Hyangyee Oh

in vivo regulation of Yorkie phosphorylation and localization

Yorkie (Yki), a transcription factor of the Fat and Hippo signaling pathways, is negatively regulated by the Warts kinase. Here, we use Phos-tag gels to characterize Warts-dependent phosphorylation of Yki in vivo, and show that Warts promotes phosphorylation of Yki at multiple sites. We also show that Warts inhibits Yki nuclear localization in vivo, and can promote binding of Yki to 14-3-3 proteins in cultured cells. In vivo assessment of the influence of individual upstream regulators of Warts reveals that some mutants (e.g. fat) have only partial effects on Yki phosphorylation, and weak effects on Yki localization, whereas other genotypes (e.g. ex fat double mutants) have stronger effects on both Yki phosphorylation and localization. We also identify serine 168 as a critical site through which negative regulation of Yki by Warts-mediated phosphorylation occurs, but find that this site is not sufficient to explain effects of Hippo signaling on Yki in vivo. These results identify modulation of subcellular localization as a mechanism of Yki regulation, and establish that this regulation occurs in vivo through multiple sites of Warts-dependent phosphorylation on Yki.

In vivo analysis of Yorkie phosphorylation sites

The co-activator Yorkie (Yki) mediates transcriptional regulation effected by the Drosophila Fat–Warts (Wts)–Hippo (Hpo) pathways. Yki is inhibited by Wts-mediated phosphorylation, and a Wts phosphorylation site at Ser168 has been identified. Here we identify two additional Wts phosphorylation sites on Yki, and examine the respective contribution of all three sites to Yki nuclear localization and activity. Our results show that although Ser168 is the most critical site, all three phosphorylation sites influence Yki localization and activity in vivo, and can be sites of regulation by Wts. Thus, investigations of the role of Yki and its mammalian homolog Yes-associated protein (YAP) in development and oncogenesis should include evaluations of additional sites. The WW domains of Yki are not required for its phosphorylation, but instead are positively required for its activity. We also identify two potential sites of phosphorylation by an unknown kinase, which could influence phosphorylation of Ser168 by Wts, suggesting that there are additional mechanisms for regulating Yki/YAP activity.

Zyxin Links Fat Signaling to the Hippo Pathway

Using genetic and molecular analyses, the authors identify Zyx as a positive regulator of Hippo signaling and characterize its role within the pathway.

Yorkie: the final destination of Hippo signaling.

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.

Phosphorylation-independent repression of Yorkie in Fat-Hippo signaling

The Fat-Hippo signaling pathway plays an important role in the regulation of normal organ growth during development, and in pathological growth during cancer. Fat-Hippo signaling controls growth through a transcriptional co-activator protein, Yorkie. A Fat-Hippo pathway has been described in which Yorkie is repressed by phosphorylation, mediated directly by the kinase Warts and indirectly by upstream tumor suppressors that promote Warts kinase activity. We present here evidence for an alternate pathway in which Yorkie activity is repressed by direct physical association with three other pathway components: Expanded, Hippo, and Warts. Each of these Yorkie repressors contains one or more PPXY sequence motifs, and associates with Yorkie via binding of these PPXY motifs to WW domains of Yorkie. This direct binding inhibits Yorkie activity independently from effects on Yorkie phosphorylation, and does so both in vivo and in cultured cell assays. These results emphasize the importance of the relative levels of Yorkie and its upstream tumor suppressors to Yorkie regulation, and suggest a dual repression model, in which upstream tumor suppressors can regulate Yorkie activity both by promoting Yorkie phosphorylation and by direct binding.

Genome-wide Association of Yorkie with Chromatin and Chromatin-Remodeling Complexes

The Hippo pathway regulates growth through the transcriptional coactivator Yorkie, but how Yorkie promotes transcription remains poorly understood. We address this by characterizing Yorkies association with chromatin and by identifying nuclear partners that effect transcriptional activation. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF), the Brahma complex, and the Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkies transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin-binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of messenger RNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. Our studies suggest a widespread role for Yorkie as a regulator of transcription and identify recruitment of the chromatin-modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie.

