Matrix attachment region binding proteins have been shown to play an important role in gene regulation by altering chromatin in a stage-and tissue-specific manner. Our previous studies report that SMAR1, a matrix-associated protein, regresses B16-F1-induced tumors in mice. Here we show SMAR1 targets the cyclin D1 promoter, a gene product whose dysregulation is attributed to breast malignancies. Our studies reveal that SMAR1 represses cyclin D1 gene expression, which can be reversed by small interfering RNA specific to SMAR1. We demonstrate that SMAR1 interacts with histone deacetylation complex 1, SIN3, and pocket retinoblastomas to form a multiprotein repressor complex. This interaction is mediated by the SMAR1(160-350) domain. Our data suggest SMAR1 recruits a repressor complex to the cyclin D1 promoter that results in deacetylation of chromatin at that locus, which spreads to a distance of at least the 5 kb studied upstream of the cyclin D1 promoter. Interestingly, we find that the high induction of cyclin D1 in breast cancer cell lines can be correlated to the decreased levels of SMAR1 in these lines. Our results establish the molecular mechanism exhibited by SMAR1 to regulate cyclin D1 by modification of chromatin.The periodic movement of the cell cycle is orchestrated by programmed oscillations in the activity of cyclins, cyclin-dependent kinases (CDKs), and their target proteins, including pRb and E2F/DP1 complexes (34). In response to the mitogenic stimuli, normal cells exit G 1 phase and enter S phase by assembling a D type of cyclins with respective CDK partners. Cyclin D1, a G 1 -phase cyclin, belongs to a family of three closely related proteins termed cyclins D1, D2, and D3. These proteins are expressed in redundant fashion in all proliferating cells and collectively control cell cycle progression along with CDK4/CDK6. Cyclin D/CDK complexes further phosphorylate retinoblastoma (Rb) protein and release an E2F transcription factor that triggers progression into S phase. The activation of CDKs is dependent on their association with cyclin partners, while inactivation is dependent on CDK inhibitors. A fine control of cyclins and CDK inhibitors is set by both transcriptional and degradation mechanisms.A complex transcriptional regulatory mechanism has been shown to exist to coordinate the specific temporal profiles of cyclins (38). Previous studies have demonstrated that autoregulatory loops occur between CDKs and their substrate, cyclin D1 (17). Cyclin D1 is induced by several proteins in proliferative signaling and transformations, including Ras, Rac, and Stat5 (27, 41). Elevation of cyclin D1 mRNA in 50 to 70% of breast cancers, while failing to develop normal mammary glands in transgenic mice lacking both cyclin D1 alleles, associates its role in cancer as well as normal breast development (35,47). Aberrant cyclin D1 expression in the malignancies is attributed to gene amplification, loss of transcriptional control, and stabilization (23,40).The molecular mechanisms involved in cyclin D1 downregulat...

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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."

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.

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