Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Aug 1.
Published in final edited form as: Mech Dev. 2016 Jul 1;141:4–13. doi: 10.1016/j.mod.2016.07.001

Loss of histone deacetylase HDAC1 induces cell death in Drosophila epithelial cells through JNK and Hippo signaling

Tianyi Zhang 1, Zhentao Sheng 1, Wei Du 1
PMCID: PMC4995152  NIHMSID: NIHMS801328  PMID: 27378074

Abstract

Inactivation of HDAC1 and its homolog HDAC2 or addition of HDAC inhibitors in mammalian systems induces apoptosis, cell cycle arrest, and developmental defects. Although these phenotypes have been extensively characterized, the precise underlying mechanisms remain unclear, particularly in in vivo settings. In this study, we show that inactivation of Rpd3, the only HDAC1 and HDAC2 ortholog in Drosophila, induced apoptosis and clone elimination in the developing eye and wing imaginal discs. Depletion of Rpd3 by RNAi cell-autonomously increased JNK activities and decreased activities of Yki, the nuclear effecter of Hippo signaling pathway. In addition, inhibition of JNK activities largely rescued Rpd3 RNAi-induced apoptosis, but did not affect its inhibition of Yki activities. Conversely, increasing the Yki activities largely rescued Rpd3 RNAi-induced apoptosis, but did not affect its induction of JNK activities. Furthermore, inactivation of Mi-2, a core component of the Rpd3-containing NuRD complex strongly induced JNK activities; while inactivation of Sin3A, a key component of the Rpd3-containing Sin3 complex, significantly inhibited Yki activities. Taken together, these results reveal that inactivation of Rpd3 independently regulates JNK and Yki activities and that both Hippo and JNK signaling pathways contribute to Rpd3 RNAi-induced apoptosis.

Keywords: HDAC1, Rpd3, NuRD, Sin3, JNK, Hippo, Yki, Drosophila, epithelial, apoptosis

1. Introduction

Histone deacetylases (HDACs) regulate transcription and other processes by removing acetyl modification from histones and other proteins (Kurdistani and Grunstein, 2003; Ng and Bird, 2000). Human or mouse has 18 HDACs that can be divided to four classes. Class I HDACs include HDAC1, HDAC2, HDAC3 and HDAC8. HDAC1 and HDAC2 have very similar protein sequences with redundant functions (Gregoretti et al., 2004; Moser et al., 2014). Loss of both HDAC1 and HDAC2 induces apoptosis, cell cycle arrest, and DNA damage in many cell types. Importantly, HDAC1 and HDAC2 specific inhibitors are potential anti-cancer drugs (Dokmanovic et al., 2007; Glozak and Seto, 2007; Johnstone, 2002; Kelly and Cowley, 2013; Minucci and Pelicci, 2006; Ropero and Esteller, 2007; West and Johnstone, 2014). However, the safety of the HDAC inhibitors is a significant concern because HDAC1 and HDAC2 also play important roles in regulating gene expression in development. Loss of HDAC1 and HDAC2 led to profound defects in tissues with epithelial structures, such as intestine and epidermis (Brunmeir et al., 2009; Gonneaud et al., 2015; Winter et al., 2013). Furthermore, the enzymatic activities of HDAC1 and HDAC2 are dependent on their incorporation into different protein complexes, such as the Sin3, NuRD and CoREST complexes, which bind and deacetylate distinct substrates (Kelly and Cowley, 2013; Moser et al., 2014). The specific roles of these different complexes in developing epithelial cells are not well understood.

Class I HDACs are highly conserved during evolution. Yeast has a single HDAC1, also known as Rpd3. Drosophila has two Class I HDACs: Rpd3 and HDAC3. Rpd3 plays important functions in regulating gene expression by interacting with transcriptional repressors such as Groucho, Polycomb and Atrophin (Chang et al., 2001; Chen et al., 1999; Tea et al., 2010; Tie et al., 2001; Zhang et al., 2013). In addition, loss of Drosophila Rpd3 inhibited cell growth and induced apoptosis (Zhu et al., 2008), suggesting that Rpd3 is required for cell survival similar to the roles of HDAC1 and HDAC2 in mammalian cells. However, the downstream pathways affected by loss of Rpd3 are largely unknown.

The key signaling pathways regulating cell proliferation, differentiation, and cell death are highly conserved between Drosophila and mammals. The well-characterized developmental programs in Drosophila combined with the sophisticated genetic, developmental, and cellular approaches make Drosophila a powerful model to investigate the functions of genes and pathways in vivo. Drosophila imaginal discs at larval stages have epithelial structures and most of cells keep proliferating, which make them as ideal models to study gene functions in proliferation, differentiation, and apoptosis (Hariharan and Bilder, 2006; Morata, 2001; Pan, 2007; Vidal and Cagan, 2006). In this study, we address how Rpd3 regulates cell survival by analyzing mosaic clones depleting Rpd3 in Drosophila imaginal discs. We show that loss of Rpd3 activates JNK signaling and inhibits Yki activities, and that both JNK and Yki contribute to Rpd3 RNAi-induced apoptosis and clone elimination. These results provide new insights into the roles of HDAC1 and HDAC2 in vivo and contribute to a better understanding on the potential effects of the Class I HDAC inhibitors on epithelial cells.

2. Materials and Methods

2.1 Drosophila stocks

Fly stocks used in this study include: Rpd3 RNAi (BL 33725) (Bloomington Stock Number), Rpd3 RNAi-2 (BL 34846), UAS-Puc (Martin-Blanco et al., 1998), pucE69 (Riesgo-Escovar et al., 1996), diap1-lacz (BL 12093), UAS-YkiS168A (BL 28818), ex-lacz (BL 44248), Mi-2 RNAi (BL 51774), Mi-2 RNAi-2 (BL33419), Sin3A RNAi (BL 32368).

2.2 Mosaic clone induction

Heat shock Flp-out system used to induce clones with ectopic expression of protein or RNAi in our studies is based on UAS/GAL4, FLP/FRT cassette, and in vivo RNAi methods (Brand and Perrimon, 1993; Ni et al., 2008; Pignoni and Zipursky, 1997). To generate the clones, 24-48 hours after egg deposition (AED) Drosophila larvae with hs-FLP Act>CD2>Gal4 UAS-GFP and UAS driven protein coding cDNA and/or RNAi were heat shocked at 34°C for 15 minutes to 1 hour, depending on the clone sizes of each genotype. The imaginal discs were dissected from larvae at 48-72 hours after the heat shock for fixation and staining. Except for the heat shock clone induction, all flies for the experiments were kept at 25°C.

