S Phase and Meristem-specific Expression of the Tobacco RNR1b Gene Is Mediated by an E2F Element Located in the 5′ Leader Sequence

Abstract

The RB/E2F pathway is involved in the control of the G 1 /S transition of the eukaryotic cell cycle where various S phase genes are activated by specific E2F factors. Ribonucleotide reductase (RNR) plays an essential role in the DNA synthesis pathway. Earlier studies showed that there are at least two RNR1 genes (RNR1a and RNR1b) and one RNR2 gene in tobacco. In synchronized tobacco BY2 cells, RNR1b gene expression is at its highest level in S phase. To investigate transcriptional regulation of the RNR1b gene, its promoter region was cloned and sequenced. Unlike its animal counterparts, the tobacco RNR1b promoter contains a consensus E2F-binding site. Surprisingly, this site is found in the leader sequence of the gene. We show here by gel shift analysis and antibody competition that one nuclear complex specifically binds this motif, and an E2F factor is part of this complex. Using reporter gene analysis, tobacco RNR1b promoter activity was detected during S phase in synchronized cells and in plant meristematic tissues. Mutation of the E2F element substantially reduced both activities. For the first time in plants, a single E2F motif found in the leader sequence plays an important role in the meristem and S phase-specific expression of the tobacco RNR1b gene.Progression of the cell cycle is associated with the phasespecific induction of genes whose products control the cell cycle or are involved in DNA replication (1). Transcription of several genes induced at the G 1 /S transition is mainly controlled by the E2F pathway (2). E2F complexes can act either as transcription repressors or activators depending on their target genes and the phase of the cell cycle. While transcription repression is generally achieved by binding of retinoblasma-type proteins (RB) 1 to E2F factors (3), repression of the PAI-1 gene is carried out by direct binding of E2F to its target motif (4). Transcriptional activation is triggered when the E2F factor is associated to its DP partner. E2F-binding sites (TTTC/GC/GCGC) were identified in numerous gene promoters (5). Some genes, such as cdc2 or B-Myb, appear to be strongly derepressed during the transition from G 0 to G 1 (i.e. quiescence to growth), while others, such as the p107 and DHFR genes, are up-regulated at the G 1 /S transition (6).E2F and DP factors (7-12) as well as RB proteins have been recently identified in plants (13), suggesting that the control of G 1 /S-phase-specific transcription in plants could be similar to that found in animals. Up to now, a few E2F target genes have been characterized in plants. The two E2F motifs identified in the tobacco RNR2 promoter were shown to specifically interact with a tobacco E2F factor and to mediate up-regulation of the promoter at the G 1 /S transition of the cell cycle (14). In addition, it was shown that Arabidopsis E2F1 factor binds an E2F motif in the promoter of a S-phase-regulated gene AtCDC6 (10). Alternatively, two E2F cis-elements were found in the promoter of tobacco gene encoding the proliferating cell nuclear antigen (PC...

Ddb1 Is Required for the Proteolysis of the Schizosaccharomyces pombe Replication Inhibitor Spd1 during S Phase and after DNA Damage

“…The mammalian RNR subunits R1 and R2 are activated in early S phase through transcription and new synthesis (25)(26)(27). The transcription of RNR is activated by the E2F family of transcription factors.…”

ATR Regulates a G2-Phase Cell-Cycle Checkpoint in Arabidopsis thaliana

“…We searched GenBank and identified the Arabidopsis homolog of RNR1, the large subunit of RNR (accession number AF092841). Transcription of RNR1 has already been shown to be regulated in a cell-cycle dependent manner in tobacco (Nicotiana tabacum) (Chabouté et al, 1998(Chabouté et al, , 2002, and transcription is induced by DNA damage in this plant (M.E. Chabouté , personal communication).…”

A Collection of Conserved Noncoding Sequences to Study Gene Regulation in Flowering Plants

“…In this analysis, upstream and downstream are used relative to the translation start site and translation stop site, respectively. This is done because it was shown previously that regulatory elements can be found in the 59 and 39 untranslated region (UTR; Chabouté et al, 2002;Liu et al, 2010;Wang and Xu, 2010). The second reason to include UTRs is that not all genes have information about their UTR available.…”