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Title: Computational analysis of transcriptional regulation
Author: Monahan, Jack Michael
ISNI:       0000 0004 9347 9064
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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It is doubtful Friedrich Miescher appreciated how groundbreaking and transformative his isolation of ’nuclein’ in 1869 would prove. Eukaryotic gene expression is a noisy process that is subject to multiple layers of regulation. Key features of this are the three-dimensional (3D) chromatin organisation of eukaryotic genomes and the post-transcriptional control of RNA fates. Eukaryotic nuclear DNA is tightly packaged as chromatin that is further folded into higher order structures. The 3D folding of eukaryotic genome lends itself to the formation of interactions between otherwise distant regions of the genome. These interactions modulate transcription. I investigated the impact of human papillomavirus (HPV) 16 integration on host chromatin organisation and transcription using the W12 model for early cervical carcinogenesis with a novel Chromosome Conformation Capture (3C) method that specifically enriches for interactions involving viral integrants. Integration occurs without disrupting host 3D chromatin structure but alters the expression of many neighbouring host genes. The advent of reliable protocols for performing single-cell RNA sequencing (scRNA-seq) has revealed that transcriptional noise is widespread and a biologically important feature in many populations of mammalian cells. Ageing is associated with the progressive decline in biological function. It has recently been described that aged somatic tissues have greater cell-to-cell transcriptional variability. Ageing is also associated with a decline in male fertility. Some have attributed this to the clonal expansion of selfish spermatogonial lineages. To address this, I explored the effect of ageing on the transcriptomes of sorted populations of mouse undifferentiated spermatogonia using bulk and single-cell RNA sequencing (RNAseq). While subtle changes in mean gene expression are detectable, it was apparent that ageing, unlike in somatic tissues, leads to a decline in cell-to-cell transcriptional variability. This may reflect the phenomenon of selfish spermatogonial selection. Finally, I explored the role of an RNA post-transcriptional modification (RPTM), N6- methyladenosine (m6A), in buffering transcriptional noise. Maternally-supplied YTHDF2 is essential for degradation of m6A-modified transcripts during the maternal-to-zygotic transition (MZT) early in mammalian embryogenesis. YTHDF2 targets increase in abundance in its absence. Using scRNA-seq data generated from control and maternal conditional knock-out mouse zygotes I show that many of these targets exhibit greater cell-to-cell transcriptional heterogeneity in the absence of YTHDF2-mediated degradation. Suggesting that YTHDF2 has a additional function in buffering transcriptional noise.
Supervisor: Enright, Anton James Sponsor: EMBL
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Cervical carcinogenesis ; chromatin interactions ; ageing ; regulation of transcriptional variability ; RNA post-transcriptional modifcations