scholarly journals Mapping native R-loops genome-wide using a targeted nuclease approach

2018 ◽  
Author(s):  
Qingqing Yan ◽  
Emily J. Shields ◽  
Roberto Bonasio ◽  
Kavitha Sarma
Keyword(s):  
Affinity Purification ◽  
Repetitive Sequences ◽  
Mapping Method ◽  
Rnase H ◽  
Micrococcal Nuclease ◽  
Genome Wide ◽  
Input Material ◽  
Prokaryotic Genomes ◽  
High Level ◽  
Nuclear Processes

AbstractR-loops are three-stranded DNA:RNA hybrids that are pervasive in the eukaryotic and prokaryotic genomes and have been implicated in a variety of nuclear processes, including transcription, replication, DNA repair, and chromosome segregation. While R-loops may have physiological roles, the formation of stable, aberrant R-loops has been observed in disease, particularly neurological disorders and cancer. Despite the importance of these structures, methods to assess their distribution in the genome invariably rely on affinity purification, which requires large amounts of input material, is plagued by high level of noise, and is poorly suited to capture dynamic and unstable R-loops. Here, we present a new method that leverages the affinity of RNase H for DNA:RNA hybrids to target micrococcal nuclease to genomic sites that contain R-loops, which are subsequently cleaved, released, and sequenced. Our R-loop mapping method, MapR, is as specific as existing techniques, less prone to recover non-specific repetitive sequences, and more sensitive, allowing for genome-wide coverage with low input material and read numbers, in a fraction of the time.

scholarly journals Experimental and Bioinformatic upgrade for genome-wide mapping of nucleosomes in Trypanosoma cruzi

2021 ◽  
Author(s):  
Paula Beati ◽  
Milena Massimino Stepnicka ◽  
Salome Vilchez Larrea ◽  
Guillermo Daniel Alonso ◽  
Josefina Ocampo
Keyword(s):  
Trypanosoma Cruzi ◽  
Cell Cycle Progression ◽  
Repetitive Sequences ◽  
Nucleosome Position ◽  
Regulatory Elements ◽  
Histone Variants ◽  
Chromatin Organization ◽  
Micrococcal Nuclease ◽  
Cell Manipulation ◽  
Genome Wide

In Trypanosoma cruzi, as in every eukaryotic cell, DNA is packaged into chromatin by octamers of histone proteins that constitute nucleosomes. Besides compacting DNA, nucleosomes control DNA dependent processes by modulating the access of DNA binding proteins to regulatory elements on the DNA; or by providing the platform for additional layers of regulation given by histone variants and histone post-translational modifications. In trypanosomes, protein coding genes are constitutively transcribed as polycistronic units. Therefore, gene expression is controlled mainly post transcriptionally. However, chromatin organization and the histone code influence transcription, cell cycle progression, replication and DNA repair. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities of these processes in trypanosomes. Digestion of chromatin with micrococcal nuclease followed by deep sequencing has been widely applied for genome-wide mapping of nucleosomes in several organisms. Nonetheless, this parasite presents numerous singularities. On one hand, special growth conditions and cell manipulation are required. On the other hand, chromatin organization shows some uniqueness that demands a specially designed analytical approach. An additional entanglement is given by the nature of its genome harboring a large content of repetitive sequences and the poor quality of the genome assembly and annotation of many strains. Here, we adapted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we developed an exhaustive and thorough computational workflow for data analysis, highlighting the relevance of using its whole genome as a reference instead of the commonly used Esmeraldo-like haplotype. Moreover, the performance of two aligners, Bowtie2 and HISAT2 was tested to find the most appropriate tool to map any genomic read to reference genomes bearing this complexity.

scholarly journals Nucleosome Positioning

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Hiromi Nishida
Keyword(s):  
Transcription Initiation ◽  
Genomic Dna ◽  
Cytosine Methylation ◽  
Nucleosome Positioning ◽  
Micrococcal Nuclease ◽  
Gene Splicing ◽  
Nucleosomal Dna ◽  
Genome Wide ◽  
Wide Range ◽  
High Level

