22-BBSRC/NSF-BIO Building synthetic regulatory units to understand the complexity of mammalian gene expression

22-BBSRC/NSF-BIO 构建合成调控单元以了解哺乳动物基因表达的复杂性

• 批准号: BB/Y008898/1
• 负责人: Douglas Higgs
• 金额: $ 107.23万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY008898%2F1
• 关键词: 22; BBSRC; NSF; BIO; Building

It is estimated that in mammals there are ~ 20,000 genes regulated by hundreds of thousands of other pieces of DNA that are still not very understood, neither in structure nor in function. Both these components of DNA contain most of the genetic code in an organism and form the genome. The genome brings these fundamental elements together within loci (genes plus important pieces of regulatory DNA) to interact and accurately switch genes on and off, thereby directing development, lineage specification and differentiation, crucial for the appropriate formation of tissues and organs in a living organism. Only when we succeed in building (synthesising) a functional cell or tissue, do we begin to understand the basis of the genome function. The red cell is almost a perfect example of a deceptively simple synthesisable cell. It is seemingly simple because it contains almost exclusively hemoglobin molecules, the protein substance that gives its red colour and is crucial for CO2/O2 exchanges in the body. No DNA is present in these cells! There, the simplicity ends. How does this remarkable machine do what it does without so much as a single base-pair of DNA, thought to be the code for life? The answer lies in what happens in the earlier cell types that reside in the bone marrow, the so-called progenitor cells, from which mature red cells evolve. These progenitor cells "know" the status of this future red cell, and then express (produce) the appropriate globins (proteins) needed until this cell becomes ready to expel its DNA and exit from the bone marrow so it can function better in circulation in the blood. To engineer that kind of program in future synthetic cells, a deep understanding of transcriptional regulation, a key molecular process that leads to protein production from genes, is required. This process is deeply embedded in the genome as well as in its unique three dimensional folding that engages unique DNA sequences in different cell types. Despite unprecedented advances in the depth of genome data, key questions of how fundamental pieces of DNA that act as switches to regulate gene expression, the so-called regulatory elements (enhancers, promoters and insulators) work at the right time and place in the cells of the body remain unanswered. It is also unknown to what extent spacing and relative position of these elements contribute to regulation of gene expression. Newly developed technology to synthesize large pieces of DNA allows us to address the relationships between genome structure and gene expression in detail by constructing loci (genes with surrounding important pieces of DNA sequences) in which the sequences and spacing of regulatory elements can be changed by design. Here we will use synthetic genomics to engineer a relatively simple mammalian locus, The alpha-globin locus present in red cells, to establish principles by which individual genes are switched on and off throughout development, lineage specification and differentiation. The alpha-globin offers a well-established and tractable model of a mammalian gene locus compared to other loci in the genome. Powered by Boeke's Lab de novo DNA design and synthesis approaches, together with the Higgs/Kassouf genomic engineering and analysis strategies, we propose to address key questions in this field by initially creating and analysing 11 new hypothesis-driven mouse genetic models based on the natural alpha-globin gene locus. We will analyse the effect of the designs we create on the state of the red cells we will produce in a dish in the lab. Based on the design and its impact on the red cell ability to produce haemoglobin, we will deduce the importance of the different pieces of DNA we add or subtract and eventually come up with clearer rules and explanation of how genes are controlled by these otherwise not well-understood pieces of DNA. The discoveries from this work would have an impact on fundamental science as well as on genomic medicine and genetic disease.

据估计，在哺乳动物中，大约有20，000个基因受数十万个其他DNA片段的调控，这些DNA片段的结构和功能都还不清楚。DNA的这两种成分都包含生物体中的大部分遗传密码，并形成基因组。基因组将这些基本元素聚集在基因座内（基因加上重要的调控DNA片段），以相互作用并准确地打开和关闭基因，从而指导发育，谱系特化和分化，这对生物体中组织和器官的适当形成至关重要。只有当我们成功地构建（合成）一个功能细胞或组织时，我们才开始理解基因组功能的基础。红细胞几乎是一个完美的例子，一个看似简单的合成细胞。它看起来很简单，因为它几乎只含有血红蛋白分子，这种蛋白质物质使其呈红色，对体内的CO2/O2交换至关重要。这些细胞中没有DNA！至此，简单性结束了。这台非凡的机器是如何在没有被认为是生命密码的DNA碱基对的情况下完成它的工作的？答案在于骨髓中早期的细胞类型发生了什么，所谓的祖细胞，成熟的红细胞从中进化而来。这些祖细胞“知道”这个未来红细胞的状态，然后表达（产生）所需的适当球蛋白（蛋白质），直到这个细胞准备好排出它的DNA并从骨髓中退出，这样它就可以在血液循环中更好地发挥作用。为了在未来的合成细胞中设计这种程序，需要对转录调控（一个导致基因产生蛋白质的关键分子过程）有深入的了解。这一过程深深嵌入基因组及其独特的三维折叠中，该三维折叠涉及不同细胞类型中的独特DNA序列。尽管基因组数据的深度取得了前所未有的进展，但作为调节基因表达的开关的基本DNA片段，即所谓的调节元件（增强子，启动子和绝缘体）如何在身体细胞中的正确时间和位置工作的关键问题仍然没有答案。也不知道这些元件的间隔和相对位置在多大程度上有助于基因表达的调节。新开发的合成大片段DNA的技术使我们能够通过构建基因座（基因周围有重要的DNA序列片段）来详细解决基因组结构和基因表达之间的关系，其中调控元件的序列和间隔可以通过设计来改变。在这里，我们将使用合成基因组学来设计一个相对简单的哺乳动物基因座，即红细胞中的α-珠蛋白基因座，以建立个体基因在整个发育、谱系特化和分化过程中打开和关闭的原则。与基因组中的其他基因座相比，α-珠蛋白提供了哺乳动物基因座的完善且易于处理的模型。由Boeke的实验室从头DNA设计和合成方法，以及Higgs/Kassouf基因组工程和分析策略提供支持，我们建议通过初步创建和分析11个新的假设驱动的小鼠遗传模型来解决该领域的关键问题。我们将分析我们创建的设计对我们将在实验室培养皿中产生的红细胞状态的影响。基于设计及其对红细胞产生血红蛋白的能力的影响，我们将推断出我们添加或减去的不同DNA片段的重要性，并最终提出更清晰的规则和解释基因是如何被这些原本不太清楚的DNA片段控制的。这项工作的发现将对基础科学以及基因组医学和遗传疾病产生影响。