Allen Laughon

Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic

The TGF-β (transforming growth factor-β)-related signalling proteins, including Decapentaplegic (Dpp) in Drosophila and bone morphogenic proteins and activin in vertebrates, affect the growth and patterning of a great variety of structures. However, the mechanisms by which these ligands regulate gene expression are not understood. Activation of complexes of type I with type II receptors results in the phosphorylation and nuclear localization of members of the SMAD protein family, which are thought to act as co-activators of transcription, perhaps in conjunction with sequence-specific cofactors. Here we show that the amino-terminal domain of the Drosophila Mothers against dpp protein (Mad), a mediator of Dpp signalling, possesses a sequence-specific DNA-binding activity that becomes apparent when carboxy-terminal residues are removed. Mad binds to and is required for the activation of an enhancer within the vestigial wing-patterning gene in cells across the entire developing wing blade. Mad also binds to Dpp-response elements in other genes. These results suggest that Dpp signalling regulates gene expression by activating Mad binding to target gene enhancers.

Interaction of Smad Complexes with Tripartite DNA-binding Sites

The Smad family of transcription factors function as effectors of transforming growth factor-β signaling pathways. Smads form heteromultimers capable of contacting DNA through the amino-terminal MH1 domain. The MH1 domains of Smad3 and Smad4 have been shown to bind to the sequence 5′-GTCT-3′. Here we show that Smad3 and Smad4 complexes can contact three abutting GTCT sequences and that arrays of such sites elevate reporter expression relative to arrays of binding sites containing only two GTCTs. Smad3/4 complexes bound synergistically to probes containing two of the four possible arrangements of three GTCT sequences and showed a correlated ability to synergistically activate transcription through these sites. Purified Smad3 and Smad4 were both able to contact three abutting GTCT sequences and reporter experiments indicated that either protein could mediate contact with all three GTCTs. In contrast, the Smad4 MH1 domain was essential for reporter activation in combination with Smad1. Together, these results show that Smad complexes are flexible in their ability to interact with abutting GTCT triplets. In contrast, Smads have high affinity for only one orientation of abutting GTCT pairs. Functional Smad-binding sites within several native response elements contain degenerate GTCT triplets, suggesting that trimeric Smad-DNA interaction may be relevant in vivo.

Repression of Dpp Targets by Binding of Brinker to Mad Sites

Signaling by decapentaplegic (Dpp), aDrosophila member of the transforming growth factor (TGF) β superfamily of growth factors, has recently been shown to activate targets such as vestigial (vg) indirectly through negative regulation of brinker (brk). Here we show that the Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding activity was localized to an amino-terminal region containing a putative homeodomain. Brk bound to a Dpp response element of the Ultrabithorax(Ubx) midgut enhancer at a sequence that overlaps a binding site for the Smad protein, Mothers Against Dpp (Mad). Furthermore, Brk was able to compete with Mad for occupancy of this binding site. This recognition of overlapping binding sites provides a potential explanation for why the G/C-rich Mad binding site consensus differs the Smad3/Smad4 binding site consensus. We also found that the Dpp response element from Ubx was more sensitive than the vgquadrant enhancer to repression by Brk. This difference correlates with short-range activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Brk binding sites may play a critical role in limiting thresholds for activation by Dpp. Finally, we provide evidence that Brk is capable of functioning as an active repressor. Thus, whereas Brk and Mad compete for regulation of Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad.

Expanded, a negative regulator of cell proliferation in drosophila, shows homology to the NF2 tumor suppressor

Genetic methods in Drosophila make it possible to identify negative regulators of cell proliferation by the overgrowth phenotype caused by loss-of-function mutations, and sequence comparisons can identify human homologs of these genes that may also function as tumor suppressor genes (Watson and Bryant, 1993). Confirming this possibility, we have found that the product of the expanded gene (ex; Boedigheimer and Laughon, 1993) shows extensive homology to the protein encoded by the human neurofibromatosis 2 (NF2) tumor suppressor gene (Trofatter et al., 1993; Rouleau et al., 1993), as well as to other members of the Band 4.1 protein family (for review see Trofatter, 1993). Null mutations in ex affect cell differentiation and proliferation in imaginal discs. Mutants die as pharate adults with highly overgrown wings, proximal leg segments and proximal antennal segments, as well as missing eyes, distal leg segments and distal antennal segments. The wing overgrowth is due to extra cell divisions occurring in the wing primordia primarily during the third larval instar stage, but the disc tissue retains its single layered epithelial structure and ability to differentiate; thus, ex may belong to the hyperplastic group of tumor suppressor genes (Watson and Bryant, 1993). The homology between Ex and NF2 is broken into seven blocks that are shared between all members of the Band 4.1 family (Fig. 1A), with variable spacing between blocks. Within these blocks the amino acid identity between Ex and other members ranges from about 20% to 50% (Fig. 1B). This family of proteins is readily divided into two subfamilies based on amino acid sequence in and around the conserved blocks. Ex