Nucleosome positioning is not only related to genomic DNA compaction but also to other biological functions. After the chromatin is digested by micrococcal nuclease, nucleosomal (nucleosome-bound) DNA fragments can be sequenced and mapped on the genomic DNA sequence. Due to the development of modern DNA sequencing technology, genome-wide nucleosome mapping has been performed in a wide range of eukaryotic species. Comparative analyses of the nucleosome positions have revealed that the nucleosome is more frequently formed in exonic than intronic regions, and that most of transcription start and translation (or transcription) end sites are located in nucleosome linker DNA regions, indicating that nucleosome positioning influences transcription initiation, transcription termination, and gene splicing. In addition, nucleosomal DNA contains guanine and cytosine (G + C)-rich sequences and a high level of cytosine methylation. Thus, the nucleosome positioning system has been conserved during eukaryotic evolution.

scholarly journals Unravelling the hidden inter and intra-varietal diversity of durum wheat commercial varieties used in Portugal

2019 ◽  
Vol 17 (04) ◽  
pp. 386-389
Author(s):  
Miguel Bento ◽  
Sónia Gomes Pereira ◽  
Wanda Viegas ◽  
Manuela Silva
Keyword(s):  
Durum Wheat ◽  
Repetitive Sequences ◽  
Inter Simple Sequence Repeat ◽  
Wheat Breeding ◽  
Genomic Diversity ◽  
Varietal Diversity ◽  
Wheat Varieties ◽  
Coding Sequences ◽  
Coding Regions ◽  
High Level

AbstractAssessing durum wheat genomic diversity is crucial in a changing environmental particularly in the Mediterranean region where it is largely used to produce pasta. Durum wheat varieties cultivated in Portugal and previously assessed regarding thermotolerance ability were screened for the variability of coding sequences associated with technological traits and repetitive sequences. As expected, reduced variability was observed regarding low molecular weight glutenin subunits (LMW-GS) but a specific LMW-GS allelic form associated with improved pasta-making characteristics was absent in one variety. Contrastingly, molecular markers targeting repetitive elements like microsatellites and retrotransposons – Inter Simple Sequence Repeat (ISSR) and Inter Retrotransposons Amplified Polymorphism (IRAP) – disclosed significant inter and intra-varietal diversity. This high level of polymorphism was revealed by the 20 distinct ISSR/IRAP concatenated profiles observed among the 23 individuals analysed. Interestingly, median joining networks and PCoA analysis grouped individuals of the same variety and clustered varieties accordingly with geographical origin. Globally, this work demonstrates that durum wheat breeding strategies induced selection pressure for some relevant coding sequences while maintaining high levels of genomic variability in non-coding regions enriched in repetitive sequences.

Distinctive patterns of age-dependent hypomethylation in interspersed repetitive sequences

2010 ◽  
Vol 41 (2) ◽  
pp. 194-200 ◽  
Author(s):  
Pornrutsami Jintaridth ◽  
Apiwat Mutirangura
Keyword(s):  
Mononuclear Cells ◽  
Repetitive Sequences ◽  
Endogenous Retrovirus ◽  
Human Endogenous Retrovirus ◽  
Cellular Functions ◽  
Peripheral Blood Mononuclear ◽  
Global Methylation ◽  
Short Interspersed Elements ◽  
Genome Wide ◽  
Age Dependent

