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Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands
Published on: August 21, 2021
Chromatin movement visualized with photoactivable GFP-labeled histone H4
Karien Wiesmeijer1, Ilke M Krouwels, Hans J Tanke
1Department of Molecular Cell Biology, Leiden University Medical Center, Postal zone S1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Differentiation; Research in Biological Diversity
|November 21, 2007
Summary
Chromatin movement within the cell nucleus is restricted, suggesting it interacts with nuclear structures like speckles. This interaction appears independent of transcriptional activity.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The cell nucleus exhibits organized structures, including discrete chromosome territories and specific protein localization.
- Nuclear organization is dynamic, adapting to cellular conditions to regulate transcription.
- Chromatin exhibits limited mobility in living cells, hinting at interactions with nuclear components.
Purpose of the Study:
- To investigate chromatin mobility within specific nuclear compartments.
- To utilize photoactivatable GFP (green fluorescent protein) fused to histone H4 as a tool for chromatin mobility analysis.
- To explore the relationship between chromatin and nuclear speckles.
Main Methods:
- Employing photoactivatable GFP fused to histone H4 to track chromatin movement.
- Utilizing two-photon excitation microscopy for selective photoactivation of GFP in defined nuclear regions.
- Co-expressing fluorescently tagged splicing factor SF2/ASF to visualize nuclear speckles.
Main Results:
- Observed constrained motion of chromatin within the nucleus.
- Demonstrated that chromatin mobility is unaffected by transcriptional inhibition.
- Provided evidence for close interactions between chromatin and nuclear speckles.
Conclusions:
- Chromatin exhibits restricted mobility, indicating association with the nuclear matrix or compartments.
- Nuclear speckles appear to be sites of interaction with chromatin.
- The observed chromatin immobility is not directly influenced by transcriptional changes.
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01:01Euchromatin
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...
Euchromatin is the less dense region of the chromatin and stains lighter. Euchromatin contains histone H3 extensively...
02:38Heterochromatin
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 9th...
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 9th...
02:38Heterochromatin
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 9th...
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 9th...
02:32Position-effect Variegation
In 1928, a German botanist Emil Heitz observed the moss nuclei with a DNA binding dye. He observed that while some chromatin regions decondense and spread out in the interphase nucleus, others do not. He termed them euchromatin and heterochromatin, respectively. He proposed that the heterochromatin regions reflect a functionally inactive state of the genome. It was later confirmed that heterochromatin is transcriptionally repressed, and euchromatin is transcriptionally active chromatin.
02:21Chromatin Packaging
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 structures.
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 structures.