Joana Sequeira-Mendes

The Functional Topography of the Arabidopsis Genome Is Organized in a Reduced Number of Linear Motifs of Chromatin States

A computational study of combinations of histone modifications and DNA features was developed to determine a high-resolution landscape of Arabidopsis chromatin states. The linear topography across the genome revealed the existence of chromatin motifs. This work provides a basis for studies on gene expression, e.g., bivalent H3K27me3/H3K4me3-containing regions, DNA replication, and epigenetics. Chromatin is of major relevance for gene expression, cell division, and differentiation. Here, we determined the landscape of Arabidopsis thaliana chromatin states using 16 features, including DNA sequence, CG methylation, histone variants, and modifications. The combinatorial complexity of chromatin can be reduced to nine states that describe chromatin with high resolution and robustness. Each chromatin state has a strong propensity to associate with a subset of other states defining a discrete number of chromatin motifs. These topographical relationships revealed that an intergenic state, characterized by H3K27me3 and slightly enriched in activation marks, physically separates the canonical Polycomb chromatin and two heterochromatin states from the rest of the euchromatin domains. Genomic elements are distinguished by specific chromatin states: four states span genes from transcriptional start sites (TSS) to termination sites and two contain regulatory regions upstream of TSS. Polycomb regions and the rest of the euchromatin can be connected by two major chromatin paths. Sequential chromatin immunoprecipitation experiments demonstrated the occurrence of H3K27me3 and H3K4me3 in the same chromatin fiber, within a two to three nucleosome size range. Our data provide insight into the Arabidopsis genome topography and the establishment of gene expression patterns, specification of DNA replication origins, and definition of chromatin domains.

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.

Looking at plant cell cycle from the chromatin window

The cell cycle is defined by a series of complex events, finely coordinated through hormonal, developmental and environmental signals, which occur in a unidirectional manner and end up in producing two daughter cells. Accumulating evidence reveals that chromatin is not a static entity throughout the cell cycle. In fact, there are many changes that include nucleosome remodeling, histone modifications, deposition and exchange, among others. Interestingly, it is possible to correlate the occurrence of several of these chromatin-related events with specific processes necessary for cell cycle progression, e.g., licensing of DNA replication origins, the E2F-dependent transcriptional wave in G1, the activation of replication origins in S-phase, the G2-specific transcription of genes required for mitosis or the chromatin packaging occurring in mitosis. Therefore, an emerging view is that chromatin dynamics must be considered as an intrinsic part of cell cycle regulation. In this article, we review the main features of several key chromatin events that occur at defined times throughout the cell cycle and discuss whether they are actually controlling the transit through specific cell cycle stages.

Genome architecture: from linear organisation of chromatin to the 3D assembly in the nucleus

The genetic information is stored in the eukaryotic nucleus in the form of chromatin. This is a macromolecular entity that includes genomic DNA and histone proteins that form nucleosomes, plus a large variety of chromatin-associated non-histone proteins. Chromatin is structurally and functionally organised at various levels. One reveals the linear topography of DNA, histones and their post-translational modifications and non-histone proteins along each chromosome. This level provides regulatory information about the association of genomic elements with particular signatures that have been used to define chromatin states. Importantly, these chromatin states correlate with structural and functional genomic features. Another regulatory layer is established at the level of the 3D organisation of chromatin within the nucleus, which has been revealed clearly as non-random. Instead, a variety of intra- and inter-chromosomal genomic domains with specific epigenetic and functional properties has been identified. In this review, we discuss how the recent advances in genomic approaches have contributed to our understanding of these two levels of genome architecture. We have emphasised our analysis with the aim of integrating information available for yeast, Arabidopsis, Drosophila, and mammalian cells. We consider that this comparative study helps define common and unique features in each system, providing a basis to better understand the complexity of genome organisation.

Regulating DNA Replication in Plants

Chromosomal DNA replication in plants has requirements and constraints similar to those in other eukaryotes. However, some aspects are plant-specific. Studies of DNA replication control in plants, which have unique developmental strategies, can offer unparalleled opportunities of comparing regulatory processes with yeast and, particularly, metazoa to identify common trends and basic rules. In addition to the comparative molecular and biochemical studies, genomic studies in plants that started with Arabidopsis thaliana in the year 2000 have now expanded to several dozens of species. This, together with the applicability of genomic approaches and the availability of a large collection of mutants, underscores the enormous potential to study DNA replication control in a whole developing organism. Recent advances in this field with particular focus on the DNA replication proteins, the nature of replication origins and their epigenetic landscape, and the control of endoreplication will be reviewed.

