor growth (GFP) and invasiveness (Mmp1). This action of both Pdp1 and Ets21C suggest a feed-forward loop downstream of Notch that in the context of polarity loss and JNK activity promotes neoplastic growth. However, given that Atf3, Pdp1 and Ets21C (but also Ftz-f1) are all upregulated in N hyperplastic conditions, their sole upregulation cannot be sufficient for neoplasia. The fact that Atf3 and Pdp1 iRegulon enrichments are not found in N could indicate that, despite being upregulated in hyperplastic N, their transcriptional activities are hindered, or that one key cooperating factor enabling their action is missing. Further studies are thus required to test this possibility and study how, in the context of normal epithelial polarity, Notch activation prevents the action of Pdp1/Ets21C/Atf3, thus preventing the transition to neoplasia (Logeay, 2022).

GENE STRUCTURE

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).

REGULATION

Promoter

The pattern of Pdp1 mRNA accumulation in the developing embryo suggests that the transcriptional regulation of Pdp1 is likely to be controlled by multiple cis-acting elements distributed over several kilobases of DNA. Accordingly, gene fragments that contain enhancers regulating Pdp1 expression have been identified. Of the restriction fragments tested, two contain enhancer activity that closely mimics endogenous Pdp1 expression. A 4.4-kb restriction fragment was identified within the first intron that directs midgut and fat body expression in the embryo and larva. This enhancer fragment drives expression in the midgut of stage 11 and 12 embryos and in later stage 14 and 16 embryos in the posterior midgut (fourth lobe), the Malpighian tubules, and the fat body in a pattern that is similar to that of the endogenous gene. This embryonic pattern of expression is maintained in first- and third-instar larvae (Reddy, 2000).

Pdp1 protein is expressed in the segmented mesoderm of stage 11 embryos. At this stage, the more dorsal mesodermal cells are the precursor cells of the fat body and the more ventral cells are the progenitor/founder cells of the body wall muscles. Since Pdp1 is expressed in the developing embryonic fat body and body wall muscles in later embryos, it is important to determine whether Pdp1 is expressed in the precursors of these tissues. Thus it is particularly significant that the 4.4-kb enhancer drives expression in the segmented dorsal mesoderm of stage 11 embryos, since this enhancer expresses in the fat body and not the body wall muscles in older embryos. Thus, it can be concluded that Pdp1 is expressed in the fat body precursor cells and in the developing fat body as it differentiates (Reddy, 2000).

A second fragment observed to have enhancer activity is an 11-kb fragment located in the second intron. This enhancer fragment is expressed in the pharyngeal muscle and the body wall muscles in the developing embryo and larva. Expression in the body wall muscles is particularly strong in a subset of ventral and dorsal muscles; weaker and/or no detectable expression is found in other muscles. In the midgut of embryos, the 11-kb enhancer shows very weak expression in the anterior midgut and strong expression in a region of the posterior midgut and the hindgut. This embryonic pattern gives rise to enhancer-driven expression in the gastric caeca, posterior midgut, and hindgut of the first- and third-instar larva. Enhancer activity is not detected in the middle midgut of embryos; however, endogenous Pdp1 is expressed in the basophilic cells of the middle midgut in the embryo. These cells are the precursors to the larval copper cells. Furthermore, the 11-kb enhancer expresses strongly in the copper cells of the middle midgut in all larval stages. These results suggest that a separate unidentified enhancer regulates expression in the basophilic and copper cells of the middle midgut in the embryo (Reddy, 2000).

Pdp1 is expressed in the precursor cells of the fat body in stage 11 embryos. The 11-kb enhancer also drives expression in the segmented mesoderm of stage 11 embryos. A close comparison shows, however, two or three small clusters of mesodermal cells in each hemisegment expressing beta-galactosidase driven by the 11-kb enhancer that are slightly ventral to those expressed by the 4.4-kb enhancer. The position of these cells corresponds to those of the muscle progenitor/founder cells at this stage. Furthermore, since the 11-kb enhancer expresses in the body wall muscles of later embryos and does not express in the fat body, it is concluded that Pdp1 is expressed in muscle precursor cells that will eventually fuse to form the body wall muscles in later embryos (Reddy, 2000).

Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors

Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions, muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search were performed for direct Ladybird targets. The data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, bric-a-brac 2 gene was identified as a new component of identity code and inflated encoding αPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells (Junion, 2007).

Uncovering how the cell fate-specifying genes exert their functions and determine unique properties of cells in a tissue is central to understanding the basic rules governing normal and pathological development. To approach the cell fate determination process at a whole genome level a search was performed for transcriptional targets of the homeobox transcription factor Lb known to be evolutionarily conserved and required for specification of a subset of cardiac and muscular precursors. To this end the targeted expression profiling and the novel ChIP-on-chip method ChEST were combined. The data revealed an unexpectedly complex gene network operating downstream from lb, which appears to act not only by regulating components of the cell identity code but also as a modulator of pan-muscular gene expression at fiber-type level. Of note, the role of Drosophila lb in regulating segment border muscle (SBM) founder motility appears reminiscent of the role of its vertebrate ortholog Lbx1, known to control the migration of leg myoblasts (Vasyutina, 2005) in mouse embryos (Junion, 2007).

Earlier genetic studies revealed that within the same competence domain the cell fate specifying factors acted as repressors to down-regulate genes determining the identity of neighboring cells. Consistent with this finding, lb was found to repress msh and kruppel (kr) during diversification of lateral muscle precursors and even skipped (eve) within the heart primordium. This study found that additional identity code components are regulated negatively by lb. In the lateral muscle domain lb acts as a repressor of the MyoD ortholog nau and the NK homeobox gene slou, both known to be required for the specification of a subset of somatic muscles. This suggests that a particularly complex network of transcription factors (Ap, Msh, Kr, Nau, Slou) controls the specification of individual muscle fates in the lateral domain. Interestingly, none of these factors is coexpressed with lb in the SBM, which appears to be a functionally distinct muscle requiring a specific developmental program. Besides factors with well-documented roles in diversification of muscle fibers, the global approach identified a few novel potential players in the muscle identity network. Among those that are are expressed in somatic muscle precursors are the Pdp1 gene encoding Par domain factor and the CG32611 gene containing a zinc finger motif (Junion, 2007).

Interestingly, in the cardiac domain the data demonstrate that lb is able to positively regulate the expression of tin and the effector of RTK pathway pointed (pnt), both involved in cardiac cell fate specification. These findings are consistent with earlier observations that the forced lb expression leads to the ectopic tin-positive cells within the dorsal vessel. Also, during early cardiogenesis lb directly represses bric a brac 2 (bab2), which emerges as a novel component of the genetic cascade controlling the diversification of cardiac cells. Thus, the ability of Lb to act either as repressor or as activator suggests a context-dependent interaction with cofactors. Of note, several miroarray identified Lb targets have also been found in the RNAi-based screen for genes involved in heart morphogenesis (Junion, 2007).

The data indicate that lb exerts its muscle identity functions via regulation of pan-muscular genes that control cell movements, cell shapes and cell-cell interactions including myoblast fusion, myotube growth, and attachment events (Junion, 2007).

Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component

Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, Per and Tim, repress Clk-Cyc-mediated transcription. To recognize additional direct Clk target genes, a genome-wide approach was designed and clockwork orange (cwo) was identified as a new core clock component. cwo encodes a transcriptional repressor functioning downstream of Clk that synergizes with Per and inhibits Clk-mediated activation. Consistent with this function, the mRNA profiles of Clk direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Impaired activity of Cwo leads to an elevated trough of per, tim, vri, and Pdp1 mRNA at ZT3 (three hours into the morning) in cwo RNAi transgenic flies compared with those of wild-type flies. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that Cwo acts preferentially in the late night to help terminate Clk-Cyc-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks (Kadener, 2007). To other studies similarly identified Clockwork orange an a transcriptional repressor that inhibits Clk-mediated activation (Matsumoto, 2007; Lim, 2007).

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

Clock (Clk) is a master transcriptional regulator of the circadian clock in Drosophila. To identify Clk direct target genes and address circadian transcriptional regulation in Drosophila, chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) were performed with a number of circadian proteins. Clk binding cycles on at least 800 sites with maximal binding in the early night. The Clk partner protein Cycle (Cyc) is on most of these sites. The Clk/Cyc heterodimer is joined 4-6 h later by the transcriptional repressor Period (Per), indicating that the majority of Clk targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. Clk has specific targets in different tissues, implying that important Clk partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation (Abruzzi, 2011).

Previous circadian models in Drosophila suggested a transcriptional cascade in which Clk directly controls a limited number of genes, including core clock genes, which then drive the oscillating expression of many different output genes. The results of this study indicate that Clk directly regulates not only the five core clock genes (i.e., pdp1, vri, tim, per, and cwo), but also many output genes, including ~60 additional transcription factors. Some of these are tissue-specific; e.g., lim1 and crp. In addition, Clk direct target gene regulation may impact timekeeping in yet unforeseen ways. For example, Clk, Per, and Cyc bind to three of the four known circadian kinases; i.e., dbt, nmo, and sgg. Although none of these mRNAs have been previously reported to cycle, both dbt and sgg have cycling Pol II, and dbt shows weak oscillations via qRT-PCR. nmo expression is enriched in circadian neurons and has been shown to cycle in l-LNvs. The data, taken together, indicate that this simple ChIP-chip strategy has uncovered important relationships and suggest that the transcriptional regulation of some of these new target genes warrants further investigation (Abruzzi, 2011).

Most of the 1500 Clk direct target genes are also bound by two other circadian transcription factors: Cyc and Per. Because a previous study showed that there is a progressive, ordered recruitment of Clk, Pol II, and Per on per and tim (Menet, 2010), the same basic mechanism is conserved on most Clk direct targets. Clk binding increases in late morning and gives rise to an increase in Pol II signal where detectable (ZT6-ZT10). Clk binding is maximal in the early night (ZT14), and both Clk binding and Pol II occupancy decrease rapidly after the repressor Per is bound to chromatin 4-6 h later, at ZT18. Interestingly, Per binds to nearly all Clk direct targets at the identical Clk/Cyc locations, suggesting Per recruitment via protein-protein interactions (Abruzzi, 2011).

The identical binding sites for Clk, Cyc, and Per suggest that binding is not background binding or 'sterile' binding with no functional consequence. This is because three components of the circadian transcription machinery are present with proper temporal regulation. Pol II cycling on ~30% of cycling Clk targets further supports this interpretation. The Pol II signal is maximal from mid- to late morning (ZT6-ZT10), which slightly anticipates the maximal transcription times of core circadian genes like per and tim. Most Pol II signals are promoter-proximal and may reflect poised Pol II complexes often found on genes that respond quickly to environmental stimuli (Abruzzi, 2011).

