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

据估计，在哺乳动物中，有大约20,000个基因受到数十万个其他DNA片段的调控，这些基因在结构和功能上仍不是很清楚。DNA的这两个组成部分都包含了生物体中的大部分遗传密码，并形成了基因组。基因组将这些基本要素聚集在基因座内(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、谱系指定和分化，这对活着的有机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。红细胞几乎是一个看似简单的可合成细胞的完美例子。它看起来很简单，因为它几乎只含有血红蛋白分子，这是一种使身体呈现红色的蛋白质物质，对体内二氧化碳/氧气的交换至关重要。这些细胞中没有DNA存在！在这里，简单到此为止。这台非凡的机器如何在没有被认为是生命密码的单一碱基对的情况下完成它的工作呢？答案在于驻留在骨髓中的早期细胞类型发生了什么，即所谓的祖细胞，成熟的红血球是从这些细胞进化而来的。这些祖细胞“知道”这个未来的红血球的状态，然后表达(产生)适当的珠蛋白(蛋白质)，直到这个细胞准备好排出它的DNA并离开骨髓，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出这样的程序，需要对转录调控有深入的了解，转录调控是从基因中产生蛋白质的关键分子过程。这一过程深深植根于基因组以及其独特的三维折叠中，该折叠在不同细胞类型中参与独特的DNA序列。尽管基因组数据的深度取得了前所未有的进展，但关键问题仍未得到解答，即DNA的基本片段如何充当调控基因表达的开关，即所谓的调控元件(增强子、启动子和绝缘体)在人体细胞中的正确时间和地点发挥作用。这些元件的间距和相对位置在多大程度上对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(带有周围重要DNA序列的基因)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阿尔法珠蛋白基因座，以建立个体基因在发育、谱系指定和分化过程中被开启和关闭的原则。与基因组中的其他基因座相比，α-珠蛋白提供了一个完善的、易处理的哺乳动物基因座模型。在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的支持下，我们建议通过初步创建和分析基于自然α-珠蛋白基因位点的11个新的假设驱动的小鼠遗传模型来解决这一领域的关键问题。我们将分析我们创造的设计对我们将在实验室的培养皿中产生的红细胞状态的影响。基于这种设计及其对红细胞产生血红蛋白能力的影响，我们将推断我们增加或减少的不同DNA片段的重要性，最终得出更清晰的规则，并解释这些原本不被很好理解的DNA片段是如何控制基因的。这项工作的发现将对基础科学以及基因组医学和遗传病产生影响。

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.