The genes encoding Arabidopsis ORC subunits are E2F targets and the two ORC1 genes are differently expressed in proliferating and endoreplicating cells

Abstract

Initiation of eukaryotic DNA replication depends on the function of pre-replication complexes (pre-RC), one of its key component being the six subunits origin recognition complex (ORC). In spite of a significant degree of conservation among ORC proteins from different eukaryotic sources, the regulation of their availability varies considerably in different model systems and cell types. Here, we show that the six ORC genes of Arabidopsis thaliana are regulated at the transcriptional level during cell cycle and development. We found that Arabidopsis ORC genes, except AtORC5, contain binding sites for the E2F family of transcription factors. Expression of AtORC genes containing E2F binding sites peaks at the G1/S-phase. Analysis of AtORC gene expression in plants with reduced E2F activity, obtained by expressing a dominant negative version of DP, the E2F heterodimerization partner, and with increased E2F activity, obtained by inactivation of the retinoblastoma protein, led us to conclude that all AtORC genes, except AtORC5 are E2F targets. Interestingly, Arabidopsis contains two AtORC1 (a and b) genes, highly conserved at the amino acid level but with unrelated promoter sequences. AtORC1b expression is restricted to proliferating cells. However, AtORC1a is preferentially expressed in endoreplicating cells based on our analysis in endoreplicating tissues and in a mutant with altered endocycle pattern. This suggests a differential expression of the two ORC1 genes in Arabidopsis.

Figure 1

Arabidopsis ORC proteins and subunit interaction map. (A) Summary of domain organization and major landmarks of AtORC proteins deduced from their cDNA sequence. Regions with the highest homology to ORC proteins from other sources appear in grey. Putative CDK phosphorylation sites (closed circles), KEN boxes (empty circles) and D-boxes (bars) are also shown. Note the six domains (hatched) shared among plant and animal ORC1, ORC4 and ORC5 proteins and CDC6 and RFC1. Accession numbers of sequences reported here are: AtORC1a (AJ421410), AtORC1b (AJ426477), AtORC4 (CAE01428), AtORC5 (CAE01429) and AtORC6 (CAE01430). Sequences of AtORC2 and AtORC3 have been reported (U40269 and AY524002, respectively). (B) Pull-down assays of in vitro translated AtORC subunits (ORC2-6) with purified GST-ORC proteins. (C) Schematic representation of the interactions observed among the different AtORC subunits. Lines indicate direct interaction in the pull-down assays.

Figure 2

Organ- and cell cycle-dependent expression of AtORC genes. (A) Expression pattern of AtORC genes in different organs. Measurements were normalized to the amount of UBQ10 or ACT2 and, then all the AtORC values made relative to the amount of AtORC1a present in the sample of aerial part of these seedlings (the lowest of all). Samples were prepared from aerial parts and root system of 12 day-old seedlings, young and mature rosette leaves, cauline leaves, stems, flowers at different stages or growth. (B) A.thaliana MM2d suspension cultured cells were sucrose-starved for 24 h and the amount of different AtORC mRNAs was determined by real-time RT–PCR, using the normalization procedure described for panel A. (C–D) A.thaliana MM2d suspension cultured cells, sucrose-starved for 24 h, were stimulated to re-enter the cell cycle, as described (19). The amount of mRNA of several cell cycle marker genes (31) was determined at the indicated times after sucrose addition by real-time RT–PCR, as described in panel A. CYCD3;1 and CYCD2;1 were used as G1 markers, CYCA3;1 and histone H4, as S-phase markers and CYCB1;1, as a G2/M marker (panel C). The mRNA levels of each AtORC gene (panel D) were determined at the indicated times after sucrose addition by real-time RT–PCR, as described in panel A. Numbers on top of the bars in panels C and D indicate the fold increase at the maximum level of expression relative to the value, in each case, obtained at time zero (arrested cells). In all cases, the RT–PCR measurements were repeated, at least, 2–3 times but error bars have been omitted for simplicity.

Figure 3

E2F binding to the AtORC gene promoters. (A) Summary of the location of consensus E2F DNA-binding sites in the AtORC promoters, relative to the ATG. Note that in the case of AtORC2, the transcription initiation start site (12) is indicated (arrow). E2F binding sites (oligonucleotides used in panel B are in parenthesis) are: TTTCCCGC (1a, 1b, 2.2, 3 and 4), TTTCCCGG (2.1), TTTGGCGG (6.1) and ATTCGCGG (6.2). (B) EMSA with purified AtE2Fc/AtDPb using the oligonucleotide indicated at the top. C, control using an oligonuclotide known to interact with E2F/DP (8). M, assay using the same probe but containing two point mutations that abolish E2F/DP binding (24). Arrow points to the DNA–protein complexes and the asterisk to the free DNA probe. The lanes lacking AtE2Fc/AtDPb proteins for each probe have been omitted.

Figure 4

E2F-mediated regulation of AtORC gene expression in planta. (A) Levels of mRNA for each AtORC gene and for AtCDC6 were determined by real-time RT–PCR in extracts of 10–12 day-old seedlings of plants expressing a dominant negative version of DP (24) and in control plants transformed with an empty vector. Measurements were carried out as described in Materials and Methods and, then the AtORC values made relative to that of AtORC1a in control plants. Asterisks indicate that the differences between the mean relative values of control and DPΔBD-expressing plants were statistically significant (P ≤ 0.025). (B) Levels of mRNA for each AtORC gene and for AtCDC6 were determined by real-time RT–PCR in extracts of 10 day-old seedlings of plants expressing the wild-type geminivirus RepA protein (RepA^wt) or the same protein bearing the E198K point mutation (RepA^E198K), and in control plants transformed with an empty vector. Measurements were carried out 7 h after induction of RepA protein by treatment with 1 µM dexamethasone and in each case the values were made relative to those obtained in the control plants. Asterisks indicate that the differences between the mean relative values in plants expressing RepA^wt and RepA^E198K were statistically significant.

Figure 5

Expression pattern of AtORC1b. The activity of the AtORC1b promoter was monitored by histochemical detection of the marker GUS gene in different organs during development. (A and B) Four day-old seedlings grown in the light or (C and D) in the dark. B and D are details of the shoot apical region in each case. (E) Primary root or (F–H) lateral roots at different stages of growth in 10 day-old seedlings. (I) Mature leaf. (J and K) Flowers at two stages of development. (L) Detail showing the anthers and pollen grains. (M–O) Pistils with embryos at different stages of development and (P) seeds in a mature silique.

Figure 6

Detection of AtORC1 mRNA in hypocotyl cells. (A–D) AtORC1 messages were revealed by whole-mount in situ hybridization in wild-type (A and B) and ctr1 mutant (C and D) 4 day-old seedlings grown in the dark. A and C correspond to the signal obtained with antisense (as) probe and B and D with the sense (s) probe. (E) Measurement of the mRNA levels of each AtORC1a and AtORC1b by real-time RT–PCR in extracts of hypocotyl cells of 4 day-old seedlings grown under light or dark conditions. Note than AtORC1a, but not AtORC1b, mRNAs increases in the dark, coinciding with occurrence of extra endoreplication cycles.