Impact of nucleosome dynamics and histone modifications on cell proliferation during Arabidopsis development.

Eukaryotic chromatin is a highly structured macromolecular complex of which DNA is wrapped around a histone-containing core. DNA can be methylated at specific C residues and each histone molecule can be covalently modified at a large variety of amino acids in both their tail and core domains. Furthermore, nucleosomes are not static entities and both their position and histone composition can also vary. As a consequence, chromatin behaves as a highly dynamic cellular component with a large combinatorial complexity beyond DNA sequence that conforms the epigenetic landscape. This has key consequences on various developmental processes such as root and flower development, gametophyte and embryo formation and response to the environment, among others. Recent evidence indicate that posttranslational modifications of histones also affect cell cycle progression and processes depending on a correct balance of proliferating cell populations, which in the context of a developing organisms includes cell cycle, stem cell dynamics and the exit from the cell cycle to endoreplication and cell differentiation. The impact of epigenetic modifications on these processes will be reviewed here.

Expression and stability of Arabidopsis CDC6 are associated with endoreplication.

Studies on the CDC6 protein, which is crucial to the control of DNA replication in yeast and animal cells, are lacking in plants. We have isolated an Arabidopsis cDNA encoding the AtCDC6 protein and studied its possible connection to the occurrence of developmentally regulated endoreplication cycles. The AtCDC6 gene is expressed maximally in early S-phase, and its promoter contains an E2F consensus site that mediates the binding of a plant E2F/DP complex. Transgenic plants carrying an AtCDC6 promoter-beta-glucuronidase fusion revealed that it is active in proliferating cells and, interestingly, in endoreplicating cells. In particular, the extra endoreplication cycle that occurs in dark-grown hypocotyl cells is associated with upregulation of the AtCDC6 gene. This was corroborated using ctr1 Arabidopsis mutants altered in their endoreplication pattern. The ectopic expression of AtCDC6 in transgenic plants induced endoreplication and produced a change in the somatic ploidy level. AtCDC6 was degraded in a ubiquitin- and proteosome-dependent manner by extracts from proliferating cells, but it was degraded poorly by extracts from dark-grown hypocotyl endoreplicating cells. Our results indicate that endoreplication is associated with expression of the AtCDC6 gene and, most likely, the stability of its product; it also apparently requires activation of the retinoblastoma/E2F/DP pathway. These conclusions may apply to endoreplicating cells in other tissues of the plant and to endoreplicating cells in other eukaryotes.

Characterization of wheat DP, a heterodimerization partner of the plant E2F transcription factor which stimulates E2F-DNA binding.

Recent studies suggest that the G1/S transition in plants depends on the activity of E2F transcription factors. In animal cells, E2Fs interact with DP proteins, whose identification in plants has been elusive, so far. Here we show that although an E2F-containing DNA-binding activity can be detected in plant cell extracts, purified E2F protein binds poorly to DNA. In a yeast two-hybrid screening, using wheat E2F as a bait, we have isolated a cDNA clone encoding a wheat DP (TmDP) protein. TmDP is expressed ubiquitously and exhibits a domain organization similar to animal DPs. Contrary to the specificity observed for the plant RBR/E2F interaction, human and plant E2F and DP proteins can interact in a heterologous manner. Purified TmDP protein stimulates E2F-DNA complex formation.

Relationship of oligomerization to DNA binding of Wheat dwarf virus RepA and Rep proteins.

Members of the genus Mastrevirus (family Geminiviridae) produce a complementary-sense (c-sense) transcription unit with the potential to encode two proteins, RepA and Rep. In the present work, we have studied the DNA-protein complexes formed by the Wheat dwarf virus (WDV) RepA protein within the WDV large intergenic region. WDV RepA forms large nucleoprotein complexes near the TATA boxes of the viral complementary-sense and virion-sense (v-sense) promoters (the RepA C- and V-complexes, respectively), a location similar to those of WDV Rep-DNA complexes but with distinct DNase I footprints. We have also studied the relationship of oligomerization of WDV RepA and Rep proteins to DNA-protein complex formation. Using chemical cross-linking, we have determined that both WDV proteins can form oligomers in solution. Interestingly, the pH is critical for the monomer-oligomer equilibrium and small changes produce a displacement in such a way that at pH /= 7.4 it is a monomer. Complex formation is also strongly affected by pH and occurs more efficiently at pH 7.0-7.4. We found that preformed oligomers interact very poorly with DNA. Thus, our data are consistent with a stepwise model for protein-DNA complex assembly in which monomers interact with DNA and then with other monomers to assemble an oligomeric structure on the DNA. These results may be relevant for studies on the DNA binding, replication, and transcription properties of geminivirus proteins.

The cloning of plant E2F, a retinoblastoma-binding protein, reveals unique and conserved features with animal G(1)/S regulators.

Association of the retinoblastoma (Rb) protein with E2F transcription factors is central to cell cycle-specific gene expression and growth in animal cells. Whether Rb-E2F complexes are also involved in plant cell growth and differentiation is still unknown since E2F proteins have not yet been identified in plants. Here we report the isolation and characterisation of a wheat E2F (TmE2F) cDNA clone. Interestingly, the overall domain organisation of plant E2F is related to the human E2F-1/2/3 subset but its primary sequence is slightly more related to the E2F-4/5 subset. TmE2F-Rb binding depends on residues, located at the C-terminus, which are different from those of animal E2Fs. However, the acidic or hydrophobic nature of certain residues is maintained, strongly suggesting that they may have a crucial role in E2F activities. Plant E2F is expressed in proliferating cultured cells and in differentiated tissues and is up-regulated early in S phase. Our studies reinforce the idea that G(1)/S regulators in plants are unrelated to those of yeast cells but similar to those of animal cells and provide new tools to analyse the links between cell cycle regulators, plant growth and developmental signals.