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Thomas D Gilmore - One of the best experts on this subject based on the ideXlab platform.
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Two Alleles of NF-kB in the Sea Anemone Nematostella vectensis Are Widely Dispersed in Nature and Encode Proteins with Distinct Activities
2016Co-Authors: James C. Sullivan¤a, Thomas D Gilmore, Francis S. Wolenski, Adam M. Reitzel¤b, Courtney E. French, Nikki Traylor-knowles, John R FinnertyAbstract:Background: NF-kB is an evolutionarily conserved transcription factor that controls the expression of genes involved in many key organismal processes, including innate immunity, development, and stress responses. NF-kB proteins contain a highly conserved DNA-binding/dimerization Domain called the Rel Homology Domain. Methods/Principal Findings: We characterized two NF-kB alleles in the sea anemone Nematostella vectensis that differ at nineteen single-nucleotide polymorphisms (SNPs). Ten of these SNPs result in amino acid substitutions, including six within the Rel Homology Domain. Both alleles are found in natural populations of Nematostella. The Relative abundance of the two NF-kB alleles differs between populations, and departures from Hardy-Weinberg equilibrium within populations indicate that the locus may be under selection. The proteins encoded by the two Nv-NF-kB alleles have different molecular properties, in part due to a Cys/Ser polymorphism at residue 67, which resides within the DNA recognition loop. In nearly all previously characterized NF-kB proteins, the analogous residue is fixed for Cys, and conversion of human RHD proteins from Cys to Ser at this site has been shown to increase DNA-binding ability and increase resistance to inhibition by thiol-reactive compounds. However, the naturally-occurring Nematostella variant with Cys at position 67 binds DNA with a higher affinity than the Ser variant. On the other hand, the Ser variant activates transcription in reporter gene assays more effectively, and it is more resistant to inhibition by a thiol-reactive compound. Reciprocal Cys,-.Ser mutations at residue 67 of the nativ
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Rel Homology Domain-containing transcription factors in the cnidarian Nematostella vectensis.
Development genes and evolution, 2006Co-Authors: James C Sullivan, Thomas D Gilmore, Demetrios Kalaitzidis, John R FinnertyAbstract:The Rel/NF-κB and NFAT families of transcription factors are Related through an N-terminal DNA-binding Domain called the Rel Homology Domain (RHD). Neither the RHD nor the NF-κB pathway has been identified in a basal (i.e., nonbilaterian) animal phylum. Using genomic and cDNA databases, we have identified two RHD Domain-containing proteins from the cnidarian Nematostella vectensis: an NF-κB-like protein (Nv-NF-κB) and an NFAT-like protein (Nv-NFAT). The gene structure and RHD predicted amino acid sequence of Nv-nfkb are similar to those of the vertebrate NF-κB p50/p52 proteins, whereas the sequence of Nv-NFAT allows only ambiguous assignment to the NFAT family. Nv-NF-κB lacks the C-terminal IκB-like sequences present in all other NF-κB proteins. There are, however, two IκB-like genes in Nematostella encoded by loci distinct from Nv-nfkb. The separate nfkb and ikb genes of Nematostella may reflect the ancestral metazoan condition, suggesting that a gene fusion event created the nfkb genes in Drosophila and vertebrates. Nematostella also has genes that encode upstream and downstream components of the vertebrate NF-κB signaling pathway. Upstream components include Toll- and tumor necrosis-like receptors and ligands, adaptor proteins (Trafs, Myd88), caspases, and a TBK-like kinase. Downstream components include the NF-κB coactivator protein Bcl-3 and several NF-κB target genes. These results demonstrate that RHD-containing transcription factors and associated pathways are evolutionarily more ancient than previously known. Moreover, they suggest models for the evolutionary diversification of the insect and vertebrate Rel/NF-κB/IκB and NFAT gene families and suggest that cnidarians possess an NF-κB-regulated developmental or stress response pathway.
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Transformation by the vRel oncoprotein requires sequences carboxy-terminal to the Rel Homology Domain.
