Gene name - Mothers against dpp
Cytological map position - 23D
Function - TGF beta signal transduction
Key words - segment polarity
Symbol - Mad
Genetic map position - 2-
Classification - novel signaling protein
Cellular location - cytoplasmic
Mothers against dpp was discovered (Raftery, 1995) in a search for elements of the decapentaplegic signaling pathway in Drosophila (for review, see Raftery, 1999). When given the problem of identifying elements in a biochemical pathway, a geneticist will usually take a genetic approach, one that has worked in previous attempts in other systems. The tactic is to disclose enhancers which exacerbate known mutations. An enhancer is a mutation in the sought-for gene that engenders a more severe phenotype than that caused by the mutation of a single, already characterized gene. Thus a search was carried out for mutations that produce a more severe phenotype (an "enhancement") when combined with known dpp mutations. Mad mutations can be placed in an allelic series based on the relative severity of the maternal effect enhancement of weak dpp alleles, thus explaining the name Mothers against dpp..
Two types of experiments were carried out. The first was designed to reveal mutations in genes expressed in zygotes that exacerbate the phenotype of embryos that have a limiting amount of DPP. Mutations that act as enhancers of such DPP limited embryos cause embryonic lethality. The possibility that some of the gene products involved in DPP signaling might be supplied during oogenesis necessitated a second experiment looking for failure to recover progeny (that is, embryonic lethals) from mothers exhibiting a limiting amount of DPP. The two experiments yielded several kinds of enhancers: 1) new dpp alleles, which along with an already limited quantity of DPP cause embryonic lethality; 2) mutations in tolloid, a gene whose product is involved in DPP processing; 3) mutations in screw whose protein product is a ubiquitously expressed member of the TGF-beta family required for specification of dorsal cell fates in the Drosophila embryo; 4) mutations in Media, another gene involved in DPP signaling, and 5) mutations in Mad. Mutations in Mad, when interacting with limiting DPP levels, produce a defective amnioserosa (see also [Image]), the extra-embryonic membrane comprising the dorsal-most cells in early embryos (Raftery, 1995).
Mad mutations are also enhancers of mutant dpp appendage phenotypes. Thus Mad mutants produce a further reduction in wing blade size, a slight reduction in the eye, and loss of tarsal claws. These Mad mutant phenotypes exhibit a close correspondence to dpp mutant phenotypes. Homozygous Mad mutant larvae also show midgut defects and a greatly reduced gastric caecae. DPP signaling from visceral mesoderm to midgut endoderm is required for proper extension of the gastric caecae in parasegment 4 and for the induction of the homeotic gene labial in the adjacent endoderm of parasegment 7. Homozygous Mad mutant embryos lack labial expression and have defects in midgut constriction engendered by labial expression. Other imaginal disc derived structural defects are evident in homozygous Mad mutants, including heldout wings, split notum, loss of distal leg segments, duplications of the third antennal segment and defects in female genitalia (Sekelsky, 1995).
To date there is no indication that the Drosophila MAD protein is nuclear: antibody staining experiments indicate a cytoplasmic localization. Neverless there is clear indication that a human MAD homolog enters the nucleus upon BMP2 signaling (Hoodless, 1996).
A simple experiment was carried out to see if MAD acts upstream or downstream of DPP. DPP was ubiquitously expressed in Mad mutants. If Mad acts upstream of DPP, then ubiquitous expression of DPP should result in labial induction in the midgut endoderm independently of Mad. In fact labial does not get induced in Mad mutants even when dpp is expressed ubiquitously, indicating that MAD acts downstream of dpp. When MAD is artifically expressed in mesoderm, it fails to rescue labial induction in embryos otherwise deficient in MAD, but artificial expression of MAD in endoderm does rescue labial induction (Newfeld, 1996). Thus MAD appears to be a component in DPP signaling acting downstream of dpp in cells that are the recipients of DPP signaling.
