Beyond the Bones: A Case Report of Extraosseous Ewing Sarcoma.

Ewing sarcoma (ES) is primarily recognized as a primary bone tumor; however, its extraosseous variant is exceptionally rare and presents unique clinical challenges. In this article, we report the case of a 22-year-old male who initially presented with abdominal swelling. Diagnostic tests included abdominal imaging and a CT scan, revealing a solid liver mass. A thorough evaluation confirmed it to be an extraosseous ES, supported by liver biopsy and immunohistochemistry demonstrating positive expression for AE1/AE3 and CD-99, along with genetic analysis revealing a rearrangement of the EWSR1 gene (translocation 22q12). The patient's treatment involved a multimodal approach, including perioperative chemotherapy, surgery, and postoperative chemotherapy, following which the patient remained in complete remission after 24 months. This case emphasizes the importance of considering rare malignancies such as ES in differential diagnoses for young patients with liver masses. It also accentuates the pivotal role of family physicians in early detection and holistic patient care, underscoring the need for comprehensive investigations when encountering persistent symptoms.

Distinct roles of Arabidopsis ORC1 proteins in DNA replication and heterochromatic H3K27me1 deposition.

Most cellular proteins involved in genome replication are conserved in all eukaryotic lineages including yeast, plants and animals. However, the mechanisms controlling their availability during the cell cycle are less well defined. Here we show that the Arabidopsis genome encodes for two ORC1 proteins highly similar in amino acid sequence and that have partially overlapping expression domains but with distinct functions. The ancestral ORC1b gene, present before the partial duplication of the Arabidopsis genome, has retained the canonical function in DNA replication. ORC1b is expressed in both proliferating and endoreplicating cells, accumulates during G1 and is rapidly degraded upon S-phase entry through the ubiquitin-proteasome pathway. In contrast, the duplicated ORC1a gene has acquired a specialized function in heterochromatin biology. ORC1a is required for efficient deposition of the heterochromatic H3K27me1 mark by the ATXR5/6 histone methyltransferases. The distinct roles of the two ORC1 proteins may be a feature common to other organisms with duplicated ORC1 genes and a major difference with animal cells.

Differences in firing efficiency, chromatin, and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins.

Eukaryotic genome replication depends on thousands of DNA replication origins (ORIs). A major challenge is to learn ORI biology in multicellular organisms in the context of growing organs to understand their developmental plasticity. We have identified a set of ORIs of Arabidopsis thaliana and their chromatin landscape at two stages of post-embryonic development. ORIs associate with multiple chromatin signatures including transcription start sites (TSS) but also proximal and distal regulatory regions and heterochromatin, where ORIs colocalize with retrotransposons. In addition, quantitative analysis of ORI activity led us to conclude that strong ORIs have high GC content and clusters of GGN trinucleotides. Development primarily influences ORI firing strength rather than ORI location. ORIs that preferentially fire at early developmental stages colocalize with GC-rich heterochromatin, but at later stages with transcribed genes, perhaps as a consequence of changes in chromatin features associated with developmental processes. Our study provides the set of ORIs active in an organism at the post-embryo stage that should allow us to study ORI biology in response to development, environment, and mutations with a quantitative approach. In a wider scope, the computational strategies developed here can be transferred to other eukaryotic systems.

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.

Sequential ChIP Protocol for Profiling Bivalent Epigenetic Modifications (ReChIP).

Identification of chromatin modifications, e.g., histone acetylation and methylation, among others, is widely carried out by using a chromatin immunoprecipitation (ChIP) strategy. The information obtained with these procedures is useful to gain an overall picture of modifications present in all cells of the population under study. It also serves as a basis to figure out the mechanisms of chromatin organization and gene regulation at the population level. However, the ultimate goal is to understand gene regulation at the level of single chromatin fibers. This requires the identification of chromatin modifications that occur at a given genomic location and within the same chromatin fiber. This is achieved by following a sequential ChIP strategy using two antibodies to distinguish different chromatin modifications. Here, we describe a sequential ChIP protocol (Re-ChIP), paying special attention to the controls needed and the required steps to obtain meaningful and reproducible results. The protocol is developed for young Arabidopsis seedlings but could be adapted to other plant materials.

Retrotransposons are specified as DNA replication origins in the gene-poor regions of Arabidopsis heterochromatin.

Genomic stability depends on faithful genome replication. This is achieved by the concerted activity of thousands of DNA replication origins (ORIs) scattered throughout the genome. The DNA and chromatin features determining ORI specification are not presently known. We have generated a high-resolution genome-wide map of 3230 ORIs in cultured Arabidopsis thaliana cells. Here, we focused on defining the features associated with ORIs in heterochromatin. In pericentromeric gene-poor domains ORIs associate almost exclusively with the retrotransposon class of transposable elements (TEs), in particular of the Gypsy family. ORI activity in retrotransposons occurs independently of TE expression and while maintaining high levels of H3K9me2 and H3K27me1, typical marks of repressed heterochromatin. ORI-TEs largely colocalize with chromatin signatures defining GC-rich heterochromatin. Importantly, TEs with active ORIs contain a local GC content higher than the TEs lacking them. Our results lead us to conclude that ORI colocalization with retrotransposons is determined by their transposition mechanism based on transcription, and a specific chromatin landscape. Our detailed analysis of ORIs responsible for heterochromatin replication has implications on the mechanisms of ORI specification in other multicellular organisms in which retrotransposons are major components of heterochromatin and of the entire genome.

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.

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.

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.

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.

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

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.

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.

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 cell's 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.

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.

Transcription initiation activity sets replication origin efficiency in mammalian cells.

Genomic mapping of DNA replication origins (ORIs) in mammals provides a powerful means for understanding the regulatory complexity of our genome. Here we combine a genome-wide approach to identify preferential sites of DNA replication initiation at 0.4% of the mouse genome with detailed molecular analysis at distinct classes of ORIs according to their location relative to the genes. Our study reveals that 85% of the replication initiation sites in mouse embryonic stem (ES) cells are associated with transcriptional units. Nearly half of the identified ORIs map at promoter regions and, interestingly, ORI density strongly correlates with promoter density, reflecting the coordinated organisation of replication and transcription in the mouse genome. Detailed analysis of ORI activity showed that CpG island promoter-ORIs are the most efficient ORIs in ES cells and both ORI specification and firing efficiency are maintained across cell types. Remarkably, the distribution of replication initiation sites at promoter-ORIs exactly parallels that of transcription start sites (TSS), suggesting a co-evolution of the regulatory regions driving replication and transcription. Moreover, we found that promoter-ORIs are significantly enriched in CAGE tags derived from early embryos relative to all promoters. This association implies that transcription initiation early in development sets the probability of ORI activation, unveiling a new hallmark in ORI efficiency regulation in mammalian cells.