Combinatorial transcriptional regulation: the interaction of transcription factors and cell signaling molecules with homeodomain proteins in Drosophila development.

Patterning and cell fate specification during development require complex interplay among multiple families of transcription factors to establish, maintain, and coordinate transcriptional cascades. During these processes, homeodomain proteins and cell signaling proteins cooperate to generate tissue-and stage-specific responses. This review of physical and genetic interactions in Drosophila melanogaster development highlights the cross-talk among these protein families. Protein-protein association can modulate regulation by both signal transduction-regulated transcription factors and homeodomain proteins, as observed in Drosophila and other organisms. Enhancers or genes regulated by multiple transcription factors provide opportunities for protein-protein binding to modulate transcription factor function. Combinatorial regulation of several enhancers by homeodomain proteins and cell signaling-regulated transcription factors is discussed; detailed maps of the genetic interactions that pattern the embryonic midgut and the larval wing imaginal disc are used to illustrate the multiplicity of potential protein-protein interactions. These interactions potentially provide direct mechanisms for communication between transcription factors as well as for generating the requisite functional specificity.

High-pressure denaturation of apomyoglobin.

The pressure denaturation of wild type and mutant apomyoglobin (apoMb) was investigated using a high-pressure, high-resolution nuclear magnetic resonance and high-pressure fluorescence techniques. Wild type apoMb is resistant to pressures up to 80 MPa, and denatures to a high-pressure intermediate, I(p), between 80 and 200 MPa. A further increase of pressure to 500 MPa results in denaturation of the intermediate. The two tryptophans, both in the A helix, remain sequestered from solvent in the high-pressure intermediate, which retains some native NOESY cross peaks in the AGH core as well as between F33 and F43. High-pressure fluorescence shows that the tryptophans remain inaccessible to solvent in the I(p) state. Thus the high-pressure intermediate has some structural properties in common with the apoMb I(2) acid intermediate. The resistance of the AGH core to pressures up to 200 MPa provides further evidence that the intrinsic stability of these alpha-helices is responsible for their presence in a number of equilibrium intermediates as well as in the earliest kinetic folding intermediate. Mutations in the AGH core designed to disrupt packing by burying a charge or increasing the size of a hydrophobic residue significantly perturbed the unfolding of native apoMb to the high-pressure intermediate. The F123W and S108L mutants both unfolded at lower pressures, while retaining some resistance to pressures below 50 MPa. The charge burial mutants, A130K and S108K, are not stable at very low pressures and both denature to the intermediate by 100 MPa, half of the pressure required for wild type apoMb. Thus a similar intermediate state is created independent of the method of perturbation, and mutations have similar effects on native state destabilization for both methods of denaturation. These data suggest that equilibrium intermediates that can be formed through different means are likely to resemble a kinetic intermediate.