Aufsatz Werner, Mette M Florian, van der Winden Johannes, Matzke Marjori, Matzke Antonius J M
Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria.
EMBO J. 2002 Dec 16;21(24):6832-41. doi: 10.1093/emboj/cdf663.
To analyze relationships between RNA signals, DNA methylation and chromatin modifications, we performed a genetic screen to recover Arabidopsis mutants defective in RNA-directed transcriptional silencing and methylation of a nopaline synthase promoter-neomycinphosphotransferase II (NOSpro- NPTII) target gene. Mutants were identified by screening for recovery of kanamycin resistance in the presence of an unlinked silencing complex encoding NOSpro double-stranded RNA. One mutant, rts1 (RNA-mediated transcriptional silencing), displayed moderate recovery of NPTII gene expression and partial loss of methylation in the target NOSpro, predominantly at symmetrical C(N)Gs. The RTS1 gene was isolated by positional cloning and found to encode a putative histone deacetylase, HDA6. The more substantial decrease in methylation of symmetrical compared with asymmetrical cytosines in rts1 mutants suggests that HDA6 is dispensable for RNA-directed de novo methylation, which results in intermediate methylation of cytosines in all sequence contexts, but is necessary for reinforcing primarily C(N)G methylation induced by RNA. Because CG methylation in centromeric and rDNA repeats was not reduced in rts1 mutants, HDA6 might be specialized for the RNA- directed pathway of genome modification.
为了分析RNA信号、DNA甲基化和染色质修饰之间的关系，我们进行了一项遗传筛选，以获得在RNA介导的转录沉默和胭脂碱合酶启动子-新霉素磷酸转移酶II（NOSpro-NPTII）靶基因甲基化方面存在缺陷的拟南芥突变体。通过在存在编码NOSpro双链RNA的非连锁沉默复合体的情况下筛选卡那霉素抗性的恢复来鉴定突变体。一个突变体rts1（RNA介导的转录沉默）表现出NPTII基因表达的中度恢复以及靶标NOSpro中甲基化的部分丧失，主要发生在对称的C(N)G位点。通过图位克隆分离出RTS1基因，发现它编码一种假定的组蛋白脱乙酰酶HDA6。与rts1突变体中不对称胞嘧啶相比，对称胞嘧啶甲基化的更显著降低表明，HDA6对于RNA介导的从头甲基化是可有可无的，RNA介导的从头甲基化会导致所有序列背景下胞嘧啶的中间甲基化，但对于增强主要由RNA诱导的C(N)G甲基化是必需的。由于rts1突变体中着丝粒和rDNA重复序列中的CG甲基化没有降低，HDA6可能专门参与基因组修饰的RNA介导途径。
