Understanding the relationship between large-scale chromatin structure and gene expression
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Date
19/04/2022Author
Groat, Elaine
Metadata
Abstract
Eukaryotic genomes are packaged into a complex DNA, RNA, and protein rich chromatin
fibre, creating an interdependent functional relationship between the structure of the
chromatin and the activity of the genetic features stored within. The composition of active
genomic regions is weighted towards transcription factors, co-activating proteins, and active
histone modifications such as acetylation. Active regions are associated with more disrupted
chromatin fibre structure, greater chromatin accessibility and cytological decompaction of
large-scale chromatin. The striking decompaction of large-scale chromatin observed through
microscopy is well documented, however, it is unclear what molecular mechanisms drive
the change in structure. The aim of this thesis was to document the changes to chromatin
structure induced by the activation of estrogen responsive loci and determine what cellular
processes are responsible for decompacting large-scale chromatin.
Experiments were performed in hormonal responsive MCF-7 breast cancer cells where
genes GREB1 and TFF1 are strongly upregulated upon stimulation by estrogen. ChIP-seq was
used to assess binding of the transcription factor, estrogen receptor α, finding estrogen
treatment increased enrichment of the transcription factor at the enhancer and promotor
of both genes with similar dynamics. However, performing ChIP-seq for active histone
modification, H3K27ac, revealed greater and more rapid deposition of H3K27ac at the TFF1
locus, which was also associated with more rapid increases in transcriptional output as
assayed by TT-seq. Fluorescence in situ hybridisation (FISH) was used to observe chromatin
decompaction at both genomic loci after estrogen stimulation, however, like H3K27ac
deposition and transcription, the chromatin decompaction at TFF1 was much more rapid
(within 15 min) than at GREB1 (3 h). This suggested that both transcription and histone
acetylation could be key drivers of large-scale chromatin decompaction.
To understand the role of transcription, I performed estrogen activation experiments on
MCF-7 cells in the presence of transcription inhibitors. Interestingly, in the presence of
either α-amanitin, which inhibits transcription elongation, or triptolide, which inhibits
transcription initiation, estrogen stimulation still caused cytological decompaction of the
TFF1 locus, though the decompaction was delayed. The delay suggests transcription does
play a role in large-scale chromatin decompaction, though it is not the sole driver. In the
presence of α-amanitin, the delay in chromatin decompaction is also associated with a delay
in H3K27ac deposition, suggesting the processes of transcription, acetylation, and
chromatin decompaction may be interlinked. Thus, in the absence of transcription,
deposition of H3K27ac, and by extension, other chromatin remodelling processes, can drive
large-scale chromatin decompaction though their efficiency is hindered by the lack of
transcription.
To further investigate the regulators of large-scale chromatin conformation, physics-based
polymer simulations of chromatin were used to predict experimentally observed chromatin
compaction states. In the Highly Predictive Heteromorphic Polymer (HiP-HoP) model,
chromatin accessibility peaks and estrogen receptor α binding peaks were used to represent
chromatin binding sites for two simulated proteins. CTCF sites marked anchor points for
loop extrusion and H3K27ac rich regions were given a more flexible chromatin fibre
structure. In previous experiments on the Pax6 locus, the variable fibre flexibility
determined by H3K27ac distribution was vital in predicting the chromatin conformation of
the locus when the gene is highly expressed. Simulations of the GREB1 and TFF1 loci
revealed that the input parameters described could predict large-scale chromatin
decompaction of both loci upon estrogen treatment, however, the decompaction at TFF1
observed experimentally in the presence of α-amanitin was not replicated in the
simulations. This could suggest that histone modification and large-scale chromatin
remodelling are only linked in the presence of transcription, though more likely suggests
improvements to the model must be made to represent chromatin interactions during drug
induced transcriptional inhibition more accurately.
Together this thesis provides an extensive examination of the changes in chromatin
structure which occur during estrogen induced transcriptional activation, providing another
example for the investigation of the relationship between large-scale chromatin structure
and gene expression. I found a link between transcriptional activation, H3K27ac deposition
and the decompaction of large-scale chromatin, a link strengthened by the delay of both
acetylation and chromatin decompaction during transcription inhibition. The results here
provide a framework to further examine the details of the molecular mechanisms driving
expression associated changes in large-scale chromatin structure.