High-Resolution, Genome-Wide Mapping of Chromatin Modifications by GMAT

AbstractSingle-cell chromatin studies provide insights into how chromatin structure relates to functions of individual cells. However, balancing high-resolution and genome wide-coverage remains challenging. We describe a computational method for the reconstruction of large 3D-ensembles of single-cell (sc) chromatin conformations from population Hi-C that we apply to study embryogenesis in Drosophila. With minimal assumptions of physical properties and without adjustable parameters, our method generates large ensembles of chromatin conformations via deep-sampling. Our method identifies specific interactions, which constitute 5–6% of Hi-C frequencies, but surprisingly are sufficient to drive chromatin folding, giving rise to the observed Hi-C patterns. Modeled sc-chromatins quantify chromatin heterogeneity, revealing significant changes during embryogenesis. Furthermore, >50% of modeled sc-chromatin maintain topologically associating domains (TADs) in early embryos, when no population TADs are perceptible. Domain boundaries become fixated during development, with strong preference at binding-sites of insulator-complexes upon the midblastula transition. Overall, high-resolution 3D-ensembles of sc-chromatin conformations enable further in-depth interpretation of population Hi-C, improving understanding of the structure-function relationship of genome organization.

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Clinical Relevance of Plasma DNA Methylation in Colorectal Cancer Patients Identified by Using a Genome-Wide High-Resolution Array

Annals of Surgical Oncology ◽

10.1245/s10434-014-4277-2 ◽

2014 ◽

Vol 22 (S3) ◽

pp. 1419-1427 ◽

Cited By ~ 26

Author(s):

Pei-Ching Lin ◽

Jen-Kou Lin ◽

Chien-Hsing Lin ◽

Hung-Hsin Lin ◽

Shung-Haur Yang ◽

...

Keyword(s):

Colorectal Cancer ◽

Dna Methylation ◽

High Resolution ◽

Cancer Patients ◽

Clinical Relevance ◽

Plasma Dna ◽

Genome Wide ◽

A Genome ◽

Colorectal Cancer Patients

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Genome-Wide, High-Resolution Detection of Copy Number, Loss of Heterozygosity, and Genotypes from Formalin-Fixed, Paraffin-Embedded Tumor Tissue Using Microarrays

Cancer Research ◽

10.1158/0008-5472.can-06-3597 ◽

2007 ◽

Vol 67 (6) ◽

pp. 2544-2551 ◽

Cited By ~ 53

Author(s):

Sharoni Jacobs ◽

Ella R. Thompson ◽

Yasuhito Nannya ◽

Go Yamamoto ◽

Raji Pillai ◽

...

Keyword(s):

High Resolution ◽

Loss Of Heterozygosity ◽

Copy Number ◽

Tumor Tissue ◽

Copy Number Loss ◽

Genome Wide ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses

Journal of Virology ◽

10.1128/jvi.01455-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 13

Author(s):

Nicholas S. Eyre ◽

Stephen M. Johnson ◽

Auda A. Eltahla ◽

Maria Aloi ◽

Amanda L. Aloia ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Insertional Mutagenesis ◽

High Resolution Imaging ◽

Ns1 Protein ◽

Reporter Virus ◽

Genome Wide ◽

Infected Cells ◽

Resolution Imaging ◽

Virus Replication Cycle

ABSTRACT Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3′ untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies. IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as “vesicle packets” (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

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