Mechanisms of the triplet repeat expansion in eukaryotic chromatin.

自1991年以来，超过10种人类遗传性疾病，包括脆性X综合征、肌强直性营养不良和Friedreichs's共济失调已被确定与（CTG）_n、（CGG）_n和（GAA）_n的扩增有关。基于这些重复序列可以形成各种不同的DNA构象（包括发夹、滑链结构、四重DNA和分子内三重结构）的特征，已经提出了几种扩展机制，但确切的机制尚不清楚。由于真核细胞DNA被包装成核小体阵列，染色质可能参与了三重重复疾病。本项目旨在阐明三重重复扩增引起遗传病的机制。为了解决这个问题，我们研究了三联体重复序列对体内染色质组织和基因表达的影响。我们开发了一种检测系统，用于检测DNA序列对酵母小染色体核小体形成的影响。在这个系统中，我们发现（CGG）12破坏了一系列定位的核小体。核小体的这种不稳定可以用稳定的高阶DNA结构（haipin，四重结构或滑移结构）的形成或核小体与序列形成的高能量成本来解释。相反，CTG重复序列促进核小体的形成。由于核小体作为基因表达的一般抑制因子，CTG重复序列可能通过染色质结构改变来调节致病基因的表达。A (GAA)_<12>序列被整合到核小体中，但核小体在邻近区域的定位发生了改变。我们还发现（CGG）_<12>，而（CTG）_<12>和（GAA）_<12>没有增加UAS-less启动子的报告子lacZ表达。这与（CGG）_<12>使核小体不稳定的发现是一致的。综上所述，三联体重复序列可以影响体内染色质组织和基因表达，这可能是导致扩增现象和疾病表型的原因之一。少

Since 1991, more than 10 human hereditary diseases, including Fragile X syndrome, myotonic dystrophy, and Friedreichs's ataxia have been identified to be associated with the expansion of (CTG)_n, (CGG)_n, and (GAA)_n. Several mechanisms of expansions have been proposed based on the features that these repeats can form a variety of alternative DNA conformations including hairpins, slipped strand structures, quadruplex DNA and intramolecular triplexes, yet exact mechanisms are unknown. Since in eukaryotic cells DNA is packaged into arrays of nucleosomes, chromatin may be involved in the triplet repeat diseases.The purpose of this project is to elucidate mechanisms of triplet repeat expansion causing genetic diseases. To address this issue, we examined effects of triplet repeat sequences on the chromatin organization and gene expression in vivo. We developed an assay system with which to examine the effects of DNA sequences on the nucleosome formation in yeast minichromosomes in vivo. Usi … More ng this system, we show that (CGG)12 disrupts an array of positioned nucleosomes. Such destabilization of nucleosomes can be accounted for in terms of formation of stable higher-order DNA structures (haipins, quadruplex or sllipped structures) or high energy cost of nucleosome formation with the sequences. In contrast, it was shown that CTG repeats promote nucleosome formation. Since nuclesomes acts as a general represser for gene expression, the CTG repeats may modulate expression of disease-causing genes via chromatin structural changes. A (GAA)_<12> sequence was incorporated in a nucleosome, but nuclesome positioning in the neighboring region was altered. We also find that (CGG)_<12>, but not (CTG)_<12> and (GAA)_<12> increased a reporter lacZ expression from UAS-less promoters. This is consistent with the finding that (CGG)_<12> destabilizes nucleosomes. In summary, triplet repeat sequences can affect the chromatin organization as well as gene expresion in vivo, which may contribute to the expansion phenomenon and the disease phynotypes. Less