Lifetime fluorescence imaging to study DNA compaction and gene activities

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(a) Schemes of labeling approaches targeting early S-phase and mid-S-phase replicating chromatin domains. The labeling protocol was adjusted for the segregation of the labeled chromosomal territories to facilitate image analysis. (b) and (c) FLIM detection of chromatin refractive index and FLIM-FRET detection of DNA compaction, respectively. Credit: by Svitlana M. Levchenko, Artem Pliss, Xiao Peng, Paras N. Prasad and Junle Qu

Studies on the compaction of genomic DNA in the cell nucleus and the dynamic reorganization during physiological processes or disease development in living cell environments are extremely difficult. This complexity stems from a high degree of compaction required to accommodate about 2 meters of genomic DNA into the cell nucleus, which is typically 5-10 microns in diameter. In addition, the compacting density of chromatin is not static, but fluctuates over time depending on the activities of the genes. Meanwhile, the 3D resolution of light microscopy is not high enough even for subdiffraction imaging modalities, thus limiting studies of the spatial geometry of complex genomic architecture and its dynamic transformations.

Lifetime fluorescence imaging to study DNA compaction and gene activitiesDNA replication sites labeled with AlexaFluo 546. (b) Color-coded image of the distribution of lifespan in the cell nucleus. (c) Schematics of the FLIM approach to measure the compaction of chromatin replication at different S-phase windows. (d) Average fluorescence lifetime of AlexaFluo546 used to label DNA replication sites in early, middle and late S phase. Error bars correspond to standard deviations. The data indicate a higher refractive index, and therefore a higher compaction density for late S-phase replicating gene-poor chromatin domains compared to early S-phase replicated gene-rich chromatin. Credit: Svitlana M. Levchenko, Artem Pliss, Xiao Peng, Paras N. Prasad and Junle Qu”/>

(a) Representative images of the fluorescence lifespan of early, middle and late S-phase DNA replication sites labeled with AlexaFluo 546. (b) Color-coded image of the distribution of lifespan in the cell nucleus. (c) Schematics of the FLIM approach to measure the compaction of chromatin replication at different S-phase windows. (d) Average fluorescence lifetime of AlexaFluo546 used to label DNA replication sites in early, middle and late S phase. Error bars correspond to standard deviations. The data indicate a higher refractive index, and therefore a higher compaction density for late S-phase replicating gene-poor chromatin domains compared to early S-phase replicated gene-rich chromatin. Credit: Svitlana M. Levchenko, Artem Pliss, Xiao Peng, Paras N. Prasad and Junle Qu

In a new article published in Light: science and applications, a team of scientists, led by Professor Junle Qu from the Center for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Shenzhen University, China, and Professor Paras N. Prasad from the Institute of Lasers, Photonics and Biophotonics, State University of New York, Buffalo, USA, has developed an alternative strategy based on Lifetime Fluorescence Imaging (FLIM) to overcome the existing limitations of conventional approaches. The authors propose two independent FLIM tests allowing fine measurements of DNA compaction. The first is based on the inverse quadratic relationship between the fluorescence lifetimes of fluorescent probes incorporated into DNA and their local refractive index, which varies due to the compacting density of the chromatin. Another FLIM approach uses Förster resonance energy transfer (FRET) between fluorescently labeled nucleotides incorporated into strands of DNA.

Lifetime fluorescence imaging to study DNA compaction and gene activities

(a) Approach schemes: cultured cells sequentially labeled with CldU at the start of S phase and with IdU at the end of S phase were followed in subsequent cell generations, allowing to visualize chromosomal territories separated with a chromatin marked in early and late S phase. . Segregation of tags allows analysis of chromatin compaction in individual chromosomal territories. (bc) Cells were fixed at various intervals after exposure to halogenated nucleotides and stained (b) for CldU (AlexaFluor 546, green) and (c) IdU (AlexaFluor647, red) as shown in panel (a) . Representative fluorescence intensity (be), lifespan images (f) and FRET efficiency (gh) of the chromosomal territory formation process after labeling of S-phase early and late replication chromatin domains. High FRET areas (red ) indicate close proximity between early and late S-phase replicated chromatin fibers. Credit: Svitlana M. Levchenko, Artem Pliss, Xiao Peng, Paras N. Prasad and Junle Qu

In this study, both FLIM assays were validated in cultured cells, where the researchers comparatively analyzed the compaction of chromatin-rich domains in genes that replicate in early S phase and those that replicate in mid to late S phase. and mainly contain coding non-sequences. The data obtained demonstrate the sensitivity of the two FLIM assays and reveal a significant difference in the compaction of gene-rich and gene-poor genomic DNA pools. They show that gene-rich DNA is weakly compacted compared to dense DNA domains lacking active genes.


Lifetime fluorescence imaging to study DNA compaction and gene activities


More information:
Svitlana M. Levchenko et al, Lifetime fluorescence imaging for the study of DNA compaction and gene activities, Light: science and applications (2021). DOI: 10.1038 / s41377-021-00664-w

Provided by Chinese Academy of Sciences

Quote: Lifetime fluorescence imaging to study DNA compaction and gene activities (2021, December 27) retrieved December 29, 2021 from https://phys.org/news/2021-12-fluorescence-lifetime -imaging-dna-compaction.html

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