dorsal

Interaction with transcription factors

The simultaneous reduction in the levels of DL and any one of several helix-loop-helix (HLH) proteins results in severe disruptions in the formation of mesoderm and neuroectoderm. The area of twistand snail expression in the presumptive mesoderm is severely reduced in dl-daughterless double heterozygotes. The same is true in dorsal/achaete-scute double heterozygotes. Certain triple heterozygous combinations essentially lack mesoderm as a result of a block in ventral furrow formation during gastrulation. HLH proteins that have been implicated previously in sex determination and neurogenesis (daughterless, achaete, and scute) are required for the formation of these embryonic tissues. Evidence suggests that DL-HLH interactions involve the direct physical association of these proteins in solution mediated by the rel and HLH domains (Gonzalez-Crespo, 1993).

Transactional activation by low levels of DL in lateral regions (the presumptive neuroectoderm) depends on cooperative DNA binding interactions between DL and bHLH proteins. The snail repressor blocks this interaction and restricts expression to the neuroectoderm (Jiang, 1993).

DL-bHLH interactions mediating gene expression in the neuroectoderm and mesoderm are fundamentally distinct. Close proximity of DL and bHLH binding sites is essential for the synergistic activation of gene expression in the lateral neuroectoderm, where there are diminishing levels of the DL regulatory gradient. In contrast, sharp on/off patterns of gene expression in the presumptive mesoderm do not require linkage of these sites. These results are consistent with two distinct modes of DL-bHLH synergy: cooperative binding to DNA (requiring linkage of sites) and synergistic contact of basal transcription factors (no linkage required) (Szymanski, 1995).

Grainyhead/NTF-1 is a cofactor in the repression of decapentaplegic (dpp) and zerknüllt by Dorsal. Repression by Dorsal appears to require accessory proteins that bind to corepression elements in Dorsal-dependent regulatory modules called ventral repression regions (VRRs). A corepression element in dpp is located within a previously identified VRR and close to essential Dorsal-binding sites. In the dpp VRR, one of the dpp response elements overlaps the binding site for a potential activator protein, suggesting that one mechanism of ventral repression may be the mutually exclusive binding of repressor and activator proteins. The Dorsal response element binding protein is identical to NTF-1/ Grainyhead, a factor originally identified as an activator of the Ultrabithorax and Dopa decarboxylase promoters (Huang, 1995).

In the same cell, one protein can activate some genes and repress others. Dorsal activates the twist gene and represses the zen gene in the ventral region of early embryos. A Drosophila HMG1 protein, called DSP1 (dorsal switch protein), converts Dorsal and NF-kappa B from transcriptional activators to repressors. This effect requires a sequence termed a negative ventral regulatory element (VRE), found adjacent to Dorsal-binding sites in the zerknüllt promoter and adjacent to the NF-kappa B-binding site in the human interferon-beta (IFN-beta) enhancer. Previous studies have shown that another type of HMG protein, HMG I(Y), can stimulate NF-kappa B activity. Thus, the HMG-like proteins DSP1 and HMG I(Y) can determine whether a specific regulator functions as an activator or a repressor of transcription (Lehming, 1994 and Ip, 1995).

Krü, snail, and knirps do not appear to require DNA binding cofactors to mediate transcriptional repression. The VRE contains binding sites for a number of proteins suggesting that the repressor may function as a complex. Some of these components may be redundant. One putative corepressor, DSP1 (dorsal switch protein 1), binds to a sequence within the VRE and functions together with DL or other REL proteins to repress transcription. DSP-1 may be necessary for optimal silencing, but does not appear to be necessary for VRE activity, because minimal VRE sequences lacking DSP-1 binding sites can still mediate ventral repression. Another protein, NTF-1/Elf-1, was recently reported to bind to the ventral repression elements of the zen and dpp promoters. However, as with DSP1, it is still unclear whether the NTF-1/Elf-1 protein itself is involved in VRE activity. Interaction of DL and corepressor require fixed spacing between DL and corepressor binding sites, as separation of DL and corepressor sites with a 5-bp spacer, abolishes the silencer activity, while a 10-bp spacer restores silencer activity (Cai, 1996).

The Dorsal Switch Protein (DSP1) was identified in a genetic screen for activities which convert Drosophila Dorsal protein from an activator of transcription to a repressor. DSP1 shares homology with the HMG-1/2 family and inhibits activation by the rel transcription factors Dorsal and NF-kappaB in transfection studies. DSP1 protein can act as a potent transcriptional repressor for multiple activator proteins. DSP1 binds directly to the TATA binding protein (TBP) and forms a stable ternary complex with TBP bound to DNA. DSP1 preferentially disrupts the DNA binding of TBP complexes containing TFIIA and displaces TFIIA from binding to TBP. Consistent with inhibition of TFIIA-bound complexes, DSP1 inhibits activated (but not basal) transcription reactions. The ability of DSP1 to interact with TBP and to repress transcription has been found to map to the C-terminal domain, which contains two HMG boxes (Kirov, 1996).

The Drosophila HMG1-like protein DSP1 was identified in yeast by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion. DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B; SP100B in turn binds to human homologs of HP1. "nuclear bodies" (NBs), found in nuclei of mammalian cells, are known to contain SP100A and PML. SP100A is an autoantigen recognized by antibodies from patients suffering from primary biliary cirrhosis, and PML has been implicated to play a role in acute promyelocytic leukemia. Both SP100A and PML are up-regulated by interferons: overexpression of PML results in slow growth. HP1 is a Drosophila protein involved in transcriptional silencing. Each of these proteins represses transcription when tethered to DNA in mammalian cells. These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression (Lehming, 1998).