To address RNA cycling, ten direct target genes with Pol II cycling were examined. Eight of these genes show oscillating mRNA with >1.5-fold amplitude, suggesting that oscillating Pol II indeed reflects cycling transcription. Because this assay may underestimate cycling transcription due to tissue heterogeneity (i.e., masking by noncycling gene expression elsewhere in the head), ~30% is a minimal estimate of Clk direct targets with cyclical mRNA (Abruzzi, 2011).

Interestingly, previous microarray studies did not detect many of these genes. One possibility is that the alternative start sites that characterize 55% of Clk direct targets are not detectable on microarrays; e.g., moe and mnt. However, several mRNAs that cycle robustly by qRT-PCR are not isoform-specific and are also not consistently identified in microarray studies. A second possibility is that the relatively low cycling amplitude of many target genes -- twofold or less, compared with the much greater amplitudes of core clock genes such as tim, per, and pdp1, assayed in parallel -- may be more difficult to detect on microarrays (Abruzzi, 2011).

Low-amplitude cycling may result from relatively stable mRNA, which will dampen the amplitude of cycling transcription. Alternatively, or in addition, low-amplitude cycling may reflect cycling in one head location and noncycling elsewhere within the head, which will dampen head RNA cycling amplitude. This is likely for many eye-specific Clk targets, which appear expressed elsewhere in the head via a Clk-independent mechanism (Abruzzi, 2011).

A third and arguably more interesting explanation for low-amplitude cycling is that Clk binds on promoters with other transcription factors within single tissues. These could include chromatin modifiers and would function together with Clk in a gene- and tissue-specific fashion. For example, a gene could be constitutively expressed at a basal level by one transcription factor, with temporal Clk binding causing a modest boost to transcription. For example, gol is a Clk target exclusively in the eye, and gol mRNA cycles with a fourfold amplitude. Rather than cycling from 'OFF' (no or very low mRNA levels) to 'ON,' however, gol mRNA levels are quite high even at the trough or lowest time points. This suggests that gol cycles from a substantial basal level in the late night and daytime to an even higher level of expression in the evening and early night. Since mRNA levels decrease by >10-fold in GMR-hid flies, trough transcription levels are not likely from other tissues. Therefore, Clk probably acts on gol and other targets not as an 'ON/OFF switch,' but rather in concert with other factors to boost a basal level of gene expression at a particular time of day and cause low-amplitude cycling within a single tissue (Abruzzi, 2011).

The large number of Clk target genes in fly heads is explained in part by tissue-specific Clk binding. Transcription assays that measure the cycling of mRNA and Pol II binding in one head tissue can be masked by noncycling expression in another. The ChIP assays, in contrast, are not plagued with the same problem. They can identify a gene bound by the cycling circadian transcription machinery even if the same gene is constitutively expressed elsewhere in the head. Surprisingly 44% of Clk direct targets were no longer detected when eyes were ablated with GMR-hid. Because many of these mRNAs are not particularly eye-enriched, it is inferred that their genes are constitutively expressed under the control of other transcription factors elsewhere in the head (Abruzzi, 2011).

The large number of target genes is also explained by the efficiency and sensitivity of the ChIP assay. It is inferred that it can detect Clk binding from a relatively low number of cells within the fly head. Lim1 is one example and is expressed predominantly in a subset of circadian neurons (l-LNvs; enriched more than four times relative to head). Preliminary cell-specific Clk ChIP-chip experiments from LNvs confirm that lim1 is an enriched Clk direct target in these cells, suggesting that this is the source of a large fraction of the binding signal in the head ChIP-chip experiments. Experiments are under way to more clearly define circadian neuron-specific Clk-binding patterns (Abruzzi, 2011).

This tissue specificity also suggests the existence of factors and/or chromatin modifications that help regulate Clk-mediated gene expression. They could enable Clk binding to specific genes in one tissue or inhibit binding in another tissue. These tissue-specific factors are strongly indicated by the pdp1 and lk6 Clk-binding patterns, which change so strikingly and specifically in GMR-hid. Although not unprecedented, tissue-specific factors that enable or inhibit specific DNA-binding locations are intriguing and warrant further investigation and identification (Abruzzi, 2011).

Targets of Activity

Pdp1 is involved in regulating expression of the Tropomyosin I gene in somatic body-wall and pharyngeal muscles by binding to DNA sequences within the muscle activator that are required for activator function. Mutations that eliminate Pdp1 binding eliminate muscle activator function and severely reduce expression of a muscle activator plus Mef2 mini-enhancer. These and previous results suggest that Pdp1 may function as part of a larger protein/DNA complex, which interacts with Mef2 to regulate transcription of Drosophila muscle genes (S. C. Lin, 1997).

Drosophila ensconsin promotes productive recruitment of Kinesin-1 to microtubules

Ensconsin is a conserved microtubule-associated protein (MAP) that interacts dynamically with microtubules, but its cellular function has remained elusive. This study shows that Drosophila ensconsin is required for all known kinesin-1-dependent processes in the polarized oocyte without detectable effects on microtubules. ensconsin is also required in neurons. Using a single molecule assay for kinesin-1 motility in Drosophila ovary extract, it was shown that recruitment to microtubules and subsequent motility is severely impaired without ensconsin. Ensconsin protein is enriched at the oocyte anterior and apically in polarized epithelial cells, although required for localization of posterior determinants. Par-1 is required for ensconsin localization and directly phosphorylates it at conserved sites. These results reveal an unexpected function of a MAP, promoting productive recruitment of a specific motor to microtubules, and an additional level of kinesin regulation. Furthermore, spatial control of motor recruitment can provide additional regulatory control in Par-1 and microtubule-dependent cell polarity (Sung, 2008).

Asymmetric cell polarization is fundamental to embryonic development and to the functions of epithelial cells. For cells that are large, like some oocytes, or have long extensions, such as neurons, the microtubule cytoskeleton contributes to polarity and provides the means for directional long-distance transport. As microtubules are inherently polarized, spatial control of their nucleation and growth can give a polarized cytoskeleton. This polarity is then 'read out' by minus-end-directed motors, primarily the dynein motor complex, and plus-end-directed motors, such as classical kinesins (Sung, 2008).

Microtubules interact with, and are regulated by, a large number of proteins. Apart from motors, diverse families of conserved microtubule-associated proteins (MAPs) have been identified. Their functions can vary, affecting microtubule growth, stability, or interactions. They may also affect the directional motors - for example, acting as 'roadblocks.' Tau, a well-studied MAP involved in neurodegeneration, interferes with kinesin-1 transport when overexpressed in cells, and with the processive movement of purified kinesin-1 motors on microtubules. This shows that Tau can block the kinesin-1 motor, but whether this is its physiological function remains a matter of debate. One complication in assigning biological function to MAPs lies in the difficulty of bridging the gap between knowledge obtained from detailed in vitro experiments that use artificially naked microtubules and incomplete motor complexes and the physiological effects that can be observed in cells and tissues (Sung, 2008).

Mammalian Ensconsin, also called E-MAP-115 for epithelial microtubule-associated protein of 115 kD, was isolated biochemically by virtue of its ability to tightly associate with microtubules). This study used the name ensconsin to avoid confusion with the unrelated EMAP (echinoderm MAP). Ensconsin binds along the length of microtubules, and early studies suggested that it might stabilize microtubules. However, this idea has not been supported by subsequent experiments involving less extreme overexpression. In addition, live imaging showed that the association between ensconsin and microtubules is very dynamic in cells and appears to be regulated by phosphorylation events. Phosphorylation of ensconsin and its microtubule association is regulated during the cell cycle. Mice mutant for ensconsin/E-MAP-115 are viable, but have defects in microtubule-rich structures in spermatogenesis. More widespread functions might be obscured by genetic redundancy, however, as mammalian genomes encode a related protein, RPRC1. Drosophila has only one gene of this family, which is investigated here (Sung, 2008).

The oocyte of Drosophila is a well-studied example of microtubule-dependent cell polarization. At mid-to-late oogenesis, localization of the anterior determinant bicoid requires the anterior microtubules and the dynein motor complex, whereas posterior determinants Staufen and oskar mRNA require kinesin-1, a plus-end-directed motor. Recent analysis indicates that oskar mRNA transport is primarily mediated by kinesin-1, itself performing a random biased walk along a weakly polarized cytoskeleton. In many contexts, the Par proteins are important for polarity, establishing mutually exclusive cortical domains, and affecting the cytoskeleton. Setting up oocyte polarity also requires Par-1 kinase, which itself is enriched at the posterior. Par-1 activity, together with the phosphorylation-induced binding of 14-3-3/Par-5 to target proteins, restricts proteins, such as Bazooka/Par-3, to the apical domain in epithelial cells. A similar mechanism may act downstream of Par-1 in the germline, but here the relevant Par-1 targets are not known. A link from Par-1 to microtubules appears conserved even if not fully understood. A mammalian homolog of Par-1, microtubule affinity regulating kinase (MARK), was identified based on its ability to phosphorylate microtubule-associated proteins and thereby affect microtubules. The MAP Tau is phosphorylated by Par-1/MARK in both mammalian and Drosophila cells. However, Tau is not required for development or for Drosophila oogenesis. Par-1/MARK may have multiple targets that impinge on the microtubule cytoskeleton. This study found that ensconsin/E-MAP-115 is a physiologically significant Par-1 target in the oocyte. Par-1 regulates ensconsin localization, which, in turn, is required for effective kinesin-1-dependent transport (Sung, 2008).

In a screen for genes affecting development of the Drosophila female germline, a PiggyBac insertion was identified in the gene CG14998. The encoded protein is similar to mouse ensconsin/E-MAP-115, with two regions of higher similarity that are called ensconsin homology region (EHR)-1 and EHR2. For both mammalian and Drosophila proteins, multiple isoforms exist due to alternate splicing immediately before and after EHR1. With a microtubule pelleting assay routinely used to identify MAPs, the endogenous CG14998 protein was found to associate with microtubules in embryo extracts as well as in ovary extract. In mouse ensconsin, the N-terminal 200 amino acids were responsible for microtubule association. Similarly, in CG14998, a truncated protein removing EHR2 but retaining EHR1 (Ens-X) still associated with microtubules when expressed in the ovary, whereas removing EHR1 but retaining EHR2 (Ens-C) abrogated microtubule binding, whether the protein was expressed at modest or high levels. Finding CG14998 to be a likely functional ortholog of mouse ensconsin/E-MAP-115, it was named ensconsin (Sung, 2008).

