Institute of Biophysics of the Czech Academy of Sciences, 612 65 Brno, Czech Republic.
Int J Mol Sci. 2024 Jul 9;25(14):7540. doi: 10.3390/ijms25147540.
Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.
尽管 DNA 甲基化广泛参与 rDNA 转录的调控，但不同胞嘧啶甲基化途径的相对参与目前仅在少数模式植物中得到描述。使用 PacBio、亚硫酸氢盐和 RNA 测序；PCR；Southern 杂交；和 FISH，我们在两个谱系，por1 和 por2 中估计了 rDNA 拷贝数变异的表观遗传后果，后者的 rDNA 拷贝数是前者的两倍多，大致均匀分布在染色体 A 和 D 的 NOR 上。por1 中的 rDNA 含量较低与 D-NOR 的大小显著减小（>90%）相关。此外，在 por2 中检测到两个（L 和 S）突出的 rDNA 变体，它们在基因间间隔区的重复组织上存在差异，而在 por1 中仅检测到 S-rDNA 变体。por1 中 S-rDNA 的转录活性与两个 A-NOR 的次生缢痕相关。相反，por2 中 S-rDNA 的沉默伴随着 A-NOR 的浓缩、D-NOR 的次生缢痕和 L-rDNA 的转录活性，表明（i）双向核仁优势和（ii）S-rDNA 与 A-NOR 以及 L-rDNA 与 D-NOR 的关联在每个 S-和 L-rDNA 阵列中都由几个亚变体组成，这些变体在遗传上（特定 SNP）和表观遗传上（转录效率和胞嘧啶甲基化）都有所不同。rDNA 沉默和甲基化之间的相关性最强的是对称的 CWG 基序，其次是 CG 基序。在 CCGs 或不对称的 CHHs 中，外部胞嘧啶没有检测到相关性，或者甲基化是位置依赖的，特别是对于富含 AT 的变体。我们得出结论，植物二倍体中 rDNA 拷贝数的变化可能伴随着快速的表观遗传反应，以维持适当数量的活性 rDNA。CWG 的甲基化动态可能是调节沉默和活跃 rDNA 状态的最主要因素。
