Year: 2013
Type: dissertation
Abstract: Ligand-activation of the constitutively expressed nuclear receptor PPARγ in normal human urothelial (NHU) cells in vitro entrains a programme of transcriptional changes resulting in expression of gene and protein markers associated with in vivo differentiated urothelium. \n \nIt was hypothesised that, after induction of differentiation, PPARγ would translocate to the nucleus and facilitate targeted expression of differentiation-associated genes through altering chromatin constitution. To address this hypothesis, differential solubility of chromatin and nuclear-matrix bound proteins were exploited to observe changes in PPARγ localisation. In addition, label-free mass spectrometric analysis of NHU extracts was undertaken to discern if differential relative abundance of chromatin-associated proteins could be detected, and next-generation sequencing technologies were employed to assess the changes induced in the chromatin environment by sequencing RNA transcripts (RNA-seq), performing high-throughput chromosome conformation (HiC), assessing transcription factor binding via formaldehyde-assisted isolation of regulatory elements (FAIRE), and histone epigenetic markers of transcription (ChIP-seq). \nPotentially novel PPARγ isoforms were observed by western blot, with little localisation alterations between differentiated and control NHU cells. Differentiation markers were downregulated after treatment with siRNA specifically targeting PPARγ2, without significant reduction in PPARγ abundance. Label-free mass spectrometry detected peptides from chromatin-associated proteins involved as having differential abundance between extracts from differentiated and control NHU cells. RNA transcriptomics revealed upregulation of novel transcription factors not previously associated with urothelial differentiation. Preliminary FAIRE results revealed the presence of regulatory elements unique to terminally differentiated cells. \n \nThis study extends the understanding of the behaviour of PPARγ in NHU differentiation, identified chromatin constituents with potential roles in differentiation and provided a rich transcriptomics resource which will be a valuable tool in assessment of the impact of transcription factor binding on local chromatin organisation during urothelial differentiation. \n
Author: Carl Fishwick
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1527223465