The microtubule cytoskeleton is complex and dynamic, with extensive regulation of microtubule growth and turnover. It is not clear, however, whether the resulting polarized microtubules are simply roads to be freely used by motors or whether there is significant crowding on them and assistance is needed to get on. In this study, this study provides evidence that a MAP may function to stimulate productive interaction of a specific directional motor, kinesin-1, with microtubules in vivo. This was demonstrated using a combination of genetic analysis and single-molecule imaging performed in cell extracts. That Ensconsin is required for efficient kinesin-1-dependent transport in vivo, irrespective of which cargo is being transported, was demonstrated by the specificity of the mutant phenotypes. This was shown in detail for the oocyte, but analysis of neuronal phenotypes suggests that the function of Ensconsin is general. The kinesin motility assay in extract allowed direct analysis of physiologically active kinesin with and without Ensconsin present under conditions close to the cellular environment, but on experimentally defined microtubule tracts. Previous studies have shown that some MAPs can regulate the dynamics of microtubules themselves, and that others can act as 'roadblocks,' inhibiting motor traffic. Aiding microtubule motor activity somewhat unexpected function for a MAP, and has not been shown previously (Sung, 2008).

The difference in the effect of Ensconsin on full-length kinesin versus the short 'constitutively active' form highlights the importance of finding appropriate ways to analyze complete and functional motor proteins in order to understand their regulation. The method described here, using functional kinesin and cell extracts, should be helpful in this regard. Importantly, the results with and without Ensconsin reveal an additional level of regulation of the inherently very active motor, kinesin-1. Binding of specific cargo is thought to stimulate motor activity by relieving autoinhibition in classical kinesins. This may ensure that only occupied kinesin motors interact with microtubules. A dedicated regulator, such as Ensconsin, can provide spatial and temporal control of the motor, regardless of the cargo. Ensconsin protein accumulation is itself spatially controlled, and microtubule association of Ensconsin may be controlled by phosphorylation, allowing recruitment of kinesin/cargo complexes to microtubules to be intricately regulated. As the truncated kinesin is active regardless of ensconsin, both in vivo and in extracts, this additional level of control also appears to operate as relief of inhibition. This implies that most kinesin in the cell is in an inactive state, and multiple positive inputs converge to allow actual motility (Sung, 2008).

Tau is a MAP that, like Ensconsin, binds along microtubules, and, in some assays, is able to stabilize microtubules. Tau has been extensively studied due to its association with neurodegeneration, but its actual biological function is still somewhat unclear. In cells, moderately overexpressed Tau inhibits the function of classical kinesin. Tau may inhibit the productive attachment of kinesin-1 motor to microtubules and thereby also processivity of movement for cargoes with more that one kinesin motor. Ensconsin does the opposite. Such potentially counteracting MAPs may allow fine-tuned control over trafficking along microtubules, which would be of particular importance in neurons and other large, polarized cells (Sung, 2008).

The present findings for Drosophila Ensconsin are likely to apply to mammalian ensconsin as well. Mammalian ensconsin was found to associate dynamically with microtubules along their length, and modest overexpression did not affect microtubule stability. Similarly, this study did not observe overt defects in microtubule density upon removal of ensconsin in vivo. Like the fly protein, mammalian ensconsin is apically localized in a polarized epithelium (Vanier, 2003) and contains potential 14-3-3 binding sites between EHR1 and EHR2, which are likely Par-1 phosphorylation sites. No clear requirement was found for Ensconsin in the follicular epithelium, but it may be subtle or redundant with other regulators. In both mammals and flies, Par-1/MARK is unlikely to be the only kinase regulating ensconsin. This is indicated by the heavy cell cycle-dependent phosphorylation of mammalian ensconsin affecting its interaction with microtubules (Masson, 1995), as well as the 'background' (Par-1-independent) phosphorylation seen in the current kinase assays with oocyte proteins. Finally, EHR1 is responsible for microtubule binding in both mammalian and Drosophila ensconsin. The second conserved domain, EHR2, may be responsible for the specific effect on kinesin. A detailed biophysical analysis will be needed to determine exactly how Ensconsin stimulates both recruitment and subsequent motility of full-length kinesin. A simple hypothesis would be recruitment by direct physical interaction, which has so far not been detected. However, given that ensconsin also has a positive effect on kinesin movement, their interaction would most likely be transient and/or regulated -- and possibly not straightforward to observe (Sung, 2008).

Localization of ensconsin is controlled at multiple levels, including mRNA localization, Par-1 phosphorylation, and a dependency on microtubules. The Ens-mut mislocalization shows that at least some of the effects of Par-1 on ensconsin are direct. The clear microtubule dependency in the oocyte indicates that Par-1 phosphorylation is not sufficient for biased Ens localization; it requires transport or other microtubule-dependent events as well. Spatially, Par-1 negatively affects Ensconsin, and this agrees with the phenotype of mislocalized Ensconsin resembling that of Par-1 loss of function. Why is Ensconsin subject to this regulation, and how may this contribute to polarity? A recent study argues convincingly that the posterior determinant oskar is localized primarily as a cargo of kinesin-1, with motor-cargo particles moving inefficiently by biased random walk toward the posterior. The implication is that microtubules in the oocyte have a net polarity in the expected direction (more minus ends anteriorly), but it is only a weak bias with many microtubules pointing in other directions. A higher level of Ensconsin at the anterior/lateral cortex can promote more productive interactions of kinesin-cargo complexes with microtubules in this region, and, hence, contribute to the bias of posteriorly directed events. At the posterior, microtubule density is relatively low, but kinesin-1 accumulates over time. Low levels of Ensconsin (thus, very little active kinesin-1) will discourage active 'backwards' transport of kinesin cargoes. The asymmetric Ensconsin distribution can thus contribute to asymmetry in microtubule-dependent transport (Sung, 2008).

The oocyte is a large and dynamically polarized structure likely utilizing multiple overlapping mechanisms of polarity establishment and maintenance. Microtubule-dependent asymmetry and polarity can be affected by microtubule density, as well as by bias in orientation. Both processes may be modulated by Par-1. The function and regulation of Ensconsin described in this study indicate that regulators of microtubule-dependent polarity need not affect microtubules themselves, but may also control the effectiveness of the directional motors that 'read out' the polarity of the microtubules (Sung, 2008).

Protein Interactions

Par-1 controls myosin-II activity through myosin phosphatase to regulate border cell migration

Localized actomyosin contraction couples with actin polymerization and cell-matrix adhesion to regulate cell protrusions and retract trailing edges of migrating cells. Although many cells migrate in collective groups during tissue morphogenesis, mechanisms that coordinate actomyosin dynamics in collective cell migration are poorly understood. Migration of Drosophila border cells, a genetically tractable model for collective cell migration, requires nonmuscle myosin-II (Myo-II). How Myo-II specifically controls border cell migration and how Myo-II is itself regulated is largely unknown. This study shows that Myo-II regulates two essential features of border cell migration: (1) initial detachment of the border cell cluster from the follicular epithelium and (2) the dynamics of cellular protrusions. It was further demonstrated that the cell polarity protein Par-1 (MARK), a serine-threonine kinase, regulates the localization and activation of Myo-II in border cells. Par-1 binds to myosin phosphatase (Flap wing) and phosphorylates it at a known inactivating site. Par-1 thus promotes phosphorylated myosin regulatory light chain, thereby increasing Myo-II activity. Furthermore, Par-1 localizes to and increases active Myo-II at the cluster rear to promote detachment; in the absence of Par-1, spatially distinct active Myo-II is lost. This study has identified a critical new role for Par-1 kinase: spatiotemporal regulation of Myo-II activity within the border cell cluster through localized inhibition of myosin phosphatase. Polarity proteins such as Par-1, which intrinsically localize, can thus directly modulate the actomyosin dynamics required for border cell detachment and migration. Such a link between polarity proteins and cytoskeletal dynamics may also occur in other collective cell migrations (Majunder, 2012).

Myo-II plays a fundamental role in establishing the front-rear axis of migrating cells to promote directional migration. Myo-II localizes to the cell rear and stimulates motility at the front, likely by local stabilization of adhesions and actomyosin bundles at the cell rear but not at the front. In contrast to single cells, the mechanisms that set up or maintain polarized actomyosin contraction during collective migration are still poorly understood. This study identified a new role for Par-1 kinase, namely that Par-1 regulates myosin phosphatase to control Myo-II activation. A model is proposed in which Myo-II is activated in a polarized manner). Myosin phosphatase, which is distributed uniformly in the cluster, is locally inactivated by Par-1 at the basolateral side (back) of the cluster. The consequent polarization of active Myo-II induces contraction and cell morphological changes critical for detachment and motility. The question of how Par-1 becomes localized to the basolateral side of border cells is largely unknown. Phosphorylation by the apical polarity protein aPKC restricts Par-1 to basolateral membranes in epithelial cells and is also critical for Par-1 function in border cells. This mechanism may thus restrict basolateral localization of Par-1 in border cells, although a role for border cell-specific factors cannot be ruled out (Majunder, 2012).

The observation that there is an increase in Sqh:GFP at the rear of the border cell cluster during detachment is consistent with a specific role for Myo-II in promoting epithelial detachment of border cells. Loss-of-function sqh mosaic clone experiments demonstrated a requirement for Myo-II in border cells and possibly adjacent epithelial follicle cells. Indeed, live imaging analyses revealed that disruption of Myo-II function inhibited the ability of border cells to detach. This raises the question of how Myo-II contributes to detachment. Activation of Rok by Rho GTPase can destabilize cell-cell junctions by inducing actomyosin contraction in normal and tumor-derived epithelial cells. In other contexts, however, Rho-dependent Myo-II stabilizes cell junctions through regulation of the junctional protein E-cadherin. The overall levels of E-cadherin were unchanged when Rok was knocked down in border cells, suggesting that activated Myo-II more likely contributes directly to detachment. It is suggested that the localized increase in active Myo-II at the rear specifically contracts the border cell cluster and helps it pull away from the epithelium. In the absence of Par-1, overall levels of activated Myo-II were decreased and Sqh:GFP foci, which correlate with active Myo-II, exhibited altered dynamics; this potentially leads to uncoordinated or decreased contractile forces and thus to defects in detachment (Majunder, 2012).

Par-1 promotes increased p-MRLC/Sqh levels and higher levels of activated myosin by phosphorylation of myosin phosphatase at a known inactivating threonine. Regulated myosin phosphatase activity is essential for many cellular processes, including cell motility and epithelial morphogenesis. Despite identification of kinases that inactivate myosin phosphatase in vitro, few have been shown to do so in vivo during migration. Notably, vertebrate MARK2 (Par-1) phosphorylates the Mbs homolog MYPT1 in vitro at several sites, including the conserved threonine examined in the current study, although this has not been confirmed in vivo. This raises the intriguing possibility that vertebrate Par-1 homologs regulate myosin phosphatase, and thus Myo-II, during cell migration. However, Par-1-mediated regulation of Myo-II phosphorylation via myosin phosphatase may be cell or context specific. In contrast to the situation in border cells, Par-1 does not colocalize with myosin phosphatase in Drosophila ovarian epithelial follicle cells; active p-MRLC/Sqh and Mbs localized to apical domains in follicle cells whereas Par-1 localizes to basolateral membrane (Majunder, 2012).

