Charles J. Underwood

Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication.

Making a Histone Mark The covalent marks on histones (the principal components of chromatin) play a critical role in the regulation of gene expression. Somehow these marks are preserved when a cell in a tissue divides so that the daughter cells maintain the gene expression program and tissue identity of the parent cell. Jacob et al. (p. 1249) show that the Arabidopsis histone methylase ATXR5 is specific for the replication-dependent histone variant H3.1 and maintains the repressive histone H3 lysine-27 methyl mark on the H3.1 variant during genome replication, thus, preserving cell-type–specific regions of heterochromatin and gene repression through cell division and beyond. The specificity of a histone methyltransferase for a histone variant maintains heterochromatin through cell division. Histone variants have been proposed to act as determinants for posttranslational modifications with widespread regulatory functions. We identify a histone-modifying enzyme that selectively methylates the replication-dependent histone H3 variant H3.1. The crystal structure of the SET domain of the histone H3 lysine-27 (H3K27) methyltransferase ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) in complex with a H3.1 peptide shows that ATXR5 contains a bipartite catalytic domain that specifically “reads” alanine-31 of H3.1. Variation at position 31 between H3.1 and replication-independent H3.3 is conserved in plants and animals, and threonine-31 in H3.3 is responsible for inhibiting the activity of ATXR5 and its paralog, ATXR6. Our results suggest a simple model for the mitotic inheritance of the heterochromatic mark H3K27me1 and the protection of H3.3-enriched genes against heterochromatization during DNA replication.

GenomeScope: fast reference-free genome profiling from short reads

Summary: GenomeScope is an open‐source web tool to rapidly estimate the overall characteristics of a genome, including genome size, heterozygosity rate and repeat content from unprocessed short reads. These features are essential for studying genome evolution, and help to choose parameters for downstream analysis. We demonstrate its accuracy on 324 simulated and 16 real datasets with a wide range in genome sizes, heterozygosity levels and error rates. Availability and Implementation: http://genomescope.org, https://github.com/schatzlab/genomescope.git. Contact: mschatz@jhu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination

During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural Arabidopsis thaliana accessions, we identified two major recombination quantitative trait loci (rQTLs) that explain 56.9% of crossover variation in Col×Ler F2 populations. We mapped rQTL1 to semidominant polymorphisms in HEI10, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null hei10 mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional HEI10 copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest HEI10-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in Arabidopsis recombination. Our work reveals that HEI10 naturally limits Arabidopsis crossovers and has the potential to influence the response to selection.

Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes

Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.

Regulation of MicroRNA-Mediated Developmental Changes by the SWR1 Chromatin Remodeling Complex

SWR1-C is required for miRNA-mediated developmental controls through transcriptional activation and generates proper balances between miRNAs and target mRNAs for plant development in Arabidopsis thaliana. The ATP-dependent SWR1 chromatin remodeling complex (SWR1-C) exchanges the histone H2A-H2B dimer with the H2A.Z-H2B dimer, producing variant nucleosomes. Arabidopsis thaliana SWR1-C contributes to the active transcription of many genes, but also to the repression of genes that respond to environmental and developmental stimuli. Unlike other higher eukaryotic H2A.Z deposition mutants (which are embryonically lethal), Arabidopsis SWR1-C component mutants, including arp6, survive and display a pleiotropic developmental phenotype. However, the molecular mechanisms of early flowering, leaf serration, and the production of extra petals in arp6 have not been completely elucidated. We report here that SWR1-C is required for miRNA-mediated developmental control via transcriptional regulation. In the mutants of the components of SWR1-C such as arp6, sef, and pie1, miR156 and miR164 levels are reduced at the transcriptional level, which results in the accumulation of target mRNAs and associated morphological changes. Sequencing of small RNA libraries confirmed that many miRNAs including miR156 decreased in arp6, though some miRNAs increased. The arp6 mutation suppresses the accumulation of not only unprocessed primary miRNAs, but also miRNA-regulated mRNAs in miRNA processing mutants, hyl1 and serrate, which suggests that arp6 has a transcriptional effect on both miRNAs and their targets. We consistently detected that the arp6 mutant exhibits increased nucleosome occupancy at the tested MIR gene promoters, indicating that SWR1-C contributes to transcriptional activation via nucleosome dynamics. Our findings suggest that SWR1-C contributes to the fine control of plant development by generating a balance between miRNAs and target mRNAs at the transcriptional level.

Genetic and epigenetic variation of transposable elements in Arabidopsis

Transposable elements are mobile genetic elements that are prevalent in plant genomes and are silenced by epigenetic modification. Different epigenetic modification pathways play distinct roles in the control of transposable element transcription, replication and recombination. The Arabidopsis genome contains families of all of the major transposable element classes, which are differentially enriched in particular genomic regions. Whole genome sequencing and DNA methylation profiling of hundreds of natural Arabidopsis accessions has revealed that transposable elements exhibit significant intraspecific genetic and epigenetic variation, and that genetic variation often underlies epigenetic variation. Together, epigenetic modification and the forces of selection define the scope within which transposable elements can contribute to, and control, genome evolution.

Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis

Significance The majority of eukaryotes reproduce sexually, creating genetic variation within populations. Sexual reproduction requires gamete production via meiotic cell division. During meiosis, homologous chromosomes pair and undergo exchange, called crossover. Crossover is vital for crop breeding and remains a major tool to combine useful traits. Despite the importance of crossovers for breeding, their levels are typically low, with one to two forming per chromosome, irrespective of physical chromosome size. Here we genetically engineer superrecombining Arabidopsis, via boosting the major procrossover pathway (using additional copies of the HEI10 E3-ligase gene), and simultaneously removing a major antirecombination pathway (using mutations in RECQ4A and RECQ4B helicase genes). This strategy has the potential to drive massive crossover elevations in crop genomes and accelerate breeding. During meiosis, homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double-strand breaks (DSBs), which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of ∼100–200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as noncrossovers. To bias DSB repair toward crossovers, we simultaneously increased dosage of the procrossover E3 ligase gene HEI10 and introduced mutations in the anticrossovers helicase genes RECQ4A and RECQ4B. As HEI10 and recq4a recq4b increase interfering and noninterfering crossover pathways, respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases distally towards the subtelomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

Epigenetic activation of meiotic recombination near Arabidopsis thaliana centromeres via loss of H3K9me2 and non-CG DNA methylation

Eukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis, centromeres are suppressed for inter-homolog crossover, as recombination in these regions can cause chromosome missegregation and aneuploidy. Plant centromeres are surrounded by transposon-dense pericentromeric heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. However, the role of these chromatin modifications in control of meiotic recombination in the pericentromeres is not fully understood. Here, we show that disruption of Arabidopsis thaliana H3K9me2 and non-CG DNA methylation pathways, for example, via mutation of the H3K9 methyltransferase genes KYP/SUVH4 SUVH5 SUVH6, or the CHG DNA methyltransferase gene CMT3, increases meiotic recombination in proximity to the centromeres. Using immunocytological detection of MLH1 foci and genotyping by sequencing of recombinant plants, we observe that H3K9me2 and non-CG DNA methylation pathway mutants show increased pericentromeric crossovers. Increased pericentromeric recombination in H3K9me2/non-CG mutants occurs in hybrid and inbred backgrounds and likely involves contributions from both the interfering and noninterfering crossover repair pathways. We also show that meiotic DNA double-strand breaks (DSBs) increase in H3K9me2/non-CG mutants within the pericentromeres, via purification and sequencing of SPO11-1-oligonucleotides. Therefore, H3K9me2 and non-CG DNA methylation exert a repressive effect on both meiotic DSB and crossover formation in plant pericentromeric heterochromatin. Our results may account for selection of enhancer trap Dissociation (Ds) transposons into the CMT3 gene by recombination with proximal transposon launch-pads.

Argonautes team up to silence transposable elements in Arabidopsis

The de novo silencing of transposable elements in plants and animals is mediated in part by RNA‐directed chromatin modification. In flowering plants, AGO4 has been seen as the key argonaute protein in the RNA‐directed DNA methylation pathway that links the plant‐specific RNA polymerase V with the de novo DNA methyltransferase DRM2 (Zhong et al, 2014). Two recent papers in The EMBO Journal strongly implicate a role for the AGO6 protein in the process of de novo silencing.