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. 2010 Jan 4;38(7):2201–2216. doi: 10.1093/nar/gkp1203

Figure 2.

Figure 2.

PRMT6 synergistically coactivates ERα transcriptional activity in the presence of SRC-1. (A) CV-1 cells were transiently co-transfected with an ERE-E1b-luciferase reporter, 5-ng expression vector for ERα, along with expression vectors for PRMT6 or SRC-1 as indicated. Following treatment with vehicle (ethanol) or 10–9 M E2, cells were assayed for luciferase activity. Numbers above bars show fold increase in luciferase activity compared to transfection with ERα alone and 10–9M E2 stimulation. Each data point represents the mean and SD of results from four transfected cultures. Results shown are from a single experiment, which is representative of three independent experiments. (B) Mammalian-2-hybrid analysis demonstrates that PRMT6 interacts with full-length SRC-1. HeLa cells were co-transfected with pG5-E1b-luciferase reporter plasmid along with expression vectors for the Gal4 DNA-binding domain alone (Gal4), Gal4-SRC-1 chimera, the VP16 transcriptional activation domain alone (VP16) or VP16-PRMT6 chimera as indicated. Cell extracts were tested for luciferase activity. Each data point represents the mean and SD of results from four transfected cultures. Results shown are from a single experiment, which is representative of three independent experiments. *P < 0.001. (C) GST pull-down assays were used to test the ability of GST-PRMT6 to interact with full length or fragments of 35S-radiolabelled SRC-1. GST alone and in vitro translated 35S-radiolabelled luciferase served as negative controls. A schematic representation of the SRC-1 protein fragments used in the assays is shown in the panel, with amino acid positions of the SRC-1 protein or SRC-1 protein fragments indicated. The major functional domains of SRC-1 are indicated, bHLH/PAS, NR Boxes, AD1 and AD2. (D) Recruitment of PRMT6 to oestrogen response elements (EREs) located in promoter regions of the GREB1 gene. Following 0-, 15- and 45-min treatment of MCF-7 cells with 10−9 M E2, recruitment of PRMT6 to EREs in the GREB1 promoter and to a site downstream of the GREB1 transcriptional start site (+6 kb) was determined by chromatin immunoprecipitation as detailed in ‘Materials and Methods’ section. Results show the average and SD of four independent experiments. (E) Recruitment of PRMT6 to an oestrogen-receptor-binding enhancer region of the PR gene. Following 0-, 15- and 45-min treatment of MCF-7 cells with 10−9 M E2, recruitment of PRMT6 to a known oestrogen-receptor-binding enhancer region of the PR gene (PR ERE) and to a site downstream of the PR gene transcriptional start site (+5 kb) was determined by chromatin immunoprecipitation as detailed in ‘Materials and methods’ section. Results show the average and SD of four independent experiments.