Daughters against dpp (Dad), whose transcription is induced by Dpp shares, weak homology with Drosophila Mad, a protein required for transduction of Dpp signals. Dad is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule, and in fact ectopic Dpp expression results in abnormally large discs and in ectopic expression of Dad. In contrast to Mad or the activated Dpp receptor, whose overexpression hyperactivates the Dpp signaling pathway, overexpression of Dad blocks Dpp activity. Dpp target gene optomotor blind is absent in Dad-overexpressing cells. Expression of Dad together with either Mad or the activated receptor rescues phenotypic defects induced by either protein alone. Dad can also antagonize the activity of a vertebrate homolog of Dpp, bone morphogenetic protein, as evidenced by induction of dorsal or neural fate following overexpression in Xenopus embryos. It is concluded that the pattern-organizing mechanism governed by Dpp involves a negative-feedback circuit in which Dpp induces expression of its own antagonist, Dad. This feedback loop appears to be conserved in vertebrate development (Tsuneizumi, 1997).
Drosophila Medea encodes a homolog of Smad4. Smad4 is relatively divergent from other vertebrate Smads and does not appear to be regulated by signal-dependent phosphorylation. However, overexpression of vertebrate Smad4 stimulates TGF-beta and activin responses. Smad4 associates with Smad1 (the mammalian homolog of Mad) in response to BMP2/4 or with Smad2 in response to TGF-beta, and dominant negative Smad4 blocks both BMP and activin responses. These observations have generated a model in which Smad4 is essential for signal transduction by all TGF-beta family members through its interaction with phosphorylated receptor-regulated Smads. Medea functions downstream of Dpp; complete removal of the Medea gene product causes the same embryonic phenotype as dpp null mutations. Mad undergoes signal-dependent translocation to the nucleus in the absence of Medea; in contrast, Medea is localized in the cytoplasm and requires Mad in order to accumulate in the nucleus. Specific mutations identified in strong alleles of Medea disrupt either Medea interaction with Mad or nuclear translocation of the Mad/Medea complex. Thus, interaction with Mad and nuclear import are critical for Medea function. However, unlike Mad, Medea is not required for expression of all Dpp-dependent genes and in its absence intracellular Dpp signaling rapidly attenuates with distance from the Dpp source. It is propose that the presence of Medea in heteromeric nuclear complexes with Mad modifies or enhances Dpp signaling (Wisotzkey, 1998).
Hox proteins control the differentiation of serially iterated structures in arthropods and chordates by differentially regulating many target genes. It is yet unclear to what extent Hox target gene selection is dependent upon other regulatory factors and how these interactions might affect target gene activation or repression. Two Smad proteins, effectors of the Drosophila Dpp/TGF-ß pathway, that are genetically required for the activation of the spalt (sal) gene in the wing, collaborate with the Hox protein Ultrabithorax (Ubx) to directly repress sal in the haltere. The repression of sal is integrated by a cis-regulatory element (CRE) through a remarkably conserved set of Smad binding sites flanked by Ubx binding sites. If the Ubx binding sites are relocated at a distance from the Smad binding sites, the proteins no longer collaborate to repress gene expression. These results support an emerging view of Hox proteins acting in collaboration with a much more diverse set of transcription factors than has generally been appreciated (Walsh, 2007).
The activation of sal in the wing and its repression in the haltere are regulated by a 1.1 kb CRE, sal1.1 (Galant, 2002). Previous studies have shown that sal1.1 is directly repressed by Ubx in the haltere (Galant, 2002). In order to test whether Mad/Med binds to and directly represses the activity of the sal1.1 CRE in the haltere, candidate Mad/Med binding sites were sought in the sal1.1 CRE. One candidate Mad/Med binding site, M1 (5'-AGACGGGCAC-3'), was identified that lies between Ubx binding sites 5 and 6 in sal1.1, using binding site prediction and electrophoretic mobility shift assays (EMSAs). The sequence of M1 deviates somewhat from published Mad/Med silencer consensus binding sites (5'-AGAC-5 bp-GNCGYC-3') (Gao, 2005; Pyrowolakis, 2004), and Mad and Med bound with >10-fold and >25-fold lower affinities, respectively, to the M1 site than to the bam (Gao, 2005) and brk (Pyrowolakis, 2004) silencer elements (Walsh, 2007).