Active Myo-II accumulates at the apical side/front of the border cell cluster in addition to its localization at the rear. Myo-II that is localized near the leading edge of single cells has been proposed to promote retraction by coordinating cell-substrate adhesions with the actin cytoskeleton. Likely roles for Myo-II at apical (front) side of the border cell cluster include retraction of protrusions, as well as resolving protrusion dynamics from the pre- to post-detachment phases of migration. The data do not explicitly support a role for Par-1 at the apical side of the cluster. Moreover, in the absence of Par-1, a low level of phosphorylated MRLC/Sqh was detected that was still partially localized. Thus, Myo-II is activated by at least one other kinase in addition to Par-1 (Majunder, 2012).

It was hypothesize that Par-1 promotes higher levels of Myo-II activity at the basolateral side (back), whereas another kinase activates Myo-II (and/or inactivates the phosphatase) specifically at the front. Rok can phosphorylate both MRLC and myosin phosphatase. Knockdown of Rok by RNAi significantly reduced p-MRLC/Sqh levels and disrupted border migration. The combined depletion of both Par-1 and Rok almost completely abolished detachment, suggesting that the two kinases converge (directly or indirectly) on the same target (Myo-II). Epithelial morphogenesis during C. elegans embryonic elongation and Drosophila larval tissue development require multiple kinases to optimally activate Myo-II. Furthermore, different kinases have been shown to regulate MRLC activation at discrete locations within single migrating cells. For example, MLCK phosphorylates MRLC at the front or leading edge, whereas Rok targets MRLC in the cell body and at the trailing edge of fibroblasts. However, it remains to be determined whether Rok and/or additional kinases have a polarized or more general role in Myo-II activation in border cells (Majunder, 2012).

This study demonstrates that maintaining localized Myo-II activity is a critical feature of collective cell detachment and motility and identifies the conserved polarity kinase Par-1 as a key new regulator of this pathway. Active Myo-II is polarized within the border cell cluster, rather than in individual border cells, emphasizing that asymmetrically activated Myo-II contributes to collective behavior. Notably, in a model of collective cancer cell invasion, high actomyosin activity at cell-matrix contacts combined with low activity at contacts between cells within the group, produced optimal contractile force around the outside and thus promoted collective cell movement. It will be important to determine whether vertebrate Par-1 homologs also regulate actomyosin contraction during processes that depend on collective cell motility, such as wound healing or tumor invasion and metastasis. Given that many metastasizing tumors detach from epithelia both as single cells and collective groups, it will be important to further probe the mechanisms of myosin-mediated contraction in this process (Majunder, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

Drosophila PDP1 mRNA can be detected as early as 3-6 hours. At 9-12 hours and continuing through hatching, seven different-sized RNAs are detected with the Pdp1 probe. By whole-mount in situ hybridization, PDP1 transcripts are first detectable at stage 9 in the developing posterior endoderm midgut primodium and, by stage 11, in both anterior and posterior endoderm midgut primordia and the cephalic mesoderm. The expression pattern is quite uniform and at a relatively high level in both midgut primordia. By stage 14, Pdp1 continues to be expressed, albeit unevenly, in the forming midgut endoderm and the cephalic mesoderm as it forms the pharyngeal muscle. Pdp1 is also expressed in the epithelial cells of the hindgut at this stage and in the Malpighian tubules in later stage embryos. Pdp1 expression can also be seen in the developing somatic body-wall muscles spanning the anterior-posterior and dorsal-ventral axes and in the fat body of stage 14 embryos. In cross sections of stage 13 embryos, PDP1 RNA can be detected in unfused somatic myoblasts that give rise to the body wall muscles. RNA expression is not detected in the muscle or fat body in twist mutant embryos since these embryos do not make mesoderm. PDP1 mRNA has not been detected in the cardial or pericardial cells of the dorsal vessel. PDP1 RNA is also expressed in the epidermis of stage 14 and stage 16 embryos, the latter also showing expression in mature muscle fibers and fat body. In late-stage embryos, a low level of Pdp1 expression is also seen in a small subset of cells in the central nervous system (S. C. Lin, 1997).

The Pdp1 gene is complex, containing at least four transcriptional start sites and producing at least six different mRNAs and Pdp1 isoforms. Five of the Pdp1 isoforms differ by the substitution or insertion of amino acids at or near the N-terminal of the protein. At least three of these alternately spliced transcripts are differentially expressed in different tissues of the developing embryo in which Pdp1 expression is correlated with the differentiation of different cell types. A sixth isoform is produced by splicing out part of the PAR and basic DNA binding domains, and DNA binding and transient transfection experiments suggest that it functions as a dominant negative inhibitor of transcription. Furthermore, two enhancers have been identified within the gene that express in the somatic mesodermal precursors to body wall muscles and fat body; together, they direct expression in other tissues that closely mimics the expression of the endogenous gene. These results show that Pdp1 is widely expressed, including in muscle, fat, and gut precursors, and is likely involved in the transcriptional control of different developmental pathways through the use of differentially expressed Pdp1 isoforms. Furthermore, the similarities between Pdp1 and the other PAR domain genes suggest that Pdp1 is the homolog of the vertebrate genes (Reddy, 2000).

Pdp1 RNA is expressed in the somatic mesoderm that gives rise to the body wall and pharyngeal muscles and fat body; the developing foregut, midgut, hindgut, and Malpighian tubules, and the epidermis. The synthesis and accumulation of mRNA do not necessarily ensure the synthesis of protein, however. Accordingly, it was of interest to determine whether Pdp1 protein correlates with the pattern of Pdp1 mRNA; an anti Pdp1-alpha antibody has been used in whole-mount embryo analysis to determine its expression pattern. Pdp1 protein is detected in stage 10 embryos in the cephalic mesoderm, the endoderm of the developing anterior and posterior midgut, and the segmented mesoderm of the extended germ band that will give rise to the fat body and somatic body wall muscles. The cephalic mesoderm staining gives rise to the pharyngeal muscles seen in the stage 14 embryos. The somatic mesoderm develops into the fat body and body wall muscles, and Pdp1 protein can be seen in their nuclei. Pdp1 expression in the developing gut includes the esophagus and the hindgut. Pdp1 is also expressed in Malpighian tubules. This pattern of Pdp1 protein expression coincides precisely with the expression of PDP1 mRNA. The expression of Pdp1 protein in the midgut is better resolved than in the RNA studies. Initially, Pdp1 is expressed uniformly throughout the midgut from stage 11 and up to early stage 13. The pattern of Pdp1 expression, however, very quickly becomes restricted in stage 14 embryos to the anterior portion of the midgut. It is this part of the midgut, which gives rise to developing gastric caeca, the large nuclei of the basophilic cells of the second (middle) midgut lobe, and the cells of the fourth (posterior) midgut lobe. The basophilic cells of the middle midgut differentiate to form the interstitial and copper cells of the late embryo and larva (Reddy, 2000).

The timing and pattern of Pdp1 protein in the different regions of the gut suggest that Pdp1 may play a fundamental role in their differentiation. Low amounts of Pdp1 protein are seen in the brain and nuclei of cells of the central nervous system (Reddy, 2000).

The Pdp1 gene encodes multiple proteins and raises the possibility that they may be differentially expressed and have unique functions. Since isoform-specific antibodies are not available, the alternately spliced exons were subloned and each was used in whole-mount in situ hybridization. The largest of the alternately spliced exon probes available (exon 3) is the 395-nt Pdp1-epsilon antisense probe. Using this exon probe, hybridization was detected in the central nervous system and the brain of stage 13-14 embryos. These results indicate that Pdp1-epsilon mRNA is expressed at a significantly lower level than total Pdp1 RNA. The alpha/beta/delta common second exon probe is 200 bp and relatively strong hybridization could be observed after 90 min of development. Indeed, the pattern of expression depicted by this exon 2 probe closely resembles the expression pattern observed for the entire Pdp1-alpha cDNA clone, which represents all Pdp1 mRNAs. Hybridization was observed in the developing anterior and posterior midgut rudiments of stage 11 embryos and the elongated midguts of stage 12 and 13 embryos, and later expression was seen in the gastric caeca and the anterior and posterior midgut. Hybridization could also be detected in the hindgut and Malpighian tubules and in the pharyngeal and body wall muscles. Hybridization was detected in the developing midgut of stage 11 and 12 embryos. By stage 13, Pdp1-gamma mRNA could be seen in the developing gastric caeca, middle midgut, Malpighian tubules, and fat body. In addition, barely detectable hybridization was occasionally observed in the pharyngeal muscles but not the body wall muscles. However, because the body wall muscles are adjacent and peripheral to the fat body and because the hybridization signal is low, it is not possible to rule out low-level expression in the body wall muscles. Expression of the Pdp1-phi (exon 4) mRNA in embryos could not be detected by PCR and thus was not hybridized to whole-mount embryos. Since the Pdp1-phi cDNA was isolated from an adult library, its expression may be limited to adult tissues (Reddy, 2000).

EFFECTS OF MUTATION

Pdp1 is a regulator of larval growth, mitosis and endoreplication

PDP1 is a basic leucine zipper (bZip) transcription factor that is expressed at high levels in the muscle, epidermis, gut and fat body of the developing Drosophila embryo. Three mutant alleles of Pdp1 have been identified, each having a similar phenotype. This study describes in detail the Pdp1 mutant allele, Pdp1^p205, which is null for both Pdp1 RNA and protein. Interestingly, homozygous Pdp1^p205 embryos develop normally, hatch and become viable larvae. Analyses of Pdp1 null mutant embryos reveal that the overall muscle pattern is normal as is the patterning of the gut and fat body. Pdp1^p205 larvae also appear to have normal muscle and gut function and respond to ecdysone. These larvae, however, are severely growth delayed and arrested. Furthermore, although Pdp1 null larvae live a normal life span, they do not form pupae and thus do not give rise to eclosed flies. The stunted growth of Pdp1^p205 larvae is accompanied by defects in mitosis and endoreplication similar to that associated with nutritional deprivation. The cellular defects resulting from the Pdp1^p205 mutation are not cell autonomous. Moreover, PDP1 expression is sensitive to nutritional conditions, suggesting a link between nutrition, PDP1 isotype expression and growth. These results indicate that Pdp1 has a critical role in coordinating growth and DNA replication (Reddy, 2006).

