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Defining the molecular events required for GM-CSF gene activation in T cells


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Brettingham-Moore, K 2007 , 'Defining the molecular events required for GM-CSF gene activation in T cells', PhD thesis, University of Tasmania.

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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-ĸB 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

Item Type: Thesis - PhD
Authors/Creators:Brettingham-Moore, K
Keywords: Granulocyte-macrophage colony-stimulating factor, Immune response, T cells, Molecular immunology, Cellular immunity
Copyright Holders: The Author
Copyright Information:

Copyright 2007 the Author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s).

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