HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H)
2021; Springer Nature; Volume: 22; Issue: 4 Linguagem: Inglês
10.15252/embr.202051298
ISSN1469-3178
AutoresJin Sun, Xia Wang, Rong‐Gang Xu, Decai Mao, Da Shen, Xin Wang, Yuhao Qiu, Yuting Han, Xinyi Lu, Yutong Li, Qinyun Che, Zheng Li, Ping Peng, Xuan Kang, Ruibao Zhu, Yu Jia, Yinyin Wang, Luping Liu, Zhijie Chang, Jun‐Yuan Ji, Zhao Wang, Qingfei Liu, Shao Li, F Sun, Jian‐Quan Ni,
Tópico(s)Invertebrate Immune Response Mechanisms
ResumoArticle17 February 2021free access Transparent process HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H) Jin Sun Jin Sun orcid.org/0000-0003-2684-0078 Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, ChinaThese authors contributed equally to this work Search for more papers by this author Xia Wang Xia Wang Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China School of Life Sciences, Peking University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Rong-Gang Xu Rong-Gang Xu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Decai Mao Decai Mao Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Sichuan Academy of Grassland Science, Chengdu, ChinaThese authors contributed equally to this work Search for more papers by this author Da Shen Da Shen Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Xin Wang Xin Wang Institute for TCM-X, MOE Key Laboratory of Bioinformatics/Bioinformatics Division, BNRIST, Department of Automation, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Yuhao Qiu Yuhao Qiu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Yuting Han Yuting Han orcid.org/0000-0001-8156-2678 Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Xinyi Lu Xinyi Lu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Yutong Li Yutong Li Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Qinyun Che Qinyun Che Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Li Zheng Li Zheng Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Ping Peng Ping Peng Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China Search for more papers by this author Xuan Kang Xuan Kang Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Advanced Institute of Translational Medicine, Tongji University, Shanghai, China Search for more papers by this author Ruibao Zhu Ruibao Zhu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China Search for more papers by this author Yu Jia Yu Jia Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China Search for more papers by this author Yinyin Wang Yinyin Wang State Key Laboratory of Membrane Biology, School of Medicine and the School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Lu-Ping Liu Lu-Ping Liu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Zhijie Chang Zhijie Chang State Key Laboratory of Membrane Biology, School of Medicine and the School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Jun-Yuan Ji Jun-Yuan Ji Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX, USA Search for more papers by this author Zhao Wang Zhao Wang School of Pharmaceutical Sciences, Tsinghua University, Beijing, China Search for more papers by this author Qingfei Liu Qingfei Liu School of Pharmaceutical Sciences, Tsinghua University, Beijing, China Search for more papers by this author Shao Li Corresponding Author Shao Li [email protected] orcid.org/0000-0002-8709-9167 Institute for TCM-X, MOE Key Laboratory of Bioinformatics/Bioinformatics Division, BNRIST, Department of Automation, Tsinghua University, Beijing, China Search for more papers by this author Fang-Lin Sun Corresponding Author Fang-Lin Sun [email protected] orcid.org/0000-0003-1174-6468 Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Advanced Institute of Translational Medicine, Tongji University, Shanghai, China Search for more papers by this author Jian-Quan Ni Corresponding Author Jian-Quan Ni [email protected] orcid.org/0000-0001-9332-8440 Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsingdao Advanced Research Institute, Tongji University, Qingdao, China Search for more papers by this author Jin Sun Jin Sun orcid.org/0000-0003-2684-0078 Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, ChinaThese authors contributed equally to this work Search for more papers by this author Xia Wang Xia Wang Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China School of Life Sciences, Peking University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Rong-Gang Xu Rong-Gang Xu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Decai Mao Decai Mao Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Sichuan Academy of Grassland Science, Chengdu, ChinaThese authors contributed equally to this work Search for more papers by this author Da Shen Da Shen Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Xin Wang Xin Wang Institute for TCM-X, MOE Key Laboratory of Bioinformatics/Bioinformatics Division, BNRIST, Department of Automation, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Yuhao Qiu Yuhao Qiu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Yuting Han Yuting Han orcid.org/0000-0001-8156-2678 Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Xinyi Lu Xinyi Lu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Yutong Li Yutong Li Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Qinyun Che Qinyun Che Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Li Zheng Li Zheng Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Ping Peng Ping Peng Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China Search for more papers by this author Xuan Kang Xuan Kang Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Advanced Institute of Translational Medicine, Tongji University, Shanghai, China Search for more papers by this author Ruibao Zhu Ruibao Zhu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China Search for more papers by this author Yu Jia Yu Jia Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China Search for more papers by this author Yinyin Wang Yinyin Wang State Key Laboratory of Membrane Biology, School of Medicine and the School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Lu-Ping Liu Lu-Ping Liu Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Zhijie Chang Zhijie Chang State Key Laboratory of Membrane Biology, School of Medicine and the School of Life Sciences, Tsinghua University, Beijing, China Search for more papers by this author Jun-Yuan Ji Jun-Yuan Ji Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX, USA Search for more papers by this author Zhao Wang Zhao Wang School of Pharmaceutical Sciences, Tsinghua University, Beijing, China Search for more papers by this author Qingfei Liu Qingfei Liu School of Pharmaceutical Sciences, Tsinghua University, Beijing, China Search for more papers by this author Shao Li Corresponding Author Shao Li [email protected] orcid.org/0000-0002-8709-9167 Institute for TCM-X, MOE Key Laboratory of Bioinformatics/Bioinformatics Division, BNRIST, Department of Automation, Tsinghua University, Beijing, China Search for more papers by this author Fang-Lin Sun Corresponding Author Fang-Lin Sun [email protected] orcid.org/0000-0003-1174-6468 Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Advanced Institute of Translational Medicine, Tongji University, Shanghai, China Search for more papers by this author Jian-Quan Ni Corresponding Author Jian-Quan Ni [email protected] orcid.org/0000-0001-9332-8440 Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China Tsingdao Advanced Research Institute, Tongji University, Qingdao, China Search for more papers by this author Author Information Jin Sun1,2, Xia Wang1,3, Rong-Gang Xu1, Decai Mao1,4, Da Shen1, Xin Wang5, Yuhao Qiu1,6, Yuting Han1, Xinyi Lu1, Yutong Li1, Qinyun Che1, Li Zheng1, Ping Peng1,6, Xuan Kang7, Ruibao Zhu1,6, Yu Jia1,6, Yinyin Wang8, Lu-Ping Liu1, Zhijie Chang8, Jun-Yuan Ji9, Zhao Wang10, Qingfei Liu10, Shao Li *,5, Fang-Lin Sun *,7 and Jian-Quan Ni *,1,11 1Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing, China 2Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, China 3School of Life Sciences, Peking University, Beijing, China 4Sichuan Academy of Grassland Science, Chengdu, China 5Institute for TCM-X, MOE Key Laboratory of Bioinformatics/Bioinformatics Division, BNRIST, Department of Automation, Tsinghua University, Beijing, China 6Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China 7Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Advanced Institute of Translational Medicine, Tongji University, Shanghai, China 8State Key Laboratory of Membrane Biology, School of Medicine and the School of Life Sciences, Tsinghua University, Beijing, China 9Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX, USA 10School of Pharmaceutical Sciences, Tsinghua University, Beijing, China 11Tsingdao Advanced Research Institute, Tongji University, Qingdao, China *Corresponding author. E-mail: [email protected] *Corresponding author. E-mail: [email protected] *Corresponding author. E-mail: [email protected] EMBO Reports (2021)22:e51298https://doi.org/10.15252/embr.202051298 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Notch signaling and epigenetic factors are known to play critical roles in regulating tissue homeostasis in most multicellular organisms, but how Notch signaling coordinates with epigenetic modulators to control differentiation remains poorly understood. Here, we identify heterochromatin protein 1c (HP1c) as an essential epigenetic regulator of gut homeostasis in Drosophila. Specifically, we observe that HP1c loss-of-function phenotypes resemble those observed after Notch signaling perturbation and that HP1c interacts genetically with components of the Notch pathway. HP1c represses the transcription of Notch target genes by directly interacting with Suppressor of Hairless (Su(H)), the key transcription factor of Notch signaling. Moreover, phenotypes caused by depletion of HP1c in Drosophila can be rescued by expressing human HP1γ, suggesting that HP1γ functions similar to HP1c in Drosophila. Taken together, our findings reveal an essential role of HP1c in normal development and gut homeostasis by suppressing Notch signaling. Synopsis Heterochromatin protein 1c regulates gut development