Investigating the chromatin dynamics of gene activation
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Date
24/05/2022Author
McGhan, Portia
Metadata
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.