Drosophila glial architecture and development: analysis using a collection of new cell-specific markers

Monoclonal antibodies and enhancer trap insertions that mark subsets of neurons have been valuable tools in the study of Drosophila neuronal development. Similarly, glial specific cell markers could prove to be valuable in investigating the development and function of glia. Here we characterize the architecture and development of distinct sets of Drosophila embryonic glia, using a reporter gene driven by fushi tarazu homeodomain binding sites. Reporter expresssion in glia is dependent on the orientation and spacing of the homeodomain binding sites, revealing potential differences in glial determination. These studies suggest that the use of transcription factor binding sites to drive reporter gene expression may prove to be a generally useful means of generating additional cell type-specific markers.

Crossveinless d is a vitellogenin-like lipoprotein that binds BMPs and HSPGs, and is required for normal BMP signaling in the Drosophila wing

The sensitivity of the posterior crossvein in the pupal wing of Drosophila to reductions in the levels and range of BMP signaling has been used to isolate and characterize novel regulators of this pathway. We show here that crossveinless d (cv-d) mutations, which disrupt BMP signaling during the development of the posterior crossvein, mutate a lipoprotein that is similar to the vitellogenins that comprise the major constituents of yolk in animal embryos. Cv-d is made in the liver-like fat body and other tissues, and can diffuse into the pupal wing via the hemolymph. Cv-d binds to the BMPs Dpp and Gbb through its Vg domain, and to heparan sulfate proteoglycans, which are well-known for their role in BMP movement and accumulation in the wing. Cv-d acts over a long range in vivo, and does not have BMP co-receptor-like activity in vitro. We suggest that, instead, it affects the range of BMP movement in the pupal wing, probably as part of a lipid-BMP-lipoprotein complex, similar to the role proposed for the apolipophorin lipid transport proteins in Hedgehog and Wnt movement.

Decapentaplegic-responsive Silencers Contain Overlapping Mad-binding Sites

Smad proteins regulate transcription in response to transforming growth factor-β signaling pathways by binding to two distinct types of DNA sites. The sequence GTCT is recognized by all receptor-activated Smads and by Smad4. The subset of Smads that responds to bone morphogenetic protein signaling recognizes a distinct class of GC-rich sites in addition to GTCT. Recent work has shown that Drosophila Mad protein, the homologue of bone morphogenetic protein rSmads, binds to GRCGNC sites through the same MH1 domain β-hairpin interface used to contact GTCT sites. However, binding to GRCGNC requires base-specific contact by two Mad proteins, and here we provide evidence that this is achieved by contact of the two Mad subunits that overlap across the two central base pairs of the site. This topology is supported by results indicating that His-93, which is located at the tip of the Mad β-hairpin, is in close proximity to base pairs 2 and 5. Also consistent with the model is disruption of binding by mutation of Glu-39 and Glu-40, which are predicted to lie at the interface of the two overlapping Mad MH1 domains. As predicted from the overlapping model, binding is disrupted by insertion of 1 bp in the middle of the site, whereas insertion of 2 bp creates abutting sites that can be bound by the Mad-Medea heterotrimer without requiring Glu-39 and Glu-40. Overlapping Mad sites predominate in decapentaplegic response elements, consistent with a high degree of specificity in response to signaling.

The isolation and characterization of a Drosophila gene encoding a putative NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase

Mammalian NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase is a bifunctional mitochondrial enzyme expressed in most established cell lines but only in developing normal tissues. We report the cloning and molecular characterization of a Drosophila gene (DNMDMC) that encodes a protein with 56% identity to the mammalian bifunctional protein. Like the mammalian bifunctional proteins, the Drosophila protein contains a putative mitochondrial targeting sequence and its transcripts are expressed in developing tissues. Unlike its mammalian homologs, DNMDMC is expressed at high levels in adult tissues. DNMDMC maps to polytene chromosome band 85C, is encoded in three exons, and is closely flanked by two additional genes.

Drosophila glial development is regulated by genes involved in the control of neuronal cell fate

The Drosophila proneural genes specify neuronal determination among cells within the ectoderm. Here we address the question of whether proneural genes also affect the specification of glia, the most abundant cell type in the nervous system. We provide evidence that the proneural gene daughterless is essential for the formation of two major classes of PNS glia. In contrast, the proneural genes in the achaete-scute complex have no detectable effect on the specification and differentiation of these PNS glia and certain CNS glia. We also show that, as with neuronal development, glial determination is restricted by the neurogenic genes neuralized, Delta, and the genes of the Enhancer of split complex. Finally, we demonstrate that prospero, a gene involved in neuronal differentiation, also affects glial development. These results demonstrate extensive overlap in the genetic control of glial and neuronal development.