Oncogene, 1993Co-Authors: Sarkar S, Thomas D GilmoreAbstract:The vRel oncoprotein of the avian Rev-T retrovirus is a member of the Rel/NF-kappa B family of transcription factors. The highly conserved amino-terminal Rel Homology (RH) Domain in these proteins is required for DNA binding, protein-protein interactions and nuclear localization, and many mutations within this Domain abolish transformation by vRel. We demonstrate here that overexpression of the vRel RH Domain alone is insufficient to induce transformation of chicken spleen cells, indicating that sequences from the nonconserved carboxy terminus are necessary for the vRel transforming function. Therefore, we constructed and assayed several vRel mutants with deletions of carboxy-terminal sequences. These mutant vRel proteins did not transform spleen cells with equal efficiency, even though they were functionally similar by several other criteria. Our results demonstrate that there are two regions (aa 389 to 432 and aa 437 to 503) within the carboxy-terminal half of vRel that are important for transformation: mutant vRel proteins containing the RH Domain and one or both of these carboxy-terminal regions can transform at roughly wild-type levels. Analysis of Gal4 fusion proteins containing carboxy-terminal sequences from the vRel mutants indicated that there is a corRelation between the ability of these mutant proteins to transform avian spleen cells and their ability to activate transcription. These observations suggest that vRel induces malignant transformation by directly altering gene expression.
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p105 the nf kappa b p50 precursor protein is one of the cellular proteins complexed with the v Rel oncoprotein in transformed chicken spleen cells
Journal of Virology, 1992Co-Authors: Anthony J Capobianco, D Chang, George Mosialos, Thomas D GilmoreAbstract:Active NF-kappa B-like transcription complexes are multimers consisting of one or two members of a family of proteins Related to the c-Rel proto-oncoprotein. We have isolated a chicken cDNA encoding p105, the precursor protein for the p50 subunit of NF-kappa B. Sequence analysis shows that chicken p105 is approximately 70% identical to the mouse and human p105 proteins, containing the Rel Homology Domain in its N-terminal 370 amino acids and several ankyrinlike repeats in the C-terminal portion of the protein. The Rel Homology Domain is particularly highly conserved between chicken and mammalian p50, and an in vitro-synthesized, truncated chicken p105 protein, containing sequences that correspond to the predicted p50 protein, bound to a consensus kappa B site in an electrophoretic mobility shift assay. In v-Rel-transformed chicken spleen cells, v-Rel is found in high-molecular-weight complexes which include cellular proteins of approximately 124 kDa (p124) and 115 kDa (p115). Here we report that in vitro-produced p105 comigrates with p124 from v-Rel-transformed spleen cells and that p105 and p124 appear to be identical by partial proteolytic mapping with V8 protease. Furthermore, both p105 and p50 can complex directly with v-Rel and chicken c-Rel in vitro. However, in vitro association with p105 by v-Rel does not necessarily corRelate with transformation, since one nontransforming v-Rel mutant can associate with p105 in vitro.
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a protein kinase a recognition sequence is structurally linked to transformation by p59v Rel and cytoplasmic retention of p68c Rel
Molecular and Cellular Biology, 1991Co-Authors: George Mosialos, Anthony J Capobianco, Peter Hamer, Richard A Laursen, Thomas D GilmoreAbstract:The Rel family of proteins includes a number of proteins involved in transcriptional control, such as the retroviral oncoprotein v-Rel, c-Rel, the Drosophila melanogaster developmental protein Dorsal, and subunits of the transcription factor NF-kappa B. These proteins are Related through a highly conserved Domain of approximately 300 amino acids, called the Rel Homology Domain, that contains dimerization, DNA binding, and nuclear targeting functions. Also within the Rel Homology Domain, there is a conserved consensus sequence (Arg-Arg-Pro-Ser) for phosphorylation by cyclic AMP-dependent protein kinase (PKA). We used linker insertion mutagenesis and site-directed mutagenesis to determine the importance of this sequence for the transformation of avian spleen cells by v-Rel and the subcellular localization of c-Rel in chicken embryo fibroblasts (CEF). The insertion of 2 amino acids (Pro-Trp) within this sequence completely abolished transformation and transcriptional repression by v-Rel and resulted in a shift in the localization of c-Rel from cytoplasmic to nuclear in CEF. When the conserved Ser within the PKA recognition sequence was replaced by Ala, there was no significant effect on transformation and transcriptional repression by v-Rel or on cytoplasmic retention of c-Rel. However, when this Ser was changed to Asp or Glu, transformation and transcriptional repression by v-Rel were significantly inhibited and c-Rel showed a diffuse nuclear and cytoplasmic localization in CEF. Although a peptide containing the recognition sequence from v-Rel can be phosphorylated by PKA in vitro, this site is not constitutively phosphorylated to a high degree in vivo in transformed spleen cells incubated with okadaic acid. Our results indicate that the transforming and transcriptional repressing activities of v-Rel and the cytoplasmic retention of c-Rel are dependent on the structure of the conserved PKA recognition motif. In addition, they suggest that phosphorylation at the conserved PKA site could have a negative effect on transformation and transcriptional repression by v-Rel and induce the nuclear localization of c-Rel.
