Mechanisms of the triplet repeat expansion in eukaryotic chromatin.

真核染色质三联体重复扩增的机制。

• 批准号: 11672193
• 负责人: SHIMIZU Mitsuhiro
• 金额: $ 1.98万
• 依托单位: Meisei University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672193/
• 关键词: triolet repeat sequences; genetic diseases; chromatin; nucleosome; DNA structure; gene expression; budding veast; 染色体; 酵母; 分子生物学; DNAの高次構造; 神経筋疾患; ゲノム

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

自1991年以来，已有10多种人类遗传性疾病，包括脆性X综合征、强直性肌营养不良和弗里德赖希共济失调等被确定与(CTG)_n、(CGG)_n和(GAA)_n的扩张有关。基于这些重复可以形成多种替代DNA构象的特征，包括发夹、滑链结构、四链体DNA和分子内三链体，已经提出了几种扩展机制，但确切的机制尚不清楚。由于在真核细胞中，DNA 被包装成核小体阵列，因此染色质可能与三联体重复疾病有关。本项目的目的是阐明三联体重复扩增引起遗传疾病的机制。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，用于检查 DNA 序列对体内酵母微小染色体核小体形成的影响。利用这个系统，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通过稳定的高阶DNA结构（发夹、四链体或滑动结构）的形成或与序列形成核小体的高能量成本来解释。相反，CTG 重复序列促进核小体形成。由于核体充当基因表达的一般抑制子，因此 CTG 重复可以通过染色质结构变化调节致病基因的表达。 (GAA)_ 12 序列被掺入核小体中，但相邻区域中的核体定位被改变。我们还发现(CGG)_<12>而非(CTG)_<12>和(GAA)_<12>增加了无UAS启动子的报告子lacZ表达。这与(CGG)_ 12 使核小体不稳定的发现一致。总之，三联体重复序列可以影响染色质组织以及体内基因表达，这可能有助于扩张现象和疾病表型。较少的

项目成果

Mitsuhiro Shimizu, Ryo Fujita, Nobuyuki Tomita Heisaburo Shindo and Robert D. Wells: "Chromatin structure of yeast minichromdsomes containing triplet repeat sequences associated with human hereditary neurological diseases"Nucleic Acids Res. Supple.. 1. 71

Mitsuhiro Shimizu、Ryo Fujita、Nobuyuki Tomita Heisaburo Shindo 和 Robert D. Wells：“含有与人类遗传性神经系统疾病相关的三联体重复序列的酵母微型染色体的染色质结构”核酸研究。

Mitsuhiro Shimizu: "Chromatin structure of yeast minichromosomes containing triplet repeat sequences associated with human hereditary neurological diseases"Nucieic Acids Res. Supple. 1. 71-72 (2001)

Mitsuhiro Shimizu：“含有与人类遗传性神经系统疾病相关的三联体重复序列的酵母微型染色体的染色质结构”核酸研究。

Mitsuhiro Shimizu: "Mechanisms of chromatin alteration in transcriptional regulation"Biophysics (in Japanese). 39. 376-380 (1999)

Mitsuhiro Shimizu：“转录调控中染色质改变的机制”生物物理学（日语）。

Mitsuhiro Shimizu: "Destabilization of nucleosomes by an unusual DNA conformation adopted by poly dA poly dT tracts in vivo"EMBO J.. 19. 3358-3365 (2000)

Mitsuhiro Shimizu：“体内聚 dA 聚 dT 束采用的不寻常 DNA 构象对核小体的不稳定”EMBO J.. 19. 3358-3365 (2000)

Mitsuhiro Shimizu, Tatsuhiro Mori, Takayuki Sakurai and Heisaburo Shindo /: "Destabilization of nucleosomes by an unusual DNA conformation adopted by poly dA poly dT tracts in vivo"EMBO J.. 19. 3358-3365 (2000)

Mitsuhiro Shimizu、Tatsuhiro Mori、Takayuki Sakurai 和 Heisaburo Shindo /：“体内聚 dA 聚 dT 束采用的不寻常 DNA 构象对核小体的不稳定”EMBO J.. 19. 3358-3365 (2000)