Project description:The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mistargeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report the surprising finding that RAG1 binds to thousands of sites in the genome of developing lymphocytes, primarily at active promoters and enhancers. The genome has responded by reducing the abundance of "cryptic" recombination signals near sites of RAG1 binding. This depletion operates specifically on the RSS heptamer, with nonamers enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes. MNase-seq profiles of mouse thymocytes
Project description:We performed ChIP-seq for the meiotic strand exchange protein DMC1, which marks an early stage in the meiotic recombination pathway, and the chromosome axis protein ASY1, which promotes interhomolog synapsis and recombination in plants, using tissue collected from immature pre-emergence spikes from wild type bread wheat cultivar Chinese Spring plants. To investigate connections between meiotic recombination and chromatin states in wheat, we also performed ChIP-seq for euchromatic (H3K4me3) and constitutive heterochromatic (H3K9me2 and H3K27me1) marks, and mapped genome-wide nucleosome occupancy via micrococcal nuclease sequencing (MNase-seq) using leaf tissue from Chinese Spring.
Project description:Integrated breeding strategies are used to increase both the yield potential and stability of crops. Most of these approaches have a direct genetic basis. The utility of epigenetics in breeding to improve complex traits such as yield and stress tolerance is not clear. A better understanding of the status of the epigenome and its contribution to the agronomic performance would help in developing strategies to incorporate the epigenetic component of complex traits in breeding,Starting from isogenic canola lines, epilines were generated by selecting recursively during three generations for lines with a higher energy use efficiency and drought tolerance. These epilines were more energy use efficient, drought tolerant, high nitrogen use efficient, and higher yielding under suboptimal conditions. Moreover, these characteristics were transgenerational inheritable. Transcriptome comparison with a line selected for energy use efficiency only revealed common differentially expressed genes related to the onset of signaling events regulating stress tolerance. Genes related to salt, osmotic, abscisic acid and drought were specifically differentially expressed in the drought tolerant epilines. The status of the epigenome, scored as differential trimethylation of lysine 4 of histone 3, supports the energy use efficient and drought tolerant phenotype by facilitating transcription of the genes that are found to be differentially expressed.From these results it can be concluded that the epigenome can be shaped by selection to increase yield and stress tolerance. This acquired knowledge will support further development of strategies to incorporate epigenetics in breeding. To investigate the epigenetic effect on histone mark distribution of the EUE/PEG selection we performed ChIP-seq analyses. Native ChIP using an anti-H3K4me3 and no antibody (background control) was done on PEG1 and control plants.
Project description:Bone marrow-derived multipotent stromal cells (BM-MSCs) exhibit therapuetically valuable properties, including the capacity to differentiate into skeletal tissues and modulate immune system activity. These properties depend on proper regulation of dynamic gene expression in response to environmental and developmental stimuli. This study used chromatin immunoprecipitation (ChIP) coupled with human promoter tiling microarray analysis (ChIP-on-chip) to profile histones H3K4me3 and H3K27me3 at promoters genome-wide. The goal of the study was to identify gene promoters marked by H3K27me3 and H3K4me3 in BM-MSCs. ChIP-on-chip performed with antibodies to H3K4me3 and H3K27me3 on BM-MSCs from 3 different donors (labeled 1632, 167696, and 8F3560) and with technical replicates.
Project description:Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. Part of the explanation probably lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicated that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses revealed that SF3B1 is specifically bound to nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affected the splicing of these exons similarly to inhibition of SF3B1 expression. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure. MNase-seq on Input and SF3B1 pull-down, mRNA-seq on control and SF3B1 si-RNA treated cells as well as on TSA (Trichostatin A) treated and untreated cells.
Project description:We performed ChIP-seq for the meiotic strand exchange protein DMC1, which marks an early stage in the meiotic recombination pathway, and the chromosome axis protein ASY1, which promotes interhomolog synapsis and recombination in plants, using tissue collected from immature pre-emergence spikes from wild type bread wheat cultivar Chinese Spring plants. To investigate connections between meiotic recombination and chromatin states in wheat, we also performed ChIP-seq for euchromatic (H3K4me3) and constitutive heterochromatic (H3K9me2 and H3K27me1) marks, and mapped genome-wide nucleosome occupancy via micrococcal nuclease sequencing (MNase-seq) using leaf tissue from Chinese Spring.
