Figure 3. m18 activates the Raet1e promoter activity through an Sp-transcription factor binding element.

(A) A graphic representation of Raet1e promoter luciferase constructs used in (B). (B) Raet1e promoter activity was measured by quantifying luminescence in lysates of cells transfected with the indicated luciferase promoter constructs in combination with m18 or vector control. Data are represented as fold increase in luminescence over vector control. Data are represented as mean±SEM. Data are representative of three independent experiments. ****p<0.00005 (1 way ANOVA with Bonferroni’s multiple comparison post-test). (C) Alignment of the WT m18RE (top), Sp consensus binding sequence (middle) and mutant m18RE (bottom). (D) Raet1e promoter activity was measured in cells co-transfected with a luciferase construct containing WT Raet1e promoter or Raet1e promoter containing a mutation in the Sp-binding site (m18RE*) and either m18 or vector control. Data shows fold increase in luminescence over vector control. Data are represented as mean (±SEM). Data are representative of three independent experiments. *p<0.05 (Student’s T-test). (E) EMSA was performed on mouse fibroblast nuclear extracts incubated with radio-labeled m18RE (left panel) or radio-labeled Sp consensus sequence (right panel). The indicated non-radiolabeled oligos were added in 1000-fold excess before separation by non-denaturing PAGE. Data is representative of three experiments. (F) ChIP was performed on MCA-205 cells using the indicated antibodies and enrichment of the Raet1e promoter was assayed by qPCR. Values were normalized to input chromatin. Data are representative of three independent experiments. (G) Mouse fibroblasts were co-transfected with m18-RFP and DN-Sp1 GFP or GFP vector control and cells expressing both GFP and RFP were analyzed for RAE-1 expression by flow cytometry. Data is representative of three experiments. (H) Mouse fibroblasts were transfected with m18-GFP and treated with Mithramycin or vehicle control were analyzed for expression of RAE-1 by flow cytometry. Data is representative of three experiments.

DOI: http://dx.doi.org/10.7554/eLife.14749.005

Figure 3—figure supplement 1. m18 induces transcription from the Raet1e promoter independent of E2F binding sites.

(A) Mouse fibroblasts were transfected with m18 or vector control plasmids along with with luciferase reporter constructs containing the RAE-1 promoter (WT Rae-1) or a Rae-1 promoter with both E2F binding elements mutated (E2F DKO). Data are represented as fold difference between promoter construct transfected with m18 over promoter construct transfected with vector control. Values are shown as mean±SEM. Data are representative of three independent experiments. n.s.: Not significant (1-way ANOVA with Bonferroni’s multiple comparison post-test).

DOI: http://dx.doi.org/10.7554/eLife.14749.006

Figure 3—figure supplement 2. MCA-205 carcinoma cell line is inducible for RAE-1 expression by m18.

(A) MCA-205 cells stably tranduced with either vector control or m18 and analyzed for RAE-1 expression by flow cytometry. Data are representative of three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.14749.007