ABSTRACT
Various stresses and DNA-damaging agents trigger transcriptional activity of p53 by post-translational modifications, making it a global regulatory switch that controls cell proliferation and apoptosis. Earlier we have shown that the novel MAR-associated protein SMAR1 interacts with p53. Here we delineate the minimal domain of SMAR1 (the arginine-serine-rich domain) that is phosphorylated by protein kinase C family proteins and is responsible for p53 interaction, activation, and stabilization within the nucleus. SMAR1-mediated stabilization of p53 is brought about by inhibiting Mdm2-mediated degradation of p53. We also demonstrate that this arginine-serine (RS)-rich domain triggers the various cell cycle modulating proteins that decide cell fate. Furthermore, phenotypic knock-down experiments using small interfering RNA showed that SMAR1 is required for activation and nuclear retention of p53. The level of phosphorylated p53 was significantly increased in the thymus of SMAR1 transgenic mice, showing in vivo significance of SMAR1 expression. This is the first report that demonstrates the mechanism of action of the MAR-binding protein SMAR1 in modulating the activity of p53, often referred to as the "guardian of the genome."
Subject(s)
Arginine/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Serine/metabolism , Tumor Suppressor Protein p53/metabolism , Amino Acid Motifs , Animals , Arginine/genetics , Cell Cycle , Cell Cycle Proteins/genetics , Cell Nucleus/metabolism , DNA-Binding Proteins/genetics , Genes, Reporter , Mice , Mice, Transgenic , Microscopy, Confocal , Nuclear Proteins/genetics , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Serine/genetics , Thymus Gland/metabolismABSTRACT
Scaffold/matrix-associated region-1-binding protein (SMAR1) specifically interacts with the MARbeta sequence, which is located 400-bp upstream of the murine TCRbeta enhancer and is highly expressed during the DP stage of thymocyte development. To further analyze the functions of SMAR1, transgenic mice were generated that express SMAR1 in a tissue-independent manner. SMAR1-overexpressing mice exhibit severely altered frequency of the T cells expressing commonly used Vbetas (Vbeta5.1/5.2 and Vbeta8.1/8.2/8.3). The rearrangements of Vbeta5.1/5.2, Vbeta8.1/8.2/8.3 loci are also reduced in SMAR1 transgenic mice. The T cells in SMAR1 transgenic mice exhibit a mild perturbation at the early DN stage. SMAR1 transgenic mice exhibit hypercellular lymph nodes and spleen accompanied with prominent architectural defects in these organs. These results indicate that SMAR1 plays an important role in the regulation of T cell development as well as V(D)J recombination besides maintaining the architecture of the lymphoid organs.
Subject(s)
Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Genes, T-Cell Receptor beta , Nuclear Proteins/genetics , T-Lymphocytes/immunology , VDJ Recombinases/metabolism , Animals , DNA/genetics , DNA/isolation & purification , DNA Primers , Gene Rearrangement, T-Lymphocyte , Humans , Lymph Nodes/immunology , Mice , Mice, Transgenic , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Spleen/immunologyABSTRACT
Chromatin modulation at various cis-acting elements is critical for V(D)J recombination during T and B cell development. MARbeta, a matrix-associated region (MAR) located upstream of the T cell receptor beta (TCRbeta) enhancer (Ebeta), serves a crucial role in silencing Ebeta-mediated TCR activation. By DNaseI hypersensitivity assays, we show here that overexpression of the MAR binding proteins SMAR1 and Cux/CDP modulate the chromatin structure at MARbeta. We further demonstrate that the silencer function of MARbeta is mediated independently by SMAR1 and Cux/CDP as judged by their ability to repress Ebeta-dependent reporter gene expression. Moreover, the repressor activity of SMAR1 is strongly enhanced in the presence of Cux/CDP. These two proteins physically interact with each other and colocalize within the perinuclear region through a SMAR1 domain required for repression. The repression domain of SMAR1 is separate from the MARbeta binding domain and contains a nuclear localization signal and an arginine-serine (RS)-rich domain, characteristic of pre-mRNA splicing regulators. Our data suggest that at the double positive stage of T cell development, cis-acting MARbeta elements recruit the strong negative regulators Cux and SMAR1 to control Ebeta-mediated recombination and transcription.
