Redistribution of DNA loop domains in human lymphocytes under blast transformation with interleukin 2.

K S Afanasieva,A V Sivolob,A V Lozovik,S R Rushkovsky,M I Chopei

doi:10.15407/ubj88.06.045

K S Afanasieva, A V Sivolob + Show 3 more

Open Access

https://doi.org/10.15407/ubj88.06.045

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Abstract

At higher order levels chromatin fibers in interphase nuclei are organized into loop domains. Gene regulatory elements (promoters and enhancers) are often located near the sites of loop attachments. Therefore, loop domains play a key role in regulation of cell transcriptional activity. We investigated the kinetics of DNA loop exit during single cell gel electrophoresis (the comet assay) of nucleoids obtained from two cell types that differ in their synthetic activity – human lymphocytes and lymphoblasts. Lymphocyte activation and transformation into lymphoblasts (blast transformation) was performed with interleukin 2. The results obtained suggest that a rearrangement of the loops occurs after lymphocyte activation. After blast transformation we observed an increase of the amount of loop domains on the surface of nucleoids against a decrease of the inner loop fraction. Therefore, the comet assay can be used for detection of large-scale changes in the cell nucleus that follow changes in cell functional state.

Highlights

E xperimental evidences in favor of DNA loopingas one of the main features of spatial organization of chromatin in cell nucleus have been obtained back to the 1970’s [1]
In our previous works, which has been done with intact human lymphocytes, we have shown that the comet assay may be successfully applied to study the DNA looping [10, 11]
Our results show that the comet assay may be applied for detection of changes in chromatin three-dimensional organization associated with different functional states of the cell

Summary

Materials and Methods

Recombinant interleukin 2 (1000 units/ml, Biotech-pharm, Russia) was added to the cell culture to induce activation and proliferation of lymphocytes This cytokine concentration was found to be the most efficient for blast transformation. Upon cultivation completion the cells were harvested by centrifugation, washed in Hanks’ salt solution and an aliquot of suspension was used for cytological estimation of blast transformation efficiency. For this purpose the cells were incubated in a hypotonic solution (0.075М KCl) at 37 °С for 30 min and in ethanol : glacial acetic acid (3 : 1) fixative solution at –20 °С for 10 min. To obtain the parameters the equation was fitted to experimental data using a least-square procedure

Results and Discussion

Parameter Lymphocytes Lymphoblasts

Lymphocytes a Lymphoblasts c