Understanding the relationship between large-scale chromatin structure and gene expression

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.