and homeostasis in Drosophila melanogaster by suppressing Notch signaling pathways. HP1c directly interacts with Su(H) to repress Notch target genes. Abnormal expression of HP1c results in developmental phenotypes resembling Notch dysregulation. Depletion of HP1c increases Notch target gene transcription. A direct interaction with Su(H) is essential for HP1c to repress Notch target genes. Expression of human HP1γ rescues phenotypes caused by HP1c depletion of in Drosophila. Introduction In addition to the primary functions of digestion and absorption, intestinal epithelium plays an important role in resistance to tissue injury, inflammation, and other adverse conditions. Disruption of intestinal homeostasis can lead to diseases such as gastrointestinal tumors. In Drosophila, intestinal stem cells (ISCs) reside along the midgut to maintain tissue homeostasis (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006). An ISC divides asymmetrically, giving rise to one ISC (for self-renewal) and one committed progenitor enteroblast (EB), which further differentiates into either an absorptive enterocyte (EC) or a secretory enteroendocrine cell (ee cell) (Lemaitre & Miguel-Aliaga, 2013). A variety of signaling pathways, including Notch, JAK/STAT, Wnt, EGFR/Ras, Hippo, Hedgehog, and BMP, are involved in regulating the self-renewal and differentiation of ISCs under either normal homeostasis or stress conditions (Biteau et al, 2011; Jiang & Edgar, 2011; Pasco et al, 2015). Of these signaling pathways, the highly conserved Notch is critical in maintaining intestinal homeostasis (Ohlstein & Spradling, 2007; Fre et al, 2011). Accordingly, dysregulation of Notch signaling causes abnormal intestinal cell lineage development and uncontrolled intestinal cell growth. Specifically, reduced Notch signaling in ISCs disrupts stem cell differentiation, resulting in fewer ECs and more ee cells, as well as the aggregation of ISCs and the formation of gut tumors (Micchelli & Perrimon, 2006; Ohlstein & Spradling, 2006; Perdigoto et al, 2011). Conversely, hyperactive Notch signaling forces ISCs to differentiate into ECs, leading to the loss of ISCs and ee cells (Ohlstein & Spradling, 2007; Goulas et al, 2012). The Notch signaling pathway is initiated by a ligand–receptor interaction between two adjacent cells. The receptor Notch then goes through two steps of a proteolytic cleavage process, resulting in the release of the intracellular domain of the Notch protein (NICD), which translocates into the nucleus. Subsequently, NICD binds to the CSL-type DNA-binding transcription factor and recruits transcription coactivators to regulate the expression of Notch target genes. In Drosophila, the CSL-type DNA-binding protein Su(H) (Suppressor of Hairless) orchestrates transcription cofactors such as Hairless, CtBP, and Groucho to control the transcription of Notch target genes (Mummery-Widmer et al, 2009; Saj et al, 2010; Guarani et al, 2011; Mulligan et al, 2011; Maier et al, 2013; Contreras-Cornejo et al, 2016; Han et al, 2016; Nagel et al, 2016). However, due to many interconnections with other signaling pathways and complicated outcomes, the mechanisms by which these transcription cofactors, particularly epigenetic factors, regulate the expression of Notch target genes in the nucleus remain unclear. The heterochromatin protein 1 (HP1) family is highly conserved epigenetic proteins in eukaryotes, and their functional diversity is further achieved through multiple variants (Canzio et al, 2014). There are five HP1 variants in Drosophila, HP1a-e, with HP1a being extensively studied as a silencing epigenetic factor (Vermaak & Malik, 2009; Canzio et al, 2014), and HP1c as a euchromatic protein (Kwon & Workman, 2011). Previous results showed that HP1c interacts with zinc finger-containing DNA-binding proteins WOC (without children) and ROW (relative of WOC) to form a complex (Font-Burgada et al, 2008; Abel et al, 2009), which may be involved in RNA polymerase II pausing (Kwon et al, 2010; Kessler et al, 2015). Therefore, HP1c has been generally recognized as a promising epigenetic regulator of gene transcription (Kwon & Workman, 2011). However, the role of HP1c in development and gene expression regulation remains poorly understood. Here, we report our analyses of the key role of HP1c in regulating development and intestinal homeostasis through Notch signaling in Drosophila. Our results indicate that HP1c physically interacts with Su(H) to suppress the transcription of Notch target genes. Results HP1c-specific phenotypes resemble Notch perturbations To elucidate functional genomic abnormalities underlying human gut tumorigenesis, we conducted genomic disorder analyses including disease-related gene prediction and differential gene expression analyses. We identified 178 highly conserved high-risk genes in both human colorectal and gastric cancers by using CIPHER (average top 5% in the rank list) (Fig 1A and B, Dataset EV1), a state-of-the-art network-based prediction for prioritizing disease genes in a genome-wide scale (Wu et al, 2008). To validate the functions