In Situ Hi-C Library Preparation for Plants to Study Their Three-Dimensional Chromatin Interactions on a Genome-Wide Scale

An increasing number of studies have revealed that long non-coding RNAs (lncRNAs) play important roles in gene regulation and nuclear organization. Although the mechanisms are still largely unknown, many lncRNAs have been shown to interact with chromatin. Thus, one approach to understanding the function of these lncRNAs is to identify their sites of genomic interaction. Hybridization capture methods using oligonucleotide probes have been used for years to study chromatin-associated RNA. Recently, several groups have developed novel methods based on proximity ligation to investigate RNA–chromatin interactions at a genome-wide scale. This review discusses these technologies and highlights their advantages and disadvantages for the consideration of potential users.

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Mapping the nuclear microenvironment of genes at a genome-wide scale

10.1101/2021.07.11.451976 ◽

2021 ◽

Author(s):

Asli Yildirim ◽

Nan Hua ◽

Lorenzo Boninsegna ◽

Guido Polles ◽

Ke Gong ◽

...

Keyword(s):

Single Cells ◽

Three Dimensional ◽

Population Based ◽

Nuclear Bodies ◽

Cell Models ◽

Chromatin Compaction ◽

Functional Potential ◽

Genome Wide ◽

A Genome ◽

Wide Scale

The folding and subnuclear compartmentalization of chromosomes relative to nuclear bodies is an integral part of gene function. However, mapping the three-dimensional (3D) organization of all genes, in single cells, on a genome-wide scale remains a major challenge. Here, we demonstrate that data-driven population-based modeling, from ensemble Hi-C data alone, can provide a detailed description of the nuclear microenvironment of genes. We define the microenvironment of a gene by its subnuclear positions with respect to different nuclear bodies, local chromatin compaction, and preferences in chromatin compartmentalization. These structural descriptors are determined in single cell models on a genome-wide scale, thereby revealing the dynamic variability of the subnuclear microenvironment of a gene across a population of cells. We demonstrate that the microenvironment of a gene is directly linked to its functional potential in gene transcription, replication, and subnuclear compartmentalization. Some chromatin regions are distinguished by their strong preferences to a single microenvironment (either transcriptionally active or silenced), due to strong associations to specific nuclear bodies. Other chromatin shows highly variable microenvironments and lacks specific preferences. We demonstrate that our method produces highly predictive genome structures, which accurately reproduce data from TSA-seq, DamID, and DNA-MERFISH imaging. Thus, our method considerably expands the range of Hi-C data analysis.

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Imaging chromatin interactions at sub-kilobase resolution Via Tn5-FISH

10.1101/601690 ◽

2019 ◽

Author(s):

Xu Zhang ◽

Jing Niu ◽

Guipeng Li ◽

Qionghai Dai ◽

Dayong Jin ◽

...

Keyword(s):

Three Dimensional ◽

Cost Effective ◽

Imaging Method ◽

Chromatin Interactions ◽

Genome Wide ◽

Cytogenetic Changes ◽

Chromatin Configuration ◽

Clinical Detection ◽

Tn5 Transposase

ABSTRACTThere is increasing interest in understanding how the three-dimensional organization of the genome is regulated. Different strategies have been employed to identify chromatin interactions genome wide. However, due to the current limitations in resolving genomic contacts, visualization and validation of these genomic loci with sub-kilobase resolution remain the bottleneck for many years. Here, we describe Tn5 transposase-based Fluorescence in situ Hybridization (Tn5-FISH), a Polymerase Chain Reaction (PCR)-based, cost-effective imaging method, which achieved the co-localization of genomic loci with sub-kilobase resolution, to fine dissect genome architecture at sub-kilobase resolution and to verify chromatin interactions detected by Chromatin Configuration Capture (3C)-derivative methods. Especially, Tn5-FISH is very useful to verify short-range chromatin interactions inside of contact domain and Topologically Associated Domain (TAD). It also offers one powerful molecular diagnosis tool for clinical detection of cytogenetic changes in cancers.

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Global Genome Conformational Programming during Neuronal Development Is Associated with CTCF and Nuclear FGFR1—The Genome Archipelago Model

International Journal of Molecular Sciences ◽

10.3390/ijms22010347 ◽

2020 ◽

Vol 22 (1) ◽

pp. 347

Author(s):

Brandon Decker ◽

Michal Liput ◽

Hussam Abdellatif ◽

Donald Yergeau ◽

Yongho Bae ◽

...

