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

据估计，哺乳动物中大约有2万个基因是由成千上万的其他DNA片段调控的，这些DNA片段的结构和功能都还不太清楚。DNA的这两种成分都包含了生物体中大部分的遗传密码，并构成了基因组。基因组将这些基本元素聚集在基因座（基因加上重要的调控DNA片段）中，相互作用并准确地开关基因，从而指导发育、谱系规范和分化，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）一个有功能的细胞或组织时，我们才开始了解基因组功能的基础。红细胞几乎是一个完美的例子，一个看似简单的可合成细胞。这看起来很简单，因为它几乎只含有血红蛋白分子，这种蛋白质物质使其呈现红色，对体内二氧化碳/氧气的交换至关重要。这些细胞里没有DNA ！到这里，简单的事情就结束了。如果没有被认为是生命密码的单一碱基对DNA，这台非凡的机器是如何做到这一点的？答案在于存在于骨髓中的早期细胞类型发生了什么，即所谓的祖细胞，成熟的红细胞就是从祖细胞进化而来的。这些祖细胞“知道”这个未来的红细胞的状态，然后表达（产生）适当的球蛋白（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。要在未来的合成细胞中设计这种程序，就需要深入了解转录调控，这是导致基因产生蛋白质的关键分子过程。这个过程深深嵌入到基因组中，以及它独特的三维折叠中，这种折叠涉及不同细胞类型中独特的DNA序列。尽管基因组数据的深度取得了前所未有的进步，但关于作为调节基因表达开关的DNA基本片段，即所谓的调节元件（增强子、启动子和绝缘子）如何在人体细胞中正确的时间和位置起作用的关键问题仍然没有答案。这些元件的间距和相对位置在多大程度上有助于基因表达的调节也是未知的。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，其中调节元件的序列和间距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，即存在于红细胞中的α -珠蛋白基因座，以建立个体基因在发育、谱系规范和分化过程中开启和关闭的原理。与基因组中的其他基因座相比，α -珠蛋白提供了一种完善且易于处理的哺乳动物基因座模型。通过Boeke的Lab de novo DNA设计和合成方法，以及Higgs/Kassouf基因组工程和分析策略，我们提出通过初步创建和分析基于天然α -球蛋白基因位点的11种新的假设驱动的小鼠遗传模型来解决该领域的关键问题。我们将分析我们创造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，说明基因是如何由这些尚未被充分理解的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。

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.