2.3 Immunostaining

Immunostaining and imaging were done with the protocols as described in our previous studies (Gordon et al., 2013; Zhang et al., 2014). Primary antibodies used in this study include: mouse anti-β-Galactosidase (1:100, DSHB), mouse anti-DLG (1:100, DSHB), rabbit anti-activated Caspase-3 (C3, 1:500, Cell Signaling), rabbit anti-Yki antibody (1:400) (Oh and Irvine, 2008). Secondary antibodies are from Jackson ImmunoResearch (1:200 to 1:400).

2.4 Drosophila genotypes used in each figures

Figure 1

A, B, E, G: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; UAS-Rpd3 RNAi/+

D, F: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+;

C: dpp-Gal4, UAS-GFP/+; UAS-Rpd3 RNAi-2/+

Figure 2

A, B: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; puc-lacz/+

C, D: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; puc-lacz UAS-Rpd3 RNAi/+

E, F: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; puc-lacz UAS-Rpd3 RNAi/UAS-Puc

G, H: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; UAS-Rpd3 RNAi/UAS-Puc

Figure 3

A, B: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; diap1-lacz/+

C, D: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; diap1-lacz UAS-Rpd3 RNAi/+

E, F: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; diap1-lacz UAS-Rpd3 RNAi/UAS-YkiS168A

G, H: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; UAS-Rpd3 RNAi/UAS-YkiS168A

Figure 4

A, B: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; diap1-lacz UAS-Rpd3 RNAi/UAS-Puc

C, D: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; puc-lacz UAS-Rpd3 RNAi/UAS-YkiS168A

E, F: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; puc-lacz UAS-Rpd3 RNAi/UAS-Puc UAS-YkiS168A

Figure 5

A, B: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; puc-lacz/UAS-Mi-2 RNAi

C, D: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; diap1-lacz/UAS-Mi-2 RNAi

E, G: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; UAS-Mi-2 RNAi/+

F, H: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; UAS-Mi-2 RNAi/UAS-Puc

Figure 6

A, B: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; diap1-lacz /UAS-Sin3A RNAi

C, D: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; puc-lacz /UAS-Sin3A RNAi

Figure S1

yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; puc-lacz/UAS-Rpd3 RNAi-2

Figure S2

yw, hsFLP, Act>CD2>Gal4, UAS-CD8GF/+P; UAS-Rpd3 RNAi puc-lacz /UAS-P35

Figure S3

yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; diap1-lacz/UAS-Rpd3 RNAi-2

Figure S4

A, B: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; ex-lacz/+; UAS-Rpd3 RNAi/+

C, D: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; UAS-Rpd3 RNAi/+

Figure S5

A, B: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; puc-lacz/UAS-Mi-2 RNAi-2

C, D: yw, hsFLP, Act>CD2>Gal4, UAS-CD8GFP/+; diap1-lacz/UAS-Mi-2 RNAi-2

3. Results

3.1 Inactivation of Rpd3 induces apoptosis

Previous study showed that Rpd3 mutant clones were tiny in Drosophila imaginal discs (Zhu et al., 2008), which makes it challenging to further analyze the underlying molecular mechanisms. To overcome this problem, we used the hs-FLP/FRT cassette and the UAS/Gal4 based "heat shock flp-out" system to deplete Rpd3 by RNAi. With this system, the Rpd3-RNAi clones in imaginal discs were labeled with the co-expressed GFP and the levels and the consequences of Rpd3 knockdown were controlled by the amount of time after heat shock. As expected, very few surviving Rpd3-RNAi clones were observed in either eye or wing discs at 72 hours after clone induction. The level of activated Caspase3 (C3) is correlated with cell apoptosis in Drosophila imaginal discs. Staining with C3 antibody showed high C3 level in the Rpd3-RNAi clones by comparing with surrounding WT cells (Fig. 1A-B). Similarly, constitutive expression of another Rpd3 RNAi construct, Rpd3 RNAi-2, that targets a distinct part of Rpd3 mRNA, also induced strong C3 level (Fig. 1C). Therefore, inactivation of Rpd3 induced cell death and caused clone elimination.

Figure 1.

Figure 1

Open in a new tab

Rpd3 RNAi induces apoptosis and clone elimination. In all Figures, the orientation of eye discs is posterior to the right and the orientation of wing discs is ventral to the right. The genotypes, stained proteins (or markers), hours after clone induction and image size scales are shown in the Figures. GFP is co-expressed with other protein or RNAi to mark the clones. Activated caspase-3 (C3) staining is used to detect apoptosis (or cell death) in developing imaginal discs. (A-B) Only a few Rpd3-RNAi clones survive at 72 hours after clone induction by heat shock and some of the cells in the clones have strong C3 signals. (C) Overexpression of Rpd3 RNAi-2 driven by dpp-Gal4 also induces strong C3 signals in central area of wing disc. (D-G) Comparing with WT control clones, Rpd3 RNAi clones did not exhibit obvious growth defects at 48 hours but had apparent defects at 63 hours after clone induction. Morphogenetic furrow (MF) is indicated by yellow arrows in D-G.

The extent of clone elimination by Rpd3 RNAi depends on the amount of time after it was induced. In comparison to the WT clones, the reduced amount of Rpd3-RNAi clones was apparent at 63 hours after clone induction, but no consistent difference was observed at 48 hours after clone induction (Fig. 1D-G). Since Rpd3-RNAi clones at 63 hours after clone induction can still be easily obtained and already exhibit significant phenotypes, we selected 63 hours as the time point to study the effect of Rpd3 RNAi on signaling pathways in the follow-up experiments.

3.2 JNK activation contributes to Rpd3 RNAi-induced apoptosis

Next, we evaluated pathways that may contribute to Rpd3 RNAi-induced apoptosis and clone elimination. Previous studies showed that JNK pathway induced apoptosis and eliminated clones in imaginal discs (Igaki, 2009). We used puc-lacZ to monitor the JNK activities since Puc, which encodes a phosphatase that inhibits JNK kinase activities, is a target of JNK pathway (Martin-Blanco et al., 1998). The background expression of puc-lacZ is uniformed and low in the regions anterior to morphogenetic furrow (MF) of eye discs and the pouch regions of wing discs, and the relative strong puc-lacZ signals are found in the regions posterior to MF of eye discs and peripodial membrane cells at the edge of the discs (Fig. 2A-B). We found that puc-lacZ was upregulated autonomously in the Rpd3-RNAi clones and non-autonomously in some WT cells adjacent to the Rpd3-RNAi clones in eye (Fig. 2C-C”’, white and yellow arrows respectively) and wing discs (Fig. 2D-D”’, white and yellow arrows). Similarly, Rpd3 RNAi-2 induced both autonomous and non-autonomous JNK activities (Fig. S1). These observations suggest that depletion of Rpd3 induces JNK activation. The apoptotic cells can induce JNK activities in certain contexts (Shlevkov and Morata, 2012). To test whether the increased puc-lacz levels in Rpd3-RNAi clones are the consequence of apoptosis, we checked the puc-lacz levels in Rpd3 RNAi clones co-expressed with UAS-P35 to block cell apoptosis. puc-lacz was still strongly induced autonomously in Rpd3-RNAi UAS-P35 clones and non-autonomously in some cells surrounding the clones (Fig. S2A-B), which suggests that the induction of JNK activities by Rpd3 RNAi is not a consequence of apoptosis.

