Quadruple 9-mer-based protein binding microarray analysis confirms AACnG as the consensus nucleotide sequence sufficient for the specific binding of AtMYB44.

AtMYB44 is a member of the R2R3 MYB subgroup 22 transcription factors and regulates diverse cellular responses in Arabidopsis thaliana. We performed quadruple 9-merbased protein binding microarray (PBM) analysis, which revealed that full-size AtMYB44 recognized and bound to the consensus sequence AACnG, where n represents A, G, C or T. The consensus sequence was confirmed by electrophoretic mobility shift assay (EMSA) with a truncated AtMYB44 protein containing the N-terminal side R2R3 domain. This result indicates that the R2R3 domain alone is sufficient to exhibit AtMYB44 binding specificity. The sequence AACnG is the type I binding site for MYB transcription factors, including all members of the subgroup 22. EMSA showed that the R2R3 domain protein binds in vitro to promoters of randomly selected Arabidopsis genes that contain the consensus binding sequence. This implies that AtMYB44 binds to any promoter region that contains the consensus sequence, without determining their functional activity or specificity. The C-terminal side transcriptional activation domain of AtMYB44 contains an asparagine-rich fragment, NINNTTSSRHNHNN (aa 215-228), which, among the members of subgroup 22, is unique to AtMYB44. A transcriptional activation assay in yeast showed that this fragment is included in a region (aa 200-240) critical for the ability of AtMYB44 to function as a transcriptional activator. We hypothesize that the C-terminal side of the protein, but not the N-terminal side of the R2R3 domain, contributes to the functional activity and specificity of AtMYB44 through interactions with other regulators generated by each of a variety of stimuli.