THE INFLUENCE OF THE PREPARATION PRETREATMENT ON IN SITU DETECTION
OF 5-METHYLCYTOSINE IN METAPHASE CHROMOSOMES AND IN INTERPHASE NUCLEI
N.A. Grudinina,1 L.K. Sasina,1 E.M. Noniashvili,1 E.G. Neronova,2 L.I. Pavlinova,1,3
I.O. Suchkova,1 G.A. Sofronov,1 E.L. Patkin 1,*
1 Department of Molecular Genetics, Institute of Experimental Medicine of the North West Branch
of the Russian Academy of Medical Sciences, St. Petersburg, 194376,
2 A.M. Nikiforov All-Russian Center of Emergency and Radiation Medicine, EMERCOM of Russia,
St. Petersburg, 197136, and 3 I.P. Pavlov Institute of Physiology RAS, St. Petersburg, 199034;
* e-mail:elp44@mail.ru
Qualitative and quantitate analysis of DNA methylation in situ at the level of cells, chromosomes and chromosomal
domains is extremely important for the diagnosis and treatment of various diseases, the study of ageing
and the consequences of environmental impacts. An important question arises, whether the revealed in situ
methylation pattern reflects DNA methylation per se and (or) availability of the DNA for antibodies, which in
turn depends on the peculiarities of chromatin structure and chromosome condensation. These events can lead
to an incorrect evaluation of the actual pattern of DNA methylation. To avoid this shortcoming as far as possible,
we have modified the most widely used method of revealing 5-methylcytosine in situ with monoclonal antibodies.
Here we have shown that the detection of DNA methylation staining of chromosomes including C-heterochromatin,
chromosomal arms and sister chromatids is drastically dependent on pretreatment of chromosomal
preparations for immunocytochemical study using fluorescent antibodies. Using undifferentiated stem cells
of mouse embryonal carcinoma line F9, it has been found that change in preparations storage results in a sharp
fluorescence decrease up to complete disappearance of the signal in centromeric heterochromatin. With the
help of the method described in the work, we have first revealed the asymmetry of sister chromatids methylation
in metaphase chromosomes of F9 cell and lymphocytes of human periphery blood. This may lead to asymmetry
of transcriptional signature of daughter cells after division. The proposed here modification of 5-methylcytosine
detection in situ provides a more complete characterization of methylation of chromosomes and chromosomal
domains, compared to previously published methods.
Key words:
overall DNA methylation, chromatin, heterochromatin, metaphase chromosomes, epigenetic
regulation, in situ analysis, immunocytochemistry, chromosomal domains, monoclonal antibodies against methylcytosine,
asymmetric sister chromatids methylation
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