DSP1, fused to a GAL4 DNA-binding domain and bound to DNA at GAL4 sites, functions as a transcriptional repressor in HeLa cells. The reporter used in this experiment bore five GAL4 sites upstream of DNA sequences, taken from the TK promoter, that bind the activators C/EBP and SP1. A GAL4 fusion bearing the complete DSP1 represses the reporter some tenfold in mammalian cells. A fusion protein containing the carboxyl half of DSP1, which includes both HMG boxes, represses even more efficiently than does the full length fusion, whereas the amino half of DSP1 increases activation, a result of unknown significance. Intact human HMG2 fused to GAL4 also works as an efficient repressor in mammalian cells. In all cases, repression is abolished by deleting the GAL4-binding sites. In no case is repression found in yeast, suggesting that the repression seen in higher eukaryotes requires proteins absent from yeast (Lehming, 1998).

To identify such proteins from higher eukaryotes, a yeast two-hybrid screen was performed by using GAL4-DSP1(178-393) as bait and challenging with a human cDNA library derived from B cells. The cDNAs were fused to DNA encoding rII, an activating region derived from GAL4. One interacting candidate was found, a previously sequenced protein called SP100B, a splice variant of the NB protein SP100A. SP100A is 479 amino acids long; SP100B contains 688 amino acids. Both proteins are identical in their first 476 amino acids. The cDNA clone represents amino acids 5-528 of SP100B, referred to here as SP100B. SP100B, but not SP100A, interacts with the carboxyl half of DSP1, as well as with human HMG2. The B-specific domain of SP100B (amino acids 477-528) is sufficient for the interaction with both DSP1(178-393) and hHMG2 in vitro. The fusion protein GAL4-SP100B, like GAL4-DSP1 and GAL4-hHMG2, works as a repressor in mammalian cells but not in yeast. The repression depends on the presence of the GAL4 DNA-binding sites in the reporter (Lehming, 1998).

A second two-hybrid screen was performed, similar to the first, except that in this case the bait was GAL4-SPl00B. Two strongly interacting candidates were recovered, each of which contained both a chromodomain (CD) and a chromo shadow domain (CSD). Both proteins, hHPlalpha and hHP1gamma, are homologs of the Drosophila protein HPl (also called Su[var]205). In a yeast two-hybrid assay system, hHPlalpha and hHP1gamma interact with SP100B. The interaction requires the CSD of hHP1 but not the CD. For SP100B, the interaction domain is located between amino acids 286 and 333, a region identical in both SP100A and SP100B. SP100B also interacts with itself: this self-association determinant is probably located in the N terminus of the protein. SP100B interacts with both hHP1 proteins in vitro. Both GAL4-hHP1 fusion proteins work as repressors when bound to GAL4 sites in mammalian cells. In all cases, repression is abolished by deleting the GAL4-binding sites. Like the interaction with SP100B, repression requires the chromo shadow and not the CD. GAL4-hHPl represses approximately tenfold more efficiently than does GAL4-DSP1(178-393) and fivefold more efficiently than does GAL4-SP100B. Thus the CSD of hHP1alpha, comprising 53 amino acids, bears two functions: it confers the repression function when fused to GAL4, and it interacts with SP100B (Lehming, 1998).

The establishment of mesoderm and neuroectoderm in the early Drosophila embryo relies on interactions between the Dorsal morphogen and basic-helix-loop-helix (bHLH) activators. Dorsal and the bHLH activator Twist synergistically activate transcription in cell culture and in vitro from a promoter containing binding sites for both factors. Somewhat surprisingly, a region of Twist outside the conserved bHLH domain is required for the synergy. The two N-terminal Gln-rich regions of Twist appear to mediate synergistic activation by Dorsal and Twist and are also required for binding to Dorsal in vitro. In Dorsal, the rel homology domain appears to be sufficient for synergy. The interaction between Dorsal and Twist does not appear to be of sufficient strength to yield cooperative binding to DNA. It is suggested that the interaction betweein Dorsal and Twist induces a conformational change in one of the factors that enables it to efficiently activate transcription (Shirokawa, 1997).

The primary developmental effect of Drosophila Creb-binding protein (CBP: Nejire) mutation involves the function of Dorsal. nejire mutants exhibit, at germband elongation, the clockwise or counter-clockwise twisting of the embryo, just behind the cephalic furrow, often with the posterior side down. The ventral and cephalic furrows appear normal, but the mesodermally derived internal tissues and a block of ectodermal cells are often missing. On the basis of this phenotype, the Drosophila CBP mutant was named nejire, which means 'twist' in Japanese. Mesoderm formation is crucial event that takes place during early embryogenesis. To initiate the differentiation of the mesoderm in Drosophila, multiple zygotic genes such as twist (twi) and snail (sna), which encode a basic-helix-loop-helix and a zinc finger transcription factor, respectively, are required. The transcription of these genes is induced by maternal Dorsal protein, a transcription factor that is homologous to the NF-kappa B family of proteins. Drosophila CBP mutants, devoid of both maternal and zygotic nejire expression, fail to express twi and therefore generate twisted embryos. This is explained by results showing that dCBP is necessary for Dorsal-mediated activation of the twi promoter. In vitro Dorsal has been shown to bind the N-proximal portion of Drosophila CBP in a phosphorylation independent manner. A region of Dorsal lying between amino acids 186 and 356 (a part of the Rel homology domain) is required for interaction with CBP (Akimaru, 1997). Further studies indicated that a CBP-Dorsal complex is formed on the twist promoter (Perkins, 1996).

