Homeotic DUX4 genes shape dynamic inter-chromosomal contacts with nucleoli in human cells

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Abstract

Nucleoli shape inter-chromosomal contacts with genes controlling differentiation and cancer genesis. DUX4 genes specify transcription factor possessing two homeodomains. Previously, using Circular Chromosome Conformation Capture (4С) approach on population of cells, it was demonstrated that DUX4 gene clusters form frequent contacts with nucleoli. It was found also that these contacts are almost completely abolished after heat shock treatment. 4C approach as all ligation-mediated methods is capable to detect rather close interactions between chromatin loops in nuclei. In order to independently confirm the formation and the frequency of the contacts in single cells we used FISH approach. Here, we show that DUX genes in single cells form stable contacts in all tested HEK293T cells. The contacts after heat shock treatment reversibly retreat up to 1–3 μm distance. We conclude that inter-chromosomal contacts shaping by nucleoli are dynamic and stable providing both the initiation and maintenance of a differentiated state.

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About the authors

Е. S. Klushevskaya

Engelhardt Institute of Molecular Biology Russian Academy of Sciences

Email: tchurikov@eimb.ru
Russian Federation, Moscow

I. R. Alembekov

Engelhardt Institute of Molecular Biology Russian Academy of Sciences

Email: tchurikov@eimb.ru
Russian Federation, Moscow

Yu. V. Kravatsky

Engelhardt Institute of Molecular Biology Russian Academy of Sciences

Email: tchurikov@eimb.ru
Russian Federation, Moscow

N. А. Tchurikov

Engelhardt Institute of Molecular Biology Russian Academy of Sciences

Author for correspondence.
Email: tchurikov@eimb.ru
Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. A physical map of the subtelomeric region of chr4 and the contact points of rRNA genes identified using the 4C method in the HEK293T cell line (coordinates in the IGB genomic browser are given for the hg19 version of the human genome). The blue color shows the contact points of the rRNA genes. The DUX genes are indicated in green. The red rectangle indicates a 5511 bp DNA region that was developed using long PCR and used for FISH.

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3. Fig. 2. FISH results. a – experiments with the original cells. b – experiments with cells after heat shock. White shows the axis when scanning fluorescence signals passing through the center of the nucleoli. b – Core scan profile without heat shock. g – core scan profile after heat shock. The DUX signal (Alexa-5) is shown in red, and the rDNA signal (Alexa-3) is green. The distances in microns are shown horizontally.

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4. Fig. 3. Diagrams showing the number of DUX probe hybridization foci per nucleus in the initial cells and in cells after heat shock (a) and % of DUX sample hybridization foci in nuclei as they move away from the center of the nucleolus (b). HS – experiments with initial cells. HS+ – experiments with cells after heat shock. 100 cells were used for the analysis. p-value < 0.05.

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Presented by Academician of the RAS P. G. Georgiev


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