Characterization of cis-acting element involved in cell cycle phase-independent activation of Arath;CycB1;1 transcription and identification of putative regulatory proteins.
Progression through the cell cycle is driven by cyclin-dependent kinases (CDKs) whose activity is controlled by regulatory subunits called cyclins. The expression of cyclins is subject to numerous controls at multiple levels, not least at the level of transcription. As a first step to unravel the mechanisms that regulate expression of B-cyclins in plants, we undertook the identification of the required promoter elements of the Arath;CycB1;1 gene. A detailed analysis of different promoter fragments consisted in analysing their ability to mediate cell cycle-dependent transcriptional oscillations of the gus reporter gene in transformed BY-2 cell lines. We showed that different promoter regions took part in transcriptional activation. Furthermore, 202 bp upstream of the ATG were sufficient to induce M-phase-specific expression. This region contains an 18 bp sequence including a Myb-binding core (AACGG) which is able to activate reporter gene without leading to M-phase-specific expression. Electrophoretic mobility shift assays showed that this 18 bp sequence specifically binds protein complexes from Arabidopsis cell suspension enriched either in G1 or G2 phase. Furthermore, the Myb core, AACGG, was characterized as necessary for the binding of proteins. DNA affinity purification of the complexes bound to the 18 bp sequence allowed the isolation of three different complexes and two proteins from these complexes were identified by mass spectrometry analyses. A new putative Myb transcription factor and a hypothetical protein, HYP containing with a leucine zipper and Myc-type dimerization domains were identified. When over-expressed in plants, HYP factor is able to trans-activate the expression of gus reporter gene downstream from the -202 promoter fragment as well as the endogenous CycB1;1 gene.
Institut de Biotechnologie des Plantes, UMR 8618, Université Paris-Sud, Orsay, France.
Séverine
S
Planchais
Claudette
C
Perennes
Nathalie
N
Glab
Vladimir
V
Mironov
Dirk
D
Inzé
Catherine
C
Bergounioux
D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D019926:Cyclin B / Q000235:genetics*; D056744:Cyclin B1; D018744:DNA, Plant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D007210:Indoleacetic Acids / Q000494:pharmacology; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020218:Response Elements / Q000187:drug effects / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
C511988:ATR1 protein, Arabidopsis; D029681:Arabidopsis Proteins; C511989:CPC protein, Arabidopsis; D002352:Carrier Proteins; D019926:Cyclin B; D056744:Cyclin B1; D018744:DNA, Plant; D004268:DNA-Binding Proteins; D007210:Indoleacetic Acids; D009841:Oligonucleotides; D010940:Plant Proteins; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase