Nucleolar rDNA folds into condensed foci with a specific combination of epigenetic marks

Abstract

Arabidopsis thaliana 45S ribosomal genes (rDNA) are located in tandem arrays called nucleolus organizing regions on the termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, a crucial structural element of the ribosome. The current model of rDNA organization suggests that inactive rRNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while the nucleolus delineates active genes. We challenge the perspective that all intranucleolar rDNA is active by showing that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones that also contain the repressive H3K9me2 histone mark. By using plant lines containing a low number of rDNA copies, we further found that the condensed foci relate to the folding of rDNA, which appears to be a common mechanism of rDNA regulation inside the nucleolus. The H3K9me2 histone mark found in condensed foci represents a typical modification of bulk inactive rDNA, as we show by genome-wide approaches, similar to the H2A.W histone variant. The euchromatin histone marks H3K27me3 and H3K4me3, in contrast, do not colocalize with nucleolar foci and their overall levels in the nucleolus are very low. We further demonstrate that the rDNA promoter is an important regulatory region of the rDNA, where the distribution of histone variants and histone modifications are modulated in response to rDNA activity.

Completing the TRB family: newly characterized members show ancient evolutionary origins and distinct localization, yet similar interactions

“…Nuclei were washed three times for 5 min in 1 × PBS supplemented with 0,05% Tween-20 (PBST), then incubated 1 h with an anti-mouse Alexa 488 antibody, Invitrogen (1:750 dilution). Slides were washed three times for 5 min in 1 × PBST, then dehydrated in ethanol series as described in Kutashev et al 2021 . Coverslips were mounted in 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI), 2 µg/mL in Vectashield and imaged using fluorescence using a Zeiss AxioImager Z1 epifluorescence microscope.…”

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

“…We hypothesize that the increase in the size of the IRFs is the result of the relaxed chromatin configuration, related to the altered methylation status of rDNA and its decompaction ( nuc1 mutants; ref. 20 ), different levels of histone variants present in rDNA and/or general redistribution of rDNA genes in the nucleolus 15 , 21 . Our analysis of replication foci clustering in the nucleus did not show significant differences between the wild-type and fas1 or nuc1 mutant plants, in line with a recent report suggesting that the S-phase progression is not delayed in the fas1 background 40 .…”

“…Here, we developed a correlative workflow to study nucleolar architecture changes during progression of DNA replication in plant cells. We have previously characterized the organization and replication of ribosomal DNA in plant nuclei 14 , 15 using fluorescence in-situ hybridization and replication labelling. We have shown that the replication of the 45S ribosomal DNA (rDNA) occurs throughout S-phase in the nucleus and nucleolus, whereby the active intranucleolar fraction of rDNA replicates in early-S-phase, larger intranucleolar clusters of rDNA genes replicate in the mid-S-phase and the perinucleolar, transcriptionally inactive fraction replicates in the late-S-phase.…”

New insights on the evolution of nucleolar dominance in newly resynthesized hexaploid wheat Triticum zhukovskyi

“…Although several theories have been proposed to explain the silencing mechanism of uniparental rRNA genes, each has its shortcomings, and no single hypothesis can account for all exceptions. The regulation of rRNA gene transcription is complex and involves multiple factors, including the recruitment of specific transcription factors and RNA polymerases, chromatin structure and epigenetic modifications, nucleolar activity, and localization of rRNA genes within the nucleolus (Abraham et al., 2020; Kutashev et al., 2021). Furthermore, the establishment and maintenance of ND adds another layer of complexity to the transcriptional regulation of rRNA genes, particularly in newly formed amphiploids and distant hybridizations where there is an imbalance of genetic information (Cheng et al., 2018; Doyle et al., 2008; Feldman et al., 2012).…”