| Using in vitro and in vivo assays, we define a network of Her /Hes dimers underlying transcriptional negative feedback within the posterior zebrafish segmentation clock. Dimerization is specific, with Hes6 serving as the hub of the network. Her1 binds DNA only as a homodimer but will also dimerize with Hes6 in a non-DNA binding dimer. Her12 and Her15 bind DNA both as homodimers and as heterodimers with Hes6. Her7 dimerizes with Hes6 and Her15, the latter as a non-DNA binding dimer, and is the only protein not to form a homodimer. We determined that there are multiple residues in the loop and second helix domains that control dimerization partner by swapping subsets of the basic-helix-loop-helix domain between Her1, Her7 and Hes6. In addition to differences in dimerization, there are also differences between the strength and specificity of the DNA binding dimers for the E box DNA sequence. Changing the robustness of the dimers may alter their repressive ability, and function, in the network. This network structure engenders specific network dynamics and node functions. Computational and genetic analyses suggest Her7 disproportionately influences the availability of Hes6 to heterodimerize with other Her proteins. Thus, Her7 acts directly within the delayed negative feedback and also modulates network topology via sequestration of the network hub, Hes6. This latter function is an emergent property of the network that contributes to normal operation of the zebrafish segmentation clock. |