An Evolutionarily Conserved Domain of roX2 RNA Is Sufficient for Induction of H4-Lys16 Acetylation on the Drosophila X Chromosome

The male-specific lethal (MSL) complex, which includes two noncoding RNA on X (roX)1 and roX2 RNAs, induces histone H4-Lys16 acetylation for twofold hypertranscription of the male X chromosome in Drosophila melanogaster. To characterize the role of roX RNAs in this process, we have identified evolutionarily conserved functional domains of roX RNAs in several Drosophila species (eight for roX1 and nine for roX2). Despite low homology between them, male-specific expression and X chromosome-specific binding are conserved. Within roX RNAs of all Drosophila species, we found conserved primary sequences, such as GUUNUACG, in the 3′ end of both roX1 (three repeats) and roX2 (two repeats). A predicted stem–loop structure of roX2 RNA contains this sequence in the 3′ stem region. Six tandem repeats of this stem–loop region (72 nt) of roX2 were enough for targeting the MSL complex and inducing H4-Lys16 acetylation on the X chromosome without other parts of roX2 RNA, suggesting that roX RNAs might play important roles in regulating enzymatic activity of the MSL complex.

Yorkie Promotes Transcription by Recruiting a Histone Methyltransferase Complex

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 Yorkies ability to activate transcription. Mammalian NcoA6, a subunit of Trr-homologous methyltransferase complexes, can similarly interact with Yorkies 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.

Variable splicing of non-coding roX2 RNAs influences targeting of MSL dosage compensation complexes in Drosophila.

The noncoding roX1 and roX2 RNAs are components of the MSL dosage compensation complex in Drosophila. We found that multiple species of roX2 RNA are produced by alternative splicing, with one major and at least 20 different minor forms associated with MSL proteins. The alternative forms are generated by variable usage of multiple 5’ and 3’ splice sites between two common exons. This alternative splicing is evolutionarily conserved in several distant Drosophila species in spite of differences in primary sequences. Transgenic constructs expressing individual major or minor D. melanogaster. roX2 species display low steady-state levels of roX2 RNA, weak accumulation of MSL complex on the X chromosome, and low rescue of male-specific roX- lethality. Increased expression of individual roX2 forms using the constitutive Hsp83 promoter results in increased transgenic rescue of roX- mutant male flies. However, although males survive they are delayed in their development. In addition, MSL complexes still show low affinity for the X chromosome and abnormal accumulation at the transgenic site of synthesis of the individual roX2 alternative splice form. Taken together, these results suggest an important role for roX2 RNA splicing in optimal MSL complex assembly or function.

MSL cis-spreading from roX gene up-regulates the neighboring genes

The male-specific lethal (MSL) complex in Drosophila melanogaster paints the male X chromosome in a manner that is both cis and trans to induce 2-fold hypertranscription of the X chromosome. To characterize the upregulation of gene expression by MSL cis-spreading, we measured the expressional change of neighboring genes by microarray when the genes were bound by MSL complexes that spread from an autosomal roX transgene. Genes within a 200kb region that includes roX transgenes were upregulated concurrently with MSL cis-spreading. Conversely, there was almost no expressional change in genes from other regions. RT-PCR and ChIP analyses confirmed that the approximately 2-fold gene hypertranscription was due to MSL cis-spreading. We also demonstrated that upregulation of the neighboring gene could rescue haplo-insufficient phenotypes of the Minute mutant, such as short bristle, delayed adult eclosion and decreased viability. These results indicate that the hypertranscription by MSL cis-spreading is a general mechanism that occurs in several tissue types. Our molecular and genetic data suggest that cis-spreading of the MSL complex from high-affinity sites including the roX gene results in upregulation of the neighboring genes, which are targets for dosage compensation in the male X chromosome.