【作者】 黄敏；

【导师】 李立家；

【作者基本信息】 武汉大学 ， 遗传学， 2013， 博士

【摘要】 45S核糖体DNA(45S rDNA)由串联重复元件组成,其编码产物核糖体RNA (rRNA)约占细胞总RNA的80%。rRNA的活跃转录会在细胞核上形成核仁。核仁是rRNA加工成熟并与特定核仁蛋白组装的场所。核仁是被高度调控的结构,它的数目、大小和形态都随着细胞的代谢水平而变化。正常细胞中,只有少部分的45S rDNA拷贝能被转录,其余的拷贝则处于相对沉默的状态。表观修饰参与调控rRNA基因转录失活状态与活跃转录状态的转变。转录沉默相关的浓缩型染色质具有DNA高甲基化、组蛋白去乙酰化以及H3K9me2等表观修饰。相反,活跃转录相关的松散型染色质具有DNA去甲基化、组蛋白H3和H4高乙酰化以及H3K4me2/H3K4me3等表观修饰。为了确保翻译系统的有序性和准确性,45S rDNA序列遵循协同进化模式。但其重复单元内不同区域的进化速率不一致。编码区的序列和二级结构都是高度保守的,而非编码区在物种间存在差异。此外,45S rDNA位点的个数、大小和分布在近源物种中也存在显著差异。该位点串联重复的特质有利于同源重组的进行,因而可能引发这些遗传不稳定性。我们早期的研究表明黑麦草45S rDNA是自发形成的染色体脆性位点。这为研究其遗传不稳定性提供了新的思路。本研究中,我们的结果表明45S rDNA位点既是复制依赖型又是转录依赖型脆性位点。ActD诱导的转录压力会导致不完整的5’ETS转录本的富集和多核仁的形成,并引发一系列表观修饰的改变,包括DNA甲基化和组蛋白H3表达量的降低以及组蛋白乙酰化和H3K4me2表达量的升高。ActD诱导的DSBs会在45S rDNA位点募集大量的yH2AX,但是下游的ecc rDNAs形成则被抑制。此外,我们在Hela和CHO中也发现了高度脱浓缩的45S rDNA脆性表型。这种脱浓缩现象及其引发的断裂与染色体滞后、染色体桥和微核的形成有关。高水平的转录压力可能是这些不稳定现象的驱动力。具体的结果如下：1.复制抑制剂APH和转录抑制剂ActD都能诱导产生植物45S rDNA的脆性表型。中期染色体上,APH诱导的45S rDNA断裂往往出现在端部或近端部,并在染色体上形成空间上完全隔离或仅有少数DNA纤维连接的断裂末端。ActD诱导的45S rDNA异常表型有时也会出现这种末端分布现象,形成明显的断裂位点。但是,大多数情况下,ActD处理会使整个45S rDNA位点都呈现高度拉仲的脱浓缩的脆性表型。45S rDNA脆性位点的细胞学形态类似于人类染色体上的普通型脆性位点的表型。APH和ActD处理也能使间期核上出现45S rDNA的脱浓缩现象,形成弥散的纤维状FISH信号。但是,这两种处理都不能影响玉米着丝粒和Knobs位点的稳定性。2.高水平的转录活性有利于自发形成或ActD诱导的45S rDNA位点的脆性表达。正常黑麦草中,自发形成的45S rDNA断裂位点具有很强的银染信号,而正常的45S rDNA位点几乎没有或只有非常弱的银染信号。类似地,ActD处理过的玉米中,具有脱浓缩脆性表型的45S rDNA位点上有大量AgNOR蛋白的共定位。同时,ActD处理会使玉米细胞内积累大量不完整的5’ETS转录本和出现多核仁现象。这表明,ActD处理在显著抑制RNA Pol I转录延伸的同时会促进rRNA基因的转录起始,从而迫使更多的原本处于浓缩状态的rRNA基因转变成脱浓缩状态并分散在核基质中,形成多核仁。这种转录相关的脱浓缩现象会妨碍中期染色体的折叠,促使45S rDNA位点出现脆性表型。3.大量的表观修饰变化参与调控ActD诱导的玉米45S rDNA位点的脆性表达过程。亚硫酸盐测序的结果表明ActD会诱导出现位点特异性的去甲基化现象。这些特异的位点分别是启动子部位的第16、24、31、35和57位CpG二核苷酸。ChIP结果表明ActD处理会使组蛋白H3的总含量出现大幅度的减少,只占正常含量的11.1%～20.5%。与H3相比,H3K9ac、H3K4me2、H4K5ac和H4K16ac的含量明显升高。相反,H3K9me2的含量却略有下降。这些表观修饰改变可以促使转录沉默的浓缩型染色质(异染色质)转变成活跃转录的松散型染色质(常染色质),最终可能会导致45S rDNA位点的染色质包装失败和脆性表达。4.DNA损伤反应也参与调控ActD诱导的玉米45S rDNA位点的脆性表达过程。yH2AX(?)间接免疫荧光染色的结果表明ActD处理会使多核仁周围出现大量的yH2AX信号。相反,正常核仁周围的信号很弱。ChIP结果也证实ActD处理会使玉米45S rDNA区域的yH2AX含量升高。yH2AX富集现象表明ActD胁迫条件下,有大量DNA断裂出现在脱浓缩的45S rDNA位点。出乎意料的是,定量结果表明ActD处理会使ecc rDNAs的含量略为降低。这表明45S rDNA位点的染色单体内同源重组修复或非同源末端连接修复被抑制。ActD诱导的DNA损伤积累和DNA修复抑制可能会使更多的45S rDNA断裂未经修复就直接进入有丝分裂中期,并在染色体上形成脆性表型。5. FISH结果表明正常培养的Hela和CHO细胞系的中期染色体上也存在少量高度脱浓缩的45S rDNA脆性表型。有丝分裂后期,45S rDNA脱浓缩会使所在的染色体处于滞后状态,甚至会造成后期染色体桥。子代细胞的反向移动会使脱浓缩的45S rDNA位点产生断裂。这种断裂会使所在的染色体片段化,并在细胞核附近形成微核。间接免疫荧光染色的结果证实在不同的细胞周期(间期、中期和后期)都存在yH2AX和UBF的共定位现象。这表明高水平的转录压力可能是造成上述45S rDNA位点不稳定性的主要原因。更多还原

【Abstract】 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.更多还原