Dorsal and Groucho

The Dorsal morphogen acts as both an activator and a repressor of transcription in the Drosophila embryo in order to regulate the expression of dorsal/ventral patterning genes. Circumstantial evidence has suggested that Dorsal is an intrinsic activator and that additional factors (corepressors) convert it into a repressor. These corepressors, however, have previously eluded definitive identification. Via the analysis of embryos lacking the maternally encoded Groucho corepressor and via protein-binding assays it has been shown that recruitment of Groucho to the template by protein:protein interactions is required for the conversion of Dorsal from an activator to a repressor. Specifically, Groucho is required for the Dorsal, mediated repression of Zerknullt and Decapentaplegic. Groucho is not required for the spatially regulated expression of genes that are activated by Dorsal such as twist and snail. Groucho is therefore a critical component of the dorsal/ventral patterning system (Dubnicoff, 1997).

In the Drosophila embryo, Dorsal, a maternally expressed Rel family transcription factor, regulates dorsoventral pattern formation by activating and repressing zygotically active fate-determining genes. Dorsal is distributed in a ventral-to-dorsal nuclear concentration gradient in the embryo, the formation of which depends upon the spatially regulated inhibition of Dorsal nuclear uptake by Cactus. Using maternally expressed Gal4/Dorsal fusion proteins, the mechanism of activation and repression by Dorsal has been explored. A fusion protein containing the Gal4 DNA-binding domain fused to full-length Dorsal is distributed in a nuclear concentration gradient that is similar to that of endogenous Dorsal, despite the presence of a constitutively active nuclear localization signal in the Gal4 domain. Whether this fusion protein activates or represses reporter genes depends upon the context of the Gal4-binding sites in the reporter. A Gal4/Dorsal fusion protein lacking the conserved Rel homology domain of Dorsal, but containing the non-conserved C-terminal domain also mediates both activation and repression, depending upon Gal4-binding site context. A region close to the C-terminal end of the C-terminal domain has homology to a repression motif in Engrailed -- the eh1 motif. Deletion analysis indicates that this region mediates transcriptional repression and binding to Groucho, a co-repressor known to be required for Dorsal-mediated repression. As has previously been shown for repression by Dorsal, activation by Dorsal, in particular by the C-terminal domain, is modulated by the maternal terminal pattern-forming system (Flores-Saaib, 2001).

The results presented here show that just as Dorsal sites function in a context-dependent manner in the presence of endogenous Dorsal, so too do Gal4 sites function in a context-dependent manner in the presence of a Gal4/Dorsal fusion protein. When Gal4/Dorsal*/nt1 binds to multiple tandemly repeated Gal4 sites upstream of a core promoter, the result is activation. In contrast, when Gal4/Dorsal*/nt1 binds a modified dpp VRR in which two critical Dorsal-binding sites have been replaced by Gal4-binding sites, the result is repression. Thus, bringing Dorsal to its target sites is sufficient for both activation and repression -- the rel homology domain (RHD) itself need not be directly engaged with the DNA. Perhaps Dorsal, other DNA-bound repressors (the assistant repressors) and co-repressors such as Gro cooperatively assemble at the ventral silencer to form a 'silencesome'. As might be expected if silencer function required the assembly of such a complex, silencing by the zen VRR is crucially dependent upon the spacing between the sites for the DNA-binding proteins. Changing the spacing (by a non-integral multiple of the DNA helical repeat distance) severely abrogates silencing, presumably by rotating DNA-bound proteins onto opposite faces of the helix. Very similar spacing effects have been observed for enhancesomes (Flores-Saaib, 2001).

The co-repressor Gro, which is required for Dorsal-mediated repression, interacts with the Dorsal RHD. This finding is consistent with the observation that truncated forms of Dorsal consisting of little more than the RHD are able to mediate partial repression of target genes such as zen and dpp. However, the repression directed by the RHD alone is weak relative to that directed by full-length Dorsal and it is therefore not surprising to discover an additional Gro-interaction domain in Dorsal, this one in the CTD. Although the CTD is not conserved between Rel family proteins, the Dorsal-related immunity factor (Dif) can partially substitute for Dorsal during embryogenesis. In addition, patterning of the chick limb may involve the regulation by NF-kappaB of the vertebrate orthologs of Dorsal-target genes. Given these similarities in function, how is it possible to explain the apparent absence of the eh1-like repression domain from Dorsal-homologs such as Dif and NF-kappaB? One possibility is that Rel family protein-mediated transcriptional repression is of relatively minor importance to pattern formation. This is possible because other redundant mechanisms involving Short gastrulation (Sog)-family inhibitors exist to ensure that Dpp-orthologs will not be active at inappropriate positions along the dorsal/ventral axis of the metazoan embryo. The additional Gro-interacting repression domain in the Dorsal CTD may have arisen relatively recently, perhaps as an evolutionary adaptation to allow more complete or more reliable repression of dpp and other genes that interact with dpp to pattern the dorsal ectoderm (Flores-Saaib, 2001).

