[Thesis]. Manchester, UK: The University of Manchester; 2016.
Many genes demonstrate highly dynamic pulsatile expression, with characteristic bursts
of activity. Dynamic expression of the human prolactin (hPrl) gene in pituitary cells
has previously been investigated identifying key temporal characteristics, influenced
by the process of chromatin remodelling. Earlier work on the related pituitary human
growth hormone (hGH) proximal promoter (-496/+1bp) indicated that it displayed similar
dynamic behaviour. The human GH gene contains an extensive long-distance regulatory
sequence, including a locus control region (-14/-32kbp) that has been shown to regulate
chromatin remodelling and confer tissue-specificity of hGH expression. In this work
I aimed to study dynamic regulation of the hGH gene promoter in detail. Initially
I investigated the efficiency of several methods to express the luciferase gene in
a 180kb hGH genomic fragment using bacterial artificial chromosome recombineering,
to allow the investigation of single cell transcription dynamics. Although a functional
recombinant BAC was not finalised during the course of the work, I carried out detailed
time course studies using shorter hGH-reporter constructs. Using quantitative microscopy
to study live single cells, I compared the dynamic characteristics of a 5kb hPrl promoter
fragment with those of -840/+1bp and -3348/+1bp hGH-luciferase promoter-reporter constructs.
Whilst previous hPrl analysis utilised a binary mathematical model assuming a simplified
two-state (ON/OFF) process of gene transcription, I validated and applied a novel
stochastic switch model (SSM), assuming instead that transcription rate can switch
between any variable states at any time. Through doing so I observed an asymmetry
in transcription rate switching, suggesting an all-or-nothing activation of a single
UP-switch, with a greater number of rate decreasing DOWN-switches. The -3348/+1bp
construct produced double the number of DOWN-switches, whilst the -840/+1bp construct
produced 1.5 DOWN-switches in a 48h period. The cycling of transcriptional activity
seen by the shorter construct was modified through the addition of forskolin, activating
cAMP signalling. However, significant modification of the transcriptionally inactive
refractory period seen with the -3348/+1bp construct (reduced from 3h to 1.9h) required
histone modification through application of trichostatin A, a HDAC inhibitor. In conclusion,
different promoter elements confer different transcriptional timing and dynamics.
A subtler transcriptional modelling, such as used here in the SSM, reveals new insights
into the phenomena of transcriptional switching, but the mechanisms involved remain
to be determined.