Josef Anrather - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Bovine Endothelial Cell Activation In Vitro by Regulated Expression of a Transdominant Inhibitor of NF- k B
2016Co-Authors: Clin J. Invest, Josef Anrather, Vilmos Csizmadia, Christine Brostjan, Miguel P. Soares, Fritz H. Bach, Hans WinklerAbstract:The activation of endothelial cells is a recurrent phenome-non linked to pathologic conditions such as inflammation, chronic arthritis, allo- and xenograft rejection. To inhibit endothelial cell activation we have constructed a transacti-vation-deficient derivative of the p65/RelA subunit of NF-k B, a transcription factor known to be crucial for the induction of adhesion molecules, cytokines and procoagulants in acti-vated endothelial cells. This protein (p65RHD) comprises the Rel Homology Domain of the RelA subunit, retaining dimerization, DNA binding, and nuclear localization func-tions, but is deficient in transcriptional activation, and acts as a competitive inhibitor of NF-k B. Our data demonstrate that p65RHD is a potent and specific inhibitor of NF-
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Site-specific Phosphorylation of the p65 Protein Subunit Mediates Selective Gene Expression by Differential NF-κB and RNA Polymerase II Promoter Recruitment
The Journal of biological chemistry, 2012Co-Authors: Karin Hochrainer, Gianfranco Racchumi, Josef AnratherAbstract:Phosphorylation of NF-κB plays an important role in modulating transcriptional activity of NF-κB independently of inhibitor of κB (IκB) proteins. For the p65 subunit, multiple phosphorylation sites have been mapped in and adjacent to both the N-terminal Rel Homology Domain and the C-terminal transactivation Domain. Their impact on NF-κB-dependent transcription, however, has never been assessed at a broader level. In this study, we evaluate the importance of differential p65 phosphorylation on four serine acceptor sites in the Rel Homology Domain for the expression of an array of NF-κB-dependent genes in endothelial cells. We find that inhibition of p65 phosphorylation on these serine residues targets NF-κB activity to distinctive gene subsets in a κB enhancer element-specific context. We show that the phosphorylation-dependent alterations in gene and protein expression are reflective of the amount of p65 and phosphorylated RNA polymerase II (p-RNAP II) bound to respective gene promoter regions. Depending on the gene subset, impaired gene expression was either a result of decreased p65 promoter recruitment or of a failure of bound p65 to recruit p-RNAP II. In conclusion, our findings demonstrate that site-specific p65 phosphorylation targets NF-κB activity to particular gene subsets on a global level by influencing p65 and p-RNAP II promoter recruitment.
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Hypo‐phosphorylation leads to nuclear retention of NF‐κB p65 due to impaired IκBα gene synthesis
FEBS letters, 2007Co-Authors: Karin Hochrainer, Gianfranco Racchumi, Josef AnratherAbstract:Subcellular localization guided by IκBα is crucial for regulation of nuclear factor-κB function. Here, we show that p65 Rel Homology Domain phosphorylation mutants are transported into the nucleus after IκBα degradation, but as a consequence of lower IκBα levels their Relocation to the cytosol is blocked. We demonstrate that phosphorylation of residues S205, S276, and S281 of p65 is not required for interaction between p65 and IκBα, but is pivotal for regulating cellular IκBα levels by positively affecting gene synthesis. Our findings indicate that reduction of phosphorylation leads to nuclear retention of p65, which might be partly responsible for altered transcriptional behavior of p65 serine mutants.