The studies presented here suggest that potent repression by Dorsal does require a region with homology to the eh1 motif. Thus, Engrailed and Dorsal may use a similar interface to recruit Gro. In this respect, it is interesting to note that Engrailed and Dorsal actually have a ~150 amino acid region of similarity, with the eh1 motif at the C-terminal end of this region. The similar region contains polyalanine stretches, which is a characteristic associated with other repression domains. Perhaps this extended region of similarity plays some role in repression beyond that played by the eh1 motif (e.g. the recruitment of another co-repressor). While Dorsal can function as either an activator or repressor, Engrailed and all other previously characterized repressors containing eh1 motifs appear to be dedicated repressors. The conserved phenylalanine in the eh1 domain is required for efficient Gro recruitment and transcriptional repression. The absence of this phenylalanine in the Dorsal motif could explain the ability of Dorsal to act as either an activator or a repressor depending upon binding site context. Perhaps this 'defect' in the Dorsal eh1 motif prevents Dorsal from recruiting Gro without help from other nearby DNA-bound repressor proteins (assistant repressors). In this respect, it is very interesting to note that Hairy family proteins, which are dedicated repressors, use a C-terminal WRPW motif to recruit Gro, while Runt family proteins, which can function as both activators and repressors, recruit Gro, at least in part, via a C-terminal WRPY motif. Perhaps the conversion of the C-terminal tryptophan to a tyrosine weakens Gro recruitment thereby allowing Runt family proteins to function as either activators or repressors depending upon binding site context (Flores-Saaib, 2001).

Consistent with previous experiments showing that the CTD contributes to transcriptional activation in Drosophila S2 cells and in vitro, this domain mediates activation in embryos. Transcriptional activation by the CTD may be mediated by the previously described interactions of this domain with TAFII110 and TAFII60. Interestingly, the deletion that removes the eh1-like motif and prevents repression by the CTD also results in reduced transcriptional activation. There are multiple possible explanations for this observation. Perhaps Gro has some role in activation in addition to repression. This is reminiscent of studies suggesting that Tup1, a possible yeast ortholog of Gro, functions in both activation and repression. Alternatively, it is possible that the activation and repression domains overlap in the CTD, but that they function via completely different co-regulators. If this is true, then one might expect the binding of the co-repressor and the co-activator to be mutually exclusive, thus ensuring that Dorsal cannot function at cross-purposes by simultaneously recruiting a co-activator and a co-repressor (Flores-Saaib, 2001).

When Gal4/CTD is targeted to the anterior end of the embryo, the resulting zone of repression does not include the anterior pole of the embryo. A key finding in the study of this phenomenon came with the discovery and analysis of capicua (cic), a gene that encodes an HMG-box family transcription factor. In addition to being required for terminal pattern formation, Cic is also required for efficient Dorsal-mediated repression. The finding that Cic appears to be degraded in response to Tor activation suggests that Cic may be a direct target of the terminal pattern forming system. Previous evidence also hinted at a role for the terminal system in modulating Dorsal-mediated activation. When an artificial anterior-to-posterior gradient of Dorsal is established in the embryo, activation of a reporter gene under the control of the proximal twi VAR does not extend to the anterior pole of the embryo. This effect has attributed to the possible presence of Tor response elements in the twi VAR. However, as reported here, even when activation is mediated by nothing but tandem Dorsal sites, this activation is still inhibited at the termini of the embryo by Tor. Likewise, Tor also blocks activation by Gal4/CTD through multiple Gal4 sites. Since these artificial reporters are unlikely to contain Tor response elements distinct from the Dorsal or Gal4 sites, it is likely that the Tor pathway interferes directly with Dorsal-mediated activation, either by modifying Dorsal itself or by modifying a co-activator required for Dorsal activity. Consistent with the possibility that Dorsal itself is the direct target of the terminal system, it has been found that elimination of Tor activity results in an increase in the lower SDS-PAGE mobility form of Dorsal. Because phosphorylation usually decreases SDS-PAGE mobility, this finding suggests that Tor activation might result in the dephosphorylation of Dorsal, either by inactivating a Dorsal kinase or by activating a Dorsal phosphatase. In addition to blocking the activation of Dorsal target genes directly, the terminal system also blocks their activation indirectly, since huckebein, a zygotic target of the terminal system, clearly directs sna repression at the poles. Thus, there appear to be multiple perhaps partially redundant mechanisms to ensure that mesodermal determinants such as twi and sna will not be inappropriately expressed at the poles (Flores-Saaib, 2001 and references therein).

The effect of a tor gain-of-function mutation on activation by Dorsal and the Gal4/Dorsal fusion is not what would be predicted based upon the simple idea that Tor inhibits Dorsal-mediated activation. Instead of resulting in a further retraction of expression from the pole of the embryo, the gain-of-function mutation causes no obvious change in the size of the anterior gap. In addition, this mutation results in an expansion towards the posterior of Gal4/CTD-driven activation and a broadening in the D4/lacZ expression domain. These findings appear to be consistent with a model in which Tor has two completely different effects on Dorsal-mediated activation, inhibiting it at the poles and strengthening it away from the poles. This is precisely what has been observed for the interaction between Bcd and the terminal system. Thus, the effects of Tor on activation may be very general. How Tor is able to function in these two opposite ways depending upon position in the embryo is not clear (Flores-Saaib, 2001 and references therein).

