engleski

Spatial organization and epigenetic state of rDNA loci in oak cycling cells

Eukaryotic genome is highly organized tridimensionally within the nucleus and this nuclear architecture is considered one of the epigenetic phenomena since it plays an important role in the modulation of gene transcription. Also, epigenetically defined chromatin structure is correlated with gene-dense or highly expressing regions, being more decondensed, and gene-poor regions of low expression, being more condensed. 18S-5.8S-26S ribosomal (18S rRNA) genes represent an ideal genomic region to study the link between spatial positioning, chromatin structure and transcriptional activity since the two classes of rRNA genes (active and inactive) co-exist within the same rDNA cluster. The nucleolus represent a cytological manifestation of rRNA gene expression and synthesis of rRNA molecules, thus association of rDNA loci with nucleolus is considered to indicate transcriptional activity. In this study, we analysed the interphase tridimensional position of the two 18S rDNA loci (the major and the minor one) in cycling cells of the common oak (Quercus robur) in respect to proximity to nucleolus and the nuclear periphery. Also, by immunofluorescence (IF) using antibodies against 5-methycytosine and histone marks H3K9ac, H3K9me and H3K4me2 and subsequent fluorescence in situ hybridization with 18S rDNA probe we established epigenetic chromatin status of the rDNA loci. The combined results revealed differential transcriptional activity of the two sites of the major 18S rDNA locus, and suggest on complete inactivity of the minor 18S rDNA locus. Using inhibitor of DNA methylation, zebularin, we modulated epigenetic state of rDNA chromatin and confirmed that the preservation of the epigenetic “imprint” is crucial for maintenance of spatial positioning and transcriptional state of the rDNA loci.

rDNA chromatin; epigenetic modulation; nucleolus

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