Figure 2

The proximal promoter of Eplin-α is regulated through actin. (A) The extended Eplin-α promoter confers regulation to a luciferase reporter gene. A genomic fragment covering nucleotides -1802 to +132 relative to the putative transcription start site of Eplin-α was cloned from murine liver tissue into the luciferase reporter plasmid pGL3. Following the indicated pretreatment with latrunculin B (5 μM, 30 min) and 7 hours of stimulation with serum (15%) or cytochalasin D (2 μM), transiently transfected NIH3T3 cells were lysed and the luciferase activity was determined as described [17]. (B) Schematic diagram of the genomic structure of the Eplin gene, and the promoter reporter constructs used. The Eplin-β reporter ranges from -1249 to +71, and the truncated Eplin-α fragments range from the indicated nucleotide to +284, relative to the transcription start site. (C) Analysis of the Eplin promoter reporter constructs by transient luciferase assays in NIH 3T3 fibroblasts. Shown is the mean relative luciferase activity, normalised to Renilla luciferase. Error bars, SEM (n = 3). (D) The proximal Eplin-α promoter is differentially regulated by actin binding drugs in mouse mammary epithelial EpH4 cells. Cells were transiently transfected with the Eplin-α (-915) promoter reporter construct, treated with cytochalasin, latrunculin, or jasplakinolide (0.5 μM, 7 h), and analysed as described [20]. Shown is the mean relative luciferase activity, with Error bars indicating half range. Asterisk, significant activation; double asterisk, significant repression (p < 0.05, unpaired student's t-test).