Investigation of the functional role of the transcription factor, CTCF, in the regulation of human Bax and p14ARF genes

• Main Author: Gretton, Svetlana
• Published: University of Essex 2013
• Subjects: 572.865
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589445

CTCF (CCCTC-binding factor) is a ubiquitously expressed, multifunctional and conserved ll-Zinc finger transcription factor. CTCF is involved in the regulation of various genes, including those responsible for proliferation and apoptosis, using different mechanisms. One such mechanisms is based on reversible poly(ADP-ribosyl)ation (PARylation) of CTCF at the CTCF target sites (CTSs) and requires cooperation between CTCF and the PARylation enzyme, PARP-l. The main aim of this study was to investigate the role ofCTCF, PARP-l and PARylation in the epigenetic regulation of a pro-apoptotic gene, Bax and tumour suppressor gene, p 14ARF. We found that in all cells, breast and non-breast, the levels of Bax mRNA and protein were similar, with chromatin in the active state. However, CTCF binding was enhanced at the Bax promoter in breast cancer cells and tumours. We propose that in breast cancer cells and tumours, possibly in cooperation with P ARP-l; CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis. Four breast cell lines with the wild type p14ARF gene but different levels of p14ARF mRNA expression were used as models to study p14ARF regulation by CTCF. The relationship was observed between the levels of p14ARF expression, active chromatin marks and CTCF and PARP-l binding. The presence of both proteins at the p14ARF CTS was associated with the active state of p14ARF, whereas the presence of CTCF but not P ARP-l was associated with the silent state of p14ARF. Additionally, in this study a novel matrix Bio Vyon™IProtein A was optimised, evaluated and used for chromatin immunoprecipitation (ChIP) assays with tissue samples. The studies regarding the role of CTCF PARylation in cell proliferation were initiated and preliminary results obtained indicating that importance of CTCF PARylation for its normal functions.