PhD defence M. (Miguel) Martinez Ara

Brief summary of the doctoral thesis:

Our genome consists of the DNA consists of the 4 nucleotides G, A, T and C. It is the basis of all living forms because it contains the instructions (the genes) to make the building blocks of life, the proteins. However, these instructions only cover the 2% of the genome. The other 98% of the genome is what is called the non-coding genome.

The non-coding genome is key for the correct functioning of cells, tissues, organs and the whole body. This is because the non-coding genome instructs where and when the instructions have to be used, or in other words where and when the genes have to be expressed. For the correct functioning of the body, different sets of genes have to be expressed in each cell. This complex spatiotemporal regulation that arises from the non-coding genome is achieved through a series of mechanisms which are collectively called gene regulation. While we understand the genetic code, the code to interpret how to build proteins from genes, we still do not know how to fully understand gene regulation and the non-coding genome. This is due to the complexity of the many processes involved in gene regulation and the lack of a universal and easy to interpret code.

Two of the most important regulatory elements from the non-coding genome involved in gene regulation are promoters and enhancers. Promoters are the regions right next to the beginning of the gene that drive their expression. They are relatively well characterized and they are easy to identify. However, Promoters do not work in isolation. They are affected by their genomic surroundings, and particularly by their surrounding enhancers. Enhancers are DNA sequences that can activate promoters from a distance, from up to a megabase (a million nucleotides) away in mammalian genomes. They are more difficult to identify, there are more enhancers than promoters and it is not trivial to understand which enhancers regulate which genes. Furthermore, enhancers are the main elements that dictate the spatiotemporal regulation of genes, instructing where and when they have to be expressed. Therefore, it is important to understand how enhancers regulate promoters. In Chapter 1 the complexity of gene regulation and in particular the current knowledge about enhancers and promoters and their interplay is further explained

In this thesis, I have explored various aspects of enhancer and promoter biology in mouse embryonic stem cells by employing massively parallel reporter assays (MPRAs). This group of technologies allows us to test the functionality of thousands to millions of DNA sequences in a single experiment, thus accelerating research in gene regulation. These MPRAs usually consists of libraries of DNA elements which are barcoded using random DNA sequences. Then these libraries are introduced in the cells of interested to characterize which DNA sequences are more or less active. This is usually done by sequencing the random DNA barcodes in the input and output of the experiment.