Pdp1 is dispensable for embryogenesis despite the fact that Pdp1 is expressed in several tissues during embryogenesis. Tissues such as the gut, fat body, and muscle, which express PDP1 at high levels as they differentiate, have normal morphology in Pdp1^p205 embryos, and all appeared to function normally in Pdp1^p205 larvae. Although maternally supplied stores of mRNA and proteins can obfuscate loss-of-function embryonic phenotypes by compensating for loss of zygotic function, this does not seem to be the case here since maternally supplied Pdp1 messenger RNA or protein is not detected in early embryos. Therefore, either Pdp1 is not necessary for embryonic development, the effects of Pdp1 loss-of-function are subtle and not detected in the assays used, or Pdp1 function is being compensated for by another gene. This latter possibility is consistent with previously reported deletion analysis of PDP1 binding sites in the context of the larger muscle enhancer of the TmI gene (Reddy, 2006).

The Pdp1^p205 mutant embryos develop and hatch normally and become larvae that are able to crawl and eat, indicating normal muscle function and at least rudimentary gut function. However, these Pdp1^p205 larvae are severely growth delayed compared to their heterozygous counterparts. This Pdp1^p205 larval phenotype is very similar to the phenotype of amino acid starved larvae and larvae derived from dTOR and slimfast mutants, all of which affect larval growth through altered endoreplication. The growth of the larva is dependent upon its nutritional state. Wild-type larvae starved of nutrients (amino acids) have retarded growth that mimics the Pdp1 phenotype, although Pdp1^p205 larvae do crawl and eat. The coordination of growth with nutritional conditions is most readily observed in the endoreplicating cells of the larva. In larvae, the initial uptake of amino acids from an exogenous food source stimulates both endoreplicative and mitotic cell cycles. In addition, a constant influx of dietary amino acids is necessary to maintain endoreplication, as evidenced by complete loss of endoreplication 24 h after food withdrawal. Mitotic cells, in contrast, require only an initial pulse of amino acids to stimulate mitotic cycling since these cells continue cycling after food withdrawal. Thus, a fundamental difference between Pdp1^p205 larvae and amino acid starved larvae is that in Pdp1^p205 larvae, endoreplication does occur after hatching as assayed via BrdU incorporation. In wild-type and Pdp1^p205 larvae that are at 48 h AEL (late first instar for wild type), endoreplication is occurring in three locations within the gut during the 6-h BrdU incorporation time (Reddy, 2006).

Since endoreplication does occur, these larvae are not starving per se; they take up amino acids. Furthermore, they appeared to have normal gut functions, and adding amino acids to the diet of Pdp1^p205 larvae did not improve growth or viability. However, although endoreplication is occurring in first instar Pdp1^p205 larvae, it is progressing at a much slower rate since BrdU incorporation could not be readily detected after a 3-h period of incubation even though these were optimal conditions for wild-type larvae. Instead, a two- to three-fold longer incubation is required in Pdp1^p205 larvae to reach a comparable level of BrdU incorporation as in wild-type larvae. Thus, it is concluded that although larval cells are endoreplicating in young Pdp1^p205 larvae (48 h AEL), they are already doing so at a much slower rate, leading to a severely reduced number of endocycles. This is unlike older larvae where little or no endoreplication is observed. Since adding exogenous sucrose improved upon the phenotype, it is suspected that although Pdp1^p205 larvae may be getting sufficient nutrients in their diet, they may suffer from a metabolic defect that results in altered carbohydrate metabolism. Such larvae may not breakdown glucose or transport glucose efficiently and thus may grow slowly and experience developmental delays which may be partly overcome by the increasing the sugar (sucrose). It is known, for instance, that insulin signaling from the brain promotes mitosis and endoreplication and is particularly sensitive to carbohydrate levels. Therefore, it is possible that extra stimulation of the insulin-signaling pathway is able to partially bypass the Pdp1 null phenotype (Reddy, 2006).

In wild-type animals, DNA synthesis (as visualized by BrdU incorporation) is also occurring in the mitotic imaginal cells of the brain, gut, and discs that will give rise to adult organs. In young Pdp1^p205 mutant larvae (48 h AEL), BrdU incorporation into these cells is dramatically reduced and/or apparently absent in some cases. This lack of cell cycle progression carried over into older larvae (72 h AEL), a timeframe during which endoreplication had essentially ceased in the mutants even though midgut, salivary gland, and fat body cells are actively undergoing endoreplication in wild-type larvae. Also striking are the severely reduced levels of mitotic cell types including the imaginal cells of the midgut, brain neuroblasts, and imaginal disc cells. Thus, in comparison with endoreplicating cells, mitotically active cells in the larva seem to be especially sensitive to the loss of PDP1 function (Reddy, 2006).

Pdp1^p205 mutants also have abnormal circadian rhythm. It was shown previously that PDP1-ε is a positive regulator of Clock and is part of a second feedback loop in the circadian clock mechanism of cyclic output regulation. Thus, Pdp1^p205 mutants do not express TIM, PER, or PDF in the pacemaker cells of the brain. In mice, it has been shown that the circadian clock controls expression of cell cycle genes that regulate mitosis. Moreover, in vertebrates and Drosophila, hormonal levels regulated by circadian output control nutritional and behavioral responses. In Drosophila, the clock-regulated output gene takeout (to) has been shown to be directly linked to feeding and starvation and has been proposed to contribute to the metabolic and behavioral changes in response to the absence of food. This raises the intriguing possibility that Pdp1 might integrate circadian rhythm with the nutritional state of the organism. For instance, PDP1 might regulate clock output genes that determine cyclic feeding behavior or metabolism. PDP1 might also help regulate cyclic mitogenic and/or nutrient sensing pathways that coordinate growth with nutritional conditions. If true, then PDP1 itself might be regulated by nutritional conditions (Reddy, 2006).

Given that Pdp1^p205 larvae are sensitive to the nutritional environment, specifically sugar levels in the growth medium, the isotype profile was analyzed under starvation conditions in wild-type larvae. Indeed, upon starvation, the PDP1-φ isotype is upregulated. This is similar to the to gene which is also upregulated during starvation. Given the growth phenotype of Pdp1^p205 larvae and the PDP1 isotype switching that occurs under starvation conditions, it is conceivable that this sensitivity to nutritional conditions is fundamental and necessary to the function of the PDP1 protein. This is especially interesting since the PDP1-φ isoform is not expressed in embryos and may indicate that this isoform is necessary in larval/adult life for the purpose of sensing nutritional environment. In the embryo, such a sensor is unnecessary because the embryo has all nutritional requirements supplied by its yolk and therefore has no nutritional interaction with the environment. The nutritional regulation of PDP1 is also provocative given that the homologous vertebrate PAR proteins are involved in the circadian regulation of genes that are involved in metabolism. This link between Pdp1, vertebrate PAR proteins, and circadian rhythm is further reinforced by the observation that, in addition to the upregulation of the PDP1-φ isoform in starved larvae, typical observations show an upregulation of the PDP1-ε isoform as well. Since PDP1-ε is expressed in the clock cells where it regulates clk and other clock output genes, it is possible that nutritional entrainment of the molecular clock in Drosophila could occur through this isotype (Reddy, 2006).

Taken together, the data suggest that PDP1 in the larva may be acting as a transcriptional regulator of growth and proliferation through sensing the nutritional environment. Indeed, it has been shown recently that VRI, which together with PDP1 regulates the Clock gene, is required for normal cell growth and proliferation. VRI, however, appears to act cell autonomously, whereas PDP1 does not. The pleiotropic phenotype exhibited by Pdp1^p205 larvae suggests that the growth defects are not cell autonomous since there is no detectable PDP1 expression in some of the affected tissues. Mosaic analysis confirms that the effects of the Pdp1^p205 mutation on cell growth and proliferation are non-autonomous. A possible model to explain how PDP1 could function to integrate the nutritional state with the regulation of growth and proliferation suggests that Pdp1 exerts its effects through a diffusible factor(s). There are two organs in which PDP1 is expressed that are known to emit diffusible factors that affect mitosis, endoreplication, or both. The fat body is known to secrete mitogenic factors required for proliferation of cells in the imaginal disc. It has long been known that growing imaginal discs in culture require medium conditioned with fat body or fat body extracts. The Imaginal Disc Growth Factors (IDGF) is a group of recently discovered peptides that are required for proliferation in the imaginal tissues and are expressed in the fat body (see Igdf1 and Igdf3). There is no evidence that these factors impact endoreplication directly in the larva, but they do cooperate with insulin-like growth factors to promote endoreplication. The fat body can, however, regulate growth and endoreplication. Reducing amino acid uptake by the fat body or inhibiting dTOR signaling in the fat body inhibits growth and endoreplication in peripheral tissues perhaps through the regulation of transport protein genes in the fat body such as slimfast, which when inhibited leads to larval growth defects and reduced endoreplication. Perhaps PDP1 is involved in regulating fat body functions associated with either IDGF, other mitogen expression, transport, or fat body response to amino acid levels (Reddy, 2006).

PDP1 might exert its effect on insulin growth factor expression or its pathway(s) through signaling from the fat body (see The Insulin receptor signaling pathway). Insulin-like growth factors are expressed in neurosecretory cells in the brain, and the insulin-signaling pathway is implicated in regulating both proliferation and endoreplication. Ectopically expressing either the insulin receptor (InR) or Drosophila S6 kinase (dS6K), two proteins in this pathway, is able to relieve endoreplication defects seen in starved larvae. Mutations in several factors in this pathway, such as dS6K, InR, InsP3 and dTOR, have larval growth defects. Insulin signaling is also implicated in the proliferation of the imaginal disc cells. The most downstream factors in this pathway regulate the S-phase promoting factor E2F and ectopically expressing E2F is also able to rescue endoreplication defects seen in starved larvae or larval growth defective mutants (Reddy, 2006 and references therein).

Since the effects of the Pdp1 mutation are pleiotropic, even though PDP1 is not expressed in all affected tissues, a working model places PDP1 downstream of nutritional signals and upstream of mitogenic signals. In this model, a nutritional signal could be received by either the fat body or the brain and, either directly or indirectly, affect PDP1 regulation of the production of further signaling molecules. PDP1 could either be required for proper organ function or could regulate factors involved in nutritional homeostasis and signaling. In the case of the fat body, PDP1 could modulate the production of IDGFs or other circulating mitogenic factors, while in the brain, PDP1 could conceivably modulate IGF production. Furthermore, incorporating data from PDP1 regulation of the circadian clock, it is possible that PDP1 could serve as a link between nutritional state, growth, and circadian rhythm (Reddy, 2006).