In order to test whether Mad/Med bound specifically to the M1 site, a series of point mutations were introduced within the M1 site, and their effect on protein binding was examined in vitro. Of four point mutations to the M1 site, the single mutation at position 808 reduced the binding of a Med fusion protein (GST-MedMH1) to M1 as compared with the wild-type sequence. The remaining three point mutations did not affect the affinity of GST-MedMH1 for the probe. These results suggest that Med might contact the sequence 5'-AGAC-3' in sal1.1. By contrast, the four individual point mutations each decreased, but did not abolish, binding of a Mad fusion protein (GST-MadN) in vitro, with the point mutation at bp 814 having the strongest effect. The weaker effect of the individual point mutations in M1 on Mad binding affinity in vitro is likely to be due to the affinity of MadN for both 5'-AGAC-3' Smad sites and GC-rich sequence. Combining these four mutations (sal798-824 kM1) had the greatest effect on GST-MadN binding to the probe. This analysis of individual point mutations indicates a putative orientation for a Mad/Med compound-binding site in the sal1.1 CRE (Walsh, 2007).
Most importantly, in transgenic flies, each point mutation of M1 introduced into an otherwise wild-type sal1.1 reporter construct caused derepression of the reporter gene lacZ in the haltere imaginal disc. The strength of derepression correlates with the decreased affinity of Mad for its binding site with the pm814 mutation, the strongest point mutation in vitro, showing the strongest level of derepression in vivo. Full derepression was observed when all four point mutations were combined into a sal1.1 reporter construct. No effect of mutations in M1 were observed on sal1.1-driven reporter gene expression in the wing as compared with the wild-type sal1.1 element or with endogenous sal expression, indicating that this site is not required for gene activation in the wing or haltere disc. Together, the biochemical, reporter gene and genetic evidence indicate that Mad/Med/Shn are directly required for sal repression in the haltere imaginal disc (Walsh, 2007).
This study demonstrates that Mad/Med and Ubx bind to adjacent sites in the sal1.1 CRE and that each protein is required for the direct repression of sal expression in the haltere. Furthermore, the sequence and spacing of Ubx and Smad binding sites are highly conserved and their proximity is required for target gene repression in the haltere. Because no evidence was found that these proteins interact directly, it is suggested this is an example of 'collaboration' or target gene co-regulation without direct cooperative interaction. These results have general implications for understanding how Hox proteins regulate diverse sets of target genes in animal development (Walsh, 2007).
The direct role for Smads in the repression of sal in the haltere is surprising in the light of previous genetic and molecular studies that had indicated that the Dpp pathway and Mad/Med were involved in sal activation in the wing. No direct evidence was found that this is the case and the fact that sal is activated in Mad and Med clones in the haltere indicates that sal is activated independently of Mad/Med in the flight appendages. The requirement for Mad/Med/Shn in shaping the pattern of sal expression in the wing appears to be indirect -- the protein complex represses the expression of brk, a repressor of sal, in cells in the central region of the developing wing and thereby permits sal expression (Walsh, 2007).
The Mad-Med-Shn complex is also active within cells in the central region of the haltere as a consequence of Dpp signaling. However, whereas sal is expressed and the sal1.1CRE is active in the wing, sal and the sal1.1 CRE are repressed in the haltere. These observations raise the question of how the Mad-Med-Shn complex selectively represses sal in the haltere but not in the wing disc? The results suggest that there are two key determinants in the selective repression of sal in the haltere. The first is collaboration with Ubx, which is expressed in the haltere and not in the wing disc. The second key determinant might be the affinity of Mad/Med binding to the sal CRE (Walsh, 2007).
