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Analysis of the functions and the promoter structures of B-cell translocation gene-2 in rat cortical neurons

University of British Columbia

Previously data from out lab have shown that BTG2 is up-regulated in both in vitro and in vivo ischemic models. We hypothesize that BTG2 may play a neuro-protective role following glutamate-mediated activation of NMDAR, and that this neuro-protection depends on the collaboration of various transcription factors and cis-acting elements that are found on the BTG2 promoter. To determine the function of BTG2 , BTG2 was either over-expressed or reduced in rat cortical neurons, and treated with either NMDA or oxygen glucose deprivation (OGD). Over-expression of BTG2 reduced NMDA-induced cell death, as measured by LDH assay, and reduced activation of Caspase 3. In addition, CyclinDl mRNA level was significantly increased following NMDA treatment in cells with reduced BTG2 expression by siRNA. Thus, BTG2 appeared to have a neuro-protective function, possibly through down-regulation of CyclinDl expression. To better understand the mechanism that regulated BTG2 expression, we studied the BTG2 promoter by cloning various lengths of BTG2 promoter into a luciferase expression vector. The role of two specific cis-acting elements, a putative P53 binding element P53RE (-53/-94) (-53 to -94 relative to ATG) and a GAGA box, were investigated. While the isolated P53RE (-537-94) sequence functioned as a cis-activating and NMDA inducible element, it became repressive in the context of BTG2 promoter. Removal of P53RE (-53A94) and/or GAGA box not only increased the BTG2 promoter activity, but also made the promoter inducible to NMDA. Consistently, reduction of P53 expression with P53 siRNA led to a significant increase in basal BTG2 expression. Immuno-coprecipitation results showed that the P53 and the GAGA box binding protein were associated together. Taken together, our results suggest that P53 may associate with GAGA box binding protein to form a complex to repress BTG2 expression. A hypothetical mode of action is then proposed, showing that binding of P53 to the P53RE (-53/-94) may activate GAGA box caused suppression, which becomes dominant for BTG2 expression. This mode illustrates a possible mechanism that an transactivating factor P53 and its cis-activating binding site can be turned into a suppressor depending on the location of P53 binding site and the transcriptional regulator(s) P53 associated with.

Text

2011-03-23

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http://hdl.handle.net/2429/32757

Neuroscience

University of British Columbia

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