Elena Ramirez-Parra

G1 to S transition: more than a cell cycle engine switch

CDK-cyclin complexes are the universal drivers of cell cycle transitions. Progression through G(1) and transition to S-phase, thereby initiating genome duplication, requires the concerted action of cyclin-dependent kinase (CDK)-cyclin complexes on specific targets. These targets belong to at least two major regulatory networks: the retinoblastoma-related (RBR)/E2F pathway and complexes that are responsible for the initiation of DNA replication. The G(1) phase is central to the integration of signals that regulate both the exit from the cell division cycle to differentiation and the reactivation of cell proliferation. Cellular factors that are involved in these pathways play a role in regulating cell size and number, and organogenesis. As a consequence, they are also involved in determining plant architecture.

Cell Type-Specific Role of the Retinoblastoma/E2F Pathway during Arabidopsis Leaf Development

Organogenesis in plants is almost entirely a postembryonic process. This unique feature implies a strict coupling of cell proliferation and differentiation, including cell division, arrest, cell cycle reactivation, endoreplication, and differentiation. The plant retinoblastoma-related (RBR) protein modulates the activity of E2F transcription factors to restrict cell proliferation. Arabidopsis contains a single RBR gene, and its loss of function precludes gamete formation and early development. To determine the relevance of the RBR/E2F pathway during organogenesis, outside its involvement in cell division, we have used an inducible system to inactivate RBR function and release E2F activity. Here, we have focused on leaves where cell proliferation and differentiation are temporally and developmentally regulated. Our results reveal that RBR restricts cell division early during leaf development when cell proliferation predominates, while it regulates endocycle occurrence at later stages. Moreover, shortly after leaving the cell cycle, most of leaf epidermal pavement cells retain the ability to reenter the cell cycle and proliferate, but maintain epidermal cell fate. On the contrary, mesophyll cells in the inner layers do not respond in this way to RBR loss of activity. We conclude that there exists a distinct response of different cells to RBR inactivation in terms of maintaining the balance between cell division and endoreplication during Arabidopsis (Arabidopsis thaliana) leaf development.

E2F Regulates FASCIATA1, a Chromatin Assembly Gene whose Loss Switches on the Endocycle and Activates Gene Expression by Changing the Epigenetic Status

Maintenance of genome integrity depends on histone chaperone-mediated chromatin reorganization. DNA replication-associated nucleosome deposition relies on chromatin assembly factor-1 (CAF-1). Depletion of CAF-1 in human cells leads to cell death, whereas in Arabidopsis (Arabidopsis thaliana), where it is involved in heterochromatin compaction and homologous recombination, plants are viable. The mechanism that makes the lack of CAF-1 activity compatible with development is not known. Here, we show that the FASCIATA1 (FAS1) gene, which encodes the CAF-1 large subunit, is a target of E2F transcription factors. Mutational studies demonstrate that one of the two E2F binding sites in its promoter has an activator role, whereas the other has a repressor function. Loss of FAS1 results in reduced type A cyclin-dependent kinase activity, inhibits mitotic progression, and promotes a precocious and systemic switch to the endocycle program. Selective up-regulation of the expression of a subset of genes, including those involved in activation of the G2 DNA damage checkpoint, also occurs upon FAS1 loss. This activation is not the result of a global change in chromatin structure, but depends on selective epigenetic changes in histone acetylation and methylation within a small region in their promoters. This suggests that correct chromatin assembly during the S-phase is required to prevent unscheduled changes in the epigenetic marks of target genes. Interestingly, activation of the endocycle switch as well as introduction of activating histone marks in the same set of G2 checkpoint genes are detected upon treatment of wild-type plants with DNA-damaging treatments. Our results are consistent with a model in which defects in chromatin assembly during the S-phase and DNA damage signaling share part of a pathway, which ultimately leads to mitotic arrest and triggers the endocycle program. Together, this might be a bypass mechanism that makes development compatible with cell division arrest induced by DNA damage stress.

Genome-wide analysis of histone H3.1 and H3.3 variants in Arabidopsis thaliana

Nucleosomes package eukaryotic DNA and are composed of four different histone proteins, designated H3, H4, H2A, and H2B. Histone H3 has two main variants, H3.1 and H3.3, which show different genomic localization patterns in animals. We profiled H3.1 and H3.3 variants in the genome of the plant Arabidopsis thaliana and found that the localization of these variants shows broad similarity in plants and animals, along with some unique features. H3.1 was enriched in silent areas of the genome, including regions containing the repressive chromatin modifications H3 lysine 27 methylation, H3 lysine 9 methylation, and DNA methylation. In contrast, H3.3 was enriched in actively transcribed genes, especially peaking at the 3′ end of genes, and correlated with histone modifications associated with gene activation, such as histone H3 lysine 4 methylation and H2B ubiquitylation, as well as RNA Pol II occupancy. Surprisingly, both H3.1 and H3.3 were enriched on defined origins of replication, as was overall nucleosome density, suggesting a novel characteristic of plant origins. Our results are broadly consistent with the hypothesis that H3.1 acts as the canonical histone that is incorporated during DNA replication, whereas H3.3 acts as the replacement histone that can be incorporated outside of S-phase during chromatin-disrupting processes like transcription.

