21ENGBIO - Engineering Nucleosome Positioning in Plants

植物生物技术在全世界29个国家种植生物技术作物方面取得了巨大成功，提高了小农的福利，减少了杀虫剂的使用。此外，植物栽培的低成本，以及它们积累的丰富和多样性的天然产物，使它们成为生产用于健康和工业的有价值分子的光合作用平台。为了改良作物和提高有用分子的产量，我们需要详细了解调控功能是如何编码到DNA序列中的，以及控制基因表达的方法。为了将真核生物的染色体装入细胞核，真核生物的DNA被包装到染色质中。染色质的基本结构单位是核小体，它由缠绕在八种组蛋白周围的一段DNA组成。核小体的位置和稳定性可以决定DNA对蛋白质和其他因素的可及性，因此在DNA复制和修复以及基因表达中具有重要作用。有证据表明，DNA序列可以被编码来影响核小体的位置和稳定性。在酵母中的演示表明，重新编码DNA以影响核小体位置或稳定性可以用来调节基因表达，但类似的工作还没有在植物中完成。在初步研究中，我们发现植物调控序列旨在指定脆弱的核小体并增加DNA的可及性，从而增加了表达。在这项提案中，我们将进一步研究这一点，目的是获得直接证据，证明核小体定位可用于调节植物合成启动子的水平。此前，我们和其他人也发现，合成基因的表达水平通常会受到邻近基因表达的显著影响。在这里，我们还将研究使用核小体不利序列来减轻这些不受欢迎的上下文影响。

Plant biotechnology has been enormously successful with biotech crops grown in 29 countries worldwide and associated with improved welfare of smallholder farmers and reduced use of pesticides. In addition, the low cost of plant cultivation together with the abundance and diversity of natural products that they accumulate has led to their development as photosynthetic platforms for producing valuable molecules used in health and industry. For both crop improvement and to improve the yields of useful molecules, we require a detailed understanding of how regulatory function is encoded into DNA sequences together with methods to control gene expression.To fit their chromosomes into the nucleus, the DNA of eukaryotes is packaged into chromatin. The basic structural unit of chromatin is the nucleosome, consisting of a segment of DNA wound around eight histone proteins. The position and stability of nucleosomes can dictate the accessibility of DNA to proteins and other factors and, therefore, have important roles in DNA replication and repair as well as in gene expression. There is evidence that DNA sequences can be coded to influence the position and stability of nucleosomes. Demonstrations in yeast have shown that recoding DNA to influence nucleosome position or stability can be used to modulate gene expression but similar work has not been done in plants. In preliminary studies, we found that plant regulatory sequences designed to specify fragile nucleosomes and increase the accessibility of the DNA gave increased expression. In this proposal, we will investigate this further with the aim of obtaining direct evidence that nucleosome positioning can be used to tune the level of plant synthetic promoters. Previously, we and others have also found that expression levels from synthetic genes are often significantly affected by the expression of closely located genes. Here, we will also investigate the use of nucleosome disfavouring sequences to mitigate these undesired context effects.