RRNA genes inside the preceding interphase. The black oval represents a chromomere in which rRNA genes are assembled into dense heterochromatin. Inside a. thaliana, insertions/deletions inside the 39 external transcribed area define rRNA gene variant kinds. (B) Localization of rRNA genes (rDNA) and Pol I. DNA-FISH using an rRNA gene probe (red signals) and immunolocalization of Flag-tagged Pol I utilizing an anti-Flag antibody (green signals) were performed in a. thaliana interphase nuclei. DNA was counterstained with DAPI (gray signals). (C) Subnuclear localization of rRNA genes, pre-rRNA transcripts, and Flag-tagged HDA6. rRNA gene or COX-2 Modulator web transcript FISH signals are shown in green, immunolocalized HDA6 is in red, and DAPI-stained DNA is in blue. Merged signals are shown within the suitable column. (D) DNA-FISH detection of rRNA genes in wild-type (Col-0) and hda6 nuclei. rRNA gene FISH signals are shown in red and are merged together with the DAPI (blue) image in the correct column. (E) Detection of rRNA gene variant varieties and their transcripts by PCR applying genomic DNA or reversetranscribed (RT) cDNA of wild-type (Col-0) or hda6 plants. The amplified area is shown in a. (F) Leaf cell homogenate of a FIB2:YFP plant stained with DAPI and subjected to fluorescence microscopy. Chloroplasts fluoresce red, DAPI-stained DNA is blue, and nucleolar FIB2:YFP is green. (G) Purified nuclei obtained by FANS. (H) Purified nucleoli obtained by FANoS. (I) PCR detection of rRNA gene variant varieties in DNA of purified nuclei (N) or nucleoli (No) of wild-type (Col-0) or hda6 plants. The PCR amplicon is shown in a.and variant 1 genes are silenced (Fig. 1E, RT CR primer locations are shown inside a). On the other hand, in hda6-6 or hda6-7 mutants, all variant subtypes are expressed (Fig. 1E). To decide whether each active and silenced rRNA genes are associated with nucleoli, we performed fluorescence-activated sorting of complete nuclei or isolated nucleoli from plants expressing the nucleolar protein FIBRILLARIN2 fused to YFP (yellow fluorescent protein) (Barneche et al. 2000). FIB2:YFP localizes specifically inside the nucleolus, as shown in Figure 1F. Fluorescence-activated nuclear sorting (FANS) of cell homogenates yielded homogeneous nuclei (Fig. 1G; Supplemental Fig. S1A). Alternatively, cell extracts had been sonicated to disrupt nuclei then subjected to fluorescence-activated nucleolar sorting (FANoS), yielding nucleoli free of charge of intact nuclei, other organelles, or cellular debris (Fig. 1H; Supplemental Fig. S1B,C). rRNA gene subtypes in isolated nuclei or nucleoli have been identified by PCR amplification employing primers flanking the variable area (see Fig. 1A). All variant forms are present in nuclei of wild-type Col-0 or hda6 mutants, as expected (Fig. 1I). Having said that, in nucleoli of wild-type plants, variant 2- and 3-type rRNA genes are enriched (Fig. 1I, best row), correlating with their selective expression (see Fig. 1E). In hda6 mutants, in which variant 1 gene silencing will not happen, variant 1 genes are also present in nucleoli (Fig. 1I, bottom row). Collectively, these results indicate that rRNA genes are present in nucleoli when active and are excluded from nucleoli when silenced.MET1-dependent CG methylation is implicated in rRNA gene subtype silencing In a. thaliana, cytosine methylation at CG motifs is maintained by MET1 (the ortholog of mammalian DNMT1), CHG methylation (exactly where H is really a, T, or C) is maintained by CMT3, and RNA-directed CHH methylation is IDO Inhibitor Storage & Stability mediated by DRM2, whose paralog, DRM1, m.