and conserved roles of these genes in gut homeostasis in vivo, we chose to use Drosophila as a model organism. Drosophila gut is an ideal system to study the molecular mechanisms of tumorigenesis because of its relative simplicity in comparison with mammalian systems, the availability of sophisticated genetic tools, and the functional conservation of genes and signaling pathways. Notch signaling plays a critical role in driving ISCs to replenish the loss of absorptive and secretory cells in both Drosophila and mammals, and dysregulated Notch signaling is closely associated with gut homeostasis (Koch & Radtke, 2007; Geissler & Zach, 2012; Nowell & Radtke, 2017). Therefore, to determine whether any of these 178 high-risk genes are involved in regulating Notch signaling, we generated Drosophila transgenic RNAi lines against their human homologs. Figure 1. HP1c is involved in Notch-dependent Drosophila notum bristle and wing development A. Workflow of the network-based analysis in silico and functional screen in vivo. B. The number of high-risk genes (DIOPT score ≥ 5) in colorectal cancer and gastric cancer from genome-wide prediction (total: 17,903 genes). C–G. Resembling the phenotype caused by Notch overexpression (D) or dSir2 mutation (E), HP1c knockdown (F), and mutant (G) flies also exhibits supernumerary anterior scutellar (aSC) bristles on the notum, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm. H–M. Compared with control flies (H and K), HP1c knockdown (J) and mutant (M) flies exhibit an ectopic vein at the distal of L4 and L5 (arrows) and a broken posterior cross vein (arrowhead), which is similar to the phenotype caused by Notch overexpression (I) and dSir2 mutant (L). Scale bars, 500 μm. Download figure Download PowerPoint Given that the development of Drosophila notum bristles is sensitive to Notch signaling, we performed a phenotypic screen to test whether depleting the fly homologs of these high-risk genes affects the development of the mechanosensory bristles on the notum using pnr-Gal4, which is specifically expressed in the central region of the notum (Fig 1A and B, and Dataset EV1). Compared to the control carrying one anterior scutellar (aSC) bristle (see dotted square in Fig 1C), gain of Notch signaling with CRISPR/dCas9-based transcriptional activation system (Jia et al, 2018), or loss of Notch signaling repressor dSir2 (Mulligan et al, 2011), caused additional aSC bristles (double-bristles) in the same region (Figs 1D and E, and EV1A). Thirty-five genes show the gain of bristles, loss of bristles, or hair cell duplication phenotypes; however, most of them are involved in DNA replication, protein synthesis, or signaling pathways such as Ras, Wnt. One of the remaining genes, HP1c, which is homologous to human HP1γ (i.e., Chromobox 3, CBX3), is known as transcriptional regulators, but the developmental role is unclear. Interestingly, from this genetic screen we observed that depleting HP1c, which is homologous to human HP1γ (i.e., Chromobox 3, CBX3), resulted in ectopic aSC bristles in the flies (Figs 1F and EV1A, Dataset EV1). This effect resembles the phenotypes caused by Notch activation and dSir2 mutation. To further validate the effects of HP1c on aSC bristles, we generated an HP1c null mutant fly using the CRISPR/Cas9 system (Ren et al, 2013; Ren et al, 2014; Ren et al, 2017). Consistent with HP1c depletion, 70% of HP1c null mutant flies also displayed extra aSC bristles (Figs 1G and EV1A). To exclude potential off-target effects, we performed rescue experiments and found that the expression of wild-type HP1c can completely rescue the ectopic aSC bristle phenotype caused by either depletion or loss of HP1c (Fig EV1-EV5B–D). These observations revealed a novel function of HP1c in regulating aSC bristle development, indicating the possibility that HP1c might interact with Notch signaling to control aSC bristle development similarly to dSir2. Click here to expand this figure. Figure EV1. HP1c is associated with Notch-dependent Drosophila notum bristle development A. Statistical results for the phenotypes with supernumerary anterior scutellar (aSC) bristles shown in Fig 1C–G. Data are evaluated with two-tailed Student’s t-test (**P < 0.01, ***P < 0.001, ****P < 0.0001, n = 6 biological replicates, mean ± s.d.). B–D. HP1c cDNA expression can rescue the ectopic bristle phenotype from both HP1c KD and mutant. Error bars indicate s.d. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test). Scale bars, 100 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. HP1c and Notch signaling are both required for intestine homeostasis in Drosophila A. The number of ISCs and ee cells per unit area (200 μm × 200 μm) in the midguts of indicated flies. More than thirty midguts were examined for each group. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test). B. Statistic results of H3S10p positive (PH3+) cell numbers in midguts from wild-type and HP1c mutant flies (n = 15 biological replicates, mean ± SEM). Data are evaluated with one-tailed Student’s t-test. C–E. Images of midguts with indicated genotypes. The signal of cleaved Caspase 3 (red) in the HP1c knockdown midgut (E) is comparable with that in the GFP knockdown control (C), while the signal dramatically increased in the HP1a knockdown midgut (D). More than twenty midguts were examined for each group. Scale bars, 20 μm. F–H. Compared with the posterior midguts of 10-day-old control flies (F), Notch KD (G) and Notch OE (H) flies exhibit abnormal ISCs and ee cells’ numbers. Arrows mark ISCs, arrowheads mark ee cells, and circles indicate ISC-ISC, ISC-EB, or EB-EB nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm. I. Quantification of clone size in Fig 2E–I. n = 45–86 clones. J. Quantification of the percentage of each cell type within clones in Fig 2E–I. n = 76–199 cells. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. HP1c negatively regulates Notch signaling and interacts with Su(H) A. Quantification of the number of ISCs and ee cells per unit area (200 μm × 200 μm) in Fig 3A–D. More than thirty-five midguts were examined for each group. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-tailed t-test). B–D. Images of wings from flies with indicated genotypes. Scale bars, 500 μm. E. qRT-PCR analysis determined the expression of Notch target genes. F. DamID results show the binding of HP1c on Notch target genes. G. NRE-luciferase results for Su(H) or HP1c KD using S2 cells. H. NRE-luciferase results for Su(H), HP1c KD or HP1c OE in the background of NICD OE (Notch signaling hyper-activation) using S2 cells. Data in (E), (F), (G), and (H) are evaluated with two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3 technical replicates, mean ± s.d.). I. Co-immunoprecipitation of HP1c from s2 cells transfected with plasmids expressing Flag-tagged Su(H). Immunoprecipitation and Western blotting were performed with the indicated antibodies. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. The role of HP1c is specific among HP1 family members and EGFR-independent A–C. Images of the posterior midguts with indicated genotypes. Compared with the control flies (A), posterior midguts of HP1c KD flies exhibit decreased progenitor cells (B), while HP1b KD has no obvious influence on progenitor numbers. (C). Scale bars, 20 μm. D. The number of progenitors per unit area (200 μm × 200 μm) in the midguts of corresponding genotype flies. More than thirty midguts were examined for each genotype. Error bars indicate SD. ***P < 0.001 (two-tailed t-test). E–H. Images of wings from flies with the indicated genotypes. Compared with control flies (E), EGFR knockdown (F) flies exhibit defects in vein formation. HP1c KD (G) and overexpression (H) cannot rescue the vein phenotype from EGFR KD. Scale bars, 500 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Model of HP1c regulating the Notch signaling pathway In the absence of NICD, both HP1c and other repressors interact with different domains of Su(H) to coordinately repress the transcription of Notch target genes. Loss of HP1c/HP1γ affects the balance of this transcriptional repression, resulting in the mild activation of Notch signaling. When NICD is present, it interacts with Su(H) to compete with and replace other transcription repressors, the loss of repressors in Su(H) complex will trigger the transcription of Notch target genes and then require more Su(H) accumulate on Notch target genes, where the increasing of HP1c to prevent the hyper-activation of Notch signaling. If we reduce HP1c in this circumstance, it can further active the transcription of Notch target genes to hyper-transcription status. Download figure Download PowerPoint Given that Notch signaling and dSir2 also regulate wing morphogenesis in Drosophila (Mulligan et al, 2011), we wondered whether this was the case for HP1c. Using the MS1096-Gal4 line, which is expressed in the entire wing imaginal disk, we observed that gain of Notch signaling caused ectopic veins from the distal fourth and fifth longitudinal veins and disrupted posterior cross veins (PCV) (Fig 1H and I). Similar phenotypes were observed in approximately 80% of the wings from dSir2 mutants (Fig 1K and L), which is consistent with the previous report (Mulligan et al, 2011). Interestingly, almost all of the flies with either the HP1c depletion or HP1c mutation displayed ectopic veins from the distal of the fourth and fifth longitudinal veins and broken PCV (Fig 1J and M). These phenotypes also resemble the effects of gain of Notch signaling or loss of dSir2, implying a similar function for HP1c and dSir2 in negatively modulating Notch signaling during notum bristle and wing development. HP1c, as well as Notch signaling, regulates Drosophila gut homeostasis Considering that HP1γ is identified as a potential high-risk gene in human colorectal and gastric cancers (Fig 1 and Dataset EV1) and that Notch signaling plays a critical role in gut homeostasis (Ohlstein & Spradling, 2007; Fre et al, 2011), we asked whether HP1c, the Drosophila homolog of HP1γ, plays any role in regulating gut homeostasis. To avoid potential effects on the early developmental stage, we depleted HP1c in a temp
Referência(s)