Cell cycle status of male and female gametes during Arabidopsis reproduction.

Fertilization in Arabidopsis (Arabidopsis thaliana) is a highly coordinated process that begins with a pollen tube delivering the 2 sperm cells into the embryo sac. Each sperm cell can then fertilize either the egg or the central cell to initiate embryo or endosperm development, respectively. The success of this double fertilization process requires a tight cell cycle synchrony between the male and female gametes to allow karyogamy (nuclei fusion). However, the cell cycle status of the male and female gametes during fertilization remains elusive as DNA quantification and DNA replication assays have given conflicting results. Here, to reconcile these results, we quantified the DNA replication state by DNA sequencing and performed microscopic analyses of fluorescent markers covering all phases of the cell cycle. We show that male and female Arabidopsis gametes are both arrested prior to DNA replication at maturity and initiate their DNA replication only during fertilization.

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.

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.

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.

Comprehensive phenotyping of circulating immune cell subsets in people living with HIV.

Systemic chronic inflammation and immune dysfunction are recognized as drivers of the development of non-AIDS related comorbidities (NARCs) in people living with HIV (PLHIV). In order to lower the risk of NARCs, it is critical to elucidate what is the contribution of alterations in the composition and function of circulating immune cells to NARCs-related pathogenesis. Findings from previous immunophenotyping studies in PLHIV are highly heterogeneous and it is not fully understood to what extent phenotypic changes on immune cells play a role in the dysregulated inflammatory response observed. In this study, three flow cytometry panels were designed and standardized to phenotypically and functionally identify the main circulating immune cell subsets in PLHIV. To reduce variability, up to 10 markers out of the approximately 20 markers in each panel were used in a custom dry format DURA Innovations (LUCID product line). Intra-assay precision tests performed for the selected cell subsets showed that the three panels had a %CV below 18% for percent of positive cells and the MFI (mean fluorescent intensity) of lineage markers. Our reported pipeline for immunophenotypic analysis facilitated the discrimination of 1153 cell populations, providing an integrated overview of circulating innate and adaptative immune cells as well as the cells' functional status in terms of activation, exhaustion, and maturation. When combined with unsupervised computational techniques, this standardized immunophenotyping approach may support the discovery of novel phenotypes with clinical relevance in NARCs and demonstrate future utility in other immune-mediated diseases.

Origin Recognition Complex (ORC) Evolution Is Influenced by Global Gene Duplication/Loss Patterns in Eukaryotic Genomes.

The conservation of orthologs of most subunits of the origin recognition complex (ORC) has served to propose that the whole complex is common to all eukaryotes. However, various uncertainties have arisen concerning ORC subunit composition in a variety of lineages. Also, it is unclear whether the ancestral diversification of ORC in eukaryotes was accompanied by the neofunctionalization of some subunits, for example, role of ORC1 in centriole homeostasis. We have addressed these questions by reconstructing the distribution and evolutionary history of ORC1-5/CDC6 in a taxon-rich eukaryotic data set. First, we identified ORC subunits previously undetected in divergent lineages, which allowed us to propose a series of parsimonious scenarios for the origin of this multiprotein complex. Contrary to previous expectations, we found a global tendency in eukaryotes to increase or decrease the number of subunits as a consequence of genome duplications or streamlining, respectively. Interestingly, parasites show significantly lower number of subunits than free-living eukaryotes, especially those with the lowest genome size and gene content metrics. We also investigated the evolutionary origin of the ORC1 role in centriole homeostasis mediated by the PACT region in human cells. In particular, we tested the consequences of reducing ORC1 levels in the centriole-containing green alga Chlamydomonas reinhardtii. We found that the proportion of centrioles to flagella and nuclei was not dramatically affected. This, together with the PACT region not being significantly more conserved in centriole-bearing eukaryotes, supports the notion that this neofunctionalization of ORC1 would be a recent acquisition rather than an ancestral eukaryotic feature.

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.

Emerging roles of chromatin in the maintenance of genome organization and function in plants.

Chromatin is not a uniform macromolecular entity; it contains different domains characterized by complex signatures of DNA and histone modifications. Such domains are organized both at a linear scale along the genome and spatially within the nucleus. We discuss recent discoveries regarding mechanisms that establish boundaries between chromatin states and nuclear territories. Chromatin organization is crucial for genome replication, transcriptional silencing, and DNA repair and recombination. The replication machinery is relevant for the maintenance of chromatin states, influencing DNA replication origin specification and accessibility. Current studies reinforce the idea of intimate crosstalk between chromatin features and processes involving DNA transactions.

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.

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.