Assaying epigenome functions of PRMTs and their substrates.

Among the widespread and increasing number of identified post-translational modifications (PTMs), arginine methylation is catalyzed by the protein arginine methyltransferases (PRMTs) and regulates fundamental processes in cells, such as gene regulation, RNA processing, translation, and signal transduction. As epigenetic regulators, PRMTs play key roles in pluripotency, differentiation, proliferation, survival, and apoptosis, which are essential biological programs leading to development, adult homeostasis but also pathological conditions including cancer. A full understanding of the molecular mechanisms that underlie PRMT-mediated gene regulation requires the genome wide mapping of each player, i.e., PRMTs, their substrates and epigenetic marks, methyl-marks readers as well as interaction partners, in a thorough and unambiguous manner. However, despite the tremendous advances in high throughput sequencing technologies and the numerous efforts from the scientific community, the epigenomic profiling of PRMTs as well as their histone and non-histone substrates still remains a big challenge owing to obvious limitations in tools and methodologies. This review will summarize the present knowledge about the genome wide mapping of PRMTs and their substrates as well as the technical approaches currently in use. The limitations and pitfalls of the technical tools along with conventional approaches will be then discussed in detail. Finally, potential new strategies for chromatin profiling of PRMTs and histone substrates will be proposed and described.

Genome-Wide Mapping of Adult Plant Resistance to Leaf Rust and Stripe Rust in CIMMYT Wheat Line Arableu#1.

Leaf (brown) rust (LR) and stripe (yellow) rust (YR), caused by Puccinia triticina and P. striiformis f. sp. tritici, respectively, significantly reduce wheat production worldwide. Disease-resistant wheat varieties offer farmers one of the most effective ways to manage these diseases. The common wheat (Triticum aestivum L.) Arableu#1, developed by the International Maize and Wheat Improvement Center and released as Deka in Ethiopia, shows susceptibility to both LR and YR at the seedling stage but a high level of adult plant resistance (APR) to the diseases in the field. We used 142 F5 recombinant inbred lines (RILs) derived from Apav#1 × Arableu#1 to identify quantitative trait loci (QTLs) for APR to LR and YR. A total of 4,298 genotyping-by-sequencing markers were used to construct a genetic linkage map. The study identified four LR resistance QTLs and six YR resistance QTLs in the population. Among these, QLr.cim-1BL.1/QYr.cim-1BL.1 was located in the same location as Lr46/Yr29, a known pleiotropic resistance gene. QLr.cim-1BL.2 and QYr.cim-1BL.2 were also located on wheat chromosome 1BL at 37 cM from Lr46/Yr29 and may represent a new segment for pleiotropic resistance to both rusts. QLr.cim-7BL is likely Lr68 given its association with the tightly linked molecular marker cs7BLNLRR. In addition, QLr.cim-3DS, QYr.cim-2AL, QYr.cim-4BL, QYr.cim-5AL, and QYr.cim-7DS are probably new resistance loci based on comparisons with published QTLs for resistance to LR and YR. Our results showed the diversity of minor resistance QTLs in Arableu#1 and their role in conferring near-immune levels of APR to both LR and YR, when combined with the pleiotropic APR gene Lr46/Yr29.

Genome-wide mapping of the dominance effects based on breed ancestry for semen traits in admixed Swiss Fleckvieh bulls.

Heterosis is the beneficial deviation of crossbred progeny from the average of parental lines for a particular trait. Heterosis is due to nonadditive genetic effects with dominance and epistatic components. Recent advances in genotyping technology have encouraged researchers to estimate and scan heterosis components for a range of traits in crossbred populations, applying various definitions of such components. In this study, we defined the intralocus (dominance) component of heterosis using local genetic ancestry and performed genome-wide association analysis for admixed Swiss Fleckvieh bulls and their parental populations, Red Holstein Friesian and Swiss Simmental, for semen traits. A linear mixed model for 41,824 SNP, including SNP additive genetic, breed additive, and breed dominance effects on 1,178 bulls (148 Red Holstein Friesian, 213 Swiss Simmental, and 817 Swiss Fleckvieh) with a total of 43,782 measurements was performed. In total, 19 significant regions for breed dominance were identified for volume (2 regions on Bos taurus autosome 10 and 22) and percentage of live spermatozoa (17 regions on Bos taurus autosome 3, 4, 5, 7, 13, 14, and 17), and genes associated with spermatogenesis, sperm motility, and male fertility traits were located there. No significant region for breed dominance was detected for total number of spermatozoa. The signals for breed dominance were relatively wide, most likely due to limited numbers of recombination events in a small number of generations (10-15 generations) of crossbreeding in the recent Swiss Fleckvieh composite.

