The Hpo pathway has emerged as a conserved signaling pathway that plays a critical role in controlling tissue growth and organ size. Despite the growing recognition of the importance of this pathway in development and cancer, the transcription factor that links the cytoplasmic components to the nuclear events has remained elusive and thus represents a major gap in the pathway. Our study demonstrates Sd is the missing transcription factor of the Hpo pathway based on several lines of genetic and biochemical evidence. First, Sd and Yki formed a transcriptional complex to activate a reporter gene in S2 cells and this transcriptional activity is inhibited by Hpo signaling. Furthermore, we demonstrated that Sd and Yki synergized in vivo to promote Hpo target gene expression and tissue overgrowth. Second and more importantly, loss of Sd function suppressed tissue overgrowth induced by Yki overexpression or loss-of-function mutations in hpo, sav, and wts. In addition, we found that Sd inactivation either by RNAi or a genetic mutation blocked the ectopic expression of Hpo responsive genes induced by excessive Yki activity. Third, RNAi knockdown of Sd phenocopied knockdown of Yki, which was manifested by reduced organ size and diminished expression of Hpo pathway-responsive genes. Fourth, a constitutively active form of Sd activated multiple Hpo pathway-responsive genes and promoted tissue overgrowth. Finally, Sd promoted Yki nuclear translocation and recruited Yki to the diap1 enhancer.
We generated several sd
null alleles to further explore the consequence of loss of Sd. We found that sd
null clones located in the wing pouch region exhibited growth deficit such that early-induced clones (48–72 hrs AEL) were eliminated by the end of late third instar. However, late-induced clones (72–96 hrs AEL) survived and exhibited diminished expression of diap1
. In contrast, early-induced clones were recovered in the notal region of wing discs and in eye discs without showing discernible change in Diap1 levels. However, a previous study showed that yki
mutant clones exhibited reduced diap1
expression in eye discs (Huang et al., 2005
). It is possible that low levels of residual Sd activity persist in sd
mutant clones, which are sufficient to support the basal expression of the Hpo target genes. Alternatively, Yki may act through another transcription factor to maintain the basal expression of Hpo target genes. Nevertheless, sd
null mutation suppressed the overgrowth phenotype and ectopic cycE
expression induced by excessive Yki activity, suggesting the residual Sd in sd
mutant clones was insufficient to support the elevated Yki activity.
The identification of Hpo pathway transcription factor provided an opportunity to assess direct transcriptional targets of the pathway. To this end, we characterized the diap1 enhancer and identified a 1.8 kb enhancer element critical for diap1 expression. This region contains a total of seventeen predicted Sd binding sites. Using the ChIP assay, we demonstrated that both Sd and Yki physically interacted with the 1.8 kb diap1 enhancer and the association of Yki with the diap1 enhancer was mediated by Sd. Our results suggest that Sd recruits Yki to the diap1 enhancer to activate its transcription.
It has been shown previously that Sd acts in conjunction with Vg to promote wing development by directly regulating the expression of wing patterning genes (Guss et al., 2001
; Halder et al., 1998
). Here we have demonstrated that Sd acts in conjunction with Yki to control organ size by regulating the expression of genes involved in cell proliferation, cell growth, and apoptosis. These observations raise an important question of how Yki-Sd and Vg-Sd transcriptional complexes specifically select their targets. One possibility is that Vg-Sd and Yki-Sd prefer to interact with distinct Sd binding sites. Indeed, a previous study showed that binding of Vg to Sd modulated the DNA binding selectivity of Sd (Halder and Carroll, 2001
). Another possibility is that target selectivity could be influenced by cofactors that bind in the vicinity of Sd binding sites. In support of this notion, previous studies have shown that wing specific enhancers contain both Sd binding sites and binding sites for transcription factors that mediate specific signaling pathways (Guss et al., 2001
; Halder et al., 1998
). It is also possible that Vg-Sd and Yki-Sd may share common targets. For example, diap1
could be activated by Vg-Sd in the wing pouch, which might explain why sd
mutant clones in this region exhibited diminished diap1
In principle, the Hpo pathway could regulate the activity of Yki-Sd transcriptional complex at several levels. For example, Hpo signaling could regulate the formation Yki-Sd complex or the recruitment of other factor(s) to the Yki-Sd transcriptional complex. Alternatively, Hpo signaling could regulate the nuclear-cytoplasmic transport of Yki. In support of the latter possibility, Yki exhibited elevated nuclear localization in wts
mutant clones (; Dong et al., 2007
). In addition, coexpression of Hpo with Yki depleted nuclear Yki in S2 cells, suggesting that Hpo signaling impedes nuclear localization of Yki and thereby limits the amount of active Yki-Sd transcriptional complex.
Mutating Yki S168 to Ala increased nuclear localization and growth promoting activity of Yki (; Dong et al., 2007
). In addition, Dong et al demonstrated that phosphorylation of Yki S168 was stimulated by Hpo (Dong et al., 2007
). Phosphorylation of Yki by Hpo signaling increased their association with 14-3-3, which was abolished by mutating Yki S168 to Ala (our unpublished observation; Dong et al., 2007
). As 14-3-3 often regulates nuclear-cytoplasmic shuttling of its interacting proteins (Muslin and Xing, 2000
), these observations suggest that Hpo signaling inhibits Yki at least in part by phosphorylating Yki S168, which promotes 14-3-3 binding and cytoplasmic sequestration of Yki ().
The Hpo pathway appears to restrict cell growth and control organ size in mammals (Camargo et al., 2007
; Dong et al., 2007
; Zhao et al., 2007
). The finding that Sd is critical for Yki-induced tissue growth has raised the interesting possibility that the effect of YAP in promoting tissue growth may rely on the TEAD/TEF family of transcription factors. Corroborating this hypothesis, TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with YAP (Vassilev et al., 2001
). Furthermore, YAP can bind to and stimulate the trans-activating activity of all four TEAD/TEF family members (Vassilev et al., 2001
). The TEAD/TEF family members exhibit overlapping but distinct spatiotemporal expression patterns and thus may have redundant but unique roles during development (Kaneko and DePamphilis, 1998
; Kaneko et al., 2007
; Yasunami et al., 1996
). It will be important to determine which TEAD/TEF family members are involved in the mammalian Hpo pathway and whether YAP employs distinct sets of TEAD/TEF transcription factors in different tissues. As abnormal activation of YAP is associated with multiple types of cancer (Dong et al., 2007
; Overholtzer et al., 2006
; Zender et al., 2006
; Zhao et al., 2007
), disrupting YAP-TEAD/TEF interaction may provide a new strategy for cancer therapeutics.