Mechanisms controlling differential promoter-occupancy by the yeast forkhead proteins Fkh1p and Fkh2p: implications for regulating the cell cycle and differentiation.

The roles of DNA and Mcm1p interactions in determining the overlapping and distinct functions of the yeast cell cycle regulatory transcription factors Fkh1p and Fkh2p were examined. Full-length recombinant Fkh1p and Fkh2p were purified and their binding to bona fide promoters examined in vitro. Each protein bound a variety of target promoters with similar specificity in vitro, consistent with the observation that these proteins bind common promoters in vivo. However, in vivo, the Fkh1p and Fkh2p occupied different target promoters to different extents, suggesting that each was primarily responsible for controlling a different set of genes. Additional in vitro studies provided a mechanistic explanation for this differential promoter-occupancy. Specifically, the Fkh2p, but not the Fkh1p, was capable of binding cooperatively with Mcm1p. The Mcm1p-Fkh2p cooperative binding was enhanced by, but did not require, the presence of a Mcm1p-binding site within a target promoter. Consistent with these data, Mcm1p was present at Fkh-controlled promoters in vivo regardless of whether they contained Mcm1p-binding sites, suggesting a role for Mcm1p at promoters not thought previously to be under Mcm1p control. Analysis of Fkh1p and Fkh2p binding to promoter targets in vivo by use of mutant strains indicated that the two proteins compete for promoter-occupancy at a number of target promoters. We postulate that Fkh1p and a stable Fkh2p/Mcm1p complex compete for binding to target promoters and that the levels and/or binding activity of Fkh1p, but not Fkh2p, are most limiting for promoter-occupancy in vivo. Interestingly, the in vitro DNA-binding assays, using a variety of promoter targets, revealed that bona fide Fkh target promoters contained two or more Fkh-binding sites that allowed the Fkh1p and Fkh2p proteins to form multiple protein-DNA complexes in vitro. Multiple Fkh-binding sites may be a distinguishing feature of bona fide Fkh promoters in yeast and other organisms.

Sir3-dependent assembly of supramolecular chromatin structures in vitro.

Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterized in vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p--chromatin interactions. Together these data indicate that rSir3p cross-links individual nucleosomal arrays into supramolecular assemblies whose physical properties transcend those of typical 10-nm and 30-nm fibers. Based on these data we hypothesize that Sir3p functions, at least in part, by mediating reorganization of the canonical chromatin fiber into functionally specialized higher order chromosomal domains.

Dynamic interaction of PTPmu with multiple cadherins in vivo.

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPmu associates with the cadherin-catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S. M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977- 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPmu interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPmu and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPmu and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPmu from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPmu in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513-1517) have asserted that the association we observed between PTPmu and the cadherin-catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPmu, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPmu obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPmu antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPmu and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.