EVOLUTIONARY HOMOLOGS

PAR-domain proteins

Two cDNAs were cloned and sequenced, representing two isoforms of the zebrafish thyrotroph embryonic factor (TEF) gene products (tef alpha and beta); both are members of the PAR subfamily of bZIP transcription factors. The two isoforms encode two potential proteins of 300 and 293 amino acids, respectively. Sequence comparison analysis indicates that the zebrafish TEFs show high homology to the PAR family of transcription factors of other species in the PAR domain, the DNA binding domain and the leucine zipper domain. Expression analysis by Northern blot and RT-PCR indicates that tef alpha and tef beta are expressed throughout the zebrafish embryonic development and in some, but not all, adult tissues (Xu, 1998).

A new member of the leucine zipper (bZIP) gene family of transcription factors, thyrotroph embryonic factor (TEF) has been identified and characterized. Analysis of the ontogeny of TEF gene expression reveals the presence of TEF transcripts, beginning on embryonic day 14, only in the region of the rat anterior pituitary gland, in which thyrotrophic cells arise. This pattern of gene expression corresponds temporally and spatially to the onset of thyroid-stimulating hormone (TSH beta) gene expression, which defines the thyrotroph phenotype. TEF can bind to and trans-activate the TSH beta promoter. In contrast to this restricted pattern of expression during embryogenesis, TEF transcripts appear in several tissues in the mature organism. On the basis of the unique homology between TEF and another member of the bZIP gene family, it has been proposed that TEF belongs to a new class of bZIP proteins, the albumin D box-binding protein (DBP). TEF and DBP transcripts are coexpressed in a pituitary cell line, and these two proteins can readily form heterodimers. The DNA-binding and dimerization domains of TEF correspond to those found in other bZIP proteins. A cluster of basic amino acids, found only in TEF and DBP, has been identified as being necessary for the proper DNA-binding site specificity of TEF. A major trans-activation domain of TEF resides outside the region of homology to other bZIP proteins. These data are consistent with a role for a member of a new class of bZIP transcription factors in activating gene expression in the developing thyrotroph (Drolet, 1991).

A chicken liver cDNA expression library was screened with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter. A transcription factor termed VBP (for vitellogenin gene-binding protein) was isolated. 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 contains a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. VBP forms a stable dimer in solution. A data base search has revealed 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).

The full-length cDNA for a transcriptional activator, DBP, has been cloned; it binds to the D site of the albumin promoter. DBP belongs to a family of related transcription factors including Fos, Jun, CREB, and C/EBP, which share a conserved basic domain. However, unlike most other members of this family, DBP does not contain a "leucine zipper" structure. Among several rat tissues tested, significant levels of its protein are only observed in liver; yet, with the exception of testis, DBP mRNA is present in all of the examined tissues. DBP as well as its mRNA accumulate to significant levels only in adult animals. During chemically induced liver regeneration, DBP expression is rapidly down-regulated, suggesting that DBP may be involved in the proliferation control of hepatocytes. This cell growth-dependent expression of DBP, in contrast to its tissue specificity, appears to be controlled at the level of mRNA accumulation (Mueller, 1990).

Alternative splicing of PAR-domain proteins

An analysis of the chicken VBP gene reveals that the two different amino-terminal sequences map to alternative first exons and that the two different carboxyl-terminal sequences reflect an optional splicing event that can occur only on transcripts that are polyadenylated at the more distal of two polyadenylation sites. An RT-PCR analysis further reveals that a total of four VBP isoforms are encoded by the combinatorial use of these two splicing options. The mRNAs for these four isoforms are differentially expressed in different tissues and cell types. Evidence is provided that one function of the amino-terminal domains is to impose cell type specificity on a core transactivation domain that is present in all four isoforms. Since it is known that VBP can heterodimerize with other members of the PAR subfamily of bZIP factors, the evidence for four VBP isoforms greatly expands the number of complexes that may be used to effect transcriptional regulation through PAR-factor binding sites (Burch, 1994).

Hepatic leukemia factor (HLF) is a member of the PAR family of transcription regulatory proteins. The rat HLF gene is transcribed from two alternative promoters, alpha and beta, with different circadian amplitudes and tissue specificities. The alpha RNA isoforms produce a 43 kDa protein, HLF43, abundant in brain, liver and kidney, as is human HLF RNA. The beta RNA HLF isoforms use a CUG codon to initiate translation of a novel 36 kDa protein, HLF36, which is shorter at its N-terminus relative to the 43 kDa form. HLF36 is expressed uniquely in the liver, where it is the most abundant HLF protein. Surprisingly, the two proteins accumulate in the liver with different circadian amplitudes and have distinct liver-specific promoter preferences in transfection experiments. Thus, HLF43 stimulates transcription from the cholesterol 7 alpha-hydroxylase promoter much more efficiently than from the albumin promoter, while the converse is true for HLF36 (Falvey, 1997).

PAR-1 phosphorylates Mind bomb to promote vertebrate neurogenesis

Generation of neurons in the vertebrate central nervous system requires a complex transcriptional regulatory network and signaling processes in polarized neuroepithelial progenitor cells. This study demonstrates that neurogenesis in the Xenopus neural plate in vivo and mammalian neural progenitors in vitro involves intrinsic antagonistic activities of the polarity proteins PAR-1 and aPKC. Furthermore, Mind bomb (Mib), a ubiquitin ligase that promotes Notch ligand trafficking and activity, is a crucial molecular substrate for PAR-1. The phosphorylation of Mib by PAR-1 results in Mib degradation, repression of Notch signaling, and stimulation of neuronal differentiation. These observations suggest a conserved mechanism for neuronal fate determination that might operate during asymmetric divisions of polarized neural progenitor cells (Ossipova, 2009).

Transcriptional regulation of PAR-domain proteins

The D-site binding protein (DBP) is a member of the PAR domain subfamily of b/ZIP proteins, whose expression in the liver is highly sensitive to the growth state of that organ. This paper examines the regulation of the DBP promoter by C/EBP alpha and examines the role of autoregulation in DBP expression. Of four previously characterized proximal promoter sites, sites I and III have been shown to bind C/EBP alpha, but cotransfection in Hep G2 cells of a C/EBP alpha expression vector is unable to transactivate the promoter. In contrast, the expression of DBP, particularly in conjunction with the related protein HLF, is able to dramatically upregulate expression directed by the proximal promoter. Deletion analysis and the use of single site reporter constructs demonstrate that sites II and IV are highly responsive to transactivation by DBP and HLF. The DBP promoter is active in the UOC-B1 cell line, which bears a 17:19 translocation resulting in the creation of an E2A:HLF fusion protein. The proteins binding to site IV are elevated in this line, suggesting that upregulation of DBP expression in response to inappropriate HLF activity may be mediated through this site (Newcombe, 1998).

The D-site binding protein (DBP) is a member of the proline- and acid-rich (PAR) domain subfamily of basic/leucine zipper proteins and is involved in transcriptional regulation in the liver. Deletion analysis of the DBP protein was carried out in an effort to define the function of the conserved PAR domain. Internal deletions of the protein, i.e. removing portions of the PAR domain, result in a substantial loss in transactivation of a high affinity DBP reporter construct when assayed in Hep G2 cells. These same sequences confer significant transactivation to GAL4 DNA binding domain fusion proteins, indicating that this region acts as part of an independent activation domain comprised of sequences in both the amino terminus and in the PAR domain of DBP. The coexpression of full-length expression constructs for both DBP and hepatic leukemia factor results in a dramatic increase in activation mediated by the GAL4-DBP fusion proteins, suggesting the involvement of a regulated coactivator in this process. DBP transactivation appears to be a p300-dependent process, since a 12 S E1A expression construct disrupts DBP-mediated transactivation, and a p300 expression vector, but not a CREB binding protein vector, is able to restore DBP transactivation. These results suggest that the PAR domain is required for DBP activation, which occurs through a regulated, p300-dependent process (Lamprecht, 1999).

Binding site specificity of PAR-domain proteins

The PAR subfamily of basic leucine zipper (bZIP) factors comprises three proteins (VBP/TEF, DBP, and HLF) that have conserved basic regions flanked by proline- and acidic-amino-acid-rich (PAR) domains and functionally compatible leucine zipper dimerization domains. VBP preferentially binds to sequences that consist of abutted GTAAY half-sites (which are referred to as PAR sites) as well as to sequences that contain either a C/EBP half-site (GCAAT) or a CREB/ATF half-site (GTCAT) in place of one of the PAR half-sites. Since the sequences that describe PAR sites and PAR-CREB/ATF chimeric sites, respectively, have both been described as high-affinity binding sites for the E4BP4 transcriptional repressor, it is inferred that these sequences may be targets for positive and negative regulation. Similarly, since the sequences described as PAR-C/EBP and PAR-CREB/ATF chimeric sites are known to be high-affinity binding sites for C/EBP and CREB/ATF factors, respectively, it is inferred that these sites may each be targets for multiple subfamilies of bZIP factors. To gain insight regarding the molecular basis for the binding-site specificity of PAR factors, extensive mutational analysis of VBP was carried out. By substituting five amino acid residues that differ between the Drosophila giant bZIP factor and the vertebrate PAR bZIP factors, it has been shown that the fork region, which bridges the basic and leucine zipper domains, contributes to half-site sequence specificity. At least two domains amino terminal to the core basic region are required for VBP to bind to the full spectrum of PAR target sites. Thus, whereas direct base contacts may be restricted to basic-region residues (as indicated by GCN4-DNA crystal structures), several other domains also influence the DNA-binding specificity of PAR bZIP proteins (Haas, 1995).

PAR and C/EBP family proteins are liver-enriched basic leucine zipper (bZip) transcription factors that bind similar sites on the promoters of albumin and cholesterol 7 alpha hydroxylase genes. However, C/EBP proteins have a more relaxed binding specificity than PAR proteins, in that they recognize many sites within promoter or randomly selected rat genomic DNA sequences that are ignored by PAR proteins. Thus, DNAse I protection experiments suggest that C/EBP recognizes a binding site every 200 to 300 bp with an affinity similar to that of the cholesterol 7 alpha hydroxylase gene promoter. The frequency of PAR protein binding sites with comparable affinities is about 20-fold lower in the rat genome. By using a PCR-based amplification assay, high affinity DNA-binding sites were selected for C/EBP beta and the PAR protein DBP from a pool of oligonucleotides. Both proteins indeed recognize similar sequences with the optimal core binding sequences 5'RTTAY.GTAAY3'. However, as expected, DBP, is considerably less tolerant to deviations from the consensus site. A single amino acid substitution mutant of C/EBP beta that increases its target site specificity has been characterized. This protein, C/EBP beta V to A, contains a valine to alanine substitution at position 13 of the basic domain (residue 216 of C/EBP beta). C/EBP beta V to A selectively binds only the subset of C/EBP sites that are also DBP sites, both as oligonucleotides and within the natural contexts of the albumin and cholesterol hydroxylase promoters (Falvey, 1996).

