Genome-wide mapping of Arabidopsis thaliana origins of DNA replication and their associated epigenetic marks. Nat Struct Mol Biol
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.
[Show abstract] [Hide abstract] ABSTRACT: Genome instability is associated with mitotic errors and cancer. This phenomenon can lead to deleterious rearrangements, but also genetic novelty, and many questions regarding its genesis, fate and evolutionary role remain unanswered. Here, we describe extreme chromosomal restructuring during genome elimination, a process resulting from hybridization of Arabidopsis plants expressing different centromere histones H3. Shattered chromosomes are formed from the genome of the haploid inducer, consistent with genomic catastrophes affecting a single, laggard chromosome compartmentalized within a micronucleus. Analysis of breakpoint junctions implicates breaks followed by repair through non-homologous end joining (NHEJ) or stalled fork repair. Furthermore, mutation of required NHEJ factor DNA Ligase 4 results in enhanced haploid recovery. Lastly, heritability and stability of a rearranged chromosome suggest a potential for enduring genomic novelty. These findings provide a tractable, natural system towards investigating the causes and mechanisms of complex genomic rearrangements similar to those associated with several human disorders.
- "Duplicated and triplicated blocks could therefore, have different origins. To address this question, we asked whether breakpoint junctions of the two different copy number states display differential association to various genomic and chromatin features such as genes and repeated elements (Lamesch et al., 2012), DNA replication origins (Costas et al., 2011), DNase I hypersensitive sites (DHS) (Zhang et al., 2012) and nine non-overlapping chromatin states that partition the Arabidopsis genome (Sequeira-Mendes et al., 2014) (Supplementary file 5). When analyzing windows of 1000 bp centered around the breakpoints of duplicated blocks, we observed an enrichment in genic DNA (from 53% background level to 70%, p < 0.01,Figure 5D,F). "
[Show abstract] [Hide abstract] ABSTRACT: The mechanism whereby the same genome can give rise to different cell types with different gene expression profiles is a fundamental problem in biology. Chromatin organization and dynamics have been shown to vary with altered gene expression in different cultured animal cell types, but there is little evidence yet from whole organisms linking chromatin dynamics with development. Here, we used both fluorescence recovery after photobleaching and two-photon photoactivation to show that in stem cells from Arabidopsis thaliana roots the mobility of the core histone H2B, as judged by exchange dynamics, is lower than in the surrounding cells of the meristem. However, as cells progress from meristematic to fully differentiated, core histones again become less mobile and more strongly bound to chromatin. We show that these transitions are largely mediated by changes in histone acetylation. We further show that altering histone acetylation levels, either in a mutant or by drug treatment, alters both the histone mobility and markers of development and differentiation. We propose that plant stem cells have relatively inactive chromatin, but they keep the potential to divide and differentiate into more dynamic states, and that these states are at least in part determined by histone acetylation levels. © 2014 American Society of Plant Biologists. All rights reserved.
- "We frequently observed QC divisions in plants treated overnight with TSA (Figures 7A to 7C), whereas QC divisions are much rarer in untreated plants. Furthermore, we observed that treatment with TSA induced ectopic expression of H2A.Z in QC cells (Figures 7D and 7E); this histone variant has been shown to be associated with replication activity (Costas et al., 2011) and is absent from QCs (Figure 7D). We also showed that this H2A.Z variant is mainly expressed in meristematic tissue (SupplementalFigure 10). "
[Show abstract] [Hide abstract] ABSTRACT: The spatial arrangement of interphase chromosomes in the nucleus is important for gene expression and genome function in animals and in plants. The recently developed Hi-C technology is an efficacious method to investigate genome packing. Here we present a detailed Hi-C map of the three-dimensional genome organization of the plant Arabidopsis thaliana. We find that local chromatin packing differs from the patterns seen in animals, with kilobasepair-sized segments that have much higher intra-chromosome interaction rates than neighboring regions, representing a dominant local structural feature of genome conformation in A. thaliana. These regions, which appear as positive strips on two-dimensional representations of chromatin interaction, are enriched in epigenetic marks H3K27me3, H3.1 and H3.3. We also identify over 400 insulator-like regions. Furthermore, although topologically associating domains (TADs), which are prominent in animals, are not an obvious feature of A. thaliana genome packing, we found over 1,000 regions that have properties of TAD boundaries, and a similar number of regions analogous to the interior of TADs. The insulator-like, TAD-boundary-like, and TAD-interior-like regions are each enriched for distinct epigenetic marks, and are each correlated with different gene expression levels. We conclude that epigenetic modifications, gene density, and transcriptional activity combine to shape the local packing of the A. thaliana nuclear genome.
- "Published ChIP-seq and ChIP-chip data (Zhang et al. 2009; Costas et al. 2011; Roudier et al. 2011; Luo et al. 2012; Park et al. 2012; Stroud et al. 2012) were originally mapped to the TAIR8 version of the A. thaliana reference genome. For ChIP-seq data, 50-bp fragments were retrieved from the TAIR8 genome and mapped to TAIR10. "