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. 2009 Sep 25;284(39):26368-76.
doi: 10.1074/jbc.M109.021360. Epub 2009 Jul 24.

Phosphorylation of histone H3 by protein kinase C signaling plays a critical role in the regulation of the developmentally important TBX2 gene

Huajian Teng  1 , Reyna Deeya Ballim, Shaheen Mowla, Sharon Prince
Affiliations

Phosphorylation of histone H3 by protein kinase C signaling plays a critical role in the regulation of the developmentally important TBX2 gene

Huajian Teng et al. J Biol Chem. .

Abstract

The mechanism(s) regulating the expression of the TBX2 gene, a key regulator of development, is poorly understood and thus limits an understanding of its function(s). Here we demonstrate that 12-O-tetradecanoylphorbol-13-acetate (TPA) induces TBX2 expression in normal human fibroblasts in a protein kinase C (PKC)-dependent and MAPK-independent manner. Our data further reveal that TPA activates transcription of TBX2 through activating MSK1, which leads to an increase in phosphorylated histone H3 and the recruitment of Sp1 to the TBX2 gene. In addition, TPA was shown to activate MSK1 in a PKC-dependent and MAPK-independent manner. This study is the first to provide evidence that phosphorylation of histone H3 leads to the transcriptional activation of the TBX2 gene and to link MSK1 to PKC.

