María de la Paz Sánchez

Genome-wide mapping of Arabidopsis thaliana origins of DNA replication and their associated epigenetic marks

Genome integrity requires faithful chromosome duplication. Origins of replication, the genomic sites at which DNA replication initiates, are scattered throughout the genome. Their mapping at a genomic scale in multicellular organisms has been challenging. In this study we profiled origins in Arabidopsis thaliana by high-throughput sequencing of newly synthesized DNA and identified ~1,500 putative origins genome-wide. This was supported by chromatin immunoprecipitation and microarray (ChIP-chip) experiments to identify ORC1- and CDC6-binding sites. We validated origin activity independently by measuring the abundance of nascent DNA strands. The midpoints of most A. thaliana origin regions are preferentially located within the 5′ half of genes, enriched in G+C, histone H2A.Z, H3K4me2, H3K4me3 and H4K5ac, and depleted in H3K4me1 and H3K9me2. Our data help clarify the epigenetic specification of DNA replication origins in A. thaliana and have implications for other eukaryotes.

Arabidopsis ORC1 is a PHD-containing H3K4me3 effector that regulates transcription

Control of gene expression depends on a complex and delicate balance of various posttranslational modifications of histones. However, the relevance of specific combinations of histone modifications is not fully defined. Downstream effector proteins recognize particular histone modifications and transduce this information into gene expression patterns. Methylation of histone H3 at lysine 4 (H3K4me) is a landmark of gene expression control in eukaryotes. Its recognition depends on the presence in the effector protein of a motif termed plant homeodomain (PHD) that specifically binds to H3K4me3. Here, we establish that Arabidopsis ORC1, the large subunit of the origin recognition complex involved in defining origins of DNA replication, functions as a transcriptional activator of a subset of genes, the promoters of which are preferentially bound by ORC1. Arabidopsis ORC1 contains a PHD and binds to H3K4me3. In addition to H4 acetylation, ORC1 binding correlates with increased H4K20me3 in the proximal promoter region of ORC1 targets. This suggests that H4K20me3, unlike in animal cells, is associated with transcriptional activation in Arabidopsis. Thus, our data provide a molecular basis for the opposite role of ORC1 in transcriptional activation in plants and repression in animals. Since only ORC1 proteins of plant species contain a PHD, we propose that plant ORC1 constitutes a novel class of H3K4me3 effector proteins characteristic of the plant kingdom.

The cell cycle and seed germination

The cell cycle is the series of molecular events that allows cells to duplicate and segregate their chromosomes to form new cells. The finding that a protein kinase, the product of the yeast cdc2 gene, was fundamental in the regulation of the G 2 /M and G 1 /S transitions, associated with unstable proteins named cyclins, opened a very exciting and dynamic research area. The number of gene products that participate in the development and regulation of the cell cycle may be in the hundreds, and there is a high degree of conservation in protein sequences and regulatory pathways among eukaryotes. Although there are clear differences between plants and animals in cell structure, organization, growth, development and differentiation, the same types of proteins and very similar regulatory pathways seem to exist. Seed germination appears to be an excellent model system for studying the cell cycle in plants. Imbibition will reactivate meristematic cells – most initially with a G 1 DNA content – into the cell cycle in preparation for seedling establishment. Early events include a thorough survey of DNA status, since the drying process and seed storage conditions reduce chromosomal integrity. The initiation of cell cycle events leading to G 1 and S phases, and of the germination process itself, may depend on a G 1 checkpoint control. Most, if not all, cell cycle proteins appear to be already present in unimbibed embryos, although there is evidence of protein turnover in the early hours, suggesting the need for de novo protein synthesis. Regulation also may occur at the level of protein modification, because existing G 1 , S and G 2 cell cycle proteins appear to be activated at precise times during germination. Thus, cell cycle control during seed germination may be exerted at multiple levels; however, knowledge of cell cycle events and their importance for germination is still scarce and fragmentary, and different species may have developed unique control mechanisms, more suited to specific germination characteristics and habitat.

