Relação entre as metilações do dna e da histona H3 com o comportamento dos sítios de rDNA 45S em cromossomos e núcleos interfásicos de Lolium e Festuca

Abstract

The grasses belonging to the Lolium-Festuca complex present a prominent role in the Brazilian agricultural scenary, mainly in the South and Southeast regions. They are known worldwide for supplying the forage deficit characteristic of winter in temperate countries. For these and other reasons, the cytogenetic studies for the species were stimulated. Several studies have demonstrated a plasticity of the 45S rDNA sites behavior, for example, variation in the number and position of the sites related the possible fragility of the loci, that, consequently, justified the presence of extended sites and gaps in the metaphases. This extended organization of the nucleolar organizing regions (NORs) can be evaluated in relation to its transcriptional activity/accessibility through epigenetic changes. Therefore, the objective of this study was to evaluate the influence of methylation on cytosine (5-mCyt), directly related to gene silencing, and the dimethylation of lysine 9 in histone H3 (H3K9me2), associated with heterochromatic structure, in the different conformations of the rDNA 45S sites, in the interphase nuclei and metaphase chromosomes of two genotypes of each species of Lolium perenne, L. multiflorum and Festuca arundinacea. Immunostaining techniques were performed in combination with FISH (fluorescent in situ hybridization) for the localization of the rDNA sites. The slides were made with pretreated and unpretreated meristemac cells, fixed in Carnoy (ethanol: acetic acid, 3: 1 v / v) and indirect immunodetection with the primary antibodies anti-H3K9me2 rabbit polyclonal and anti-5-methyl cytosine mouse monoclonal. The detection was done using secondary antibodies anti-rabbit FITC-conjugated and anti-mouse FITC-conjugated. FISH Technique was performed sequentially on immunostaining using 45S rDNA probes obtained from Triticum aestivum L. (pTa 71). From the combination of these two techniques it was possible to perceive a direct relation between the epigenetic marks and the different conformations of the NORs. In the interphase nuclei, predominantly intra and perinucleolar sites were observed, which were mostly hypomethylated and/or hyper/hypomethylated, decondensed or partially condensed. The extranuclear sites were predominantly hypermethylated for both epigenetic marks and, consequently, were seen in the form of heterochromatic condensed blocks. Hypermethylated, hypomethylated and non-methylated sites were evidenced in the metaphases. The 45S rDNA sites with gaps identified in the metaphases were always hypomethylated or unmethylated for H3K9 and also for 5-mCyt, which justifies the decondensed and able to the transcription. The frequency of sites with hypermethylated gaps was very low. Thus, the structural differences visualized in these sites in the three species studied are directly related to the analyzed epigenetic marks, justifying the different conformations in the interphase nuclei and throughout the cell cycle.