Figure 5: Sp1 recruits Hey1 to the USP11 promoter to induce transcriptional repression. | Nature Communications

Figure 5: Sp1 recruits Hey1 to the USP11 promoter to induce transcriptional repression.

From: USP11 regulates PML stability to control Notch-induced malignancy in brain tumours

Figure 5

(a) Schematic representation of the 5′-regulatory region of USP11 gene, the luciferase reporter and the ChIP primers used in this study. The positions of two E boxes and four Sp1-binding regions are indicated. (b) qChIP assay in U87 cells transfected with control vector or Flag-Hey1 using control IgG or anti-Flag antibody for immunoprecipitation and indicated primer set for quantitative PCR. (c) qChIP analysis in U87 cells stably expressing indicated shRNAs using control IgG or Hey1 antibody for immunoprecipitation. Primer set IV was used for qChIP assays thereafter. (d) Reporter activity assay of U87 cells transfected with control vector or Flag-Hey1 together with indicated reporter constructs. The luciferase activities in the Flag-Hey1-expressing cells were normalized with those in the control cells. (e) qChIP analysis in U87 cells with control or Sp1 antibody. (f) qChIP analysis in U87 cells expressing control or Sp1 shRNAs with indicated antibodies. The expression levels of Sp1 protein are shown on the top. (g) Re-ChIP analysis in U87 cells with indicated antibodies. (h) Promoter activity assay of U87 cells transfected with the wild-type luciferease reporter construct shown in a together with indicated siRNAs. The relative Hey1 and Sp1 mRNA levels in each group are shown on the right. (i) A model for the mechanism by which Hey1 represses USP11 transcription (see text). (j) qChIP analysis in U87 cells transfected with Hey1 shRNAs with indicated antibodies. Data in all panels are mean±s.d. (**P<0.01 and ***P<0.001 by t-test) of three independent experiments.

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