Molecular and functional characterisations of UTE-1 binding proteins involved in the regulation of the human TIMP1 gene
Molecular and functional characterisations of UTE-1 binding proteins involved in the regulation of the human TIMP1 gene
Within the TIMP1 promoter, a DNA binding element essential for the promoter activity, named upstream TIMP-1 element (UTE-1), has been identified. This DNA region is the binding site of an unknown 30 kDa protein. The aim of this thesis was to identify this protein and its role in the control of TIMP1 expression in HSC.
The key finding of this work was the identification of RUNX proteins as the transcription factors that bind to the UTE-1. RUNX proteins are three important transcription regulators involved in the pathology of several cancers. This study demonstrated that HSC expressed several spliced forms of RUNX1 and RUNX2 and that, at least, RUNX1 was associated with the TIMP1 promoter in situ. Reporter gene assays demonstrated that overexpression of the main variant of RUNX1 (RUNX1B) inhibited TIMP1 transcription whereas RUNIX1A, which lacked the C-terminal region, and RUNX2 functioned as simulators. In addition JunD and MOZ-induced transcription of TIMP1 was inhibited by RUNX1B but not RUNX1A suggesting that the C-terminal domain of RUNX1B was responsible for the repression. This effect was associated with a lack of interaction between RUNX1B and JunD whereas RUNX1A could directly interact with JunD. These data suggested that, RUNX1B recruits, via its C-terminal domain, transcriptional repressors within the TIMP1 promoter, resulting in the inhibition of both interaction with JunD and activation of the transcription. In contrast, RUNX1B repression of transcription is reverted in the presence of CBP. Hence, RUNX1B have stimulatory or inhibitory effect on TIMP1 transcription depending on the nature of its interaction with other cofactors.
We also demonstrated the utility of baculovirus as a vector for the delivery, to both mammalian cell lines and primary cells, of a shRNA-generating cassette and the consequent RNAi-mediated silencing of the Lamin gene.
University of Southampton
Bertrand, Marie-Anne
42b7f9dd-6817-40a6-b222-f8fdc2892fa9
2004
Bertrand, Marie-Anne
42b7f9dd-6817-40a6-b222-f8fdc2892fa9
Bertrand, Marie-Anne
(2004)
Molecular and functional characterisations of UTE-1 binding proteins involved in the regulation of the human TIMP1 gene.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Within the TIMP1 promoter, a DNA binding element essential for the promoter activity, named upstream TIMP-1 element (UTE-1), has been identified. This DNA region is the binding site of an unknown 30 kDa protein. The aim of this thesis was to identify this protein and its role in the control of TIMP1 expression in HSC.
The key finding of this work was the identification of RUNX proteins as the transcription factors that bind to the UTE-1. RUNX proteins are three important transcription regulators involved in the pathology of several cancers. This study demonstrated that HSC expressed several spliced forms of RUNX1 and RUNX2 and that, at least, RUNX1 was associated with the TIMP1 promoter in situ. Reporter gene assays demonstrated that overexpression of the main variant of RUNX1 (RUNX1B) inhibited TIMP1 transcription whereas RUNIX1A, which lacked the C-terminal region, and RUNX2 functioned as simulators. In addition JunD and MOZ-induced transcription of TIMP1 was inhibited by RUNX1B but not RUNX1A suggesting that the C-terminal domain of RUNX1B was responsible for the repression. This effect was associated with a lack of interaction between RUNX1B and JunD whereas RUNX1A could directly interact with JunD. These data suggested that, RUNX1B recruits, via its C-terminal domain, transcriptional repressors within the TIMP1 promoter, resulting in the inhibition of both interaction with JunD and activation of the transcription. In contrast, RUNX1B repression of transcription is reverted in the presence of CBP. Hence, RUNX1B have stimulatory or inhibitory effect on TIMP1 transcription depending on the nature of its interaction with other cofactors.
We also demonstrated the utility of baculovirus as a vector for the delivery, to both mammalian cell lines and primary cells, of a shRNA-generating cassette and the consequent RNAi-mediated silencing of the Lamin gene.
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Published date: 2004
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Local EPrints ID: 465595
URI: http://eprints.soton.ac.uk/id/eprint/465595
PURE UUID: 74cc9550-1d85-41d5-b0a5-e3f67b6697fc
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Date deposited: 05 Jul 2022 01:56
Last modified: 16 Mar 2024 20:16
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Author:
Marie-Anne Bertrand
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