Figure 2.

Figure 2

Open in a new tab

Rpd3 RNAi induces ectopic JNK activities, which contributes to apoptosis. (A-B) puc-lacz is expressed at low and uniformed levels in regions anterior to MF of eye discs and in wing discs, and at relatively higher levels in regions posterior to MF of eye discs. (C-D) Rpd3 RNAi induces both autonomous and nonautonomous JNK activities (shown by puc-lacz expression) in eye and wing discs. C"-C"' and D"-D"' are enlarged from the rectangle parts of C and D respectively. The autonomous and nonautonomous puc-lacz are pointed by white and yellow arrows. (E-F) Co-expression of JNK inhibitor Puc inhibits autonomous but not nonautonomous Rpd3 RNAi-induced puc-lacz in eye and wing discs. (G-H) Co-expression of Puc significantly decreases Rpd3 RNAi-induced apoptosis in eye and wing discs (compare with Fig. 1A-B).

To determine whether JNK activation contributes to Rpd3 RNAi-induced apoptosis, we inhibited JNK signaling by overexpressing Puc in Rpd3-RNAi clones. As shown by puc-lacZ levels in the eye (Fig. 2E) and wing discs (Fig. 2F), co-expression of Puc completely blocked the Rpd3 RNAi-induced autonomous JNK activities within the clones, but not the non-autonomous JNK activities in cells surrounding clones. Importantly, co-expression of Puc in Rpd3-RNAi clones also significantly decreased C3 staining in both eye (Fig. 2G) and wing discs (Fig. 2H). These results suggest that the autonomous and non-autonomous JNK activities are induced independently by Rpd3 RNAi and that the autonomous JNK signaling activation contributes to Rpd3 RNAi-induced apoptosis. However our study did not directly exam the contribution of non-autonomous JNK signaling to Rpd3 RNAi-induced apoptosis. It is worth pointing out that similar autonomous and non-autonomous JNK signaling induction were observed in a previous study of scrib or dlg mutant clones and that the non-autonomous JNK signaling was found to contribute to the elimination of mutant neighbors (Ohsawa et al., 2011). Therefore it is possible that the non-autonomous JNK signaling may also contribute to the elimination of Rpd3-inactivated cells.

3.3 Decreased Yki activities also contributes to Rpd3 RNAi-induced apoptosis

Co-expression of Puc significantly reduced but did not completely block apoptosis in Rpd3-RNAi clones, indicating that Rpd3 RNAi may affect additional pathway(s) regulating apoptosis and survival. The Hippo pathway plays important roles in regulating cell proliferation and cell survival in Drosophila imaginal discs (Harvey and Tapon, 2007; Irvine and Harvey, 2015; Pan, 2010). The major output of the Hippo pathway is mediated through the transcriptional co-activator Yorkie (Yki), and upregulation of its transcriptional activities promotes proliferation and inhibits apoptosis. Therefore, we analyzed the effects of Rpd3 RNAi on the expression of diap1-lacZ, a reporter that is ubiquitously expressed in imaginal discs and directly activated by Yki (Wu et al., 2008; Zhang et al., 2008). The expression of diap1-lacZ in eye discs and wing discs is pretty uniformed except some cells with higher expression in wing discs (Fig. 3A-B). In both eye and wing discs, the expression of diap1-lacZ was significantly downregulated in Rpd3-RNAi clones compared to the WT cells surrounding the clones (white arrows in Fig. 3C-D”’). Similar effects on diap1-lacZ were observed with Rpd3 RNAi-2 in both eye and wing discs (Fig. S3A-B). Furthermore, Rpd3 RNAi also downregulated another Yki target, ex-lacZ, in both eye and wing discs (Fig. S4A-B). Interestingly, some WT cells adjacent to the Rpd3-RNAi clones in the regions anterior to MF of eye discs or in wing discs have higher diap1-lacz or ex-lacz expression than the WT cells away from the clones (yellow and blue arrows in Fig. 3C-D and Fig. S4A-B). We quantified the levels of diap1-lacz in cells within the clone, adjacent to clones (1-2 cell range surrounding the clones), and away from clones (3-4 cell range surrounding the clones). Compared to cells away from the clones, cells within the Rpd3-RNAi clones showed significantly lower diap1-lacz levels while cells adjacent to the Rpd3-RNAi clones exhibited significantly higher diap1-lacz levels (Fig. 3I-J). In contrast, there were no differences in diap1-lacZ levels between these three types of cells in WT control discs. These results suggest that Rpd3 inactivation inhibits Yki activities autonomously but can also upregulate Yki activities non-cell autonomously in some neighboring cells.

Figure 3.

