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

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.

It is estimated that in mammals there are ~ 20,000 genes regulated by ~900,000 enhancer-like elements interspersed with ~30,000 CTCF bound elements many of which act as insulators. The genome brings these fundamental elements together within loci of 10s to 1000s kb to interact and accurately switch genes on and off, thereby directing development, lineage specification and differentiation. At each locus, changes in gene expression in space and time are associated with changes in the three-dimensional chromatin structure, as different sets of dispersed elements interact. Despite unprecedented advances in the depth of genome data, key questions of how fundamental regulatory elements (enhancers, promoters and insulators) combine to control transcription of individual genes at the right time and place remain unanswered. Newly developed technology to synthesize large regulatory domains allows us to address the relationships between genome structure and gene expression in detail by constructing loci in which the sequences and spacing of regulatory elements can be changed by design.This proposal will use synthetic genomics to engineer a relatively simple mammalian regulatory domain at the well-characterised alpha-globin locus to establish principles by which individual genes are switched on and off throughout development, lineage specification and differentiation.