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Dosage manage in yeast, mouse, and human cells at the same time (Sandmeier et al. 2002; McStay and Grummt 2008; Grummt and Langst 2013). To examine the subnuclear distribution of active and silenced rRNA genes, we adapted fluorescence-activated sorting technologies to isolate whole A. thaliana nuclei or nucleoli liberated from sonicated nuclei. Exploiting sequence variation among differentially expressed rRNA gene subtypes and mutations that disrupt rRNA gene chromatin modifications or copy quantity, we show that rRNA genes occupy alternative subnuclear compartments depending on their activity state.Benefits and Discussion Partitioning of active versus inactive rRNA genes between the nucleolus and nucleoplasm A. thaliana has NORs on chromosomes 2 and 4, each consisting of ;375 rRNA genes and spanning ;four Mb (Copenhaver et al. 1995; Copenhaver and Pikaard 1996). Pol I, which transcribes 45S rRNA genes, localizes towards the nucleolus of interphase cells (Fig. 1B, green signal). The nucleolus is rich in RNA and proteins but has small DNA, hence appearing as a black hole in nuclei stained with the DNA-binding dye DAPI (Fig. 1B, gray signal). 45S prerRNA transcripts are detected all through the nucleolus by RNA-FISH (fluorescent in situ hybridization) (Fig. 1C, bottom row). Having said that, one of the most prominent 45S rRNA gene DNA-FISH signals are external for the nucleolus (Fig. 1B [red signals], C [top row, green signals]; note that NOR associations can result in fewer than four signals). rRNA genes inside the nucleolus are decondensed and more tough to detect by DNA-FISH, depending on the extent of their dispersal (e.g., cf. the two nuclei in Fig. 1B). HISTONE DEACETYLASE 6 (HDA6) is essential for uniparental rRNA gene silencing in hybrid Arabidopsis suecica and for developmentally regulated silencing of variant 1 rRNA genes in nonhybrid A. thaliana (Earley et al. 2006, 2010). HDA6 localizes throughout nuclei, which includes the nucleolus (Fig. 1C). In hda6 mutants, NORs decondense (Probst et al. 2004; Earley et al. 2006), and rRNA gene FISH signals inside the nucleolus CD5L Protein medchemexpress enhance (Fig. 1D). In leaves of wild-type plants (ecotype Col-0), variant two and three rRNA gene subtypes are expressed,In eukaryotes, 45S ribosomal RNA (rRNA) genes are tandemly arrayed at nucleolus organizer regions (NORs) (see[Keywords: transcription; gene silencing; DNA methylation; histone deacetylation; chromatin assembly; RNA polymerase I; ribosomal RNA gene] eight These authors contributed equally to this work. Present addresses: 9Laboratoire Genome et Developpement des Plantes, UMR 5096 CNRS-University of Perpignan through Domitia, Perpignan, France; ten Division of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Linnean Center for Plant Biology, Uppsala, Sweden; 11 Monsanto Enterprise, St. Louis, MO 63107, USA 12 Corresponding authors E-mail [email protected] E-mail [email protected] Short article is on the internet at genesdev.org/cgi/doi/10.1101/gad.221648.113. Freely readily available online via the Genes Improvement Open Access VIP Protein Purity & Documentation selection.GENES Improvement 27:1545?550 ?2013 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/13; genesdev.orgPontvianne et al.Figure 1. Partitioning of active and silent rRNA genes between the nucleolus and nucleoplasm. (A) Relationships in between the nucleolus, NORs, and 45S rRNA gene repeats. The drawing at the leading depicts a metaphase chromosome using the remnants of a nucleolus associated together with the secondary constriction, the location of active.