Fragility Characteristics Of45S RDNA Sites And Its Regulatory Mechanisms

The45S ribosomal DNA (45S rDNA) site is arranged as tandem repeats and codes for ribosomal RNA (rRNA) that constitutes about approximately80%of the total RNA in a cell. In interphase nuclei, the active rRNA transcription generates a nucleolus, where rRNA maturation and its assembly with specific nucleolar proteins occur. The nucleolus is a highly regulated structure, whose number, size and shape vary based on cellular metabolism. In normal cells, only a small fraction of rRNA genes are transcriptionally active, whereas the remaining copies are restricted to the relatively inactive state. The switch model for rRNA gene off/on transcription is mediated by different epigenetic modifications. The "off state-associated condensed chromatin involves DNA hypermethylation, histone hypoacetylation and H3K9me2of rRNA genes. In contrast, the "on" state-associated open chromatin involves DNA demethylation, histone H3and H4hyperacetylation and H3K4me2/H3K4me3of rRNA genes. The rDNA is evolving via concerted evolution to provide an accurate and efficient translation system. However, different rDNA repeat regions exhibit distinct evolutionary rates. The sequence and secondary structure of coding regions are highly conserved, while the non-coding regions vary among the species. Also, the number, size, and distribution of45S rDNA site are much variable among closely related species. It seems that the highly repetitive nature of45S rDNA makes it as an ideal substrate for homologous recombination, which thus may trigger the occurrence of such genetic instability. Our previous study demonstrated that45S rDNA sites were chromosome fragile sites expressed spontaneously in Lolium. It shed new light on the causes of45S rDNA genetic instability. Here, our data identified45S rDNA regions as fragile sites derived from replication-associated as well as transcription-dependent defects, and established that the ActD-induced transcription stress gave rise to accumulation of incomplete5’ETS transcripts and multiple nucleoli, accompanied by remarkable epigenetic alterations, including decreased DNA methylation, decreased levels of histone H3, and increased histone acetylation and levels of H3K4me2. Also, ActD-induced DSBs accumulated yH2AX at45S rDNA sites, whereas the ecc rDNAs formation, a downstream event of DSBs repair, was inhibited. Furthermore, we identified highly decondensed45S rDNA fragile phenotypes in Hela and CHO cell lines, and established that45S rDNA decondensation and breakage gave rise to lagging chromosomes, anaphase bridges and micronuclei. High transcriptional activity might contribute to such instability phenomena. The results are as follows:1. Plant45S rDNA fragile phenotypes were induced by replication inhibitor (APH) and transcription inhibitor (ActD). In metaphase, the APH-induced45S rDNA defects often occurred at or close to a45S rDNA terminus, thus giving rise to spatially separated ends linked with no or only a few thin rDNA fiber threads. ActD-induced45S rDNA aberrations sometimes also exhibited the terminal distribution with apparent breakage site. In most cases, ActD treatment induced highly stretched and decondensed fragile phenotypes of the whole45S rDNA site. The cytological appearances of45S rDNA fragile sites were similar to those of common fragile sites reported in human metaphase chromosomes. APH and ActD also resulted in45S rDNA decondensation in interphase nuclei, which occurred as fiber-like thread signals after FISH. However, both agents exerted no obvious effect on centromeres and Knobs in maize.2. High transcriptional activity contributed to the occurrence of spontaneous or ActD-induced45S rDNA fragile phenotypes. The intense staining of AgNOR proteins colocalized at the spontaneously formed45S rDNA lesions, whereas no signal or only very weak signals detected at normal45S rDNA sites in untreated ryegrass. Similarly, the AgNOR staining signals still co-localized with the decondensed45S rDNA fragile sites from maize treated with ActD. Meanwhile, ActD treatment accumulated incomplete5’ETS transcripts and induced multiple nucleoli formation in maize. It suggested that ActD treatment could stimulate rRNA gene transcription initiation events although Pol I elongation was strongly inhibited, thus forcing parts of the condensed rRNA genes to be decondensed and dispersed throughout the nucleoplasm, which contributed to the formation of multiple nucleoli. The transcription-associated decondensation might interfere with metaphase chromosome packaging, resulting in45S rDNA fragile phenotypes.3. Remarkable epigenetic alterations were involved in ActD-induced45S rDNA fragile expression process in maize. Bisulfite genomic sequencing revealed ActD treatment induced site-specific hypomethylation at five CpG dinucleotides (positions16,24,31,35and57) within the promoter region. ChIP analysis showed total levels of histone H3were reduced to a lower density ranging from nearly11.1%to20.5%in all analyzed regions in the presence of ActD. Compared to H3, the levels of H3K9ac, H3K4me2, H4K5ac and H4K16ac increased significantly at every analyzed amplicon. In contrast, the density of H3K9me2was slightly decreased. These epigenetic alterations accounted for the switching from "off state-associated condensed chromatin (heterochromatin) to "on" state-associated open chromatin (euchromatin), ultimately resulting in45S rDNA chromatin-packing defects and fragile expression.4. DNA damage response pathways were also involved in ActD-induced45S rDNA fragile expression process in maize. Indirect immunofluorescence staining with an antibody against yH2AX showed weak staining signals surrounded the nucleolus without treatment but intense staining signals surrounded multiple nucleoli after treatment with ActD. ChIP analysis also revealed a significant increase of yH2AX within the45S rDNA regions. The ActD-induced yH2AX accumulation indicated the presence of DNA breaks across the highly decondensed45S rDNA chromatins. Surprisingly, our quantitative results indicated a slight decrease of ecc rDNAs in ActD treated samples, suggesting that the homologous intra-strand recombination or nonhomologous end-joining within45S rDNA repeats was repressed. The ActD-induced DNA damage accumulation and DNA repair repression might cause more unrepaired45S rDNA breakage entry into metaphase, contributing to fragile phenotypes formed in chromosomes.5. FISH mapping showed highly decondensed45S rDNA fragile phenotypes occurred at a lower frequency on metaphase chromosome spreads in Hela and CHO cell lines without treatment. In anaphase,45S rDNA decondensation gave rise to lagging chromosomes, and were prone to forming anaphase bridges. The oppositely oriented movement of daughter cells might result in a breakage occurring to the decondensed45S rDNA sites. The breakage accounted for the occurrence of chromosome fragments and gave rise to micronuclei adjacent to the main nuclei. Indirect immunofluorescence staining results showed that yH2AX was co-localized with UBF during different phases of the cell cycle. It seemed that high transcriptional activity contributed to the occurrence of45S rDNA instability mentioned above.