Investigating the chromatin dynamics of gene activation

Abstract

Enhancers are cis-regulatory elements which contribute to the activation of gene expression. The spatio-temporal control of gene expression is particularly important during embryonic development, when the expression of developmental regulators is tightly controlled. Sonic hedgehog (Shh) is an important signalling protein which is vital for the patterning of the embryo. Shh is expressed throughout the developing central nervous system, gut and the posterior limb bud. This complex pattern of expression is regulated by the activity of multiple tissue-specific enhancers which are spread through a 1 Mb genomic desert. Many of these enhancers activate Shh expression over large genomic distances, with some enhancers being located within the intron of neighbouring genes. The textbook model for enhancer-mediated gene activation suggests that an enhancer moves within close proximity to its target promoter, recruiting transcription factors and RNA polymerase II to the gene and promoting transcription. However, recent studies have brought this model into question. Understanding the dynamics of enhancers and promoters during transcriptional activation is vital for comprehending how gene expression is regulated by enhancers. Advances in techniques enabling the labelling and tracking of non-repetitive loci in live cells have allowed this to start to be addressed. To study how Shh expression is regulated by its enhancers in live cells, firstly I needed to develop a system where Shh expression could be activated in cultured cells. It was known that Shh expression could be activated in mouse embryonic stem cells (mESCs) through retinoic acid treatment; however, the enhancer responsible for activating Shh expression and the cell type the mESCs differentiate into were previously unknown. I investigated changes in chromatin accessibility and modifications to show that Shh expression is activated from endodermal enhancers when mESCs are differentiated with retinoic acid. RNA-seq confirmed that the resulting cells express several markers of early endoderm and mesoderm lineages. The identification of enhancers which activate Shh expression in this mESC differentiation system allowed the tagging and tracking of these loci using a CRISPR-based live cell DNA imaging system. I developed a system that is versatile, simple and stable, with a view to decrease the number of guide RNAs required in order to visualise non-repetitive loci. The dynamics of the Shh promoter and were determined in cells where Shh was transcriptionally silent or active. I found that the dynamics of these cis-regulatory elements were sub-diffusive despite gene activity. Overall, through quantitative CRISPR-imaging, I found direct measurements for chromatin mobility of cis-regulatory elements in living cells under different states of activity.