Project description:Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic role in transcription and chromatin dynamics remains poorly understood. Here, we investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae. Our data show that Set1 and Jhd2 predominantly co-regulate transcription. We find combined activities of Set1 and Jhd2 via H3K4 methylation contribute to positive or negative transcriptional regulation at shared target genes. Providing mechanistic insights, our data reveal that Set1 and Jhd2 together control nucleosomal occupancy during transcriptional co-regulation. Moreover, we find a remarkable genome-wide co-regulation of nucleosome and chromatin structure by Set1 and Jhd2 at different groups of transcriptionally active or inactive genes and at different regions within yeast genes. Overall, our study prompts a model wherein combined actions of Set1 and Jhd2 via H3K4 methylationâdemethylation control chromatin dynamics during various facets of transcriptional regulation. Genome-wide nucleosome maps were generated from three different yeast strains representing wild type control, set1 null and jhd2 null mutants. Three independent biological samples were grown for each strain, nucleosomes were prepared by micrococcal nuclease digestion, libraries were prepared, mononculeosomal DNA was isolated, sequenced, and analyzed separately.
Project description:Nucleosome structure and positioning play pivotal roles in gene regulation, DNA repair and other essential processes in eukaryotic cells. Nucleosomal DNA is thought to be uniformly inaccessible to DNA binding and processing factors, such as MNase. Here, we show, however, that nucleosome accessibility and sensitivity to MNase varies. Digestion of Drosophila chromatin with two distinct concentrations of MNase revealed two types of nucleosomes: sensitive and resistant. MNase-resistant nucleosome arrays are less accessible to low concentrations of MNase, whereas MNase-sensitive arrays are degraded by high concentrations. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C containing dinucleotides. In contrast, MNase-sensitive nucleosomes form on A/T rich sequences represented by transcription start and termination sites, enhancers and DNase hypersensitive sites. Lowering of cell growth temperature to ~10°C stabilizes MNase-sensitive nucleosomes suggesting that variations in sensitivity to MNase are related to either thermal fluctuations in chromatin fiber or the activity of enzymatic machinery. In the vicinity of active genes and DNase hypersensitive sites nucleosomes are organized into synchronous, periodic arrays. These patterns are likely to be caused by “phasing” nucleosomes off a potential barrier formed by DNA-bound factors and we provide an extensive biophysical framework to explain this effect. Mnase-seq, Mnase-ChIP-seq of Drosophila melanogaster embryo and S2 cells chromatin
Project description:The RAG1/RAG2 endonuclease initiates V(D)J recombination at antigen receptor loci but also binds to thousands of places outside of these loci. RAG2 localizes directly to lysine 4 trimethylated histone 3 (H3K4me3) through a PHD finger. The relative contribution of RAG2-dependent and RAG1-intrinsic mechanisms in determining RAG1 binding patterns is not known. Through analysis of deep RAG1 ChIP-seq data, we provide a quantitative description of the forces underlying genome-wide targeting of RAG1. Surprisingly, sequence-specific DNA binding contributes minimally to RAG1 targeting outside of antigen receptor loci. Instead, RAG1 binding is driven by two distinct modes of interaction with chromatin: the first is driven by H3K4me3, promoter-focused, and dependent on the RAG2 PHD, and the second is defined by H3K27Ac, enhancer-focused, and dependent on "non-core" portions of RAG1. Based on this and additional chromatin and genomic features, we formulated a predictive model of RAG1 targeting to the genome. RAG1 binding sites predicted by our model correlate well with observed patterns of RAG1-mediated breaks in human pro-B acute lymphoblastic leukemia. Overall, this study provides an integrative model for RAG1 genome-wide binding and off-target activity, and reveals a novel role for the RAG1 non-core region in RAG1 targeting. ChIP-seq profiles of RAG1 from mouse thymocytes, and H3K27Ac from human REH cell line