Hormone Symphony During Root Growth and Development

Hormones regulate plant growth and development in response to external environmental stimuli via complex signal transduction pathways, which in turn form complex networks of interaction. Several classes of hormones have been reported, and their activity depends on their biosynthesis, transport, conjugation, accumulation in the vacuole, and degradation. However, the activity of a given hormone is also dependent on its interaction with other hormones. Indeed, there is a complex crosstalk between hormones that regulates their biosynthesis, transport, and/or signaling functionality, although some hormones have overlapping or opposite functions. The plant root is a particularly useful system in which to study the complex role of plant hormones in the plastic control of plant development. Physiological, cellular, and molecular genetic approaches have been used to study the role of plant hormones in root meristem homeostasis. In this review, we discuss recent findings on the synthesis, signaling, transport of hormones and role during root development and examine the role of hormone crosstalk in maintaining homeostasis in the apical root meristem. Developmental Dynamics, 2012.

The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression

Elucidating molecular links between cell‐fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell‐fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem‐cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS‐box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem‐cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS‐domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4‐dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning.

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.

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.

Progress in understanding DNA replication control

Completion of genome duplication during the S-phase of the cell cycle is crucial for the maintenance of genomic integrity. In eukaryotes, chromosomal DNA replication is accomplished by the activity of multiple origins of DNA replication scattered across the genome. Origin specification, selection and activity as well as the availability of replication factors and the regulation of DNA replication licensing, have unique and common features among eukaryotes. Although the initial studies on the semiconservative nature of chromosome duplication were carried out in the mid 1950s in Vicia faba, since then plant DNA replication studies have been scarce. However, they have received an unprecedented drive in the last decade after the completion of sequencing the Arabidopsis thaliana genome, and more recently of other plant genomes. In particular, the past year has witnessed major advances with the use of genomic approaches to study chromosomal replication timing, DNA replication origins and licensing control mechanisms. In this minireview article we discuss these recent discoveries in plants in the context of what is known at the genomic level in other eukaryotes. These studies constitute the basis for addressing in the future key questions about replication origin specification and function that will be of relevance not only for plants but also for the rest of multicellular organisms.

The impact of Polycomb group (PcG) and Trithorax group (TrxG) epigenetic factors in plant plasticity

Current advances indicate that epigenetic mechanisms play important roles in the regulatory networks involved in plant developmental responses to environmental conditions. Hence, understanding the role of such components becomes crucial to understanding the mechanisms underlying the plasticity and variability of plant traits, and thus the ecology and evolution of plant development. We now know that important components of phenotypic variation may result from heritable and reversible epigenetic mechanisms without genetic alterations. The epigenetic factors Polycomb group (PcG) and Trithorax group (TrxG) are involved in developmental processes that respond to environmental signals, playing important roles in plant plasticity. In this review, we discuss current knowledge of TrxG and PcG functions in different developmental processes in response to internal and environmental cues and we also integrate the emerging evidence concerning their function in plant plasticity. Many such plastic responses rely on meristematic cell behavior, including stem cell niche maintenance, cellular reprogramming, flowering and dormancy as well as stress memory. This information will help to determine how to integrate the role of epigenetic regulation into models of gene regulatory networks, which have mostly included transcriptional interactions underlying various aspects of plant development and its plastic response to environmental conditions.

A Molecular Switch for Initiating Cell Differentiation in Arabidopsis

BACKGROUND The onset of differentiation entails modifying the gene expression state of cells, to allow activation of developmental programs that are maintained repressed in the undifferentiated precursor cells [1, 2]. This requires a mechanism to change gene expression on a genome-scale. Recent evidence suggests that in mammalian stem cells, derepression of developmental regulators during differentiation involves a shift from stalled to productive elongation of their transcripts [3-5], but factors mediating this shift have not been identified and the evidence remains correlative. RESULTS We report the identification of the MINIYO (IYO) gene, a positive regulator of transcriptional elongation that is essential for cells to initiate differentiation in Arabidopsis. IYO interacts with RNA polymerase II and the Elongator complex and is required to sustain global levels of transcriptional elongation activity, specifically in differentiating tissues. Accordingly, IYO is expressed in embryos, meristems, and organ primordia and not in mature tissues. Moreover, differential subcellular protein distribution further refines the domain of IYO function by directing nuclear accumulation, and thus its transcriptional activity, to cells initiating differentiation. Importantly, IYO overexpression induces premature cell differentiation and leads to meristem termination phenotypes. CONCLUSIONS These findings identify IYO as a necessary and sufficient factor for initiating differentiation in Arabidopsis and suggest that the targeted nuclear accumulation of IYO functions as a transcriptional switch for this fate transition.