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cis-Acting Element-specific Transcriptional Activity of Differentially Phosphorylated Nuclear Factor-κB
The Journal of biological chemistry, 2004Co-Authors: Josef Anrather, Gianfranco Racchumi, Costantino IadecolaAbstract:Abstract Phosphorylation of nuclear factor-κB (NF-κB) subunits emerges as a mechanism by which transcriptional activity of nuclear NF-κB complexes is regulated in an inhibitor κB-independent fashion. As the main transactivator, the p65 subunit of NF-κB has an outstanding position in the hierarchy of NF-κB proteins. p65 is a multiply phosphorylated protein with phosphorylation sites in the C-terminal transactivation Domain and the N-terminal Rel Homology Domain (RHD). In this study, we describe two previously non-reported phospho-acceptor sites within the p65 RHD. We show that differential phosphorylation of serine residues within the RHD modulates transcriptional activity in a cis-acting element and promoter-specific context, thus leading to a phosphorylation state-dependent gene expression profile. RelA-/- mouse embryonic fibroblasts reconstituted with wild-type p65 or p65 phosphorylation-deficient mutants showed a distinctive expression profile of synthetic κB-dependent reporters as well as endogenous genes. Hypophosphorylated p65 did not display cis-acting element-specific changes in DNA binding or dimerization behavior. This study shows for the first time that site-specific phosphorylation can target a transcription factor to a particular subset of genes.
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Regulation of NF-κB RelA Phosphorylation and Transcriptional Activity by p21 ras and Protein Kinase Cζ in Primary Endothelial Cells
The Journal of biological chemistry, 1999Co-Authors: Josef Anrather, Vilmos Csizmadia, Miguel P. Soares, Hans WinklerAbstract:The activity of the transcription factor NF-κB is thought to be regulated mainly through cytoplasmic retention by IκB molecules. Here we present evidence of a second mechanism of regulation acting on NF-κB after Release from IκB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-κB through a pathway involving activation of protein kinase Cζ (PKCζ) and p21ras. We show that transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel Homology Domain but not the C-terminal transactivation Domain. Inhibition of phosphorylation by dominant negative mutants of PKCζ or p21ras results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation.
John R Finnerty - One of the best experts on this subject based on the ideXlab platform.
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Two Alleles of NF-kB in the Sea Anemone Nematostella vectensis Are Widely Dispersed in Nature and Encode Proteins with Distinct Activities
2016Co-Authors: James C. Sullivan¤a, Thomas D Gilmore, Francis S. Wolenski, Adam M. Reitzel¤b, Courtney E. French, Nikki Traylor-knowles, John R FinnertyAbstract:Background: NF-kB is an evolutionarily conserved transcription factor that controls the expression of genes involved in many key organismal processes, including innate immunity, development, and stress responses. NF-kB proteins contain a highly conserved DNA-binding/dimerization Domain called the Rel Homology Domain. Methods/Principal Findings: We characterized two NF-kB alleles in the sea anemone Nematostella vectensis that differ at nineteen single-nucleotide polymorphisms (SNPs). Ten of these SNPs result in amino acid substitutions, including six within the Rel Homology Domain. Both alleles are found in natural populations of Nematostella. The Relative abundance of the two NF-kB alleles differs between populations, and departures from Hardy-Weinberg equilibrium within populations indicate that the locus may be under selection. The proteins encoded by the two Nv-NF-kB alleles have different molecular properties, in part due to a Cys/Ser polymorphism at residue 67, which resides within the DNA recognition loop. In nearly all previously characterized NF-kB proteins, the analogous residue is fixed for Cys, and conversion of human RHD proteins from Cys to Ser at this site has been shown to increase DNA-binding ability and increase resistance to inhibition by thiol-reactive compounds. However, the naturally-occurring Nematostella variant with Cys at position 67 binds DNA with a higher affinity than the Ser variant. On the other hand, the Ser variant activates transcription in reporter gene assays more effectively, and it is more resistant to inhibition by a thiol-reactive compound. Reciprocal Cys,-.Ser mutations at residue 67 of the nativ
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Rel Homology Domain-containing transcription factors in the cnidarian Nematostella vectensis.