Transcriptional targets of PAR-domain proteins

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).

The human GH gene family includes the pituitary-specific hGH-1, placental-specific chorionic somatomammotropin (hCS-5, hCS-2, and hCS-1), and hGH-2 genes. These duplicated, nearly identical genes are localized on approximately 50 kb of DNA on chromosome 17q23-q24. An enhancer (CSEn2), located downstream of the hCS-2 gene, participates in mediating placental-specific hCS gene expression. CSEn2 activity derives from the cooperative binding of transcription factor-1, TEF-1, and a placental-specific factor CSEF-1 to multiple enhansons (Enh1-Enh5) that are related to the SV40 GT-IIC and SphI/SphII enhansons. Two copies of CSEn2 or a single copy of CSEn2 linked to either of the other two enhancers in the hGH/hCS locus (CSEn1 and CSEn5) act cooperatively to enhance hCS promoter activity in choriocarcinoma (BeWo) cells, but silence the promoter in pituitary GC cells. Mutation of Enh4, an essential GT-IIC-like enhanson in the context of the intact enhancer, abolishes silencer activity, and multimerized GT-IIC enhansons mimic the intact CSEn enhancer/silencer activities in BeWo and GC cells, respectively. TEF-1 has been identified as the GT-IIC-binding factor in pituitary cells. The data suggest that TEF-1 may be involved in pituitary-specific repression of placental GH/CS gene transcription through long-range interactions between the multiple CS enhancers present on the GH/CS gene locus (Jiang, 1997).

The avian leukosis virus (ALV) long terminal repeat (LTR) contains a compact transcription enhancer that is active in many cell types. A major feature of the enhancer is multiple CCAAT/enhancer element motifs that could be important for the strong transcriptional activity of this unit. The contributions of the three CCAAT/enhancer elements to LTR function were examined in B cells, since this cell type is targeted for ALV tumor induction following integration of LTR sequences next to the c-myc proto-oncogene. One CCAAT/enhancer element, termed a3, is the most critical for LTR enhancement in transiently transfected B lymphoma cells, while in chicken embryo fibroblasts all three elements contribute equally to enhancement. Vitellogenin gene-binding protein (VBP), a member of the PAR subfamily of C/EBP factors, is a major component of the nuclear proteins binding to the a3 CCAAT/enhancer element. VBP activates transcription through the a3 CCAAT/enhancer element, supporting the idea that VBP is important for LTR enhancement in B cells. A member of the Rel family of proteins, RelA, is also identified as a component of the a3 protein binding complex in B cells. While RelA does not bind directly to the LTR CCAAT/enhancer elements, it does interact with VBP to potentiate VBP DNA binding activity. The synergistic interaction of VBP and RelA increases CCAAT/enhancer element-mediated transcription, indicating that both factors may be important for viral LTR regulation and also for expression of many cellular genes (Curristin, 1997).

The regulatory regions of the genes for coagulation Factors VIII and IX contain binding sites for both liver-enriched and ubiquitous transcriptional regulators. The role of the liver-enriched protein, hepatic leukemia factor (HLF), in mediating transcriptional regulation of the Factor VIII and IX genes was examined. Using transient transfection assays in HepG2 hepatoma cells, the ability of HLF alone and in synergistic combination with the D-box binding protein (DBP), another proline and acidic-rich (PAR) protein family member, to transactivate these promoters was examined. HLF is capable of binding to multiple sites in both the Factor VIII and Factor IX promoters. At least some of the synergistic activation of the Factor VIII promoter seen with HLF and DBP cotransfection can be attributed to increased binding of HLF-DBP heterodimers to two Factor VIII promoter sites. An E2A-HLF chimera, derived from a t(17;19) translocation in pre-B acute lymphoblastic leukemia (ALL) cells, is capable of mediating expression from the Factor VIII and Factor IX promoters in both hepatoma cells and pre-B ALL cells. These observations indicate that the PAR family of transcription factors plays an important and complex role in regulating expression of the Factor VIII and Factor IX genes, involving the binding of both homodimeric and heterodimeric complexes of HLF and DBP to several sites in the promoters. Finally, these studies reaffirm the potential role of dimeric transcription factor complexes in mediating interactions with specific promoter elements, which, in the case of the Factor VIII promoter, results in dramatically enhanced binding of HLF-DBP heterodimers to two cis-acting sequences. These observations further an understanding of the role played by members of the PAR family of transcription factors in regulating expression of the Factor VIII and Factor IX genes (Begbie, 1999).

PAR-domain proteins and circadian rhythms

D-element binding protein (DBP), the founding member of the PAR family of basic leucine zipper (bZip) transcription factors, is expressed according to a robust daily rhythm in the suprachiasmatic nucleus and several peripheral tissues. Other members of this family include TEF (thyroid embryonic factor), its avian ortholog VBP (vitellogenin promoter-binding protein), and HLF (hepatocyte leukemia factor). All of these proteins share high amino acid sequence similarities within a amino-terminal activation domain, a PAR domain rich in proline and acidic amino acid residues, and a carboxy-terminal moiety encompassing the bZip region necessary for DNA binding and dimerization. In vitro all PAR bZip proteins avidly bind the consensus DNA recognition sequence 5'-RTTAYGTAAY-3' as homo- or hetero-dimers. In rat and mouse liver the expression of all three PAR bZip proteins is subject to strong circadian regulation, peak and trough levels being reached in the early evening and morning, respectively. In the case of Dbp the amplitude of circadian mRNA oscillation can largely account for the daily amplitude in protein oscillation. The mRNA accumulation oscillates not only in peripheral tissues such as liver, but also in neurons of the SCN, believed to harbor the central circadian pacemaker. Moreover, run-on experiments in isolated nuclei and physical mapping of nascent RNA chains suggest that circadian transcription plays a pivotal role in rhythmic DBP expression (Ripperger, 2000 and references therein).

Previous studies with mice that have been deleted for the Dbp gene have established that DBP participates in the regulation of several clock outputs, including locomotor activity, sleep distribution, and liver gene expression. Evidence that circadian Dbp transcription requires the basic helix-loop-helix-PAS protein CLOCK, an essential component of the negative-feedback circuitry generating circadian oscillations in mammals and fruit flies. Genetic and biochemical experiments suggest that CLOCK regulates Dbp expression by binding to E-box motifs within putative enhancer regions located in the first and second introns. Similar E-box motifs have been found previously in the promoter sequence of the murine clock gene mPeriod1. Hence, the same molecular mechanisms generating circadian oscillations in the expression of clock genes may directly control the rhythmic transcription of clock output regulators such as Dbp (Ripperger, 2000).

Transcript levels of DBP, a member of the PAR leucine zipper transcription factor family, exhibit a robust rhythm in suprachiasmatic nuclei, the mammalian circadian center. DBP is able to activate the promoter of a putative clock oscillating gene, mPer1, by directly binding to the mPer1 promoter. The mPer1 promoter is cooperatively activated by DBP and CLOCK-BMAL1. However, dbp transcription is activated by CLOCK-BMAL1 through E-boxes and inhibited by the mPER and mCRY proteins, as is the case for mPer1. Thus, a clock-controlled dbp gene may play an important role in central clock oscillation (Yamaguchi, 2000).

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 were 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 is similar in both genotypes, the amplitude of the circadian modulation of sleep time, as well as the consolidation of sleep episodes, is reduced in dbp-/- under both LD and DD conditions. Quantitative EEG analysis has demonstrated 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).

To study the molecular mechanisms of circadian gene expression, attempts have been 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 is 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).

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, 2000).

PAR-domain proteins and cancer

Oncogenic conversion of transcription factors by chromosomal translocations is implicated in leukemogenesis. The t(17;19) in acute lymphoblastic leukemia produces a chimeric transcription factor consisting of the amino-terminal portion of HLH proteins E12/E47 (products of the E2A gene) fused to the basic DNA-binding and leucine zipper dimerization motifs of a novel hepatic protein called hepatic leukemia factor (Hlf). Hlf, which is not normally transcribed in lymphoid cells, belongs to the recently described PAR subfamily of basic leucine zipper (bZIP) proteins, which also includes Dbp and Tef/Vbp. Wild-type Hlf is able to bind DNA specifically as a homodimer or as a heterodimer with other PAR factors. Structural alterations of the E2a-Hlf fusion protein markedly impair its ability to bind DNA as a homodimer, as compared with wild-type Hlf. However, E2a-Hlf can bind DNA as a heterodimer with other PAR proteins, suggesting a novel mechanism for leukemogenic conversion of a bZIP transcription factor (Hunger, 1992).

Genes encoding transcription factors are frequently altered by chromosomal translocations in acute lymphoblastic leukemia (ALL), suggesting that aberrant transcriptional regulation plays a prominent role in leukemogenesis. E2A-hepatic leukemia factor (HLF), a chimeric transcription factor created by the t(17;19), consists of the amino terminal portion of E2A proteins, including two experimentally defined transcriptional activation domains (TADs), fused to the HLF DNA binding and protein dimerization basic leucine zipper (bZIP) domain. To understand the mechanisms by which E2A-HLF induces leukemia and the crucial functions contributed by each constituent of the chimera, it is essential to define the normal transcriptional regulatory properties of HLF and related bZIP proteins. To address these questions, the human homologue of TEF/VBP, a bZIP protein closely related to HLF was cloned. Using a binding site selection assay, it was found that TEF bound preferentially to the consensus sequence 5'-GTTACGTAAT-3', which is identical to the previously determined HLF recognition site. TEF and HLF activate transcription of consensus site-containing reporter genes in several different cell types with similar potencies. Using GAL4 chimeric proteins, a TAD was mapped to an approximately 40 amino acid region of TEF and HLF within which the two proteins share 72% amino acid identity and 85% similarity. The TEF/HLF activation domain (THAD) has a predicted helical secondary structure, but shares no sequence homology with previously reported TADs. The THAD contains most, if not all, of the transcriptional activation properties present in both TEF and HLF and its deletion completely abrogates transcriptional activity of TEF and HLF in both mammalian cells and yeast. Thus, TEF and HLF share indistinguishable DNA-binding and transcriptional regulatory properties, whose alteration in leukemia may be pathogenetically important (Hunger, 1996).