Interspersed repetitive sequences (IRSs) are a major contributor to genome size and may contribute to cellular functions. IRSs are subdivided according to size and functionally related structures into short interspersed elements, long interspersed elements (LINEs), DNA transposons, and LTR-retrotransposons. Many IRSs may produce RNA and regulate genes by a variety of mechanisms. The majority of DNA methylation occurs in IRSs and is believed to suppress IRS activities. Global hypomethylation, or the loss of genome-wide methylation, is a common epigenetic event not only in senescent cells but also in cancer cells. Loss of LINE-1 methylation has been characterized in many cancers. Here, we evaluated the methylation levels of peripheral blood mononuclear cells of LINE-1, Alu, and human endogenous retrovirus K (HERV-K) in 177 samples obtained from volunteers between 20 and 88 yr of age. Age was negatively associated with methylation levels of Alu (r = −0.452, P < 10−3) and HERV-K (r = −0.326, P < 10−3) but not LINE-1 (r = 0.145, P = 0.055). Loss of methylation of Alu occurred during ages 34–68 yr, and loss of methylation of HERV-K occurred during ages 40–63 yr and again during ages 64–83 yr. Interestingly, methylation of Alu and LINE-1 are directly associated, particularly at ages 49 yr and older (r = 0.49, P < 10−3). Therefore, only some types of IRSs lose methylation at certain ages. Moreover, Alu and HERV-K become hypomethylated differently. Finally, there may be several mechanisms of global methylation. However, not all of these mechanisms are age-dependent. This finding may lead to a better understanding of not only the biological causes and consequences of genome-wide hypomethylation but also the role of IRSs in the aging process.

scholarly journals On emerging nuclear order

2011 ◽  
Vol 192 (5) ◽  
pp. 711-721 ◽  
Author(s):  
Indika Rajapakse ◽  
Mark Groudine
Keyword(s):  
Nuclear Organization ◽  
Chromatin State ◽  
Interphase Chromosome ◽  
Chromosome Arrangement ◽  
Technological Advances ◽  
Genome Wide ◽  
Nuclear Processes ◽  
Genomic Function

Although the nonrandom nature of interphase chromosome arrangement is widely accepted, how nuclear organization relates to genomic function remains unclear. Nuclear subcompartments may play a role by offering rich microenvironments that regulate chromatin state and ensure optimal transcriptional efficiency. Technological advances now provide genome-wide and four-dimensional analyses, permitting global characterizations of nuclear order. These approaches will help uncover how seemingly separate nuclear processes may be coupled and aid in the effort to understand the role of nuclear organization in development and disease.

scholarly journals H3 K79 dimethylation marks developmental activation of the β-globin gene but is reduced upon LCR-mediated high-level transcription

Blood ◽  
2008 ◽  
Vol 112 (2) ◽  
pp. 406-414 ◽  
Author(s):  
Tomoyuki Sawado ◽  
Jessica Halow ◽  
Hogune Im ◽  
Tobias Ragoczy ◽  
Emery H. Bresnick ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Genome Wide ◽  
Activation Of Transcription ◽  
High Level ◽  
The Relationship ◽  
Mutant Genes ◽  
Gene Expression Levels

Abstract Genome-wide analyses of the relationship between H3 K79 dimethylation and transcription have revealed contradictory results. To clarify this relationship at a single locus, we analyzed expression and H3 K79 modification levels of wild-type (WT) and transcriptionally impaired β-globin mutant genes during erythroid differentiation. Analysis of fractionated erythroid cells derived from WT/Δ locus control region (LCR) heterozygous mice reveals no significant H3 K79 dimethylation of the β-globin gene on either allele prior to activation of transcription. Upon transcriptional activation, H3 K79 di-methylation is observed along both WT and ΔLCR alleles, and both alleles are located in proximity to H3 K79 dimethylation nuclear foci. However, H3 K79 di-methylation is significantly increased along the ΔLCR allele compared with the WT allele. In addition, analysis of a partial LCR deletion mutant reveals that H3 K79 dimethylation is inversely correlated with β-globin gene expression levels. Thus, while our results support a link between H3 K79 dimethylation and gene expression, high levels of this mark are not essential for high level β-globin gene transcription. We propose that H3 K79 dimethylation is destabilized on a highly transcribed template.

scholarly journals Genome-wide characterization and expression profiling of B3 superfamily during flower induction stage in pineapple (Ananas comosus L.)

