Wnt signaling is crucial in the organization and maintenance of the human intestinal epithelium, and somatic mutations that result in deregulated Wnt signaling are an early event in the development of colorectal cancer. The Wnt ligand ultimately results in the stabilization of cytoplasmic -catenin, which is then free to enter the nucleus and activate transcription through its interaction with the transcription factor TCF4. Our laboratory recently found that KLF4, a transcription factor highly expressed in the adult intestine and critical for intestinal differentiation, interacts with -catenin and inhibits Wnt signaling. In this study, we characterize the molecular mechanisms of KLF4-mediated inhibition of Wnt/-catenin signaling. We find that the KLF4 directly interacts with the C-terminal transactivation domain of -catenin and inhibits p300/CBP recruitment by -catenin. KLF4 inhibits p300/CBP-mediated -catenin acetylation as well as histone acetylation on Wnt target genes. In addition, we observe that KLF4 directly interacts with TCF4 independently of -catenin and that KLF4 and TCF4 are expressed in similar patterns within the large intestine, with greatest staining near the epithelial surface. These results provide a deeper understanding of the regulation of -catenin in the intestine and will have important implications in cancer and stem cell research.The Wnt/-catenin signaling pathway plays important roles in early development (19, 31), stem cell renewal (22,34), and tumorigenesis (20,35). In addition, Wnt signaling is crucial in the organization and maintenance of the human intestinal epithelium (46, 51), and somatic mutations that result in deregulated Wnt signaling are an early event in the development of colorectal cancer (37,39,47). In this pathway, many different components work together to transduce an external signal into changes in gene expression within the target cell. Upon binding its receptor, the Wnt ligand ultimately results in the stabilization of cytoplasmic -catenin (44), which is then free to enter the nucleus and activate transcription through its interaction with the TCF/LEF family of transcription factors (1,18,36).In the adult intestine, TCF4 is the primary TCF/LEF family member involved in mediating active Wnt/-catenin signaling (25,26). In contrast, TCF1 and TCF3 are primarily transcriptional repressors (14, 49), whereas LEF1 is not expressed in the adult intestine (59). In the absence of -catenin, TCF4 recruits corepressors such as CtBP (4), HDAC1 (3, 24), and Groucho/ TLE (5, 28, 50), in order to silence expression of target genes. However, binding of -catenin results in displacement of these corepressors (7). -Catenin then recruits coactivators through its N-terminal and C-terminal transactivation domains. Its Nterminal transactivation domain interacts with BCL9/Legless, which in turn recruits Pygopus (2, 27, 45, 55), whereas its C-terminal domain recruits p300/CBP. p300 and CBP are closely related proteins that promote transcriptional activation through several mechan...