Regulation of Dorsal by calcium levels and PKA

Increased cytoplasmic calcium concentration and the expression of constitutively active Toll receptors can induce the relocalization of Dorsal. Activation of endogenous Protein kinase A and expression of wild-type Toll receptors, have only a marginal effect on the cellular distribution of the Dorsal protein. Treatment of cells with activators of Protein kinase C and radical oxygen intermediates, both of which activate Nuclear factor kappa B, also has little effect on Dorsal protein localization. Thus different threshold levels of Dorsal activation can be established by distinctly regulated signal transduction pathways (Kubota, 1993).

Control over the nuclear import of transcription factors represents a level of gene regulation integral to cellular processes such as differentiation and transformation. The Drosophila transcription factor Dorsal shares with other family members of rel oncogene a phosphorylation site for the cAMP-dependent protein kinase (PKA) located 22 amino acids N-terminal to the nuclear localization signal (NLS) at amino acids 335-340. This study examines the nuclear import kinetics of Dorsal fusion proteins in rat hepatoma cells in vivo and in vitro. Nuclear uptake is not only NLS-dependent, but also strongly dependent on the PKA site, where by using site-directed mutagenesis, the substitution of Ser312 by either Ala or Glu severely reduces nuclear accumulation. Either exogenous cAMP or PKA catalytic subunit significantly enhance the nuclear import of wild-type proteins both in vivo and in vitro. Using a direct binding assay, the molecular basis of PKA site enhancement of Dorsal fusion protein nuclear import was determined to be PKA site-mediated modulation of NLS recognition by the importin 58/97 complex. The physiological relevance of these results is supported by the observation that Drosophila embryos expressing PKA site Dorsal mutant variants are impaired in development. It is concluded that the Dorsal NLS and PKA site constitute a phosphorylation-regulated NLS essential to Dorsal function and able to function in heterologous mammalian cell systems, where phosphorylation modulates the affinity of NLS recognition by importin (Briggs, 1998).

In Drosophila, dorsal-ventral polarity is determined by a maternally encoded signal transduction pathway that culminates in the graded nuclear localization of the Rel protein, Dorsal. Dorsal is retained in the cytoplasm by the IkappaB protein, Cactus. Signal-dependent phosphorylation of Cactus results in the degradation of Cactus and the nuclear targeting of Dorsal. An in-depth study of the functional importance of Dorsal phosphorylation is presented. Dorsal is phosphorylated by the ventral signal while associated with Cactus, and Dorsal phosphorylation is essential for its nuclear import. In vivo phospholabeling of Dorsal is limited to serine residues in both ovaries and early embryos. A protein bearing mutations in six conserved serines abolishes Dorsal activity; it is constitutively cytoplasmic, and appears to eliminate Dorsal phosphorylation, but still interacts with Cactus. Two individual serine-to-alanine mutations have produced unexpected results. In a wild-type signaling background, a mutation in the highly conserved PKA site (S312) produces only a weak loss-of-function; however, it completely destabilizes the protein in a cactus mutant background. Significantly, the phosphorylation of another completely conserved serine (S317) regulates the high level of nuclear import found in ventral cells. It is concluded that the formation of a wild-type Dorsal nuclear gradient requires the phosphorylation of both Cactus and Dorsal. The strong conservation of the serines suggests that phosphorylation of other Rel proteins is essential for their proper nuclear targeting (Drier, 1999a).

Two of the serines that were changed to alanines are predicted to be phosphorylated by specific kinases. S79 is part of a predicted casein kinase II (CK II) site, and interestingly, this is the only mutant that retains wild-type function, suggesting that if the serine is phosphorylated, the modification is not essential. S312 is part of a PKA recognition site. This site may or may not be phosphorylated, but it is not the target of the signal-dependent phosphorylation that occurs on Dorsal. Rather, if S312 is phosphorylated, the modification may occur already in the ovary, and stabilize the protein. A serine (S317) that is not part of any known kinase recognition site is the likely target of the signal-dependent phosphorylation. This raises the question as to what kinase is responsible for the modification. Tube and Pelle are components of the dorsal-ventral signal transduction pathway that function downstream from the Toll receptor. They interact directly with Dorsal, albeit in the amino-terminal domain 1 of the Rel homology region (RHR). This interaction represents an essential step in the transduction of the ventral signal. Pelle is a serine-threonine kinase and may be directly responsible for the phosphorylation of Dorsal. However, as S317 is in domain 2 of the RHR, it is possible that additional kinases, such as the IB kinases, phosphorylate Dorsal and that Pelle controls their activity (Drier, 1999a).

Drosophila mediator complex is used by Dorsal

To decipher the mechanistic roles of Mediator proteins in regulating developmental specific gene expression and compare them to those of TATA-binding protein (TBP)-associated factors (TAFs), a multiprotein complex containing Drosophila Mediator (dMediator) homologs was isolated and analyzed. dMediator interacts with several sequence-specific transcription factors and basal transcription machinery and is critical for activated transcription in response to diverse transcriptional activators. The requirement for dMediator does not depend on a specific core promoter organization. By contrast, TAFs are preferentially utilized by promoters having a specific core element organization. Therefore, Mediator proteins are suggested to act as a pivotal coactivator that integrates promoter-specific activation signals to the basal transcription machinery (Park, 2001).

