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Jin, Y, Zhong H, Vershon AK.  1999.  The Yeast a1 and Alpha2 Homeodomain Proteins do not Contribute Equally to Heterodimeric DNa Binding. Mol Cell Biol. 19:585-593. Abstract
In diploid cells of the yeast Saccharomyces cerevisiae, the alpha2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression. Although both proteins bind to the DNA, in the alpha2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro. This result brings up the question of the contribution of each protein in the heterodimer complex to the DNA-binding affinity and specificity. To determine the requirements for the a1-alpha2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-alpha2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro. Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the alpha2 or a1 protein. Interestingly, an alpha2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein. These results suggest that the specificity of alpha2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts. We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex. In contrast to the lack of a defective phenotype produced by mutations in the alpha2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-alpha2-mediated repression and DNA binding. This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex.
Zhong, H, Vershon AK.  1997.  The Yeast Homeodomain Protein MATalpha2 Shows Extended DNa Binding Specificity in Complex with Mcm1. J Biol Chem. 272:8402-8409. Abstract
The MATalpha2 (alpha2) repressor interacts with the Mcm1 protein to turn off a-cell type-specific genes in the yeast Saccharomyces cerevisiae. We compared five natural alpha2-Mcm1 sites with an alpha2-Mcm1 symmetric consensus site (AMSC) for their relative strength of repression and found that the AMSC functions slightly better than any of the natural sites. To further investigate the DNA binding specificity of alpha2 in complex with Mcm1, symmetric substitutions at each position in the alpha2 half-sites of AMSC were constructed and assayed for their effect on repression in vivo and DNA binding affinity in vitro. As expected, substitutions at positions in which there are base-specific contacts decrease the level of repression. Interestingly, substitutions at other positions, in which there are no apparent base-specific contacts made by the protein in the alpha2-DNA co-crystal structure, also significantly decrease repression. As an alternative method to examining the DNA binding specificity of alpha2, we performed in vitro alpha2 binding site selection experiments in the presence and absence of Mcm1. In the presence of Mcm1, the consensus sequences obtained were extended and more closely related to the natural alpha2 sites than the consensus sequence obtained in the absence of Mcm1. These results demonstrate that in the presence of Mcm1 the sequence specificity of alpha2 is extended to these positions.
Oh, H, Slattery M, Ma L, White KP, Mann RS, Irvine KD.  2014.  Yorkie Promotes Transcription by Recruiting a Histone Methyltransferase Complex.. Cell reports. AbstractWebsite
Hippo signaling limits organ growth by inhibiting the transcriptional coactivator Yorkie. Despite the key role of Yorkie in both normal and oncogenic growth, the mechanism by which it activates transcription has not been defined. We report that Yorkie binding to chromatin correlates with histone H3K4 methylation and is sufficient to locally increase it. We show that Yorkie can recruit a histone methyltransferase complex through binding between WW domains of Yorkie and PPxY sequence motifs of NcoA6, a subunit of the Trithorax-related (Trr) methyltransferase complex. Cell culture and in vivo assays establish that this recruitment of NcoA6 contributes to Yorkie's ability to activate transcription. Mammalian NcoA6, a subunit of Trr-homologous methyltransferase complexes, can similarly interact with Yorkie's mammalian homolog YAP. Our results implicate direct recruitment of a histone methyltransferase complex as central to transcriptional activation by Yorkie, linking the control of cell proliferation by Hippo signaling to chromatin modification.
Oh, H, Irvine KD.  2010.  Yorkie: the final destination of Hippo signaling. Trends in Cell Biology. 20:410-7. AbstractWebsite
The Hippo signaling pathway is a key regulator of growth during animal development, whereas loss of normal Hippo pathway activity is associated with a wide range of cancers. Hippo signaling represses growth by inhibiting the activity of a transcriptional co-activator protein, known as Yorkie in Drosophila and Yap in vertebrates. In the 5 years since the first report linking Yorkie to Hippo signaling, intense interest in this pathway has led to rapid increases in our understanding of the action and regulation of Yorkie/Yap, which we review here. These studies have also emphasized the complexity of Yorkie/Yap regulation, including multiple, distinct mechanisms for repressing its transcriptional activity, and multiple DNA-binding partner proteins that can direct Yorkie to distinct downstream target genes.