Nuclear lamins are not required for lamina-associated domain organization in mouse embryonic stem cells.

In mammals, the nuclear lamina interacts with hundreds of large genomic regions, termed lamina-associated domains (LADs) that are generally in a transcriptionally repressed state. Lamins form the major structural component of the lamina and have been reported to bind DNA and chromatin. Here, we systematically evaluate whether lamins are necessary for the LAD organization in murine embryonic stem cells. Surprisingly, removal of essentially all lamins does not have any detectable effect on the genome-wide interaction pattern of chromatin with emerin, a marker of the inner nuclear membrane. This suggests that other components of the lamina mediate these interactions.

CUT&RUN Profiling of the Budding Yeast Epigenome.

Mapping the epigenome is key to describe the relationship between chromatin landscapes and the control of DNA-based cellular processes such as transcription. Cleavage under targets and release using nuclease (CUT&RUN) is an in situ chromatin profiling strategy in which controlled cleavage by antibody-targeted Micrococcal Nuclease solubilizes specific protein-DNA complexes for paired-end DNA sequencing. When applied to budding yeast, CUT&RUN profiling yields precise genome-wide maps of histone modifications, histone variants, transcription factors, and ATP-dependent chromatin remodelers, while avoiding cross-linking and solubilization issues associated with the most commonly used chromatin profiling technique Chromatin Immunoprecipitation (ChIP). Furthermore, targeted chromatin complexes cleanly released by CUT&RUN can be used as input for a subsequent native immunoprecipitation step (CUT&RUN.ChIP) to simultaneously map two epitopes in single molecules genome-wide. The intrinsically low background and high resolution of CUT&RUN and CUT&RUN.ChIP allows for identification of transient genomic features such as dynamic nucleosome-remodeling intermediates. Starting from cells, one can perform CUT&RUN or CUT&RUN.ChIP and obtain purified DNA for sequencing library preparation in 2 days.

Genome-Wide Mapping and Microscopy Visualization of Protein-DNA Interactions by pA-DamID.

Several methods have been developed to map protein-DNA interactions genome-wide in the last decades. Protein A-DamID (pA-DamID) is a recent addition to this list with distinct advantages. pA-DamID relies on antibody-based targeting of the bacterial Dam enzyme, resulting in adenine methylation of DNA in contact with the protein of interest. This m6A can then be visualized by microscopy, or mapped genome-wide. The main advantages of pA-DamID are an easy and direct visualization of DNA that is in contact with the protein of interest, unbiased mapping of protein-DNA interactions, and the possibility to select specific subpopulations of cells by flow cytometry before further sample processing. pA-DamID is particularly suited to study proteins that form large chromatin domains or that are part of distinct nuclear structures such as the nuclear lamina. This chapter describes the pA-DamID procedure from cell harvesting to the preparation of microscopy slides and high-throughput sequencing libraries.

Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments.

Numerous anti-cancer drugs perturb thymidylate biosynthesis and lead to genomic uracil incorporation contributing to their antiproliferative effect. Still, it is not yet characterized if uracil incorporations have any positional preference. Here, we aimed to uncover genome-wide alterations in uracil pattern upon drug treatments in human cancer cell line models derived from HCT116. We developed a straightforward U-DNA sequencing method (U-DNA-Seq) that was combined with in situ super-resolution imaging. Using a novel robust analysis pipeline, we found broad regions with elevated probability of uracil occurrence both in treated and non-treated cells. Correlation with chromatin markers and other genomic features shows that non-treated cells possess uracil in the late replicating constitutive heterochromatic regions, while drug treatment induced a shift of incorporated uracil towards segments that are normally more active/functional. Data were corroborated by colocalization studies via dSTORM microscopy. This approach can be applied to study the dynamic spatio-temporal nature of genomic uracil.

