Analysis of the amplification promoting sequence (EXP) in the ribosomal RNA gene

核糖体RNA基因重复序列（rDNA）是真核生物染色体中最具特征的区域之一。在大多数生物体中，重复序列由超过100个串联单元组成，占据了染色体的大部分。重复基因家族，如核糖体RNA基因（rDNA）被认为是缺失重组的脆弱位点。而对于rDNA，细胞具有基因扩增系统，拷贝数恢复到野生型水平。我们找到了一个顺式必要序列（EXP）。在本研究中，我分析了EXP的分子机制。首先，我们利用几种酵母对EXP进行了系统发育足迹分析，并找到了一个保守区域。有趣的是，该区域是一个双向非编码转录启动子，早在20年前就被报道过。我们测试了启动子的特征，发现转录对扩增至关重要。有趣的是，当rDNA拷贝数减少时，转录被激活，并从该区域移除内聚蛋白。结果，不相等的姐妹染色单体重组增加，并诱导扩增。然后，当拷贝数达到野生型水平左右时，Sir2蛋白抑制转录，并通过恢复内聚蛋白的功能限制不相等的姐妹染色单体重组。类似的，我们发现了拷贝数调控系统，即Sir2通过改变内聚蛋白关联，通过E-pro转录调控rDNA拷贝数。

The ribosomal RNA gene repeats (rDNA) is one of the most characteristic regions in eukaryotic chromosomes. The repeats consist of more than 100 tandem units occupying large part of the chromosome in most of organisms. Repeated genes families such as the ribosomal RNA genes (rDNA) are thought to be fragile sites for deletional recombination. However, as for the rDNA, cells have gene amplification system and recover copy number to the wild type level. We found a cis-essential sequence (EXP) for the amplification. In this research, I analyzed molecular mechanism of the EXP.First, we did the phylogenetic footprinting assay in the EXP using several kinds of yeasts and found a conserved region. Interestingly, the region was a bi-directional noncoding transcriptional promoter that is reported〜20 years ago. We tested the character of the promoter and found the transcription was essential for the amplification. Interestingly, when the rDNA copy number reduces, the transcription is activated and it removes cohesin from the region. As the result, unequal sister-chromatid recombination increases and amplification is induced. Then, when the copy number reaches around the wild type level, Sir2 protein represses the transcription and by the function of recovered cohesin the unequal sister-chromatid recombination is restricted. Like these, we found the copy number regulation system, that is, Sir2 regulates rDNA copy number through E-pro transcription by changing cohesin association.