NF-kappaB-Regulated Differential Gene Transcription: A Systems Biology Analysis

Abstract

The NF-kappaB transcription factor is expressed in the majority of mammalian cells and regulates a large number of genes with important functions in a variety of cellular processes including cell growth, division, apoptosis and inflammatory responses. Perturbation of NF-kappaB response has been implicated in a variety of diseases such as asthma and inflammatory bowel disease, in addition to various forms of cancer.Through experiments at the single cell level it has been shown that NF-kappaB displays complex temporal activation, notably including nucleo-cytoplasmic oscillations. It has been observed that these oscillations occur in a heterogeneous manner; as such they are masked when measured at the population level. In contrast, pulsed TNFalpha treatment at 100 min intervals produces regular and synchronous nuclear peaks of NF-kappaB. Such pulsatile stimulation may reflect more accurately physiological conditions.The work in this project uses a Systems Biology approach consisting of bioinformatic, mathematical, and experimental methodologies to investigate how NF-kappaB can regulate such a diverse set of gene responses. Previously published studies have proposed that target gene expression levels following NF-kappaB activation (continuous TNFalpha) can be explained by a combination of key parameters, including transcript degradation rate, transcript structure, and transcription initiation rate. Initial work in this project highlighted that these explanatory factors are not sufficient to describe the observed temporal order of gene transcription. The roles of miRNAs and NF-kappaB subunit phosphorylation in regulation were additionally explored.A large set of genes was identified that are activated more strongly by pulsed TNFalpha than by continuous TNFalpha treatment. This suggests a new unreported mechanism of gene regulation, the possible causes of which are examined in this thesis. The gene list was refined by altering pulse frequency, which revealed an enrichment of NF-kappaB targets correlated with the regularity of these pulses. Temperature shift and anti- inflammatory drug treatment (Diclofenac) were shown to have a profound effect on NF-kappaB oscillation frequency. These perturbations provide an alternative method to study the effects of NF-kappaB oscillation frequency on specific target genes, independent of a pulse regime. Integration and analysis of these datasets suggested that a core, frequency-encoded set of genes regulated by NF-kappaB might exist. It is proposed that such genes may respond optimally to specific frequencies of NF-kappaB activation, implying a potential frequency threshold. The presence of such genes may explain the need for the complex systems that control NF-kappaB timing. It was noted that there was an enrichment of genes encoding transcription factors within the frequency encoding set, in addition to proteins which are known to be involved in the control of inflammation.

Date of Award	1 Aug 2015
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Dean Jackson (Supervisor) & Michael White (Supervisor)