Genome-Wide Mapping of Yeast Retrotransposon Integration Target Sites.

Transposable elements (TEs) are present in virtually all organisms. TE integration into genomes contributes to their structure and evolution, but can also be harmful in some cases. Deciphering where and how TE integration is targeted is fundamental to understand their intricate relationship with their host and explore the outcome of TE mobility on genome evolution and cell fitness. In general, TEs display integration site preference, which differs between elements. High-throughput mapping of de novo insertions by deep sequencing has recently allowed identifying genome-wide integration preferences of several TEs. These studies have provided invaluable clues to address the molecular determinants of integration site preference. Here, we provide a step-by-step methodology to generate massive de novo insertion events and prepare a library of genomic DNA for next-generation sequencing. We also describe a primary bioinformatic procedure to map these insertions in the genome. The whole procedure comes from our recent work on the integration of Ty1 in Saccharomyces cerevisiae, but could be easily adapted to the study of other TEs.

Charge and Polarity Preferences for N-Glycosylation: A Genome-Wide In Silico Study and Its Implications Regarding Constitutive Proliferation and Adhesion of Carcinoma Cells.

The structural and functional diversity of the human proteome is mediated by N- and O-linked glycosylations that define the individual properties of extracellular and membrane-associated proteins. In this study, we utilized different computational tools to perform in silico based genome-wide mapping of 1,117 human proteins and unravel the contribution of both penultimate and vicinal amino acids for the asparagine-based, site-specific N-glycosylation. Our results correlate the non-canonical involvement of charge and polarity environment of classified amino acids (designated as L, O, A, P, and N groups) in the N-glycosylation process, as validated by NetNGlyc predictions, and 130 literature-reported human proteins. From our results, particular charge and polarity combinations of non-polar aliphatic, acidic, basic, and aromatic polar side chain environment of both penultimate and vicinal amino acids were found to promote the N-glycosylation process. However, the alteration in side-chain charge and polarity environment of genetic variants, particularly in the vicinity of Asn-containing epitope, may induce constitutive glycosylation (e.g., aberrant glycosylation at preferred and non-preferred sites) of membrane proteins causing constitutive proliferation and triggering epithelial-to-mesenchymal transition. The current genome-wide mapping of 1,117 proteins (2,909 asparagine residues) was used to explore charge- and polarity-based mechanistic constraints in N-glycosylation, and discuss alterations of the neoplastic phenotype that can be ascribed to N-glycosylation at preferred and non-preferred sites.

Computationally Tractable Multivariate HMM in Genome-Wide Mapping Studies.

Hidden Markov model (HMM) is widely used for modeling spatially correlated genomic data (series data). In genomics, datasets of this kind are generated from genome-wide mapping studies through high-throughput methods such as chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq). When multiple regulatory protein binding sites or related epigenetic modifications are mapped simultaneously, the correlation between data series can be incorporated into the latent variable inference in a multivariate form of HMM, potentially increasing the statistical power of signal detection. In this chapter, we review the challenges of multivariate HMMs and propose a computationally tractable method called sparsely correlated HMMs (scHMM). We illustrate the method and the scHMM package using an example mouse ChIP-seq dataset.

High-resolution mapping of heteroduplex DNA formed during UV-induced and spontaneous mitotic recombination events in yeast.

In yeast, DNA breaks are usually repaired by homologous recombination (HR). An early step for HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, there will be mismatches within the heteroduplex DNA (hetDNA). In wild-type strains, these mismatches are repaired by the mismatch repair (MMR) system, producing a gene conversion event. In strains lacking MMR, the mismatches persist. Most previous studies involving hetDNA formed during mitotic recombination were restricted to one locus. Below, we present a global mapping of hetDNA formed in the MMR-defective mlh1 strain. We find that many recombination events are associated with repair of double-stranded DNA gaps and/or involve Mlh1-independent mismatch repair. Many of our events are not explicable by the simplest form of the double-strand break repair model of recombination.

Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast.

Meiosis is a specialized form of cell division resulting in reproductive cells with a reduced, usually haploid, genome complement. A key step after premeiotic DNA replication is the occurrence of homologous recombination at multiple places throughout the genome, initiated with the formation of DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like protein Spo11. DSBs are distributed non-randomly in genomes, and understanding the mechanisms that shape this distribution is important for understanding how meiotic recombination influences heredity and genome evolution. Several methods exist for mapping where Spo11 acts. Of these, sequencing of Spo11-associated oligonucleotides (Spo11 oligos) is the most precise, specifying the locations of DNA breaks to the base pair. In this chapter we detail the steps involved in Spo11-oligo mapping in the SK1 strain of budding yeast Saccharomyces cerevisiae, from harvesting cells of highly synchronous meiotic cultures, through preparation of sequencing libraries, to the mapping pipeline used for processing the data.

Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania.

BACKGROUND: 5-Hydroxymethyluracil (5hmU) is a thymine base modification found in the genomes of a diverse range of organisms. To explore the functional importance of 5hmU, we develop a method for the genome-wide mapping of 5hmU-modified loci based on a chemical tagging strategy for the hydroxymethyl group. RESULTS: We apply the method to generate genome-wide maps of 5hmU in the parasitic protozoan Leishmania sp. In this genus, another thymine modification, 5-(ß-glucopyranosyl) hydroxymethyluracil (base J), plays a key role during transcription. To elucidate the relationship between 5hmU and base J, we also map base J loci by introducing a chemical tagging strategy for the glucopyranoside residue. Observed 5hmU peaks are highly consistent among technical replicates, confirming the robustness of the method. 5hmU is enriched in strand switch regions, telomeric regions, and intergenic regions. Over 90% of 5hmU-enriched loci overlapped with base J-enriched loci, which occurs mostly within strand switch regions. We also identify loci comprising 5hmU but not base J, which are enriched with motifs consisting of a stretch of thymine bases. CONCLUSIONS: By chemically detecting 5hmU we present a method to provide a genome-wide map of this modification, which will help address the emerging interest in the role of 5hmU. This method will also be applicable to other organisms bearing 5hmU.

Plasmodium falciparum epigenome: A distinct dynamic epigenetic regulation of gene expression.

Histone modification profiles are predictive of gene expression and most of the knowledge gained is acquired through studies done in higher eukaryotes. However, genome-wide studies involving Plasmodium falciparum, the causative agent of malaria, have been rather few, at lower resolution (mostly using ChIP-on-chip), and covering limited number of histone modifications. In our recent study [1], we have performed extensive genome-wide analyses of multiple histone modifications including the active (H3K4me2, H3K4me3, H3K9ac, H3K14ac, H3K27ac and H4ac), inactive (H3K9me3 and H3K27me3), elongation (H3K79me3) and regulatory element (H3K4me1) in a stage-specific manner. Furthermore, we used a ligation-based method suitable for sequencing homopolymeric stretches as seen in P. falciparum for next-generation sequencing library amplification [2], enabling highly quantitative analysis of the extremely AT-rich P. falciparum genome. Our recently published study suggests that transcription regulation by virtue of poised chromatin and differential histone modifications is unique to P. falciparum [1]. Here we describe the experiments, quality controls and chromatin immunoprecipitation-sequencing data analysis of our associated study published in Epigenetics and Chromatin [1]. Stage-specific ChIP-sequencing data for histone modifications is submitted to Gene Expression Omnibus (GEO) database under the accession number GSE63369.

Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major.

Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance.

Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers.

