Abstract
beta-Catenin is a multifunctional mediator of cellular signaling and an oncogene. Nuclear beta-catenin, when complexed with members of the T-cell factor (TCF)/leukocyte enhancer factor family of DNA-binding proteins, mediates transcriptional activation important for embryonic development and adult cell homeostasis. Deregulation of intracellular levels of beta-catenin is an early event in the development of a variety of cancers. We observed that the proteins silencing mediator for retinoid and thyroid hormone receptor (SMRT) and the nuclear receptor corepressor (NCoR) are negative regulators of transcription induced by the beta-catenin-TCF4 complex. Overexpression of SMRT and NCoR attenuated the transcription of beta-catenin-TCF4-specific reporter gene and of CCND1, an endogenous beta-catenin target gene. Knockdown of endogenous SMRT or NCoR by short interfering RNA augmented the beta-catenin-TCF4-mediated reporter gene expression. Glutathione S-transferase pulldown experiments showed there was a direct physical association of SMRT and NCoR with both beta-catenin and TCF4. DNA-protein interaction studies revealed that the interactions between either SMRT or NCoR and beta-catenin or TCF4 occurred at the promoter regions of CCND1 and other target genes. These findings demonstrate an important role for corepressors SMRT and NCoR in the regulation of beta-catenin-TCF4-mediated gene transcription.
Highlights
When nuclear -catenin is of low abundance, T-cell factor (TCF)/LEF proteins that have DNA binding domains, but lack transcriptional activation function, act as transcriptional repressors, recruiting Groucho/transducin-like enhancer proteins, which in turn recruit histone deacetylase (HDAC) to repress the target gene promoters [4, 5]
We show that the SMRT and nuclear receptor corepressor (NCoR) directly interact with -catenin and TCF4, both in vitro and in vivo, and that overexpression of either SMRT or NCoR attenuates the transactivation of -catenin-TCF4 target genes, such as CCND1 by a mechanism dependent on the TCF4-binding element (TBE)
SMRT and NCoR Affect TCF4-mediated Reporter Gene Expression—To study the effect of corepressors SMRT and NCoR on TCF4-mediated transcription, we first analyzed the effect s-SMRT and NCoR have on TCF4-dependent reporter activity in CV-1 cells
Summary
Plasmids—The Renilla null luciferase reporter was purchased from Promega Corp. (Madison, WI), and pFR-LUC was from Stratagene (La Jolla, CA). Western Blotting—Cell extracts were prepared, and Western blotting analysis was performed as described previously [49, 50] using the following antibodies: anti--catenin (BD Transduction Laboratories), anti-TCF4 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-CCND1 (NeoMarker, Freeman, CA), anti-FLAG M2 (Sigma), anti-SMRT (BD Transduction Laboratories), anti-GAL4(DBD) (RK5C1) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-VP16 [] (Santa Cruz Biotechnology), anti-histone H1 (AE-4) (Santa Cruz Biotechnology), and anti--actin (Sigma). Cell lysates were centrifuged, resuspended in wash buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1ϫ complete mini protease inhibitor mixture), and incubated for 10 min at 4 °C. Sonicated samples were centrifuged, precleared by incubation with normal IgG and protein G-agarose/salmon sperm DNA beads, and subjected to immunoprecipitation with normal IgG or specific antibodies against TCF4 (Upstate Biotechnology, Lake Placid, NY), -catenin (BD Transduction Laboratories), SMRT (Santa Cruz Biotechnology), and NCoR (Santa Cruz Biotechnology) with rotation overnight at 4 °C. In figures the following symbols are used to represent p values: *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001; and ****, p Ͻ 0.0001
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