Fig. 2.

Targeting MSK1 to the endogenous c-fos promoter induces H3 S10 and S28 phosphorylation and activates its expression. (A) Schematic diagram of the c-fos promoter showing the locations of cis-regulatory elements and primers used for ChIP analysis. CA- or KD-MSK1 was fused to the DBD of NF1 (residues 1–257) to directly target the H3 kinase to the endogenous c-fos promoter through its NF1-binding site. (B) Expression and kinase activity of different MSK1 constructs in 293T cells were examined by Western blotting using the indicated antibodies. Ponceau staining of core histones was used as loading control. (C) CA/KD-MSK1 constructs were transfected into 293T cells and their effects on expression of endogenous c-fos gene were determined by quantitative RT-PCR. c-fos mRNA levels were normalized to that of HPRT. (D) ChIP assays using an HA antibody showed that CA and KD versions of NF1-MSK1 fusions were both targeted to the promoter of endogenous c-fos gene. (E) ChIP analyses using antibodies against phosphorylated and total H3 showed induced H3 S10 and S28 phosphorylation at the c-fos promoter by NF1-CA-MSK1. Quantitative RT-PCR and ChIP–quantitative PCR results are represented as means ± SD (n = 3) and are representative of at least three independent experiments.