Heparan sulfate proteoglycans （HSPGs） are critically involved in a variety of biological events. The functions of HSPGs are determined by the nature of the core proteins and modifications of heparan sulfate （HS） glycosaminoglycan （GAG） chains. The distinct O-sulfo- transferases are important for nonrandom modifications at specific positions. Two HS 3-0 sulfotransferase （Hs3st） genes, Hs3st-A and Hs3st-B, were identified in Drosophila. Previous experiments using RNA interference （RNAi） suggested that Hs3st-B was required for Notch signaling. Here, we generated a null mutant of Hs3st-B via ends-out gene targeting and examined its role（s） in development. We found that homozygous Hs3st-B mutants have no neurogenic defects or alterations in the expression of Notch signaling target gene. Thus, our results strongly argue against an essential role for Hs3st-B in Notch signaling. Moreover, we have generated two independent Hs3st-A RNAi lines which worked to deplete Hs3st-A. Importantly, Hs3st-A RNAi combined with Hs3st-B mutant flies did not alter the expression of Notch signaling components, arguing that both Hs3st-A and Hs3st-B were not essential for Notch signaling. The establishment of Hs3st-B mutant and effective Hs3st-A RNAi lines provides essential tools for further studies of the physiological roles of Hs3st-A and Hs3st-B in development and homeostasis.
The proliferation and differentiation of adult stem cells must be tightly controlled in order to maintain resident tissue homeostasis. Dysfunction of stem cells is implicated in many human diseases, including cancer. However, the regulation of stem cell proliferation and differentiation is not fully understood. Here we show that the sterile-like 20 kinase, Tao, controls tissue homeostasis by regulating the Hippo pathway in the Drosophila adult midgut. Depletion of Tao in the progenitors leads to rapid intestinal stem cell （ISC） proliferation and midgut homeostasis loss. Meanwhile, we find that the Janus kinase （JAK）/signal transducer and activator of transcription （STAT） signaling activity and cytokine production are significantly increased, resulting in stimulated ISC proliferation. Furthermore, expression of the Hippo pathway downstream targets, Diapl and bantam, is dramatically increased in Tao knockdown intestines. Consistently, we show that the Yorkie （Yki） acts downstream of Tao to regulate ISC proliferation. Together, our results provide insights into our understanding of the mechanisms of stem cell proliferation and tissue homeostasis control.
The Hedgehog （Hh） signaling pathway plays important roles in developmental processes including pattern formation and tissue homeostasis. The seven-pass transmembrane receptor Smoothened （Smo） is the pivotal transducer in the pathway; it, and thus the pathway overall, is regulated by ubiquitin-mediated degradation, which occurs in the absence of Hh. In the presence of Hh, the ubiquitination levels of Smo are decreased, but the molecular basis for this outcome is not well understood. Here, we identify the deubiquitinase UCHL5 as a positive regulator of the Hh pathway. We provide both genetic and biochemical evidence that UCHL5 interacts with and deubiquitinates Smo, increasing stability and promoting accumulation at the cell membrane. Strikingly, we find that Hh enhances the interaction between UCHL5 and Smo, thereby stabilizing Smo. We also find that proteasome subunit RPN13, an activator of UCHL5, could enhance the effect of UCHL5 on Smo protein level. More importantly, we find that the mammalian counterpart of UCHL5, UCH37, plays the same role in the regulation of Hh signaling by modulating hSmo ubiquitination and stability. Our findings thus identify UCHL5/UCH37 as a critical regulator of Hh signaling and potential therapeutic target for cancers.
The highly conserved Notch signaling is precisely regulated at different steps in a series of developmental events. However, little is known about the regulation of Notch receptor at transcriptional level. Here, we demonstrate that dBrmsl is involved in regulating Notch signaling in Drosophila wing. We show that knockdown of dBrmsl by RNA interference （RNAi） in wing disc suppresses the expression of Notch signaling target genes wingless （wg）, cut and Enhancer of split m8 [E（spl）m8]. Consistently, the levels of Wg and Cut are reduced in the dBrmsl mutant clones. Importantly, loss of dBrmsl leads to significant reduction of Notch proteins. Furthermore, depletion of dBrmsl results in apparent downregulation of Notch transcription in the wing disc. Moreover, we find that dBrmsl is functionally conserved with human Breast cancer metastasis suppressor 1 like （hBRMSIL） in the modulation of Notch signaling. Taken together, our data provide important insights into the biological function of dBrmsl in regulating Notch signaling.
