MECHANISM OF LEF-1:BETA-CATENIN TRANSACTIVATION

Salk Institute For Biological Studies, La Jolla CA

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Abstract

The Wnt/Wingless (Wg) signaling pathway plays a critical role in specifying cell fate decisions and cell proliferation in embryogenesis in many organisms, and inappropriate activation of the pathway plays an important in the etiology of several human cancers including colon carcinoma and melanoma. Signaling through this pathway stabilizes beta- catenin, which enters the nucleus and forms a heterodimer with LEF/TCF HMG proteins, providing a constitutively-active trans-activating domain. The principle aim of LEF/TCF HMG proteins, providing a constitutively- active trans-activation domain. The principle aim of these studies is to characterize the mechanism of trans-activation by LEF-1:beta cat on nucleosomal templates. Using a chromatin-based in vitro transcription system, we have shown that the amino terminus of LEF-1 inhibits binding to chromatin in a manner that is reversed by beta-catenin. We find that LEF: beta-cat transcription requires the co-activator p300 as well as other, as yet unidentified, co-activators present in nuclear extracts, and we have also identified in activity that blocks LEF: beta-cat activation in vitro. We propose to explore this regulated binding to chromatin and define the role of CBP/P300 and other transcription co-factors in LEF: beta-cat activation. First, we will map the chromatin recognition determinants in LEF-1, and assess whether the N-terminus of LEF-1 can act in trans or with other DNA-binding domains to impede chromatin recognition. First, we will map the chromatin recognition determinants in LEF-1, and assess whether the N-terminus of LEF-1 can act in trans or with other DNA-binding domains to impede chromatin recognition., we will also map the sites of acetylation in the absence and presence of beta- catenin. Structural studies of LEF-1 will be carried out with Dr. J. Noel. Second, we will assess the function of the beta-catenin trans-activation domain by mapping the regions of P300 that are recognized of the beta- cat CTARM and those that mediate beta-cat transcription in vitro. Using GST-pull down experiments with fractions that stimulate LEF: beta-cat activity in vitro, we will identify and characterize other factors that may interact with this region of beta-catenin to stimulate transcription. Third, we will examine the ability of different chromatin remodeling complexes to enhance LEF: beta-cat transcription and the binding of the LEF: beta- cat complex to nucleosomal DNA. We find that the catalytic subunit of ISWI remodeling complexes binds avidly to the CTARM, and will identify the interacting complex and test its ability to support LEF: beta- cat activation in vitro and in vivo. Finally, we will purify and characterize an inhibitory activity that blocks transcription by LEF: beta-cat in vitro. Collectively, these studies should enhance our understanding of the mechanism of transcription regulation by LEF/TCF: beta-catenin signaling complex.