2020 ◽  
Author(s):  
Cheng Cheng Ruan ◽  
Zhe Chen ◽  
Fu Chu Hu ◽  
Xiang He Wang ◽  
Li Jun Guo ◽  
...  
Keyword(s):  
Development Process ◽  
Purifying Selection ◽  
Ananas Comosus ◽  
Flower Bud ◽  
Genome Wide ◽  
Localization Analysis ◽  
Ethephon Treatment ◽  
High Level ◽  
Differentiation Period ◽  
Induction Stage

AbstractThe B3 superfamily is a plant-specific family, which involves in growth and development process. By now, the identification of B3 superfamily in pineapple (Ananas comosus) has not reported. In this study, 57 B3 genes were identified and further phylogenetically classified into five subfamilies, all genes contained B3 domain. Chromosomal localization analysis revealed that 54 of 57 AcB3 genes were located on 21 chromosomes.Collinearity analysis indicated that the segmental duplication was the main event in the evolution of B3 gene superfamily and most of them were under purifying selection. Moreover, there were 7 and 39 pairs of orthologous B3s were identified between pineapple and Arabidopsis or rice, respectively, which indicated the closer genetic relationship between pineapple and rice. Most genes had cis-element of abscisic acid, ethylene, MeJA, light, and abiotic stress. qRT-PCR showed that the expression level of most AcB3 genes were up-regulated within 1 d after ethephon treatment and expressed high level in flower bud differentiation period in stem apex. This study provide a comprehensive understanding of AcB3s and a basis for future molecular studies of ethephon induced pineapple flowering.

scholarly journals High similarity among ChEC-seq datasets

2021 ◽  
Author(s):  
Chitvan Mittal ◽  
Matthew J. Rossi ◽  
B. Franklin Pugh
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Regulators ◽  
Micrococcal Nuclease ◽  
High Similarity ◽  
Dna Interactions ◽  
Promoter Regions ◽  
Genome Wide ◽  
A Genome ◽  
Protein Dna Interactions ◽  
Negative Controls

AbstractChEC-seq is a method used to identify protein-DNA interactions across a genome. It involves fusing micrococcal nuclease (MNase) to a protein of interest. In principle, specific genome-wide interactions of the fusion protein with chromatin result in local DNA cleavages that can be mapped by DNA sequencing. ChEC-seq has been used to draw conclusions about broad gene-specificities of certain protein-DNA interactions. In particular, the transcriptional regulators SAGA, TFIID, and Mediator are reported to generally occupy the promoter/UAS of genes transcribed by RNA polymerase II in yeast. Here we compare published yeast ChEC-seq data performed with a variety of protein fusions across essentially all genes, and find high similarities with negative controls. We conclude that ChEC-seq patterning for SAGA, TFIID, and Mediator differ little from background at most promoter regions, and thus cannot be used to draw conclusions about broad gene specificity of these factors.

scholarly journals BisMapR: a strand-specific, nuclease-based method for genome-wide R-loop detection

2021 ◽  
Author(s):  
Phillip Wulfridge ◽  
Kavitha Sarma
Keyword(s):  
Genome Stability ◽  
Antisense Transcription ◽  
Gene Promoters ◽  
Normal Cells ◽  
Disease States ◽  
Detection Strategy ◽  
Genome Wide ◽  
Loop Detection ◽  
Nucleic Acid Structures ◽  
Nuclear Processes

AbstractR-loops are three stranded nucleic acid structures with essential roles in many nuclear processes. However, their unchecked accumulation as seen in some neurodevelopmental diseases and cancers and is associated with compromised genome stability. Genome-wide profiling of R-loops in normal cells and their comparison in disease states can help identify precise locations of pathogenic R-loops and advance efforts to attenuate deviant R-loops while preserving biologically important ones. Toward this, we have developed an antibody-independent R-loop detection strategy, BisMapR, that combines nuclease-based R-loop isolation with non-denaturing bisulfite chemistry to produce high-resolution, genome-wide R-loop profiles that retain strand information. Furthermore, BisMapR achieves greater resolution and is faster than existing strand-specific R-loop profiling strategies. We applied BisMapR to reveal discrete R-loop behavior at gene promoters and enhancers. We show that gene promoters exhibiting antisense transcription form R-loops in both directions. and uncover a subset of active enhancers that, despite being bidirectionally transcribed, form R-loops exclusively on one strand. Thus, BisMapR reveals a previously unnoticed feature of active enhancers and provides a tool to systematically examine their mechanisms in gene expression.

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