Abstract
Introduction . Inactive X chromosome (Xi) is associated with noncoding XIST RNA, series of proteins and contains multiple epigenetic modifications that altogether determine a silence of the most of X-linked genes. Recently the data were obtained that tumor suppressor BRCA1 is also associated with Xi. The purpose of this study was to reveal the colocalization of BRCA1 and XIST RNA and precise spatial organization on Xi with the high resolution of confocal microscopy.Materials and methods . The object of the study is IMR90hTERT diploid immortalized fibroblast cell line. For BRCA1 and XIST RNA colocalization analysis on Xi the method of fluorescent hybridization in situ associated with immunofluorescent cell staining (immunoFISH) and confocal microscopy were used. For BRCA1 and heterochromatin protein-1 colocalization study the method of double immunofluorescent staining and common fluorescent microscopy were applied. Results . The study using confocal fluorescent microscopy with higher resolution has demonstrated at first the colocalization of BRCA1 with XIST RNA region of Xi revealed with XIST RNA probes and with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. Altogether, the data obtained suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the regulation of XIST RNA association with Xi. Moreover, according to the results of confocal microscopy, BRCA1 also colocalizes with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. This indicates a possible involvement of this protein in the replication of pericentromeric repeats in cellular chromosomes. Colocalization of BRCA1 with heterochromatin protein-1α presented in pericentromeric regions of all chromosomes supports this suggestion.Conclusions . Altogether, the data obtained in this study suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the association with noncoding inhibiting XIST RNA and in replication of heterochromatin regions.
Highlights
Inactive X chromosome (Xi) is associated with noncoding XIST RNA, series of proteins and contains multiple epigenetic modifications that altogether determine a silence of the most of X-linked genes
The purpose of this study was to reveal the colocalization of BRCA1 and XIST RNA and precise spatial organization on Xi with the high resolution of confocal microscopy
For BRCA1 and XIST RNA colocalization analysis on Xi the method of fluorescent hybridization in situ associated with immunofluorescent cell staining and confocal microscopy were used
Summary
Для локализации BRCA1 и РНК XIST на Xi использовали метод флуоресцентной гибридизации in situ, сопряженный с иммунофлуоресцентным окрашиванием клеток, и конфокальную микроскопию. C помощью BrdU выявлена также локализация BRCA1 c реплицирующимися участками Xi и аутосомами в поздней S-фазе клеточного цикла. В целом приведенные данные позволяют предположить участие BRCA1 в ингибировании экспрессии генов на Xi, обусловленном влиянием BRCA1 на ассоциацию РНК XIST с Xi. Помимо этого, впервые с использованием конфокальной микроскопии выявлена колокализация BRCA1 с реплицирующимися Xi и аутосомами, идентифицированными с использованием BrdU, в поздней S-фазе клеточного цикла, что указывает на возможное участие этого белка в репликации перицентромерных повторов хромосом. Полученные данные указывают на участие BRCA1 в ингибировании экспрессии генов на Xi в результате регуляции ассоциации некодирующей ингибирующей РНК XIST с Xi, а также в репликации гетерохроматиновых участков хромосом. Blokhin National Medical Research Center of Oncology of the Ministry of Health of Russia; 24 Kashyrskoe Sh., Moscow 115478, Russia
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