Foxler, Daniel (2012) Genetic and functional characterisation of the LIMD1 promoter and gene product: from lung cancer to the hypoxic response. PhD thesis, University of Nottingham.
LIM domain containing protein 1 (LIMD1) is a tumour suppressor located at 3p21.3, a region that harbours multiple tumour suppressor genes and is commonly subject to homozygous deletions and loss of heterozygosity in many cancers. The mechanism of LIMD1 tumour suppressive activities are not fully elucidated, however to date it has been shown to bind to the retinoblastoma protein (pRb) and repress E2F driven transcription as well as being a critical component of miRNA mediated gene silencing. Recent work has also identified LIMD1 as a possible negative regulator of hypoxia inducible factor α (HIF1α) and the hypoxic response. In lung cancer, LIMD1 protein expression is down regulated in up to 79% of tumours when compared to normal tissue with gene deletion and loss of heterozygosity accounting for 32 and 12% respectively, leaving 30% of tumours with unexplained mechanism of LIMD1 protein loss.
In an aim to identify other possible mechanisms of LIMD1 loss, scrutinisation of the LIMD1 promoter identified a CpG Island in the 5’ promoter region, within which a small region was found to be critical for transcriptional activation. This region was methylated in the non-LIMD1 expressing MDA-MB435 cell line, but became hypomethylated and LIMD1 expressed following treatment with the DNA methylation inhibitor 5-Aza-2’-deoxycytidine. In primary lung tumours, analysis of genomic DNA also identified increased methylation of this region as well as a reduction in LIMD1 mRNA levels when compared to matched normal lung tissue. Furthermore, in silico analysis identified a conserved binding motif for the Ets transcription factor PU.1. Experimentally PU.1 was verified as binding to the LIMD1 promoter with siRNA mediated depletion of PU.1 significantly reducing endogenous LIMD1 protein levels, thus identifying two possible novel mechanisms of LIMD1 silencing. Transcription of LIMD1, like that of other HIF1α regulatory proteins, was enhanced when cells were exposed to hypoxia (1% O2), facilitated by HIF1α binding a hypoxic responsive element (HRE) within the promoter. At the molecular level, in vivo LIMD1 forms an endogenous complex with proline hydroxylase 2 (PHD2) and the von Hippel-Lindau (VHL) protein, with LIMD1 loss decreasing the efficiency of HIF1α degradation and impeding the resultant cellular adaptation to chronic hypoxia.
In summary these studies identified epigenetic silencing of LIMD1 as a possible explanation for LIMD1 protein loss in transformed cells. Furthermore, LIMD1 transcription was identified as being activated by PU.1 and enhanced by HIF1α, and a revised, LIMD1 integrated, model of HIF1α regulation is proposed.
|Item Type:||Thesis (PhD)|
|Faculties/Schools:||UK Campuses > Faculty of Medicine and Health Sciences > School of Biomedical Sciences|
|Deposited By:||Mr Daniel Foxler|
|Deposited On:||12 Sep 2012 09:47|
|Last Modified:||07 Jan 2013 14:59|
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