Protein complexes binding to cis elements of the plant histone gene promoters: Multiplicity, phosphorylation and cell cycle alteration

The S phase-specific expression of histone genes provides an interesting model for studying activation of gene transcription during the cell cycle. In plants, however, trans-acting factors responsible for histone gene transcription are poorly documented. Using combined gel shift, UV cross-linking and competition analysis, we carried out a systematic study to identify and characterize proteins binding with the previously established cis elements of the plant histone gene promoters. Nuclear extracts prepared from the highly synchronizable tobacco BY2 cells were used. We confirmed the presence of proteins binding to the hexamer (ACGTCA) motif which has been previously identified as the binding site of wheat HBP-1 proteins. Interestingly, multiple proteins were found to bind specifically with the nonamer (CAATCCAAC) element and their DNA-binding activity was abolished upon in vitro protein phosphatase treatment. This later result imply phosphorylation/dephosphorylation as a potential posttranslational control for DNA-binding activity of nonamer-binding proteins. In addition, the DNA-binding activity of these nonamer-binding proteins was found to be positively correlated with the S phase-specific expression of the histone genes in the synchronized cells, suggesting their function in the activation of transcription during the G1/S transition. Finally, several proteins were observed to bind specifically with an A/T-rich hexamer (TAATAT) motif. Their DNA-binding activity, however, was insensitive to phosphatase activity in vitro and relatively constitutive during the cell cycle. This A/T-rich hexamer as a new cis-acting element of plant histone genes is discussed.