whole_Brettingham-MooreKate2007_thesis.pdf (14 MB)
Defining the molecular events required for GM-CSF gene activation in T cells
thesisposted on 2023-05-26, 23:39 authored by Brettingham-Moore, K
Granulocyte macrophage colony stimulating factor (GM-CSF) is a potent regulator of haemopoiesis and is vital for immune function. GM-CSF is rapidly, but transiently activated in response to T cell activating signals. It is now well established that activation of the GM-CSF gene in T cells is accompanied by distinct changes in chromatin structure across the promoter region. The aim of this thesis was to investigate these chromatin remodelling events and their association with gene transcription. Analysis of GM-CSF promoter accessibility and transcription in response to various pharmacological stimulations demonstrated that the processes of transcription and promoter chromatin remodelling were distinct, with each requiring different factors and signals. While chromatin remodelling was found to be dependent on factors activated downstream of PKC signalling, transcription required both PKC and calcium signalling pathways. Nuclear activation of the NF-¿í‚àèB transcription factor, c-Rel was strongly correlated with chromatin remodelling events. In contrast NFAT transcription factors were demonstrated to be required for GM-CSF transcription but not chromatin remodelling. In addition, remodelling of the GM-CSF promoter was found to be relatively stable in contrast to the more transient profile observed for transcription. The ATPase component of the SWI/SNF chromatin remodelling complex has previously been shown to associate with the GM-CSF promoter in vitro. To determine whether Brg1 was involved in activation of the GM-CSF gene in vivo, T cells were transfected with an ATPase defective Brg1 mutant construct. Analysis of these cells demonstrated that efficient activation of the GM-CSF gene is dependent on Brg1. Surprisingly, chromatin immunoprecipitation experiments revealed that Brg1 is bound to the GM-CSF promoter in resting T cells and is depleted concomitant with chromatin remodelling. These data lead to the hypothesis that Brg1 is involved in forming a basal chromatin state that is transcriptionally competent. In support of this, Brg1 is not bound to the GM-CSF promoter in B cells, which do not express GM-CSF. However a competent chromatin environment can be created in these cells by increasing histone acetylation levels. Data presented here are consistent with a model in which the basal state of the GM-CSF promoter is maintained in a transcriptionally competent state in resting T cells via histone acetylation and Brg1 recruitment. Such a chromatin environment may ensure that GM-CSF can be activated rapidly in response to T cell activation signals. Microarray analysis was subsequently used to identify genes which may be similarly poised to respond to T cell activation signals by the constitutive recruitment of Brg1. A number of cytokine genes were identified as Brg1 targets. One of these, Interferon gamma (IFNy) was found to share a similar activation profile to GM-CSF and data presented here suggests it may be regulated by a common mechanism. As observed for GM-CSF, Brg1 is constitutively poised at the IFNy promoter in resting EL-4 T cells and lost from the promoter concomitant with gene activation.
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