The E2A-HLF fusion gene, created by the t(17;19)(q22;p13) chromosomal translocation in pro-B lymphocytes, encodes an oncogenic protein in which the E2A trans-activation domain is linked to the DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF), a member of the proline- and acidic amino acid-rich (PAR) subfamily of bZIP transcription factors. This fusion product binds to its DNA recognition site not only as a homodimer but also as a heterodimer with HLF and two other members of the PAR bZIP subfamily: thyrotroph embryonic factor (TEF) and albumin promoter D-box binding protein (DBP). Thus, E2A-HLF could transform cells by direct regulation of downstream target genes, acting through homodimeric or heterodimeric complexes, or by sequestering normal PAR proteins into nonfunctional heterocomplexes (dominant-negative interference). To distinguish among these models, mutant E2A-HLF proteins were constructed in which the leucine zipper domain of HLF was extended by one helical turn or altered in essential charged amino acids, enabling the chimera to bind to DNA as a homodimer but not as a heterodimer with HLF or other PAR proteins. When introduced into NIH 3T3 cells in a zinc-inducible vector, each of these mutants induces anchorage-independent growth as efficiently as unaltered E2A-HLF, indicating that the chimeric oncoprotein can transform cells in its homodimeric form. Transformation also depends on an intact E2A activator region, providing further support for a gain-of-function contribution to oncogenesis rather than one based on a dominant-interfering or dominant-negative mechanism. Thus, the tumorigenic effects of E2A-HLF and its mutant forms in NIH 3T3 cells favor a straightforward model in which E2A-HLF homodimers bind directly to promoter/enhancer elements of downstream target genes and alter the patterns of gene expression in early B-cell progenitors (Inukai, 1997).

REFERENCES

Search PubMed for articles about Drosophila PAR-domain protein 1

Abruzzi, K. C., et al. (2011). Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression. Genes Dev. 25(22): 2374-86. PubMed Citation: 22085964

Begbie, M., Mueller, C. and Lillicrap, D. (1999). Enhanced binding of HLF/DBP heterodimers represents one mechanism of PAR protein transactivation of the factor VIII and factor IX genes. DNA Cell. Biol. 18: 165-73. PubMed Citation: 10073576

Burch, J. B. E. and Davis, D. L. (1994). Alternative promoter usage and splicing options result in the differential expression of mRNAs encoding four isoforms of chicken VBP, a member of the PAR subfamily of bZIP transcription factors. Nuc. Acids Res. 22: 4733-4741. PubMed Citation: 7984425

Curristin, S. M., et al. (1997). VBP and RelA regulate avian leukosis virus long terminal repeat-enhanced transcription in B cells. J. Virol. 71(8): 5972-5981. PubMed Citation: 9223487

Cyran, S. A., et al. (2003). vrille, Pdp1, and dClock form a second feedback loop in the Drosophila circadian clock. Cell 112: 329-341. 12581523

Drolet, D. W., Scully, K. M., Simmons, D. M., Wegner, M., Chu, K., Swanson, L. W. and Rosenfeld, M. G. (1991). TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper protein. Genes Dev. 5: 1739-1753. PubMed Citation: 1916262

Falvey, E., Marcacci, L. and Schibler. U. (1996). DNA-binding specificity of PAR and C/EBP leucine zipper proteins: a single amino acid substitution in the C/EBP DNA-binding domain confers PAR-like specificity to C/EBP. Biol. Chem. 377: 797-809. PubMed Citation: 8997490

Falvey, E., Fleury-Olela, F. and Schibler, U. (1997). The rat hepatic leukemia factor (HLF) gene encodes two transcriptional activators with distinct circadian rhythms, tissue distributions and target preferences. EMBO J. 14: 4307-4317. PubMed Citation: 7556072

Fonjallaz, P. V., Ossipow, V., Wanner, G. and Schibler, U. (1996). The two PAR leucine zipper proteins, TEF and DBP, display similar circadian and tissue-specific expression, but have different target promoter preferences. EMBO J. 15: 351-362. PubMed Citation: 8617210

Franken, P., et al. (2000). The transcription factor DBP affects circadian sleep consolidation and rhythmic EEG activity. J. Neurosci. 20(2): 617-25. PubMed Citation: 10632591

Gremke, L., et al. (1993). Coordinate regulation of Drosophila tropomyosin gene expression is controlled by multiple muscle-type-specific positive and negative enhancer elements. Dev. Biol. 159: 513-527. PubMed Citation: 8405675

Haas, N. B., Cantwell, C. A., Johnson, P. F. and Burch, J. B. E. (1995). DNA-binding specificity of the PAR basic leucine zipper protein VBP partially overlaps those of the C/EBP and CREB/ATF families and is influenced by domains that flank the core basic region. Mol. Cell. Biol. 15: 1923-1932. PubMed Citation: 7891686

Hunger, S. P., Ohyashiki, K., Toyoma, K. and Cleary, M. L. (1992). HLF, a novel hepatic bZIP protein, shows altered DNA-binding properties following fusion to E2A in t(17:19) acute lymphoblastic leukemia. Genes Dev. 6: 1608-1620. PubMed Citation: 1516826

Hunger, S. P., Li, S. B., Fall, M. Z., Naumovski, L. and Cleary, M. L. (1996). The proto-oncogene HLF and the related basic leucine zipper protein TEF display highly similar DNA-binding and transcriptional regulatory properties. Blood 87: 4607-4617. PubMed Citation: 8639829

Inukai, T., et al. (1997). Cell transformation mediated by homodimeric E2A-HLF transcription factors. Mol. Cell. Biol. 17(3): 1417-1424. PubMed Citation: 9032268

Iyer, S. V., Davis, D. L., Seal, S. N. and Burch, J. B. E. (1991). Chicken vitellogenin gene-binding protein, a leucine zipper transcription factor that binds to an important control element in the chicken vitellogenin II promoter, is related to rat DBP. Mol. Cell. Biol. 11: 4863-4875. PubMed Citation: 1922023

Jiang, S. W. and Eberhardt, N. L. (1997). The human chorionic somatomammotropin enhancers form a composite silencer in pituitary cells in vitro. Mol. Endocrinol. 11(9): 1233-1244. PubMed Citation: 9259315

Junion, G., Bataille, L., Jagla, T., Da Ponte, J. P., Tapin, R. and Jagla, K. (2007). Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors. Genes Dev. 21(23): 3163-80. PubMed citation: 18056427

Kadener, S., Stoleru, D., McDonald, M., Nawathean, P. and Rosbash. M. (2007). Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component. Genes Dev. 21(13): 1675-86. Medline abstract: 17578907

Lamprecht, C. and Mueller, C. R. (1999). D-site binding protein transactivation requires the proline- and acid-rich domain and involves the coactivator p300. J. Biol. Chem. 274(25): 17643-8

Lavery, D. J., et al. (1999). Circadian expression of the steroid 15 alpha-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP. Mol. Cell. Biol. 19(10): 6488-99

Lim, C., Chung, B. Y., Pitman, J. L., McGill, J. J., Pradhan, S., Lee, J., Keegan, K. P., Choe, J. and Allada, R. (2007). Clockwork orange encodes a transcriptional repressor important for circadian-clock amplitude in Drosophila. Curr. Biol. 17(12): 1082-9. Medline abstract: 17555964

Lin, M.-H., et al. (1996). Myocyte-specific enhancer factor 2 acts cooperatively with a muscle activator region to regulate Drosophila tropomyosin gene muscle expression. Proc. Natl. Acad. Sci. 93: 4623-28

Lin, M.-H., et al. (1997). Ectopic expression of MEF2 in the epidermis induces epidermal expression of muscle genes and abnormal muscle development in Drosophila. Dev. Biol. 182: 240-255

Lin, S. C., et al. (1997). PDP1, a novel Drosophila PAR domain bZIP transcription factor expressed in developing mesoderm, endoderm and ectoderm, is a transcriptional regulator of somatic muscle genes. Development 124(22): 4685-4696.

Logeay, R., Geminard, C., Lassus, P., Rodriguez-Vazquez, M., Kantar, D., Heron-Milhavet, L., Fischer, B., Bray, S. J., Colinge, J. and Djiane, A. (2022). Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila. Development. PubMed ID: 35005772

Lopez-Molina, L., et al. (1997). The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior. EMBO J. 16(22): 6762-71

Majumder, P., Aranjuez, G., Amick, J. and McDonald, J. A. (2012). Par-1 controls myosin-II activity through myosin phosphatase to regulate border cell migration. Curr. Biol. 22(5): 363-72. PubMed Citation: 22326025

Matsumoto, A., et al. (2007). A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock. Genes Dev. 21(13): 1687-700. Medline abstract: 17578908

Menet, J. S., et al. (2010). Dynamic PER repression mechanisms in the Drosophila circadian clock: From on-DNA to off-DNA. Genes Dev 24: 358-367. PubMed Citation: 20159956

Mueller, C. R., Maire, P. and Schibler, U. (1990). DBP, a liver-enriched transcriptional activator, is expressed late in ontogeny and its tissue specificity is determined posttranscriptionally. Cell 61: 279-291

Newcombe, K., Glassco, T. and Mueller, C. (1998). Regulation of the DBP promoter by PAR proteins and in leukemic cells bearing an E2A/HLF translocation. Biochem. Biophys. Res. Commun. 245: 633-9

Ossipova, O., Ezan, J. and Sokol, S. Y. (2009). PAR-1 phosphorylates Mind bomb to promote vertebrate neurogenesis. Dev. Cell 17(2): 222-33. PubMed Citation: 19686683

Reddy, K. L., et al. (2000). The Drosophila PAR domain protein 1 (Pdp1) gene encodes multiple differentially expressed mRNAs and proteins through the use of multiple enhancers and promoters. Dev. Biol. 224: 401-414. PubMed Citation: 10926776

Reddy, K. L., Rovani, M. K., Wohlwill, A., Katzen, A. and Storti, R. V. (2006). The Drosophila Par domain protein I gene, Pdp1, is a regulator of larval growth, mitosis and endoreplication. Dev. Biol. 289(1): 100-14. 16313897

Ripperger, J. A., et al. (2000). CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. Genes Dev. 14: 679-689.

Singh, H., Clerc, R. G. and LeBowitz, J. H. (1989). Molecular cloning of sequence-specific DNA binding proteins using recognition site probes. BioTechniques 7: 252-261.

Sung, H. H., et al. (2008). Drosophila ensconsin promotes productive recruitment of Kinesin-1 to microtubules. Dev. Cell. 15(6): 866-76. PubMed Citation: 19081075

Xu, X., et al. (1998). Cloning and characterization of two isoforms of the zebrafish thyrotroph embryonic factor (tef alpha and tefbeta). Biochim. Biophys. Acta. 1395(1): 13-20

Yamaguchi, S., et al. (2000). Role of DBP in the circadian oscillatory mechanism. Mol. Cell. Biol. 20: 4773-81.