On the opportunistic nature of transcription and replication initiation in the metazoan genome

Cellular identity and its response to external or internal signalling variations are encoded in a cells genome as regulatory information. The genomic regions that specify this type of information are highly variable and degenerated in their sequence determinants, as it is becoming increasingly evident through the application of genome‐scale methods to study gene expression. Here, we speculate that the same scenario applies to the regulatory regions controlling where DNA replication starts in the metazoan genome. We propose that replication origins cannot be defined as unique genomic features, but rather that DNA synthesis initiates opportunistically from accessible DNA sites, making cells highly robust and adaptable to environmental or developmental changes.

Links between genome replication and chromatin landscapes.

Post-embryonic organogenesis in plants requires the continuous production of cells in the organ primordia, their expansion and a coordinated exit to differentiation. Genome replication is one of the most important processes that occur during the cell cycle, as the maintenance of genomic integrity is of primary relevance for development. As it is chromatin that must be duplicated, a strict coordination occurs between DNA replication, the deposition of new histones, and the introduction of histone modifications and variants. In turn, the chromatin landscape affects several stages during genome replication. Thus, chromatin accessibility is crucial for the initial stages and to specify the location of DNA replication origins with different chromatin signatures. The chromatin landscape also determines the timing of activation during the S phase. Genome replication must occur fully, but only once during each cell cycle. The re-replication avoidance mechanisms rely primarily on restricting the availability of certain replication factors; however, the presence of specific histone modifications are also revealed as contributing to the mechanisms that avoid re-replication, in particular for heterochromatin replication. We provide here an update of genome replication mostly focused on data from Arabidopsis, and the advances that genomic approaches are likely to provide in the coming years. The data available, both in plants and animals, point to the relevance of the chromatin landscape in genome replication, and require a critical evaluation of the existing views about the nature of replication origins, the mechanisms of origin specification and the relevance of epigenetic modifications for genome replication.

A Rapid and Efficient ChIP Protocol to Profile Chromatin Binding Proteins and Epigenetic Modifications in Arabidopsis

Chromatin immunoprecipitation (ChIP) is a widely used and very powerful procedure to identify the proteins that are associated with the DNA to regulate developmental processes. These proteins can be transcription factors, or specific histone variants and modified histones, which are all crucial for gene regulation. In order to obtain reliable results, ChIP must be carried out under highly reproducible conditions. Here, we describe a simple and fast ChIP protocol adapted for Arabidopsis seedlings, which can serve as a basis for other species, organs or more sophisticated procedures, such as the sequential ChIP. We also provide user-oriented troubleshooting to increase the chances of successful applications.

The CDK regulators Cdh1 and Sic1 promote efficient usage of DNA replication origins to prevent chromosomal instability at a chromosome arm

Robustness and completion of DNA replication rely on redundant DNA replication origins. Reduced efficiency of origin licensing is proposed to contribute to chromosome instability in CDK-deregulated cell cycles, a frequent alteration in oncogenesis. However, the mechanism by which this instability occurs is largely unknown. Current models suggest that limited origin numbers would reduce fork density favouring chromosome rearrangements, but experimental support in CDK-deregulated cells is lacking. We have investigated the pattern of origin firing efficiency in budding yeast cells lacking the CDK regulators Cdh1 and Sic1. We show that each regulator is required for efficient origin activity, and that both cooperate non-redundantly. Notably, origins are differentially sensitive to CDK deregulation. Origin sensitivity is independent on normal origin efficiency, firing timing or chromosomal location. Interestingly, at a chromosome arm, there is a shortage of origin firing involving active and dormant origins, and the extent of shortage correlates with the severity of CDK deregulation and chromosome instability. We therefore propose that CDK deregulation in G1 phase compromises origin redundancy by decreasing the number of active and dormant origins, leading to origin shortage and increased chromosome instability.

Links of genome replication, transcriptional silencing and chromatin dynamics.

Genome replication in multicellular organisms involves duplication of both the genetic material and the epigenetic information stored in DNA and histones. In some cases, the DNA replication process provides a window of opportunity for resetting chromatin marks in the genome of the future daughter cells instead of transferring them identical copies. This crucial step of genome replication depends on the correct function of DNA replication factors and the coordination between replication and transcription in proliferating cells. In fact, the histone composition and modification status appears to be intimately associated with the proliferation potential of cells within developing organs. Here we discuss these topics in the light of recent advances in our understanding of how genome replication, transcriptional silencing and chromatin dynamics are coordinated in proliferating cells.