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 bind target DNA in the form of monomers, cooperative dimers or heterodimers is important in relation to their transcriptional regulatory properties. The N-Oct3 neuron-specific protein binds to an octamer-like sequence (AAATAATGC) within the (-102/-72) neuronal promoter region of the human aromatic L-amino acid decarboxylase (AADC) gene. In this atypical case the POUh and POUs tetrameric subsites are spaced one nucleotide apart and in switched order as compared with the consensus octamer. Moreover this POU binding motif overlaps the hepatocyte nuclear factor HNF-3 beta binding site (TGCTCAGTAAA) which itself contains a heptamer-like sequence (CTCAGTA). Using the isolated DNA binding domains (DBD) of the two proteins, it is shown that, when binding to this unusual recognition sequence, N-Oct3 either exhibits noncooperative homodimerization or allows the simultaneous binding of the second transcription 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-MS was used in conjunction with molecular modelling to show that the protein conformational change resulting from binary N-Oct3/DNA complex formation occurs within the linker peptide joining the POUs and POUh subdomains. Furthermore, modelling the N-Oct3/HNF-3 beta/DNA ternary complex predicts a nucleotide 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.
Millevoi
S
S
Institut de Pharmacologie et de Biologie Structurale, CNRS, Toulouse, France.
Thion
L
L
Joseph
G
G
Vossen
C
C
Ghisolfi-Nieto
L
L
Erard
M
M
D000495:Allosteric Site; D000595:Amino Acid Sequence; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D051542:Hepatocyte Nuclear Factor 3-beta; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D050814:Octamer Transcription Factor-3; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020218:Response Elements; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; D009687:Nuclear Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta; D004247:DNA; D001142:Aromatic-L-Amino-Acid Decarboxylases