Previous studies in yeast and human cells have suggested that transcriptional activator proteins interact with Mediator complexes. The requirement of dMediator for the activated transcription in response to Gal4-VP16 indicates that dMediator may also serve as a binding target of transcriptional activators. Because several coactivators, such as TAFs and the GCN5 histone acetyltransferase (HAT) complex, have been suggested to interact directly with transcriptional activators, the relative binding affinities of these coactivator complexes with the VP16 protein were examined. After incubation of nuclear extracts with an excess of GST fusion protein beads containing either wild-type or mutant (Delta456FP442) VP16 activation domain, the supernatants were analyzed by immunoblotting with Abs against the components of the coactivator complexes. Almost all of the dMediator proteins in the nuclear extract (TRAP80, MED6, and Trfp) were removed by incubating with GST-VP16 but not with GST-VP16^{Delta456FP442}. However, the amounts of dGCN5, dTAF_II40, dTAF_II250, and dTBP in the extract were not reduced at all by the incubation. When the proteins bound to the beads were analyzed, a large amount of dMediator was retained only in the GST beads containing the functional VP16 activation domain. The TFIID and dGCN5 HAT complexes did bind to the wild-type VP16 beads, but the amounts were less than 2% of the total amounts present in the extract. These data indicate that, among known transcriptional coactivator complexes, Mediator is most strongly bound to and most readily recruited to the activation domain (Park, 2001).

In addition to the model VP16 activator derived from herpesvirus, dMediator interacts with Drosophila transcriptional activators Dorsal and heat shock factor (dHSF). When dMediator complex was incubated with FLAG-Dorsal or GST-dHSF fusion protein beads, more than 20% of the dMediator input was retained specifically on the beads even after extensive washing. To extend this study to other sequence-specific transcription factors important for Drosophila development, dMediator was immobilized on protein G-agarose beads through anti-dSOH1 Ab and the binding of diverse ³⁵S-labeled Drosophila transcription factors was examined. Bicoid, Krüppel, and Fushi-tarazu are retained specifically on the dMediator beads; Twist and Hunchback are not. Therefore, dMediator functions as a binding target for many, but not all, developmental specific transcription factors (Park, 2001).

To evaluate the requirement of dMediator for activated transcription in response to the Drosophila activator proteins that interact with dMediator, the ability of dMediator-deficient nuclear extracts to support transcriptional activation by the Dorsal and Gal4-dHSF proteins was examined. The addition of Dorsal or Gal4-dHSF to mock-depleted extract causes 20- and 25-fold increases, respectively, in transcription levels from the adenovirus early region 4 (E4) promoter linked to the appropriate DNA binding site. However, the level of transcriptional activation is reduced significantly (five- and three-fold activations, respectively) in nuclear extract that has been depleted by anti-dSOH1 Ab. Therefore, dMediator is absolutely required for transcriptional activation by all the activators tested. Addition of purified dMediator back to depleted extracts partially recovers activation by Dorsal and Gal4-dHSF in much the same way as it does in the case of Gal4-VP16. dMediator is not required for transcriptional repression by the sequence-specific transcription factor Even-skipped (Park, 2001).

dMediator is generally required for transcriptional activation from both TATA-containing and TATA-less promoters through direct communication with transcriptional activators. The function of dMediator seems to be exclusively related to sequence-specific transcription factors placed at upstream enhancer elements. However, the requirement of TAFs, or at least dTAF_II250, in activated transcription appears to be redundant in the in vitro transcription system used and affected by such factors as the core promoter organization or nucleosomal structure of transcriptional templates. Several TAF components in the TFIID complex indeed have biochemical activities and structural motifs adequate for the recognition of specialized settings of transcription templates. For example, certain TAFs recognize the Inr and DPE sequences located in many Drosophila core promoters and increase the stability of TFIID-promoter interactions. In addition, TFIID contains dTAF_II250, which has a HAT catalytic activity and also possesses a histone octamer-like module comprising the histone H2B-, H3-, and H4-like TAFs. Although not experimentally demonstrated, these TAFs may have some roles in the transcriptional regulation of nucleosomal templates (Park, 2001).

The sequence-specific transcription factors which interact physically with dMediator include VP16, Dorsal, dHSF, Bicoid, Krüppel, and Fushi-tarazu. These factors contain different types of activation domains (acidic and glutamine-rich domains). Most of these transcription factors have been shown to activate transcription either constitutively or inducibly. It is noteworthy that dHSF interacts with and requires dMediator for transcriptional activation because previous reports have shown that transcriptional activation by HSF in yeast does not require the function of the Mediator protein Srb4. However, the recent finding that activation by HSF depends on another Mediator protein, Rgr1 (Trap170), suggests that some function of Mediator is required for HSF-mediated transcriptional activation in yeast, as well. Since Rgr1, but not Srb4, is conserved between yeast and Drosophila, transcriptional activation by HSF might utilize the conserved Rgr1 components of the Mediator complexes (Park, 2001).

Although some human Mediator complexes appear to have a negative effect on activated transcription, dMediator does not exhibit such an activity in an in vitro transcription system reconstituted with Drosophila transcription factors. In addition, Even-skipped, a well-known Drosophila transcriptional repressor, does not interact with, or depend for its transcriptional repression on dMediator. Previous reports have shown that the repression domain of Even-skipped directly targets TBP. It has also been confirmed that the TFIID complex in the nuclear extract specifically interacts with Even-skipped under the same conditions in which Even-skipped fails to interact with dMediator. Although Krüppel has a well-characterized repressor function in Drosophila development, it can also act as a transcriptional activator under certain conditions. Therefore, it is more plausible that the dMediator-Krüppel interaction observed is a part of the mechanism for transcriptional activation rather than transcriptional repression. Taken together with the fact that dMediator is dispensable for basal transcription, the lack of defect of the dMediator-depleted nuclear extracts on transcriptional repression by Even-skipped protein suggests that dMediator is required mainly for the mediation of transcriptional activation signals to the basal transcription machinery. Very recently, developmental roles of certain dMediator proteins found in the Drosophila genome database have begun to be also identified in genetic studies. Genetic interactions between dMediator proteins and a homeotic regulator Sex combs reduced implicate dMediator proteins as a transcriptional activator-specific target critical for Drosophila development (Park, 2001).