Figure 3

Open in a new tab

Rpd3 RNAi suppresses Yki activities, which contributes to apoptosis. (A-B) diap1-lacz is expressed uniformed in most areas of WT eye or wing discs. (C-D) Rpd3 RNAi suppresses Yki activities (shown by diap1-lacz) autonomously in eye and wing discs. C"-C"' and D"-D"' are enlarged from the rectangle parts of C and D respectively. MF is pointed by white arrowhead. White arrows point to the Rpd3-RNAi clones, yellow arrows point to WT cells adjacent to the clones, and blue arrows point to WT cells away from the clones. Rpd3-RNAi clones in both eye and wing discs have lower diap1-lacz levels than surround WT cells. Some WT cells adjacent to Rpd3-RNAi clones anterior to MF in eye discs or in wing discs have higher diap1-lacz levels than the WT cells away from the clones. (E-F) Co-expression of YkiS168A reverses diap1-lacz downregulation by Rpd3 RNAi and makes the diap1-lacz expression in the clones higher than in WT cells in eye and wing discs. (G-H) Co-expression of YkiS168A blocks Rpd3 RNAi induced apoptosis in eye and wing discs (compare with Fig. 1A-B). DAPI staining shows the morphologies of the discs in E and F. (I-J) Quantifications of the diap1-lacz levels in cells within the clones (In), adjacent to clones (1-2 cell range surrounding the clones, Adjacent), and away from clones (3-4 cell range surrounding the clones, Away) in the regions anterior of MF of eye discs and pouch regions of wing discs. The level of diap1-lacz is defined by the mean intensity of the selected area with Photoshop. For each genotype, three eye or wing discs were used for analysis and 2 to 3 clones with clear boundaries from each disc were selected. For each clone, the intensities of In, Adjacent, and Away cells are divided by the intensity of Away cells to get the relative levels. The Average and STDEV values of diap1-lacz levels of In and Adjacent cells of each genotype and the P values between two groups of data were calculated in Excel. In both eye and wing discs, no significant differences in diap1-lacz levels were observed between these three groups in WT discs (X indicates P>0.1), while the levels of diap1-lacz in Rpd3-RNAi or Rpd3-RNAi UAS-Puc clones are significantly lower and the levels of diap1-lacz in the cells adjacent to clones are significantly higher than those in cells away from clones respectively (** indicates P<0.001). There are no significant differences of the downregulated diap1-lacz levels in Rpd3-RNAi clones and Rpd3-RNAi UAS-Puc clones, or the upregulated diap1-lacz levels in the cells adjacent to Rpd3-RNAi clones and Rpd3-RNAi UAS-Puc clones.

Yki/Yap activities are generally regulated at the level of nuclear localization although additional mechanisms such as protein degradation may also contribute to the regulation (Oh and Irvine, 2010). We used an antibody specifically against Yki (Oh and Irvine, 2008) to determine whether Rpd3 RNAi affects Yki protein levels and nuclear localization. While Yki levels were not obviously changed in Rpd3-RNAi clones, the levels of Yki in the nuclei appeared to be slightly decreased in Rpd3-RNAi clones in comparison to those in cells adjacent to the Rpd3- RNAi clones (Fig. S4C-D, compare Yki levels pointed by yellow and white arrows). These results suggest that Rpd3 RNAi may modulate Yki activity through altering the nuclear Yki levels.

To determine whether downregulation of Yki activities contribute to the apoptosis caused by Rpd3 RNAi, the hyper-activated form of Yki, YkiS168A, was co-expressed with Rpd3 RNAi. The autonomous diap1-lacZ levels in Rpd3 RNAi UAS-YkiS168A clones were significantly higher than those in WT cells (Fig. 3E-F), suggesting that the downregulation of Yki activities by Rpd3 RNAi was rescued by overexpression of YkiS168A in these clones. Consistent with the functions of Yki in inhibiting apoptosis, co-expression of YkiS168A strongly inhibited apoptosis as shown by reduced C3 staining induced by Rpd3 RNAi (Fig. 3G-H). These results suggest that the inhibition of autonomous Yki activities also contributes to the apoptosis in Rpd3-RNAi cells.

3.4 Inactivation of Rpd3 affects JNK and Yki activities independently.

Previous studies indicate that there is complex crosstalk between the JNK and Hippo pathways. Since inhibition of JNK or activation of Yki activities significantly rescued Rpd3 RNAi-induced apoptosis, we further characterized the regulatory relationships between JNK and Yki activities in Rpd3-RNAi cells. To determine whether JNK activation affects Rpd3 RNAi-reduced Yki activities, we analyzed the effects of Puc overexpression on diap-lacZ expression in Rpd3-RNAi clones. Lower level of diap1-lacZ was still observed in Rpd3-RNAi UAS-Puc clones than WT cells (white arrows in Fig. 4A-B”’). Furthermore, the WT cells adjacent to some Rpd3-RNAi UAS-Puc clones have higher diap1-lacz expression than the WT cells away from the clones (yellow and blue arrows in Fig. 4A-B). These phenotypes are similar to the phenotypes of Rpd3-RNAi clones. The quantifications of the levels of diap1-lacz showed that cells within the Rpd3-RNAi UAS-Puc clones had lower while cells adjacent to the clones had higher diap1-lacz levels in comparison to cells away from clones (Fig. 3I-J). Furthermore, no significant difference in diap1-lacz levels were observed between cells within the Rpd3-RNAi clones and cells within the Rpd3-RNAi UAS-Puc clones or between cells adjacent to the Rpd3-RNAi clones and cells adjacent to the Rpd3-RNAi UAS-Puc clones (Fig. 3I-J). Therefore, inhibition of Rpd3 RNAi-induced JNK activities does not affect Rpd3-RNAi mediated changes in Yki activities.

Figure 4.

Figure 4

Open in a new tab

Rpd3 RNAi independently affects JNK and Yki activities. White arrows point to the Rpd3-RNAi clones and yellow arrows point to WT cells surrounding the clones. (A-B) Inhibition of JNK activities by co-expression of Puc does not change the Rpd3 RNAi-induced autonomous downregulation of diap1-lacz in eye and wing discs. A"-A"' and B"-B"' are enlarged from the rectangle parts of A and B respectively. MF is pointed by white arrowhead. White arrows point to the Rpd3-RNAi UAS-Puc clones, yellow arrows point to WT cells adjacent to the clones, and blue arrows point to WT cells away from the clones. Similar to Rpd3-RNAi clones, Rpd3-RNAi UAS-Puc clones in both eye and wing discs have lower diap1-lacz levels than surround WT cells, and some WT cells adjacent to Rpd3-RNAi UAS-Puc clones anterior to MF in eye discs or in wing discs have higher diap1-lacz levels than the WT cells away from the clones. (C-D) Co-expression of YkiS168A does not change the Rpd3 RNAi-induced autonomous or nonautonomous puc-lacz expression in eye and wing discs. (E-F) Co-expression of both YkiS168A and Puc suppresses Rpd3 RNAi-induced autonomous but not nonautonomous puc-lacz, which is similar to Rpd3-RNAi UAS-Puc clones (Fig. 2C-D). DAPI staining in D and F shows the morphologies of the wing discs.

To test whether increasing Yki activities affects Rpd3 RNAi-induced JNK activities, we analyzed puc-lacz levels in UAS-YkiS168A Rpd3-RNAi co-expressed cells. Both autonomous and non-autonomous puc-lacZ expression were still strongly induced in Rpd3-RNAi UAS-YkiS168A clones (Fig. 4C-D). Furthermore, the non-autonomous puc-lacZ was still induced in cells surrounding Rpd3-RNAi UAS-Puc UAS-YkiS168A clones (Fig. 4E-F), which is similar to the effects of Rpd3-RNAi UAS-Puc clones (Fig. 2C-D). These results suggest that Rpd3 RNAi-induced JNK activities are not related to the downregulation of Yki activities. Taken together, these results suggest that Rpd3 inactivation affects JNK and Yki activities independently.