Development genes and evolution, 2006Co-Authors: James C Sullivan, Thomas D Gilmore, Demetrios Kalaitzidis, John R FinnertyAbstract:The Rel/NF-κB and NFAT families of transcription factors are Related through an N-terminal DNA-binding Domain called the Rel Homology Domain (RHD). Neither the RHD nor the NF-κB pathway has been identified in a basal (i.e., nonbilaterian) animal phylum. Using genomic and cDNA databases, we have identified two RHD Domain-containing proteins from the cnidarian Nematostella vectensis: an NF-κB-like protein (Nv-NF-κB) and an NFAT-like protein (Nv-NFAT). The gene structure and RHD predicted amino acid sequence of Nv-nfkb are similar to those of the vertebrate NF-κB p50/p52 proteins, whereas the sequence of Nv-NFAT allows only ambiguous assignment to the NFAT family. Nv-NF-κB lacks the C-terminal IκB-like sequences present in all other NF-κB proteins. There are, however, two IκB-like genes in Nematostella encoded by loci distinct from Nv-nfkb. The separate nfkb and ikb genes of Nematostella may reflect the ancestral metazoan condition, suggesting that a gene fusion event created the nfkb genes in Drosophila and vertebrates. Nematostella also has genes that encode upstream and downstream components of the vertebrate NF-κB signaling pathway. Upstream components include Toll- and tumor necrosis-like receptors and ligands, adaptor proteins (Trafs, Myd88), caspases, and a TBK-like kinase. Downstream components include the NF-κB coactivator protein Bcl-3 and several NF-κB target genes. These results demonstrate that RHD-containing transcription factors and associated pathways are evolutionarily more ancient than previously known. Moreover, they suggest models for the evolutionary diversification of the insect and vertebrate Rel/NF-κB/IκB and NFAT gene families and suggest that cnidarians possess an NF-κB-regulated developmental or stress response pathway.
Gourisankar Ghosh - One of the best experts on this subject based on the ideXlab platform.
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Role of lysine methylation of NF-κB in differential gene regulation
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Maojing Yang, Gourisankar Ghosh, De Bin Huang, Han Wei, Gulcin Ozer, George R. StarkAbstract:Lysine methylation of the p65 subunit of nuclear factor κB (NF-κB) on K218 and K221 together or K37 alone strongly enhances gene expression in response to cytokines. We analyzed the effects of K-to-Q mutations in the Rel Homology Domain of p65 on the response to IL-1β in 293 cells with low levels of p65. The K218/221Q mutation greatly reduced the expression of 39 of 82 genes, whereas the K37Q mutation reduced the expression of 23 different genes. Enhanced expression of the lysine demethylase FBXL11, which catalyzes the demethylation of K218 and K221 specifically, inhibited the expression of most of the genes that were inhibited by the DKQ mutation. CHIP-Seq analysis showed that the K218/221Q mutation greatly reduces the affinity of p65 for many promoters and that the K37Q mutation does not. Structural modeling showed that the newly introduced methyl groups of K218 and K221 interact directly with DNA to increase the affinity of p65 for specific κB sites. Thus, the K218/221Q and K37Q mutations have dramatically different effects because methylations of these residues affect different genes by distinct mechanisms.
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the nfkb1 and nfkb2 proteins p105 and p100 function as the core of high molecular weight heterogeneous complexes
Molecular Cell, 2009Co-Authors: Olga V Savinova, Alexander Hoffmann, Gourisankar GhoshAbstract:Summary Nfkb1 and Nfkb2 proteins p105 and p100 serve both as NF-κB precursors and inhibitors of NF-κB dimers. In a biochemical characterization of endogenous cytoplasmic and purified recombinant proteins, we found that p105 and p100 assemble into high-molecular-weight complexes that contribute to the regulation of all NF-κB isoforms. Unlike the classical inhibitors IκBα, -β, and -ɛ, high-molecular-weight complexes of p105 and p100 proteins bind NF-κB subunits in two modes: through direct dimerization of Rel Homology Domain-containing NF-κB polypeptides and through interactions of the p105 and p100 ankyrin repeats with preformed NF-κB dimers, thereby mediating the bona fide IκB activities, IκBγ and IκBδ. Our biochemical evidence suggests an assembly pathway in which kinetic mechanisms control NF-κB dimer formation via processing and assembly of large complexes that contain IκB activities.