The different responses of the brk and sal genes to Mad/Med/Shn suggests how the different affinities of proteins for binding sites might determine how available transcriptional regulatory inputs are integrated by CREs. Mad/Med binding to the brk CRE is of high affinity (Pyrowolakis, 2004) and apparently sufficient to impart repression, whereas that to the sal CRE is of much lower affinity and insufficient to impart repression in the wing. In the haltere, although Mad-Med-Shn or Ubx binding are alone insufficient, they act together either via simultaneous or sequential occupancy of their binding sites to repress sal (Walsh, 2007).
The requirement for two or more regulators to act together to control gene expression, i.e. combinatorial regulation, is fundamental to the generation of the great diversity of gene expression patterns by a finite set of transcription factors. Several previous studies have revealed the dual requirement for Hox and Smad functions for the activation of a target gene. Studies have suggested a general combinatorial mechanism for gene activation in which apparently separate transcriptional inputs act synergistically in gene activation and, in at least one case, the Hox response element and Dpp response element are separable. In this study, however, a requirement was observed for strict evolutionary conservation of the close topology of Hox and Smad binding sites in the sal CRE. It is suggested that collaboration is a distinct mode of combinatorial regulation in which two or more regulatory proteins must bind to nearby sites, but not necessarily to each other (Walsh, 2007).
The integration of Hox and Smad inputs could work through a number of possible mechanisms in the absence of direct physical interaction. One appealing possibility that might explain the requirement for the close proximity of binding sites is that Ubx and Mad-Med-Shn might interact with, and could therefore cooperatively recruit, the same co-repressor(s) for the repression of sal. Alternatively, if Mad-Med-Shn and Ubx bind sequentially to sal1.1, they might recruit different co-repressors and thereby orchestrate the assembly of a co-repressor complex. A third possibility is that because the Ubx and Mad/Med sites are embedded within a larger block of conserved regulatory DNA sequence in the sal1.1 CRE, the binding of other interacting transcription factors might also be involved in the repression of sal by Ubx and Mad-Med-Shn (Walsh, 2007).
These and recent results raise the question of whether collaboration is a general feature of target gene selection by Hox proteins. It is suggested that collaboration might be a widespread requirement for Hox function in vivo. This proposal is prompted by three observations: (1) Hox proteins alone have low DNA-binding specificity; (2) some, and perhaps all, Hox proteins might act as both repressors and activators; (3) Hox proteins regulate a great diversity of target genes that are also regulated by other transcription factors. In order to be such versatile regulators, it would be too great a constraint to require that Hox proteins always interact cooperatively with the diverse repertoire of transcription factors with which they act. Indeed, it may be argued that too much weight has been ascribed to the cooperative binding of Hox proteins and co-factors to DNA (Walsh, 2007).
Previously, much attention has focused on Exd and Hth, which interact with Hox proteins and bind cooperatively to DNA, thereby increasing Hox DNA-binding selectivity. However, it was only recently shown that the binding of these complexes alone was not sufficient to regulate target gene expression. Rather, Hox-Exd-Hth collaborate with and require the segmentation proteins Slp and En to repress the target gene Dll. This study has shown that the Exd- and Hth-independent target gene repression of sal requires collaboration between Ubx and Mad-Med-Shn. Although still a tiny sample of target genes, cases of transcription factors of various structural types acting as collaborators with Hox proteins are now available. The picture of Hox proteins relying on dedicated interacting co-factors such as Exd and Hth is expanding to a larger pool of collaborating transcription factors that modulate target gene selection (Walsh, 2007).
Indeed, collaboration might be the key to another unresolved mystery of the Hox proteins - the regulation of Hox protein activity. Some Hox proteins appear to act in both gene activation and repression; this is certainly the case for Ubx. This versatility would appear to be crucial to their role as sculptors of major features of body patterns, but how does the same transcription factor act positively in some contexts but negatively in others? There is evidence to suggest that the identity of the collaborating proteins and/or CRE sequences determines the 'sign' of Hox action (Walsh, 2007).