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Figures

FIGURE 1.
FIGURE 1.
Induction of TBX2 expression by TPA in human fibroblasts. WI-38 (A and C) and SVWI-38 and CT-1 (B and D) cells were treated with either vehicle (control) or TPA (100 nm) for the indicated times. Whole cell lysates and total RNA were harvested and subjected to Western blot (A and B) or real-time PCR (C and D) analyses, respectively. The bar graph in A compares the intensity of the TBX2 band in each sample normalized to the tubulin loading control.
FIGURE 2.
FIGURE 2.
Induction of TBX2 gene expression by TPA in WI-38 cells is PKC-dependent but MAPK-independent. A, cells were treated with either vehicle (control) or TPA (100 nm) for the indicated times and whole cell lysates harvested and subjected to Western blot analysis with the indicated antibodies. B, cells were pretreated with vehicle or inhibitors to PKC (20 μm GF109203X), ERK1/2 (25 μm PD98095), JNK (20 μm SP600125), and p38 (10 μm SB203580) for 1 h and then treated with TPA (100 nm) for 8 h. Western blots were carried out to detect levels of TBX2, and the bar graph compares the intensity of the TBX2 band in each sample normalized to the tubulin loading control. C, cells were treated as for B and Western blotting carried out with the indicated antibodies. D, WI-38 cells were treated with vehicle or TPA for 8 h, and nuclear proteins were prepared and subjected to Western blotting to detect phosphorylated JNK. N.S. represents a nonspecific band, which is included to show equal loading.
FIGURE 3.
FIGURE 3.
TPA, c-Jun, and JunB do not affect human TBX2 promoter activity. A and B, TPA-stimulated expression of c-Jun and JunB in human fibroblasts. WI-38, SVWI-38, and CT-1 cells were treated with either vehicle (control) or TPA (100 nm) for the indicated times. Whole cell lysates (the same used in Fig. 1, A and B) were subjected to Western blot analysis with the indicated antibodies. C, TBX2 promoter-luciferase reporter (500 ng) or a 4×AP-1-luciferase construct (50 ng) containing 4 AP-1 binding sites was co-transfected into SVWI-38 and CT-1 cells with the internal control pRL-TK (50 ng) in the presence or absence of TPA (100 nm for 12 h). Luciferase activity was measured 30 h post-transfection and normalized to Renilla luciferase activity. Relative luciferase activity was calculated by setting untreated promoter activity to 100%. Mean values (±) were calculated from three independent experiments. D, TBX2 promoter-luciferase reporter (500 ng) or the 4×AP-1-luciferase construct (50 ng) was co-transfected into HT1080 cells either with the empty pCMV (200 ng) vector or c-Jun or JunB (200 ng) expression vectors, and luciferase activity determined as in C above. Relative luciferase activity was calculated by setting the effect of empty pCMV vector on promoter activity to 100%. Mean values (±) were calculated from three independent experiments.
FIGURE 4.
FIGURE 4.
Induction of TBX2 expression by TPA in WI-38 cells is associated with histone H3-Ser-10 phosphorylation and recruitment of Sp1 to the TBX2 promoter. A, TPA-stimulated increase of global histone H3-Ser-10 phosphorylation and activation of MSK1. Cells were treated with either vehicle (control) or TPA (100 nm) for the indicated times. Whole cell lysates were harvested and subjected to Western blot analysis to detect phosphorylated histone H3-Ser-10 and MSK1 and tubulin was included as a loading control. For observing phosphorylated MSK1, proteins were separated on 6% SDS-PAGE and the patterns of electrophoretic retardation were observed. B, cells were pretreated with vehicle or inhibitors to PKC (20 μm GF109203X), ERK1/2 (25 μm PD98095), JNK (20 μm SP600125), and p38 (10 μm SB203580) for 1 h and then treated with TPA (100 nm) for 8 h. Western blots were carried out as described for A above. C, top schematic illustrates the region of TBX2 proximal promoter showing the putative Sp1 site (boxed). Arrows represent primer pairs used for PCR. Cells were treated with either vehicle (control) or TPA (100 nm) for 8 h, and ChIP assays were performed with antibodies against Sp1 or phospho-H3. Co-immunoprecipitated DNA was assayed by PCR with primer pairs indicated. Inputs and no antibody are positive and negative controls, respectively. D, co-immunoprecipitated DNA was assayed by real-time PCR and was normalized to input. Fold change values were calculated by setting untreated samples to 1. E, siRNA-mediated down-regulation of MSK1 abrogates the TPA-induced increase in phosphorylated histone H3-Ser-10 and activated TBX2 expression. WI-38 cells were treated with siRNA specific for MSK1 or a control siRNA for 72 h. Cells were treated with TPA (100 nm) for 8 h prior to harvesting protein, which were analyzed by Western blotting using antibodies specific for MSK1, phosphorylated histone H3-Ser-10, TBX2, and tubulin.
FIGURE 5.
FIGURE 5.
TPA has no effect on Sp1 expression but stabilizes its binding to the TBX2 promoter. A, WI-38 cells were treated with either vehicle (control) or TPA (100 nm) for the indicated times and cell lysates subjected to Western blot analysis to detect Sp1. B, as for A above but nuclei were prepared from WI-38 cells treated with either vehicle (control) or TPA (100 nm) for 8 h. N.S. represents a nonspecific band that is included to show equal loading. C, as for A above but cells were pretreated with vehicle or inhibitors to PKC (20 μm GF109203X), ERK1/2 (25 μm PD98095), JNK (20 μm SP600125), and p38 (10 μm SB203580) for 1 h prior to TPA treatment (8 h). D, for DAI analysis, biotinylated DNA fragments were generated by PCR using indicated primer pairs and immobilized on streptavidin beads. After incubation with nuclear extracts (40 μg), the DNA-bound Sp1 complexes were analyzed by gel electrophoresis followed by immunoblotting using an antibody to Sp1. Competition assays were performed in the presence of a 5-fold excess of unlabeled wild-type or mutant CCAAT-box DNA fragments.
FIGURE 6.
FIGURE 6.
Proposed model for TBX2 regulation by TPA. In response to TPA treatment, PKC activates MSK1 which in turn phosphorylates histone H3 leading to chromatin remodeling at the TBX2 promoter. This results in the recruitment of Sp1 to the TBX2 promoter and subsequent activation of TBX2 gene expression. Indicated in the model is the possibility that the SP600125 inhibitor may block MSK1 acitivity.
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