Keyword(s):

Neuronal Development ◽

Embryonic Stem ◽

Loop Formation ◽

Hoxa Cluster ◽

Chromatin Interactions ◽

Topologically Associating Domains ◽

Genome Wide ◽

A Genome ◽

Wide Scale ◽

Genome Interactions

During the development of mouse embryonic stem cells (ESC) to neuronal committed cells (NCC), coordinated changes in the expression of 2851 genes take place, mediated by the nuclear form of FGFR1. In this paper, widespread differences are demonstrated in the ESC and NCC inter- and intra-chromosomal interactions, chromatin looping, the formation of CTCF- and nFGFR1-linked Topologically Associating Domains (TADs) on a genome-wide scale and in exemplary HoxA-D loci. The analysis centered on HoxA cluster shows that blocking FGFR1 disrupts the loop formation. FGFR1 binding and genome locales are predictive of the genome interactions; likewise, chromatin interactions along with nFGFR1 binding are predictive of the genome function and correlate with genome regulatory attributes and gene expression. This study advances a topologically integrated genome archipelago model that undergoes structural transformations through the formation of nFGFR1-associated TADs. The makeover of the TAD islands serves to recruit distinct ontogenic programs during the development of the ESC to NCC.

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Amplification-free Library Preparation Improves Quality of Hi-C Analysis

10.1101/562280 ◽

2019 ◽

Author(s):

Longjian Niu ◽

Wei Shen ◽

Yingzhang Huang ◽

Na He ◽

Yuedong Zhang ◽

...

Keyword(s):

High Throughput Sequencing ◽

Three Dimensional ◽

Pcr Amplification ◽

Interaction Network ◽

Human Cells ◽

Chromatin Interaction ◽

Library Preparation ◽

Amplification Bias ◽

Chromatin Interactions

AbstractPCR amplification of Hi-C libraries introduces unusable duplicates and results in a biased representation of chromatin interactions. We present a simplified, fast, and economically efficient Hi-C library preparation procedure that generates sufficient non-amplified ligation products for deep sequencing from 30 million Drosophila cells. Comprehensive analysis of the resulting data indicates that amplification-free Hi-C preserves higher complexity of chromatin interaction and lowers sequencing depth dramatically for the same number of unique paired reads. For human cells which has a large genome, this method recovers an amount of ligated fragments enough for direct high-throughput sequencing without amplification on as low as 250 thousand of cells. Comparison with published in situ Hi-C on millions of human cells reveals that amplification introduces distance-dependent amplification bias, which results in increasing background noise level against genomic distance. With amplification bias avoided, our method may produce a chromatin interaction network more faithfully reflecting the real three-dimensional genomic architecture.

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F-Box Genes in the Wheat Genome and Expression Profiling in Wheat at Different Developmental Stages

Genes ◽

10.3390/genes11101154 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1154

Author(s):

Min Jeong Hong ◽

Jin-Baek Kim ◽

Yong Weon Seo ◽

Dae Yeon Kim

Keyword(s):

Developmental Stages ◽

Brachypodium Distachyon ◽

Triticum Aestivum L ◽

Wheat Genome ◽

Post Translational Modification ◽

Genome Wide ◽

A Genome ◽

High Sequence Homology ◽

Wide Scale

Genes of the F-box family play specific roles in protein degradation by post-translational modification in several biological processes, including flowering, the regulation of circadian rhythms, photomorphogenesis, seed development, leaf senescence, and hormone signaling. F-box genes have not been previously investigated on a genome-wide scale; however, the establishment of the wheat (Triticum aestivum L.) reference genome sequence enabled a genome-based examination of the F-box genes to be conducted in the present study. In total, 1796 F-box genes were detected in the wheat genome and classified into various subgroups based on their functional C-terminal domain. The F-box genes were distributed among 21 chromosomes and most showed high sequence homology with F-box genes located on the homoeologous chromosomes because of allohexaploidy in the wheat genome. Additionally, a synteny analysis of wheat F-box genes was conducted in rice and Brachypodium distachyon. Transcriptome analysis during various wheat developmental stages and expression analysis by quantitative real-time PCR revealed that some F-box genes were specifically expressed in the vegetative and/or seed developmental stages. A genome-based examination and classification of F-box genes provide an opportunity to elucidate the biological functions of F-box genes in wheat.