Like yeast Mediator, dMediator bind with the CTD repeats of Drosophila Pol II. This implies that though dMediator was purified separately from Pol II, these two complexes indeed interact with each other and act together during transcriptional initiation. Besides the physical interaction with Pol II, dMediator also has some binding affinity for TBP, TFIIB, TFIIE, TFIIF, and TFIIS. Such interactions may be involved in the regulation of Pol II preinitiation complex assembly. Related with this idea, it has been reported that in yeast, recruitment of general transcription factors such as TBP, TFIIB, and TFIIH to active promoters requires the function of Mediator. Also, TFIIE interacts with the Mediator protein Gal11. Further analyses will be required to clarify whether these interactions, observed both in yeast and Drosophila, participate in the control of the stepwise preinitiation complex assembly in the course of transcription activation or simply reflect the affinities between the components of preassembled Pol II holoenzymeG (Park, 2001).

dMediator contains the protein kinase component Cdk8, which can phosphorylate serine residues in the CTD. This catalytic kinase subunit seems responsible, at least in part, for the Pol II phosphorylation by dMediator. In particular, dMediator and TFIIH synergistically phosphorylate the serine 5 residue of the carboxy-terminal Pol II repeats, suggesting the presence of a functional interaction between these complexes. Given that Pol II phosphorylation at serine 5 by TFIIH has been correlated with transcriptional activation processes, the synergy in the serine 5 phosphorylation by TFIIH and dMediator may be intimately linked with the regulatory effects that the Mediator complex exerts on Pol II transcription (Park, 2001).

The MADF-BESS domain factor Dip3 potentiates synergistic activation by Dorsal and Twist

The transcription factors Dorsal and Twist regulate dorsoventral axis formation during Drosophila embryogenesis. Dorsal and Twist bind to closely linked DNA elements in a number of promoters and synergistically activate transcription. A novel protein named Dorsal-interacting protein 3 (Dip3) has been identified that may play a role in this synergy. Dip3 functions as a coactivator to stimulate synergistic activation by Dorsal and Twist, but does not stimulate simple activation of promoters containing only Dorsal or only Twist binding sites. In addition, Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor. The possibility is assessed that the MADF and BESS domains are related to the SANT domain, a well-characterized motif found in many transcriptional regulators and coregulators (Bhaskar, 2002).

NF-kappaB, IkappaB, and IRAK control glutamate receptor density at the Drosophila NMJ

NF-κB signaling has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. However, the cellular and molecular activity of NF-κB signaling within the nervous system remains to be clearly defined. This study shows that the NF-κB and IκB homologs Dorsal and Cactus surround postsynaptic glutamate receptor (GluR) clusters at the Drosophila NMJ. Mutations in dorsal, cactus, and IRAK/pelle kinase specifically impair GluR levels, assayed immunohistochemically and electrophysiologically, without affecting NMJ growth, the size of the postsynaptic density, or homeostatic plasticity. Additional genetic experiments support the conclusion that cactus functions in concert with, rather than in opposition to, dorsal and pelle in this process. Finally, evidence is provided that Dorsal and Cactus act posttranscriptionally, outside the nucleus, to control GluR density. Based upon these data it is speculated that Dorsal, Cactus, and Pelle function together, locally at the postsynaptic density, to specify GluR levels (Heckscher, 2007).

NF-κB signaling has been implicated in the mechanisms of neural plasticity, learning, epilepsy, neurodegeneration, and the adaptive response to neuronal injury. The data presented in this study advance the understanding of neuronal NF-κB signaling in two ways. First, multiple lines of evidence are presented that NF-κB/Dorsal signaling is required for the control of GluR density at the NMJ. These data provide a synaptic function for NF-κB signaling that may be directly relevant to the diverse activities ascribed to NF-κB in the nervous system. Second, molecular and genetic evidence is provided that Dorsal, Cactus, and Pelle may function posttranscriptionally, at the postsynaptic side of the NMJ, to specify GluR density during postembryonic development (Heckscher, 2007).

Several independent lines of experimentation suggest that Cactus, Dorsal, and Pelle function together at the PSD to specify GluR density. Evidence is provided that Cactus and Dorsal localize to a similar postsynaptic domain. In addition, overexpression of a GFP-tagged Pelle protein that is sufficient to rescue a pelle mutation, can traffic to the PSD where Cactus and Dorsal reside. Next, genetic evidence is presented that cactus, dorsal, and pelle function together, in the same genetic pathway, to control GluR density. It is particularly surprising that mutations in cactus behave similarly to dorsal and pelle. In other systems (embryonic patterning and immunity), Cactus inhibits Dorsal-mediated transcription by binding and sequestering cytoplasmic Dorsal protein. As a result, in these other systems, cactus mutations cause phenotypes that are opposite to those observed in dorsal mutations. This study used the same cactus and dorsal mutations that previously have been observed to generate the predicted opposing phenotypes during embryonic patterning, and yet it was observed that cactus phenocopies the dorsal mutations. In addition, genetic epistasis experiments indicate that these genes function together to facilitate GluR density. Thus, at the NMJ, Cactus functions in concert with, rather than in opposition to, Dorsal (Heckscher, 2007).

