Atypical binding of the neuronal POU protein N-Oct3 to noncanonical DNA targets - Implications for heterodimerization with HNF-3 beta

The capacity of POU proteins to recognize different DNA sequences and to bi nd target DNA in the form of monomers, cooperative dimers or heterodimers i s important in relation to their transcriptional regulatory properties. The N-Oct3 neuron-specific protein binds to an octamer-like sequence (AAATAATG C) within the (-102/-72) neuronal promoter region of the human aromatic L-a mino acid decarboxylase (AADC) gene. In this atypical case the POUh and POU s tetrameric subsites are spaced one nucleotide apart and in switched order as compared with the consensus octamer. Moreover this POU binding motif ov erlaps the hepatocyte nuclear factor HNF-3 beta binding site (TGCTCAGTAAA) which itself contains a heptamer-like sequence (CTCAGTA). Using the isolate d DNA binding domains (DBD) of the two proteins, it is shown that, when bin ding to this unusual recognition sequence, N-Oct3 either exhibits noncooper ative homodimerization or allows the simultaneous binding of the second tra nscription activator HNF-3 beta. CD studies indicate that the binding of N- Oct3 monomers/dimers and N-Oct3-HNF-3 beta heterodimers to the DNA induces conformational changes of both protein and DNA, Partial proteolysis/MALDI-M S was used in conjunction with molecular modelling to show that the protein conformational change resulting from binary N-Oct3/DNA complex formation o ccurs within the linker peptide joining the POUs and POUh subdomains. Furth ermore, modelling the N-Oct3/HNF-3 beta /DNA ternary complex predicts a nuc leotide rearrangement in the overlap region and an interaction between both transcription factors. In the light of our findings, which illustrate both site-dependent and site-independent protein and DNA conformational changes , general implications for the allosteric function of DNA response elements in transcriptional regulation are discussed.