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the nfkb1 and nfkb2 proteins p105 and p100 function as the core of high molecular weight heterogeneous complexes
Molecular Cell, 2009Co-Authors: Olga V Savinova, Alexander Hoffmann, Gourisankar GhoshAbstract:Nfkb1 and Nfkb2 proteins p105 and p100 serve both as NF-kappaB precursors and inhibitors of NF-kappaB dimers. In a biochemical characterization of endogenous cytoplasmic and purified recombinant proteins, we found that p105 and p100 assemble into high-molecular-weight complexes that contribute to the regulation of all NF-kappaB isoforms. Unlike the classical inhibitors IkappaBalpha, -beta, and -epsilon, high-molecular-weight complexes of p105 and p100 proteins bind NF-kappaB subunits in two modes: through direct dimerization of Rel Homology Domain-containing NF-kappaB polypeptides and through interactions of the p105 and p100 ankyrin repeats with preformed NF-kappaB dimers, thereby mediating the bona fide IkappaB activities, IkappaBgamma and IkappaBdelta. Our biochemical evidence suggests an assembly pathway in which kinetic mechanisms control NF-kappaB dimer formation via processing and assembly of large complexes that contain IkappaB activities.
Albert J. Courey - One of the best experts on this subject based on the ideXlab platform.
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Dorsal interacting protein 3 potentiates activation by Drosophila Rel-Homology Domain proteins
Developmental and comparative immunology, 2008Co-Authors: Girish S. Ratnaparkhi, Hao A. Duong, Albert J. CoureyAbstract:Dorsal interacting protein 3 (Dip3) contains a MADF DNA-binding Domain and a BESS protein interaction Domain. The Dip3 BESS Domain was previously shown to bind to the Dorsal Rel Homology Domain. We show here that Dip3 also binds to the Relish Rel Homology Domain and enhances Rel family transcription factor function in both dorsoventral patterning and the immune response. While Dip3 is not essential, Dip3 mutations enhance the embryonic patterning defects that result from dorsal haplo-insufficiency, indicating that Dip3 may render dorsoventral patterning more robust. Dip3 is also required for optimal resistance to immune challenge since Dip3 mutant adults and larvae infected with bacteria have shortened lifetimes Relative to infected wild-type flies. Furthermore, the mutant larvae exhibit significantly reduced expression of antimicrobial defense genes. Chromatin immunoprecipitation experiments in S2 cells indicate the presence of Dip3 at the promoters of these genes, and this binding requires the presence of Rel proteins at these promoters.
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The Dorsal Rel Homology Domain plays an active role in transcriptional regulation.
Molecular and cellular biology, 2002Co-Authors: Songtao Jia, Rubén D. Flores-saaib, Albert J. CoureyAbstract:The Dorsal morphogen directs formation of the Drosophila dorsoventral axis by both activating and repressing transcription. It contains an N-terminal Rel Homology Domain (RHD), which is responsible for DNA binding and regulated nuclear import, and a C-terminal Domain (CTD) that contains activation and repression motifs. To determine if the RHD has a direct role in transcriptional control, we analyzed a series of RHD mutations in S2 cells and embryos. Two classes of mutations (termed class I and class II mutations) that alter activation without affecting DNA binding or nuclear import were identified. The two classes appear to define distinct protein interaction surfaces on opposite faces of the RHD. Class I mutations enhance an apparently inhibitory interaction between the RHD and the CTD and eliminate both activation and repression by Dorsal. In contrast, class II mutations result in increased activation in S2 cells but seveRely decreased activation in embryos and have little effect on repression. Analysis of the cuticles of class II mutant embryos suggests that, in the absence of Dorsal-mediated activation, Dorsal-mediated repression is not sufficient to pattern the embryo. These results provide some of the first evidence that the RHD plays an active role in transcriptional regulation in intact multicellular organisms.
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A Direct Contact between the Dorsal Rel Homology Domain and Twist May Mediate Transcriptional Synergy
Molecular and cellular biology, 1997Co-Authors: Jill M. Shirokawa, Albert J. CoureyAbstract: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. Here we show that 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. In Dorsal, the Rel Homology Domain appears to be sufficient for synergy. Protein-protein interaction assays show that Twist and Dorsal bind to one another in vitro. However, this interaction does not appear to be of sufficient strength to yield cooperative binding to DNA. Nonetheless, the regions of Twist and Dorsal required for the binding interaction are also required for synergistic transcriptional activation.