One explanation for this observation could be that Dorsal does not function as a nuclear transcription factor during the control of GluR levels. In support of this idea it has been demonstrated that (1) Dorsal protein is not detected in the nucleus, (2) reporters of Dorsal-dependent transcription fail to show activity in muscle nuclei, and (3) mutation of the Dorsal transactivation domain, dl^U5 does not impair GluR abundance even though this same mutation has been shown to impair transcription-dependent patterning during embryogenesis. An alternative explanation for the observation that dorsal and cactus have similar phenotypes at the NMJ could be that Cactus and Dorsal act synergistically to control the transcription of GluRs at the NMJ. Indeed, there is evidence in other systems that IκB can shuttle with NF-κB to the nucleus. A previous study shows Cactus accumulation in Drosophila larval muscle nuclei in a dorsal mutant background (Cantera, 1999). However, this result could not be repeated despite examination of Cactus localization in five allelic combinations of dorsal. Furthermore, the data from vertebrate systems suggest that IκB should shuttle into the nucleus with NF-κB, not in its absence. Thus, a model is favored in which Dorsal and Cactus function together at the postsynaptic membrane to facilitate GluR abundance during development (Heckscher, 2007).

If this model is correct, then it is predicted that NF-κB does not control GluR density through transcriptional regulation. This prediction is supported by two experimental observations: (1) GluR transcript levels (assessed by QT PCR) are not statistically different from wild-type in dorsal and cactus mutations that cause an ~50% decrease in GluR abundance; (2) it was demonstrated that overexpression of a myc-tagged GluRIIA cDNA using a heterologous, muscle-specific promoter is not able to restore synaptic GluRIIA levels in either the cactus or dorsal mutant backgrounds. These data are consistent with Dorsal and Cactus acting posttranscriptionally to control GluR density at the NMJ. There are two general mechanisms by which GluR levels could be controlled posttranscriptionally: (1) altered receptor delivery to the NMJ or (2) altered receptor internalization/degradation. If receptor internalization/degradation were enhanced in the cactus, dorsal, or pelle mutant backgrounds, one might expect GluRIIA-myc overexpression to overcome this change and restore normal receptor levels. In addition, less myc-tagged protein might be seen in the mutants in comparison to wild-type. This is not what was observed. Therefore, the hypothesis is favored that Cactus, Dorsal, and Pelle function together to promote the delivery of glutamate receptors to the NMJ during development (Heckscher, 2007).

The possibility that Cactus, Dorsal, and Pelle act posttranscriptionally to control GluR density raises many questions. For example, do Dorsal and Cactus exist as a protein complex at the PSD? If so, is this complex regulated and how might such a complex influence GluR density? Since pelle kinase-dead mutants impair GluR density, it is possible that Dorsal and Cactus recruit Pelle to the PSD. If so, what are the targets of Pelle kinase that are relevant to establishing or maintaining the proper density of glutamate receptors at the PSD? Finally, the demonstration that cytoplasmic NF-κB/Dorsal can influence GluR density does not rule out the possibility that NF-κB/Dorsal may also translocate to the muscle nucleus at the Drosophila NMJ under certain stimulus conditions. Indeed, in both the vertebrate central and peripheral nervous systems NF-κB is found within neuronal and muscle nuclei, and nuclear translocation can be stimulated by neuronal activity, glutamate, injury, and disease. For nuclear entry of Dorsal, two events must occur: (1) Cactus must be degraded and (2) Dorsal must be phosphorylated. It remains possible that one or both of these criteria are not met during the normal development of the Drosophila NMJ but could be met under as-yet-to-be-identified stimulus conditions. The possibility that NF-κB acts both locally at the synapse and globally via the nucleus is not unique to this signaling pathway. A similar organization has been documented for wingless/wnt signaling where noncanonical cytoplasmic signaling can impact cytoskeletal organization while canonical signaling involves the nuclear translocation of downstream beta-catenin and TCF-dependent gene transcription (Heckscher, 2007).

It remains unknown how NF-κB signaling is activated at the Drosophila NMJ. In Drosophila embryonic patterning and innate immunity, NF-κB signaling is initiated through activation of Toll or Toll-like receptors. There are nine Toll and Toll-like receptors encoded in the Drosophila genome. However, none of these receptors appear to be present in Drosophila larval muscle. The Toll receptor is expressed in a subset of embryonic muscle fibers, but is absent from larval muscle. None of the Toll-like receptors are expressed in Drosophila embryonic muscle and none appear to be expressed in larval muscle. An alternative possibility is that TNF-α receptors activate NF-κB in Drosophila muscle as has been observed in vertebrate skeletal muscle. Indeed, a TNF-α receptor homolog (Wengen) has been identified, and it is expressed in Drosophila skeletal muscle. The possibility that TNF-α signaling is mediated via NF-κB is intriguing given the recent demonstration that TNF-α regulates GluR abundance in the vertebrate central nervous system. In both cultured neurons and hippocampal slices glial-derived TNF-α signaling is required for the increase in postsynaptic AMPA receptor abundance observed following chronic activity blockade. Thus, the current data in combination with work in the vertebrate CNS raise the possibility that a conserved TNFα/NF-κB signaling system controls GluR abundance at both neuromuscular and central synapses during development and in response to chronic activity blockade (Heckscher, 2007).

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