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Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

Nucleic Acids Research ◽

10.1093/nar/gku576 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9838-9853 ◽

Cited By ~ 6

Author(s):

Saeed Kaboli ◽

Takuya Yamakawa ◽

Keisuke Sunada ◽

Tao Takagaki ◽

Yu Sasano ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Interaction ◽

Interaction Network ◽

Essential Genes ◽

Genetic Interactions ◽

Lethal Gene ◽

Synthetic Lethal ◽

Genome Wide ◽

A Genome ◽

Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

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Using 3D epigenomic maps of primary olfactory neuronal cells from living individuals to understand gene regulation

Science Advances ◽

10.1126/sciadv.aav8550 ◽

2018 ◽

Vol 4 (12) ◽

pp. eaav8550 ◽

Cited By ~ 12

Author(s):

Suhn K. Rhie ◽

Shannon Schreiner ◽

Heather Witt ◽

Chris Armoskus ◽

Fides D. Lay ◽

...

Keyword(s):

Cell Model ◽

Expression Profiles ◽

Neuronal Cells ◽

Three Dimensional ◽

Gene Expression Profiles ◽

Regulatory Elements ◽

A Genome ◽

Wide Scale ◽

Cell Model System ◽

Distal Regulatory Elements

As part of PsychENCODE, we developed a three-dimensional (3D) epigenomic map of primary cultured neuronal cells derived from olfactory neuroepithelium (CNON). We mapped topologically associating domains and high-resolution chromatin interactions using Hi-C and identified regulatory elements using chromatin immunoprecipitation and nucleosome positioning assays. Using epigenomic datasets from biopsies of 63 living individuals, we found that epigenetic marks at distal regulatory elements are more variable than marks at proximal regulatory elements. By integrating genotype and metadata, we identified enhancers that have different levels corresponding to differences in genetic variation, gender, smoking, and schizophrenia. Motif searches revealed that many CNON enhancers are bound by neuronal-related transcription factors. Last, we combined 3D epigenomic maps and gene expression profiles to predict enhancer-target gene interactions on a genome-wide scale. This study not only provides a framework for understanding individual epigenetic variation using a primary cell model system but also contributes valuable data resources for epigenomic studies of neuronal epithelium.

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Machine-learning annotation of human splicing branchpoints

10.1101/094003 ◽

2016 ◽

Cited By ~ 3

Author(s):

Bethany Signal ◽

Brian S Gloss ◽

Marcel E Dinger ◽

Timothy R Mercer

Keyword(s):

Machine Learning ◽

Learning Algorithm ◽

Gene Splicing ◽

Genetic Encoding ◽

Genome Wide ◽

Common Genetic Variants ◽

A Genome ◽

Wide Scale ◽

The Impact ◽

Splicing Patterns

ABSTRACTBackgroundThe branchpoint element is required for the first lariat-forming reaction in splicing. However due to difficulty in experimentally mapping at a genome-wide scale, current catalogues are incomplete.ResultsWe have developed a machine-learning algorithm trained with empirical human branchpoint annotations to identify branchpoint elements from primary genome sequence alone. Using this approach, we can accurately locate branchpoints elements in 85% of introns in current gene annotations. Consistent with branchpoints as basal genetic elements, we find our annotation is unbiased towards gene type and expression levels. A major fraction of introns was found to encode multiple branchpoints raising the prospect that mutational redundancy is encoded in key genes. We also confirmed all deleterious branchpoint mutations annotated in clinical variant databases, and further identified thousands of clinical and common genetic variants with similar predicted effects.ConclusionsWe propose the broad annotation of branchpoints constitutes a valuable resource for further investigations into the genetic encoding of splicing patterns, and interpreting the impact of common- and disease-causing human genetic variation on gene splicing.

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Emerging Technologies for Genome-Wide Profiling of DNA Breakage

Frontiers in Genetics ◽

10.3389/fgene.2020.610386 ◽

2021 ◽

Vol 11 ◽

Author(s):

Matthew J. Rybin ◽

Melina Ramic ◽

Natalie R. Ricciardi ◽

Philipp Kapranov ◽

Claes Wahlestedt ◽

...

Keyword(s):

Genome Instability ◽

Dna Double Strand Breaks ◽

Single Nucleotide ◽

Strand Breaks ◽

Single Strand Breaks ◽

Genome Wide ◽

A Genome ◽

Wide Scale ◽

Nucleotide Resolution ◽

Genomic Regions

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

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