Role of an Atypical E2F Transcription Factor in the Control of Arabidopsis Cell Growth and Differentiation

The balance between cell proliferation and differentiation is crucial in multicellular organisms, where it is regulated by complex gene expression networks. This is particularly relevant in plants because organogenesis is a continuous postembryonic process. Here, we investigate the function of Arabidopsis thaliana E2Ff, an atypical member of the E2F family of transcription factors, which acts independently of a dimerization partner. We have focused our analysis on roots and hypocotyls, organs where (1) cell proliferation and differentiation are spatially and/or temporally separated, (2) growth depends on cell expansion in the longitudinal axis, and (3) the AtE2Ff promoter is active. AtE2Ff overexpression produced a reduction in the size of differentiated cells of these organs. Cells of mutant e2ff-1 plants with reduced levels of AtE2Ff mRNA were larger, especially in the hypocotyl, suggesting a role as a growth regulator. These effects of AtE2Ff are not associated with changes in nuclear ploidy levels or in the expression of cell cycle marker genes. However, expression of a subset of cell wall biogenesis genes is misregulated in an AtE2Ff-dependent manner, and based on chromatin immunoprecipitation experiments, they seem to be direct E2F targets. Our results highlight the complex regulatory function exerted by E2F and suggest a possible role of AtE2Ff in repressing cell wall biosynthesis genes during cell elongation in differentiated cells.

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.

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

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.

Auxin and Epigenetic Regulation of SKP2B, an F-Box That Represses Lateral Root Formation

In plants, lateral roots originate from pericycle founder cells that are specified at regular intervals along the main root. Here, we show that Arabidopsis (Arabidopsis thaliana) SKP2B (for S-Phase Kinase-Associated Protein2B), an F-box protein, negatively regulates cell cycle and lateral root formation as it represses meristematic and founder cell divisions. According to its function, SKP2B is expressed in founder cells, lateral root primordia and the root apical meristem. We identified a novel motif in the SKP2B promoter that is required for its specific root expression and auxin-dependent induction in the pericycle cells. Next to a transcriptional control by auxin, SKP2B expression is regulated by histone H3.1/H3.3 deposition in a CAF-dependent manner. The SKP2B promoter and the 5′ end of the transcribed region are enriched in H3.3, which is associated with active chromatin states, over H3.1. Furthermore, the SKP2B promoter is also regulated by H3 acetylation in an auxin- and IAA14-dependent manner, reinforcing the idea that epigenetics represents an important regulatory mechanism during lateral root formation.

Chromatin dynamics during the plant cell cycle

Cell cycle progression depends on a highly regulated series of events of which transcriptional control plays a major role. In addition, during the S-phase not only DNA but chromatin as a whole needs to be faithfully duplicated. Therefore, both nucleosome dynamics as well as local changes in chromatin organization, including introduction and/or removal of covalent DNA and histone modifications, at genes with a key role in cell proliferation, are of primary relevance. Chromatin duplication during the S-phase and the chromosome segregation during mitosis are cell cycle stages critical for maintenance of epigenetic marks or for allowing the daughter products to acquire a distinct epigenetic landscape and, consequently, a unique cell fate decision. These aspects of chromatin dynamics together with the strict coupling of cell proliferation, cell differentiation and post-embryonic organogenesis have a profound impact on plant growth, development and response to external signals.

ANTI-SILENCING FUNCTION1 Proteins Are Involved in Ultraviolet-Induced DNA Damage Repair and Are Cell Cycle Regulated by E2F Transcription Factors in Arabidopsis

ASF1A and ASF1B genes are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation. ANTI-SILENCING FUNCTION1 (ASF1) is a key histone H3/H4 chaperone that participates in a variety of DNA- and chromatin-related processes, including DNA repair, where chromatin assembly and disassembly are of primary relevance. Information concerning the role of ASF1 proteins in the post-ultraviolet (UV) response in higher plants is currently limited. In Arabidopsis (Arabidopsis thaliana), an initial analysis of in vivo localization of ASF1A and ASF1B indicates that both proteins are mainly expressed in proliferative tissues. In silico promoter analysis identified ASF1A and ASF1B as potential targets of Elongation Factor2 (E2F) transcription factors. These observations were experimentally validated, both in vitro, by electrophoretic mobility shift assays, and in vivo, by chromatin immunoprecipitation assays and expression analysis using transgenic plants with altered levels of different E2F transcription factors. These data suggest that ASF1A and ASF1B are regulated during cell cycle progression through E2F transcription factors. In addition, we found that ASF1A and ASF1B are associated with the UV-B-induced DNA damage response in Arabidopsis. Transcript levels of ASF1A and ASF1B were increased following UV-B treatment. Consistent with a potential role in UV-B response, RNA interference-silenced plants of both genes showed increased sensitivity to UV-B compared with wild-type plants. Finally, by coimmunoprecipitation analysis, we found that ASF1 physically interacts with amino-terminal acetylated histones H3 and H4 and with acetyltransferases of the Histone Acetyl Transferase subfamily, which are known to be involved in cell cycle control and DNA repair, among other functions. Together, we provide evidence that ASF1A and ASF1B are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation.