DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis.

During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes.We thank Korbinian Schneeberger and Beth Rowan for advice implementing TIGER and Ler polymorphism data, Donna Bond for pJawohl-Act2, Quentin Gouil for the bisulfite sequencing protocol, Simon Andrews and Felix Krueger for advice using SeqMonk, Gregory Copenhaver and Avi Levy for fluorescent lines, Raphael Mercier for zip4-2 fancm-1, Chris Franklin for the ASY1 antibody, and the Gurdon Institute Imaging Facility for access to microscopes. Research was supported by a Broodbank Fellowship (to N.E.Y.), a Royal Society University Research Fellowship (to I.R.H.), grant GAT2962 from the Gatsby Charitable Foundation (to I.R.H.), and Biotechnology and Biological Sciences Research Council grant BB/L006847/1 (to I.R.H.).This is the final version of the article. It first appeared from Cold Spring Habour Laboratory Press via http://dx.doi.org/10.1101/gad.270876.11

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis.

Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.This work was supported by an EMBL long-term postdoctoral fellowship to B.Y.C., Sir Henry Wellcome Fellowships to B.Y.C. and N.I., a Wellcome Trust PhD scholarship to J.D.J., a Wellcome Trust Fellowship to A.E.F. (088789), and UK Biotechnology and Biological Sciences Research Council grants to I.B. (BB/L000334/ 1) and A.E.F. (BB/J007072/1). Work in the Baulcombe laboratory is supported by The Gatsby Charitable Foundation and the European Research Council Advanced Investigator grant TRIBE. D.C.B. is the Royal Society Edward Penley Abraham Research Professor. We wish to thank Professor Stuart G. Siddell, University of Bristol, for providing the murine 17 clone 1 cellsThis is the final version of the article. It was first available from Cold Springs Harbor Press via http://dx.doi.org/10.1261/rna.052548.11

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.Research in the Henderson laboratory was supported by a Royal Society University Research Fellowship, Gatsby Charitable Foundation grant 2962, BBSRC grant BB/N007557/1 and National Natural Science Foundation of China grant 61403318. KC was funded by an EMBO long term postdoctoral fellowship ALTF 807-2009. PAZ was supported by a Polish Mobility Plus Fellowship 605/MOB/2011/0. GPC is funded by a National Science Foundation Grant (MCB-1121563). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It first appeared from PLOS at http://dx.doi.org/10.1371/journal.pgen.1006179

TP53 mutant MDM2-amplified cell lines selected for resistance to MDM2-p53 binding antagonists retain sensitivity to ionizing radiation

Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR

Diaryl- and triaryl-pyrrole derivatives:Inhibitors of the MDM2-p53 and MDMX-p53 protein-protein interactions

Screening identified 2-(3-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile as an MDM2–p53 inhibitor (IC(50) = 12.3 μM). MDM2–p53 and MDMX–p53 activity was seen for 5-((1-(4-chlorophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (MDM2 IC(50) = 0.11 μM; MDMX IC(50) = 4.2 μM) and 5-((1-(4-nitrophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (MDM2 IC(50) = 0.15 μM; MDMX IC(50) = 4.2 μM), and cellular activity consistent with p53 activation in MDM2 amplified cells. Further SAR studies demonstrated the requirement for the triarylpyrrole moiety for MDMX–p53 activity but not for MDM2–p53 inhibition

On the encounter between the GASP galaxy JO36 and the radio plume of GIN 049

We report on the serendipitous discovery of an unprecedented interaction between the radio lobe of a radio galaxy and a spiral galaxy. The discovery was made thanks to LOFAR observations at 144 MHz of the galaxy cluster Abell 160 ($z=0.04317$) provided by the LOFAR Two-metre Sky Survey. The new low-frequency observations revealed that one of the radio plumes of the central galaxy GIN 049 overlaps with the spiral galaxy JO36. Previous studies carried out with MUSE revealed that the warm ionized gas in the disk of JO36, traced by the H$\alpha$ emission, is severely truncated with respect to the stellar disk. We further explore this unique system by including new uGMRT observations at 675 MHz to map the spectral index. The emerging scenario is that JO36 has interacted with the radio plume in the past 200-500 Myr. The encounter resulted in a positive feedback event for JO36 in the form of a star formation rate burst of $\sim14$ $M_\odot$ yr$^{-1}$. In turn, the galaxy passage left a trace in the radio-old plasma by re-shaping the old relativistic plasma via magnetic draping.Comment: 20 pages, 11 figures. Accepted for publication on ApJ on September 4th, 202

Identification of a novel ligand for the ATAD2 bromodomain with selectivity over BRD4 through a fragment growing approach

Structure-guided expansion of a fragment hit for the ATAD2 bromodomain enabled improvement in ATAD2 inhibition and selectivity over BRD4.</p

The MeerKAT international GHz tiered extragalactic exploration (MIGHTEE) survey

The MIGHTEE large survey project will survey four of the most well-studied extragalactic deep fields, totalling 20 square degrees to µJy sensitivity at Giga-Hertz frequencies, as well as an ultra-deep image of a single ∼1 deg2 MeerKAT pointing. The observations will provide radio continuum, spectral line and polarisation information. As such, MIGHTEE, along with the excellent multi-wavelength data already available in these deep fields, will allow a range of science to be achieved. Specifically, MIGHTEE is designed to significantly enhance our understanding of, (i) the evolution of AGN and star-formation activity over cosmic time, as a function of stellar mass and environment, free of dust obscuration; (ii) the evolution of neutral hydrogen in the Universe and how this neutral gas eventually turns into stars after moving through the molecular phase, and how efficiently this can fuel AGN activity; (iii) the properties of cosmic magnetic fields and how they evolve in clusters, filaments and galaxies. MIGHTEE will reach similar depth to the planned SKA all-sky survey, and thus will provide a pilot to the cosmology experiments that will be carried out by the SKA over a much larger survey volume

Arabidopsis meiotic crossover hot spots overlap with H2A.Z nucleosomes at gene promoters

PRDM9 directs human meiotic crossover hotspots to intergenic sequence motifs, whereas budding yeast hotspots overlap low nucleosome density regions in gene promoters. To investigate hotspots in plants, which lack PRDM9, we used coalescent analysis of Arabidopsis genetic variation. Crossovers increase towards gene promoters and terminators, and hotspots are associated with active chromatin modifications, including H2A.Z, histone H3K4me3, low nucleosome density and low DNA methylation. Hotspot-enriched A-rich and CTT-repeat DNA motifs occur upstream and downstream of transcriptional start respectively. Crossovers are asymmetric around promoters and highest over CTT-motifs and H2A.Z-nucleosomes. Pollen-typing, segregation and cytogenetic analysis show decreased crossovers in the arp6 H2A.Z deposition mutant, at multiple scales. During meiosis H2A.Z and DMC1/RAD51 recombinases form overlapping chromosomal foci. As arp6 reduces DMC1/RAD51 foci, H2A.Z may promote formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hotspots within eukaryotes and PRDM9 is a derived state within vertebrates