Generic spinal motor neuron identity is established by cooperative binding of programming transcription factors (TFs), Isl1 and Lhx3, to motor-neuron-specific enhancers. How expression of effector genes is maintained following downregulation of programming TFs in maturing neurons remains unknown. High-resolution exonuclease (ChIP-exo) mapping revealed that the majority of enhancers established by programming TFs are rapidly deactivated following Lhx3 downregulation in stem-cell-derived hypaxial motor neurons. Isl1 is released from nascent motor neuron enhancers and recruited to new enhancers bound by clusters of Onecut1 in maturing neurons. Synthetic enhancer reporter assays revealed that Isl1 operates as an integrator factor, translating the density of Lhx3 or Onecut1 binding sites into transient enhancer activity. Importantly, independent Isl1/Lhx3- and Isl1/Onecut1-bound enhancers contribute to sustained expression of motor neuron effector genes, demonstrating that outwardly stable expression of terminal effector genes in postmitotic neurons is controlled by a dynamic relay of stage-specific enhancers.

Quantification and genome-wide mapping of DNA double-strand breaks.

DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair.

A comprehensive epigenome map of Plasmodium falciparum reveals unique mechanisms of transcriptional regulation and identifies H3K36me2 as a global mark of gene suppression.

BACKGROUND: Role of epigenetic mechanisms towards regulation of the complex life cycle/pathogenesis of Plasmodium falciparum, the causative agent of malaria, has been poorly understood. To elucidate stage-specific epigenetic regulation, we performed genome-wide mapping of multiple histone modifications of P. falciparum. Further to understand the differences in transcription regulation in P. falciparum and its host, human, we compared their histone modification profiles. RESULTS: Our comprehensive comparative analysis suggests distinct mode of transcriptional regulation in malaria parasite by virtue of poised genes and differential histone modifications. Furthermore, analysis of histone modification profiles predicted 562 genes producing anti-sense RNAs and 335 genes having bidirectional promoter activity, which raises the intriguing possibility of RNA-mediated regulation of transcription in P. falciparum. Interestingly, we found that H3K36me2 acts as a global repressive mark and gene regulation is fine tuned by the ratio of activation marks to H3K36me2 in P. falciparum. This novel mechanism of gene regulation is supported by the fact that knockout of SET genes (responsible for H3K36 methylation) leads to up-regulation of genes with highest occupancy of H3K36me2 in wild-type P. falciparum. Moreover, virulence (var) genes are mostly poised and marked by a unique set of activation (H4ac) and repression (H3K9me3) marks, which are mutually exclusive to other Plasmodium housekeeping genes. CONCLUSIONS: Our study reveals unique plasticity in the epigenetic regulation in P. falciparum which can influence parasite virulence and pathogenicity. The observed differences in the histone code and transcriptional regulation in P. falciparum and its host will open new avenues for epigenetic drug development against malaria parasite.

A novel locus for a hereditary recurrent neuropathy on chromosome 21q21.

Hereditary recurrent neuropathies are uncommon. Disorders with a known molecular basis falling within this group include hereditary neuropathy with liability to pressure palsies (HNPP) due to the deletion of the PMP22 gene or to mutations in this same gene, and hereditary neuralgic amyotrophy (HNA) caused by mutations in the SEPT9 gene. We report a three-generation family presenting a hereditary recurrent neuropathy without pathological changes in either PMP22 or SEPT9 genes. We performed a genome-wide mapping, which yielded a locus of 12.4 Mb on chromosome 21q21. The constructed haplotype fully segregated with the disease and we found significant evidence of linkage. After mutational screening of genes located within this locus, encoding for proteins and microRNAs, as well as analysis of large deletions/insertions, we identified 71 benign polymorphisms. Our findings suggest a novel genetic locus for a recurrent hereditary neuropathy of which the molecular defect remains elusive. Our results further underscore the clinical and genetic heterogeneity of this group of neuropathies.

How to stop: the mysterious links among RNA polymerase II occupancy 3' of genes, mRNA 3' processing and termination.

Eukaryotic genes are transcribed by RNA polymerase II (RNAP II) through cycles of initiation, elongation and termination. Termination remains the least understood stage of transcription. Here we discuss the role of RNAP II occupancy downstream of the 3'ends of genes and its links with termination and mRNA 3' processing.