3.5 Rpd3 regulates JNK and Yki activities through different complexes

Rpd3 functions by forming several conserved protein complexes, including the CoREST, NuRD and Sin3 complexes (Kelly and Cowley, 2013; Moser et al., 2014). To investigate whether the effects of Rpd3 inactivation are mediated by these complexes, the core components of these three complexes, CoRest, Mi-2 and Sin3A, were knocked down by RNAi. We tested three different CoRest RNAi constructs, but none caused clone elimination or diap1-lacz/puc-lacz expression alteration in imaginal discs (data not shown). This result is consistent with recent studies showing that mutation of CoRest did not cause obvious growth defects in Drosophila wings (Curtis et al., 2013). Therefore we focused our analysis on the effects of NuRD and Sin3 on the regulation of JNK and Yki activities. We found that induction of Mi-2 RNAi for 63 hours eliminated the clones, so we changed in clone induction condition to 56 hours. Similar to those induced by Rpd3 RNAi (Fig. 2C-D), Mi-2 RNAi induced strong autonomous and non-autonomous puc-lacZ expression (Fig. 5A-B). Interestingly, Mi-2 RNAi did not significantly affect diap1-lacZ levels in eye disc (Fig. 5C) and may slightly increase diap1-lacZ in some wing disc clones (Fig. 5D, compared with the WT diap1-lacz wing disc in Fig. 3B). Knockdown of Mi-2 by another RNAi, Mi-2 RNAi-2, also strongly upregulated puc-lacZ levels but did not change diap1-lacZ levels (Fig. S5). These results suggest that the NuRD complex mainly mediates the effects of Rpd3 RNAi on JNK signaling. Consistent with this, while Mi-2-RNAi clones were largely eliminated at 72 hours after clone induction, inhibition of JNK activities by co-expressing Puc strongly protected the Mi-2-RNAi clones from elimination (Fig. 5E-H).

Figure 5.

Figure 5

Open in a new tab

Inhibition of NuRD complex strongly induces JNK activities. (A-B) Mi-2 RNAi strongly induces autonomous and nonautonomous puc-lacz expression in eye and wing discs. A"-A"' and B"-B"' are enlarged from the rectangle parts of A and B respectively. White arrows point to the Mi-2-RNAi clones and yellow arrows point to WT cells surrounding the clones. (C-D) Mi-2 RNAi does not induce strong effects on diap1-lacZ expression in eye or wing discs. In some clones in wing discs (pointed by yellow arrow), Mi-2 RNAi induces weak diap1-lacZ. (E-H) Co-expression of Puc prevents Mi-2-RNAi-induced clone elimination at 72 hours after clone induction. DAPI staining shows the morphologies of the discs in E-H.

In contrast, inactivation of Sin3 complex by Sin3A RNAi inhibited Yki activities, as shown by reduced diap1-lacZ levels within the Sin3A-RNAi clones (Fig. 6A-B, compared to Fig. 3A-B). In addition, Sin3A RNAi weakly affected JNK activity as shown by puc-lacZ levels (Fig. 6C-D). Based on these results, we propose that Rpd3 regulates JNK activities mainly through NuRD complex and regulates Yki activities mainly through Sin3 complex although some level of cross regulation exists (Fig. 6E).

Figure 6.

Figure 6

Open in a new tab

Inhibition of Sin3 complex decreases Yki activities. White arrows point to the clones. (A-B) Sin3A RNAi suppresses diap1-lacz expression in the clones in eye and wing discs. (C-D) Sin3A RNAi weakly induces puc-lacz expression in eye and wing discs. (E) A scheme to summarize the regulation of JNK and Yki activities by Rpd3 related complexes in the epithelial cells of eye and wing discs. In WT cells, Rpd3 suppresses JNK activities mainly through NuRD complex and maintains Yki activities mainly through Sin3 complex. NuRD has weak suppression on Yki activities in some clones in wing discs, while Sin3 has weak suppression on JNK activities.

4. Discussion

Our studies here showed that inhibition of Rpd3 induced JNK signaling and inhibited Yki activities. These changes likely contribute to increased cell death and clone elimination in Rpd3-RNAi clones in developing imaginal discs. Our results also suggest that Rpd3 RNAi affects JNK and Yki activities independently and mainly through the inhibition of the NuRD and the Sin3 complexes, respectively.

Yki (or Yap in mammals) is the key transcriptional co-activator downstream of the Hippo signaling pathway that plays critical roles regulating cell proliferation and survival in many different cell types. It has been shown that Yki regulates proliferation and survival by modulating the expression of CycE, Myc, and DIAP1 (Harvey and Tapon, 2007; Pan, 2007). Here we showed that Yki activities were downregulated within the Rpd3-RNAi clones but upregulated non-autonomously in some adjacent cells. Importantly, restoring the Yki activities in Rpd3-RNAi clones inhibited apoptosis and prevented clone elimination. Therefore, the cell-autonomous inhibition of Yki activities contributes to the induction of apoptosis by Rpd3 RNAi.

Interestingly, recent studies suggest that HDAC inhibitors also affect Hippo pathway in cultured human cancer cells. In one study, combining HDAC inhibitor and BET inhibitor significantly reduced Yap mRNA levels (Heinemann et al., 2015). In another study, HDAC inhibitor was found to induce expression of secreted factors that led to the upregulation of Yap transcriptional output by stabilizing TAZ, another Yki ortholog in human (Basu et al., 2013). The conclusion from the study by Heinemann et al is consistent with the cell autonomous effect of Rpd3 RNAi in downregulating Yki activities and the study by Basu et al is potentially related to the non-autonomous effect of Rpd3 RNAi of upregulating Yki activities in adjacent cells. However, since these studies in cancer cells used general HDAC inhibitors, it will be interesting to further characterize the autonomous and non-autonomous effects of specific inhibition of HDAC1 and HDAC2 in mammalian systems.