For instance, there is no evidence that the mere binding of Hox-Exd-Hth to a site determines the sign of Hox activity. These co-factors are involved in both Hox target gene activation (e.g., dpp in the midgut) and target repression (e.g.,Dll in the embryonic abdomen). But, in the latter case, En and Slp, two proteins that each harbor motifs for interaction with the co-repressor Groucho, are required collaborators for Dll repression. The roles of En and Slp in this instance might not be so much a matter of facilitating Hox target selection, but rather in regulating the sign of the output of the collaboration (Walsh, 2007).
Similar to the Hox proteins, the Smads can either activate or repress target genes. Furthermore, it has been demonstrated that the topology of Smad binding sites on DNA appears to be critical for determining whether a target gene is activated or repressed. In Drosophila, the topology of Mad and Med binding sites is critical for the recruitment of the co-repressor Shn. The recruitment of Shn was shown here to be necessary for sal repression. These two examples suggest that the positive or negative regulatory activity of a Hox protein depends on the context of surrounding binding sites and how they influence the activity of collaborating factors (Walsh, 2007).
The dependence of Hox proteins upon co-factors and collaborators indicates that, at the molecular level, Hox proteins are not 'master' regulatory proteins that dictate how target genes behave. Rather, they exert their great influence by virtue of their simple binding specificity, broad domains of expression and versatile, collaborative properties (Walsh, 2007).
Bases in 5' UTR -346
Bases in 3' UTR - 937
MAD and its homolog in vertebrates (Smad1) are essential for signaling in DPP and BMP-2/4 pathways and can elicit biological responses characteristic of BMP-2/4 (Newfeld, 1996). SMAD proteins share a high degree of homology in their amino-terminal MH1 (Mad homology) and carboxy-terminal MH2 domains. The MH2 domain is considered the effector domain, whose activity is opposed by its physical interaction with the MH1 domain. SMAD4 is a central signaling molecule of several TGFbeta-related pathways. In contrast to the pathway-restricted SMADs, SMAD4 rapidly associates with both SMAD1 in response to BMPR-1 signaling and SMAD2 in response to TbetaR-I and ActR-IB signaling. Unlike SMAD4, SMAD1 and SMAD2 contain consensus phosphorylation sites for receptor type I Ser/Thr kinases within their MH2 domains. The model emerging from recent biochemical and crystallographic studies implies that phosphorylation of the receptor-regulated SMADs relieves them of the MH1 inhibitory effect, allowing their interaction with SMAD4 and subsequent translocation to the nucleus (Sirard, 1998 and references).
Signal transduction specificity in the transforming growth factor-beta (TGF-beta) system is determined by ligand activation of a receptor complex, which then recruits and phosphorylates a subset of SMAD proteins, including Smads 1 and 2. In vertebrates, Smad1, and presumably its close homologs Smad5 and Smad8, are phosphorylated by BMP receptors and mediate BMP responses. Smad2 and its close homolog Smad3 are phosphorylated by TGF-beta receptors and mediate TGF-beta and activin responses. In Drosophila, Mad (a close homolog of Smad1) mediates the effects of the BMP-like factor, Dpp. After phosphorylation by receptors, Smads 1 and 2 associate with Smad4 and move into the nucleus where they regulate transcription. A discrete surface structure has been identified in Smads 1 and 2 that mediates and specifies their receptor interactions. This structure is the L3 loop, a 17 amino acid region that protrudes from the core of the conserved SMAD C-terminal domain. The L3 loop sequence is invariant among TGF-beta and bone morphogenetic protein (BMP)-activated SMADS, but differs at two positions between these two groups. Swapping these two amino acids in Smads 1 and 2 induces a gain or loss, respectively, in their ability to associate with the TGF-beta receptor complex and causes a switch in the phosphorylation of Smads 1 and 2 by the BMP and TGF-beta receptors, respectively. A full switch in phosphorylation and activation of Smads 1 and 2 is obtained by swapping these two amino acids while, in addition, swapping four amino acids near the C-terminal receptor phosphorylation sites. These studies identify the L3 loop as a determinant of specific SMAD-receptor interactions, and indicate that the L3 loop, together with the C-terminal tail, specifies SMAD activation (Lo, 1998).
date revised: 21 APR 97
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