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Related Concept Videos

Duplication of Chromatin Structure02:05

Duplication of Chromatin Structure

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The process of chromosome duplication during cell division requires genome-wide disruption and re-assembly of chromatin. The chromatin structure must be accurately inherited, reassembled, and maintained in the daughter cells to ensure lineage propagation.
The basic unit of the chromatin is the nucleosome, consisting of DNA wrapped around octameric histone proteins and short stretches of linker DNA separating individual nucleosomes. The histone proteins within the nucleosome have their...
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Heterochromatin02:38

Heterochromatin

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The extent of chromatin compaction can be studied by staining chromatin using specific DNA binding dyes. Under the microscope, the dense-compacted regions that take up more dye are called heterochromatin. Heterochromatin is further classified into two forms – constitutive heterochromatin and facultative heterochromatin.
Constitutive heterochromatin: It is a highly compact region of chromatin that is mostly concentrated in the centromere and telomere. Unlike euchromatin, the amino acid at...
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Chromatin Packaging02:21

Chromatin Packaging

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Each human somatic cell contains 6 billion base-pairs of DNA. Each base-pair is 0.34 nm long, which means that each diploid cell contains a staggering 2 meters of DNA. How is such a long DNA strand packed inside a nucleus measuring only 10 - 20 microns in diameter? 
The chromatin
In combination with specialized DNA binding protein called Histones, the DNA double helix forms a compact DNA: protein complex called chromatin. The chromatin itself is further compacted into higher-order...
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Chromatin Packaging01:32

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Each human somatic cell contains 6 billion base pairs of DNA. Each base pair is 0.34 nm long, meaning each diploid cell contains a staggering 2 meters of DNA. This long DNA strand is packed inside a nucleus measuring only 10-20 microns in diameter with the help of specialized DNA-binding proteins called histones. Together they form a compact DNA-protein complex called chromatin. The chromatin is further compacted into higher-order structures. The highest level of compaction is achieved during...
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Euchromatin01:01

Euchromatin

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The extent of chromatin compaction can be studied by staining chromatin using specific DNA binding dyes. Under the microscope, the dense-compacted regions take up more dye, appearing darker, while the less-compact areas take up less dye and appear lighter. Based on the compaction level, chromatins are classified into two primary forms – euchromatin and heterochromatin.
Euchromatin is the less dense region of the chromatin and stains lighter. Euchromatin contains histone H3 extensively...
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Chromatin Immunoprecipitation- ChIP02:36

Chromatin Immunoprecipitation- ChIP

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Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or histone proteins. It also helps determine the type of histone modifications such as acetylation, phosphorylation, or methylation.
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
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Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization
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Analyzing RNA-DNA Triplex Formation in Chromatin.

Rodrigo Maldonado1, Gernot Längst2

  • 1Laboratorio de Biología Celular y Molecular Aplicada, Universidad Mayor, Temuco, Chile.

Methods in Molecular Biology (Clifton, N.J.)
|July 19, 2020
PubMed
Summary

Non-coding RNAs (ncRNAs) interact with chromatin via sequence-specific triple helix formation. This study details a method to quantify RNA-triplex formation influenced by nucleosome positioning, crucial for understanding RNA-chromatin interactions.

Keywords:
ChromatinElectrophoretic Mobility Shift AssayNon-coding RNANucleosomeTriple helix structuresTriplex Forming OligoTriplex Targeting site

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Non-coding RNAs (ncRNAs) play critical roles in gene regulation and nuclear organization by interacting with chromatin.
  • RNA-DNA triple helix formation is a key mechanism for sequence-specific RNA-chromatin interactions.
  • Previous work demonstrated the in vivo existence and stabilization of these triplexes by histone tails.

Purpose of the Study:

  • To develop and present a detailed protocol for investigating RNA-chromatin interactions.
  • To determine how nucleosome positioning affects RNA triple helix formation.
  • To quantify the specificity and binding affinity of triplex sequences in relation to nucleosomes.

Main Methods:

  • Development of an in vitro assay to study triple helix formation.
  • Comparative quantification of triplex formation.
  • Analysis of triplex targeting sequences relative to nucleosome position.

Main Results:

  • The study provides a protocol to assess the influence of nucleosome positioning on triple helix formation.
  • The assay allows for comparative quantification of triplex formation and specificity.
  • This method is essential for identifying potential in vivo RNA-chromatin interaction sites.

Conclusions:

  • Nucleosome positioning is a critical factor influencing RNA-chromatin interactions via triple helix formation.
  • The developed assay is vital for characterizing the biophysical parameters of these interactions.
  • This research facilitates the identification of functional RNA-chromatin binding sites in the cellular environment.