JNK is a conserved pathway that induces inflammation and other stress responses (Ip and Davis, 1998; Liu and Rondinone, 2005). JNK can induce cell death by inducing the expression of pro-apoptotic gene hid and also through hid-independent mechanisms in Drosophila (Kanda et al., 2011; Wu et al., 2009). In our studies, co-expression of Puc inhibited JNK signaling and largely suppressed apoptosis within Rpd3-RNAi clones even though the reduced Yki activities in the clones were not affected. These results show that activation of JNK signaling also contributes to Rpd3 RNAi-induced apoptosis and clone elimination. Interestingly, loss of HDAC1 and HDAC2 in mouse guts caused upregulation of genes related to inflammation (Turgeon et al., 2013; Turgeon et al., 2014). Since JNK pathway plays important roles in inflammation, it will be interesting to study whether JNK signaling is induced and contributes to HDAC1 and HDAC2 inactivation-induced inflammation in mice.

Recent studies in Drosophila revealed that the relationship between JNK and Yki is complex and that they can positively or negatively regulate each other. On one hand, proper levels of JNK activities can upregulate Yki through Ajuba (Sun and Irvine, 2013). On the other hand, Yki can activate JNK through transcriptional up-regulation of Rho1 and JNK activities are required for Yki-induced overgrowth (Ma et al., 2015). In contrast to these positive regulations, JNK can also inhibit Yki activities in certain developmental and cellular contexts. For example, loss of cell polarity molecule Scrib induces JNK activities and suppresses Yki activities in imaginal discs, and blocking JNK in scrib mutant cells increased Yki activities (Chen et al., 2012). Interestingly, our studies show that inhibition of the ectopic JNK activities in Rpd3-RNAi clones did not affect the downregulation of Yki activities. Similarly, ectopic activation of Yki by co-expressing dominant active Yki did not affect JNK activation in Rpd3-RNAi clones. Therefore, Rpd3 regulates JNK and Yki activities independently and that both pathways directly contribute to the clone elimination and apoptosis induced by Rpd3 RNAi. It appears that the crosstalk between JNK and Hippo pathways are context dependent. Loss of Scrib or Rpd3 may generate different cellular environments to affect the crosstalk between these two pathways.

One intriguing phenotype is that Rpd3-RNAi induced strong non-autonomous JNK and Yki activities in cells adjacent to the clones. Although JNK signaling induces apoptosis in some contexts, Rpd3-RNAi induced non-autonomous JNK signaling did not cause apoptosis, probably due to the presence of strong Yki activity. Interestingly, strong non-autonomous JNK and Yki activities were also induced surrounding the scrib mutant clones (Chen et al., 2012; Ohsawa et al., 2011) and that non-autonomous JNK and Yki activities surrounding scrib mutant clones promoted cell competition and triggers phagocytosis to eliminate the mutant cells (Chen et al., 2012; Ohsawa et al., 2011). The non-autonomous Yki activities was also induced in several other conditions, such as clones with vps25, E-cad, or α-catenin mutation (Herz et al., 2006; Yang et al., 2015) or clones with overexpression of Src64, proapoptotic gene reaper, or the JNK signaling ligand eiger (Enomoto and Igaki, 2013; Sun and Irvine, 2011). While the exact mechanism of induction is not clear, it has been proposed that the non-autonomous Yki activities are induced in responses to growth defects in the clones or an imbalance of cell tension (Grusche et al., 2011; Sun and Irvine, 2011; Yang et al., 2015).

We further explored the mechanisms of Rpd3's effects on JNK and Yki activities by knockdown of core components of Rpd3-containing complexes, such as NuRD and Sin3. Interestingly, inhibition of NuRD complex by Mi-2 RNAi strongly induced JNK activities, while inactivating the Sin3 complex suppressed autonomous Yki activities. These observations suggest that the effect of Rpd3 RNAi on Yki activities is mainly mediated by the disruption of the Sin3 complex and that on JNK activation is mainly mediated by the disruption of the NuRD complex. Although we found that the functions of Rpd3 on JNK and Hippo pathways are mainly through different complexes, further studies are needed to understand the detailed molecular mechanisms of how Rpd3 regulates JNK and Hippo pathways.

In summary, our studies show that loss of Rpd3 has strong effects on JNK and Hippo pathways in Drosophila imaginal discs. Our results not only indicate new mechanisms of cell death caused by Rpd3 inhibition but also provide insights on the clinical application of HDAC inhibitors as anti-cancer drugs. The effects of HDAC inhibitors in killing cancer cells may correlate with their JNK and/or Hippo activities. Furthermore, HDAC inhibitors may have side effects on normal epithelial cells by deregulating the activities of these two pathways. Future studies on mammalian models will evaluate our findings in Drosophila.

Supplementary Material

Highlights.

  • * Inactivation of fly HDAC1/2 ortholog Rpd3 induces apoptosis and clone elimination.

  • * Clone elimination is mediated by increased JNK signaling and reduced Yki activity

  • * Rpd3-inactivation induces JNK signaling primarily through NuRD complex

  • * Rpd3-inactivation inhibits Yki activity primarily through Sin3 complex

Acknowledgements

We thank Dr. Kenneth Irvine for generously supplying antibody. We thank the Bloomington Drosophila Stock Center (supported by NIH P40OD018537) and the Developmental Studies Hybridoma bank at the University of Iowa for providing fly stocks and antibodies. We also thank Xun Pei in Du lab for assistance in immunostaining and fly stock maintenance. This work was supported in part by grants from National Institute of Health (NIH CA149275 and NIH GM074197).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Author Contributions

T.Z. and W.D. designed the experiments. T.Z. and Z.S. did the experiments and collected data presented in this manuscript. T.Z. and W.D. wrote the manuscript.

Conflict of interest: The authors declare no conflict of interest.

