Using a synthetic plant promoter to assess E2F-dependent transcriptional activation in Arabidopsis thaliana

Synthetic promoters specifically targeted by a single family of transcription factors (TFs) can reveal important clues on TFs roles and regulation. In this study, a synthetic promoter construct has been used to analyse the transcriptional activation by E2F factors in Arabidopsis thaliana. The typical E2F factors are key components of the cyclin D/retinoblastoma/E2F pathway which controls cell proliferation and other cellular processes in both plants and animals [1]. The typical E2Fs activate gene expression binding DNA together with Dimerisation Partner (DP) proteins but can acquire repressive roles by interacting with retinoblastoma-related (RBR) proteins. Moreover, atypical E2Fs that lack transactivating ability and act independently of DPs and RBR increase the complexity of E2F-mediated transcriptional regulation [2]. To analyse E2F-dependent activation in planta we transformed Arabidopsis thaliana with a GUS reporter construct controlled by a synthetic promoter (EM35S) composed of 10 repetitions of a canonical E2F binding site (TTTCGCGC) placed upstream to a minimal –60 CaMV 35S promoter. Analyses of the transformed lines revealed a cell cycle-dependent activation of the reporter gene in the G1/S phase in proliferating cells but disclosed a differential expression in the meristems of shoots versus roots which is likely linked to the contrasting action of activating and repressing E2Fs. Moreover, the constitutive over-expression of an exogenous typical E2F factor from carrot [1] is capable to overcome the endogenous control of the synthetic promoter and an assessment of the regulatory circuits controlling the typical E2Fs of Arabidopsis thaliana suggests a regulation of the activating E2Fs through direct phosphorylation.