Figure 8

NRF-1 loss attenuates GABPβ promoter activity and GABPβ/BRCA1 expression; NRF-1 is consistent between cell lines. (a) The transcriptional activity of the GABPβ promoter constructs -268, which contains the NRF-1 binding site, and -251 which does not, was assessed in MCF-7 cells via dual luciferase assay in the presence of siRNA against GAPDH (siGAPDH, negative control) and NRF-1 (siNRF-1). Promoter activity is expressed as relative light units. (b) The protein levels of NRF-1, GABPβ, GABPα, BRCA1 and TBP (internal control) were assessed by Western blot in whole cell lysates prepared from MCF-7 cells treated with siGAPDH or siNRF-1. (c) NRF-1 levels were determined by Western blot in MCF-7 and SK-BR-3 whole cell lysates. TBP was used as an internal control. Apparent molecular weight markers (kDa) are indicated to the right of the panels. (d) The activity of two GABPβ promoter constructs, Gb-270 multimer (which contains a triple repeat of the Gb-270 sequence specified in Figure 6) and -268 (see part a), was examined via dual luciferase assay in MCF-7 and SK-BR-3 cells following exogenous NRF-1 expression. Promoter activation by NRF-1 is expressed as a fold relative to empty vector controls in each cell line. (e) A ChIP assay was performed using MCF-7 and SK-BR-3 chromatin and antibodies against acetylated histone H3K9 (acH3), haemagglutinin (HA, negative control), RNA polymerase II (RNA pol II), histone deacetylase I (HDAC), NRF-1 and Oct-4 (transcription factor, negative control). PCR products obtained using primers specific to the GABPβ promoter (refer to Methods) are shown.