References

  1. Basu D, Reyes-Mugica M, Rebbaa A. Histone acetylation-mediated regulation of the Hippo pathway. PLoS One. 2013;8:e62478. doi: 10.1371/journal.pone.0062478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–15. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
  3. Brunmeir R, Lagger S, Seiser C. Histone deacetylase HDAC1/HDAC2-controlled embryonic development and cell differentiation. Int J Dev Biol. 2009;53:275–89. doi: 10.1387/ijdb.082649rb. [DOI] [PubMed] [Google Scholar]
  4. Chang YL, Peng YH, Pan IC, Sun DS, King B, Huang DH. Essential role of Drosophila Hdac1 in homeotic gene silencing. Proc Natl Acad Sci U S A. 2001;98:9730–5. doi: 10.1073/pnas.171325498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen CL, Schroeder MC, Kango-Singh M, Tao C, Halder G. Tumor suppression by cell competition through regulation of the Hippo pathway. Proc Natl Acad Sci U S A. 2012;109:484–9. doi: 10.1073/pnas.1113882109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen G, Fernandez J, Mische S, Courey AJ. A functional interaction between the histone deacetylase Rpd3 and the corepressor groucho in Drosophila development. Genes Dev. 1999;13:2218–30. doi: 10.1101/gad.13.17.2218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Curtis BJ, Zraly CB, Dingwall AK. Drosophila LSD1-CoREST demethylase complex regulates DPP/TGFbeta signaling during wing development. Genesis. 2013;51:16–31. doi: 10.1002/dvg.22346. [DOI] [PubMed] [Google Scholar]
  8. Dokmanovic M, Clarke C, Marks PA. Histone deacetylase inhibitors: overview and perspectives. Mol Cancer Res. 2007;5:981–9. doi: 10.1158/1541-7786.MCR-07-0324. [DOI] [PubMed] [Google Scholar]
  9. Enomoto M, Igaki T. Src controls tumorigenesis via JNK-dependent regulation of the Hippo pathway in Drosophila. EMBO Rep. 2013;14:65–72. doi: 10.1038/embor.2012.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Glozak MA, Seto E. Histone deacetylases and cancer. Oncogene. 2007;26:5420–32. doi: 10.1038/sj.onc.1210610. [DOI] [PubMed] [Google Scholar]
  11. Gonneaud A, Turgeon N, Boudreau F, Perreault N, Rivard N, Asselin C. Distinct Roles for Intestinal Epithelial Cell-Specific Hdac1 and Hdac2 in the Regulation of Murine Intestinal Homeostasis. J Cell Physiol. 2015 doi: 10.1002/jcp.25090. [DOI] [PubMed] [Google Scholar]
  12. Gordon GM, Zhang T, Zhao J, Du W. Deregulated G1-S control and energy stress contribute to the synthetic-lethal interactions between inactivation of RB and TSC1 or TSC2. J Cell Sci. 2013;126:2004–13. doi: 10.1242/jcs.121301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gregoretti IV, Lee YM, Goodson HV. Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis. J Mol Biol. 2004;338:17–31. doi: 10.1016/j.jmb.2004.02.006. [DOI] [PubMed] [Google Scholar]
  14. Grusche FA, Degoutin JL, Richardson HE, Harvey KF. The Salvador/Warts/Hippo pathway controls regenerative tissue growth in Drosophila melanogaster. Dev Biol. 2011;350:255–66. doi: 10.1016/j.ydbio.2010.11.020. [DOI] [PubMed] [Google Scholar]
  15. Hariharan IK, Bilder D. Regulation of imaginal disc growth by tumor-suppressor genes in Drosophila. Annu Rev Genet. 2006;40:335–61. doi: 10.1146/annurev.genet.39.073003.100738. [DOI] [PubMed] [Google Scholar]
  16. Harvey K, Tapon N. The Salvador-Warts-Hippo pathway - an emerging tumour-suppressor network. Nat Rev Cancer. 2007;7:182–91. doi: 10.1038/nrc2070. [DOI] [PubMed] [Google Scholar]
  17. Heinemann A, Cullinane C, De Paoli-Iseppi R, Wilmott JS, Gunatilake D, Madore J, Strbenac D, Yang JY, Gowrishankar K, Tiffen JC, Prinjha RK, Smithers N, McArthur GA, Hersey P, Gallagher SJ. Combining BET and HDAC inhibitors synergistically induces apoptosis of melanoma and suppresses AKT and YAP signaling. Oncotarget. 2015 doi: 10.18632/oncotarget.4242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Herz HM, Chen Z, Scherr H, Lackey M, Bolduc C, Bergmann A. vps25 mosaics display non-autonomous cell survival and overgrowth, and autonomous apoptosis. Development. 2006;133:1871–80. doi: 10.1242/dev.02356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Igaki T. Correcting developmental errors by apoptosis: lessons from Drosophila JNK signaling. Apoptosis. 2009;14:1021–8. doi: 10.1007/s10495-009-0361-7. [DOI] [PubMed] [Google Scholar]
  20. Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK)--from inflammation to development. Curr Opin Cell Biol. 1998;10:205–19. doi: 10.1016/s0955-0674(98)80143-9. [DOI] [PubMed] [Google Scholar]
  21. Irvine KD, Harvey KF. Control of organ growth by patterning and hippo signaling in Drosophila. Cold Spring Harb Perspect Biol. 2015:7. doi: 10.1101/cshperspect.a019224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov. 2002;1:287–99. doi: 10.1038/nrd772. [DOI] [PubMed] [Google Scholar]
  23. Kanda H, Igaki T, Okano H, Miura M. Conserved metabolic energy production pathways govern Eiger/TNF-induced nonapoptotic cell death. Proc Natl Acad Sci U S A. 2011;108:18977–82. doi: 10.1073/pnas.1103242108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kelly RD, Cowley SM. The physiological roles of histone deacetylase (HDAC) 1 and 2: complex co-stars with multiple leading parts. Biochem Soc Trans. 2013;41:741–9. doi: 10.1042/BST20130010. [DOI] [PubMed] [Google Scholar]
  25. Kurdistani SK, Grunstein M. Histone acetylation and deacetylation in yeast. Nat Rev Mol Cell Biol. 2003;4:276–84. doi: 10.1038/nrm1075. [DOI] [PubMed] [Google Scholar]
  26. Liu G, Rondinone CM. JNK: bridging the insulin signaling and inflammatory pathway. Curr Opin Investig Drugs. 2005;6:979–87. [PubMed] [Google Scholar]
  27. Ma X, Chen Y, Xu W, Wu N, Li M, Cao Y, Wu S, Li Q, Xue L. Impaired Hippo signaling promotes Rho1-JNK-dependent growth. Proc Natl Acad Sci U S A. 2015;112:1065–70. doi: 10.1073/pnas.1415020112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martin-Blanco E, Gampel A, Ring J, Virdee K, Kirov N, Tolkovsky AM, Martinez-Arias A. puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila. Genes Dev. 1998;12:557–70. doi: 10.1101/gad.12.4.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Minucci S, Pelicci PG. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer. 2006;6:38–51. doi: 10.1038/nrc1779. [DOI] [PubMed] [Google Scholar]
  30. Morata G. How Drosophila appendages develop. Nat Rev Mol Cell Biol. 2001;2:89–97. doi: 10.1038/35052047. [DOI] [PubMed] [Google Scholar]
  31. Moser MA, Hagelkruys A, Seiser C. Transcription and beyond: the role of mammalian class I lysine deacetylases. Chromosoma. 2014;123:67–78. doi: 10.1007/s00412-013-0441-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ng HH, Bird A. Histone deacetylases: silencers for hire. Trends Biochem Sci. 2000;25:121–6. doi: 10.1016/s0968-0004(00)01551-6. [DOI] [PubMed] [Google Scholar]
  33. Ni JQ, Markstein M, Binari R, Pfeiffer B, Liu LP, Villalta C, Booker M, Perkins L, Perrimon N. Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster. Nat Methods. 2008;5:49–51. doi: 10.1038/nmeth1146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Oh H, Irvine KD. In vivo regulation of Yorkie phosphorylation and localization. Development. 2008;135:1081–8. doi: 10.1242/dev.015255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Oh H, Irvine KD. Yorkie: the final destination of Hippo signaling. Trends Cell Biol. 2010;20:410–7. doi: 10.1016/j.tcb.2010.04.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ohsawa S, Sugimura K, Takino K, Xu T, Miyawaki A, Igaki T. Elimination of oncogenic neighbors by JNK-mediated engulfment in Drosophila. Dev Cell. 2011;20:315–28. doi: 10.1016/j.devcel.2011.02.007. [DOI] [PubMed] [Google Scholar]
  37. Pan D. Hippo signaling in organ size control. Genes Dev. 2007;21:886–97. doi: 10.1101/gad.1536007. [DOI] [PubMed] [Google Scholar]
  38. Pan D. The hippo signaling pathway in development and cancer. Dev Cell. 2010;19:491–505. doi: 10.1016/j.devcel.2010.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pignoni F, Zipursky SL. Induction of Drosophila eye development by decapentaplegic. Development. 1997;124:271–8. doi: 10.1242/dev.124.2.271. [DOI] [PubMed] [Google Scholar]
  40. Riesgo-Escovar JR, Jenni M, Fritz A, Hafen E. The Drosophila Jun-N-terminal kinase is required for cell morphogenesis but not for DJun-dependent cell fate specification in the eye. Genes Dev. 1996;10:2759–68. doi: 10.1101/gad.10.21.2759. [DOI] [PubMed] [Google Scholar]
  41. Ropero S, Esteller M. The role of histone deacetylases (HDACs) in human cancer. Mol Oncol. 2007;1:19–25. doi: 10.1016/j.molonc.2007.01.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shlevkov E, Morata G. A dp53/JNK-dependant feedback amplification loop is essential for the apoptotic response to stress in Drosophila. Cell Death Differ. 2012;19:451–60. doi: 10.1038/cdd.2011.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sun G, Irvine KD. Regulation of Hippo signaling by Jun kinase signaling during compensatory cell proliferation and regeneration, and in neoplastic tumors. Dev Biol. 2011;350:139–51. doi: 10.1016/j.ydbio.2010.11.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sun G, Irvine KD. Ajuba family proteins link JNK to Hippo signaling. Sci Signal. 2013;6:ra81. doi: 10.1126/scisignal.2004324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tea JS, Chihara T, Luo L. Histone deacetylase Rpd3 regulates olfactory projection neuron dendrite targeting via the transcription factor Prospero. J Neurosci. 2010;30:9939–46. doi: 10.1523/JNEUROSCI.1643-10.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tie F, Furuyama T, Prasad-Sinha J, Jane E, Harte PJ. The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3. Development. 2001;128:275–86. doi: 10.1242/dev.128.2.275. [DOI] [PubMed] [Google Scholar]
  47. Turgeon N, Blais M, Gagne JM, Tardif V, Boudreau F, Perreault N, Asselin C. HDAC1 and HDAC2 restrain the intestinal inflammatory response by regulating intestinal epithelial cell differentiation. PLoS One. 2013;8:e73785. doi: 10.1371/journal.pone.0073785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Turgeon N, Gagne JM, Blais M, Gendron FP, Boudreau F, Asselin C. The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis. Am J Physiol Gastrointest Liver Physiol. 2014;306:G594–605. doi: 10.1152/ajpgi.00393.2013. [DOI] [PubMed] [Google Scholar]
  49. Vidal M, Cagan RL. Drosophila models for cancer research. Curr Opin Genet Dev. 2006;16:10–6. doi: 10.1016/j.gde.2005.12.004. [DOI] [PubMed] [Google Scholar]
  50. West AC, Johnstone RW. New and emerging HDAC inhibitors for cancer treatment. J Clin Invest. 2014;124:30–9. doi: 10.1172/JCI69738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Winter M, Moser MA, Meunier D, Fischer C, Machat G, Mattes K, Lichtenberger BM, Brunmeir R, Weissmann S, Murko C, Humer C, Meischel T, Brosch G, Matthias P, Sibilia M, Seiser C. Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis. EMBO J. 2013;32:3176–91. doi: 10.1038/emboj.2013.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wu S, Liu Y, Zheng Y, Dong J, Pan D. The TEAD/TEF family protein Scalloped mediates transcriptional output of the Hippo growth-regulatory pathway. Dev Cell. 2008;14:388–98. doi: 10.1016/j.devcel.2008.01.007. [DOI] [PubMed] [Google Scholar]
  53. Wu Y, Zhuang Y, Han M, Xu T, Deng K. Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye. BMC Dev Biol. 2009;9:53. doi: 10.1186/1471-213X-9-53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yang CC, Graves HK, Moya IM, Tao C, Hamaratoglu F, Gladden AB, Halder G. Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules. Proc Natl Acad Sci U S A. 2015;112:1785–90. doi: 10.1073/pnas.1420850112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zhang L, Ren F, Zhang Q, Chen Y, Wang B, Jiang J. The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control. Dev Cell. 2008;14:377–87. doi: 10.1016/j.devcel.2008.01.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zhang T, Liao Y, Hsu FN, Zhang R, Searle JS, Pei X, Li X, Ryoo HD, Ji JY, Du W. Hyperactivated Wnt Signaling Induces Synthetic Lethal Interaction with Rb Inactivation by Elevating TORC1 Activities. PLoS Genet. 2014;10:e1004357. doi: 10.1371/journal.pgen.1004357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhang Z, Feng J, Pan C, Lv X, Wu W, Zhou Z, Liu F, Zhang L, Zhao Y. Atrophin-Rpd3 complex represses Hedgehog signaling by acting as a corepressor of CiR. J Cell Biol. 2013;203:575–83. doi: 10.1083/jcb.201306012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhu CC, Bornemann DJ, Zhitomirsky D, Miller EL, O'Connor MB, Simon JA. Drosophila histone deacetylase-3 controls imaginal disc size through suppression of apoptosis. PLoS Genet. 2008;4:e1000009. doi: 10.1371/journal.pgen.1000009. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS801328-supplement.pdf (2.6MB, pdf)

RESOURCES