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. Author manuscript; available in PMC: 2020 Jul 16.
Published in final edited form as: Dev Cell. 2016 Jun 20;37(6):507–519. doi: 10.1016/j.devcel.2016.05.013

Stuxnet Facilitates the Degradation of Polycomb Protein during Development

Juan Du 1,2,4, Junzheng Zhang 1,2,4,5, Tao He 1, Yajuan Li 1, Ying Su 1,6, Feng Tie 3, Min Liu 1,2, Peter J Harte 3, Alan Jian Zhu 1,2,*
PMCID: PMC7365346  NIHMSID: NIHMS790141  PMID: 27326929

SUMMARY

Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. Here, we report a Drosophila gene stuxnet (stx) that controls Pc protein stability. We find that heightened stx activity leads to homeotic transformation, reduced Pc activity and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyper-activation of Pc. Our biochemical analyses provide mechanistic basis for the interaction between stx and Pc; Stx facilitates Pc degradation in the proteasome independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells, and induces homeotic transformation in Drosophila. Our results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity.

Graphical Abstract

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eTOC Blurb

Polycomb-group (PcG) proteins are conserved epigenetic regulators of development. Du et al. show that the Stuxnet protein is a conserved regulator of Polycomb homeostasis and PcG activity. Stuxnet relies on a ubiquitin-like domain, bypassing a ubiquitination step, to directly target Polycomb proteins for proteasomal degradation.

INTRODUCTION

Polycomb group (PcG) genes were first identified in Drosophila for their roles in maintaining correct expression patterns of homeotic genes. PcG-mediated transcription silencing was later proven to be a well conserved regulatory mechanism throughout metazoan (Simon and Kingston, 2009). Classical PcG targets, such as Hox genes, play important roles in biological processes ranging from stem cell maintenance to genomic imprinting (Müller and Verrijzer, 2009). Recent genome-wide studies unveiled additional PcG targets, many of which encode transcription factors and cell signaling proteins that regulate a diverse array of downstream effectors (Schwartz and Pirrotta, 2013). Thus, PcG may act in a much broader spectrum of cellular processes than previously anticipated.

PcG silencing depends primarily on the activities of two Polycomb repressive complexes (PRC). In Drosophila, the PRC1 is composed of Pc (Polycomb), Ph (Polyhomeotic), Psc (Posterior sex combs) and Sce (Sex combs extra). The main subunits of the PRC2 include Esc (Extra sex combs), E(z) (Enhancer of zeste), Su(z)12 (Suppressor of zeste 12) and Caf1 (Chromatin assembly factor 1). Relying on the presence of a conserved enzymatic SET domain in E(z), PRC2 catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3). Pc then employs its chromo domain to recognize H3K27me3 mark, resulting in recruitment of PRC1 to PcG targets (Geisler and Paro, 2015). Mechanisms utilized by PRC1 to silence target genes include histone H2A mono-ubiquitination, chromatin compaction and direct interaction with the general transcription machinery (Cheutin and Cavalli, 2014).

While intensive studies have been focused on uncovering mechanisms by which PcG proteins epigenetically repress target gene expression, few are devoted to define how the PcG activities are regulated. Nevertheless, several transcription factors and miRNAs are known to directly modulate PcG expression (Gil and O'Loghlen, 2014). Feedback regulatory loops may also be important to maintain proper expression of PcG, which themselves are subject to epigenetic repression (Fauvarque and Dura, 1993; Ali and Bender, 2004; Park et al., 2012). Furthermore, post-translational modifications on several PcG proteins have been reported, and the importance of such modifications has only been revealed recently (Niessen et al., 2009; Caretti et al., 2011). For example, SUMOylation is shown to modulate PcG activity by affecting chromatin targeting of the Pc protein (Gonzalez et al., 2014), and O-GlcNAcylation has been demonstrated to prevent aggregation of PRC1 subunit Ph in Drosophila (Gambetta and Müller, 2014).

In this report, we describe that a gene CG32676, which we named stuxnet (stx), functions through ubiquitin-independent degradation (UID) to control Pc protein stability and thereby PcG-mediated epigenetic repression. We show further that vertebrate Stx regulates orthologous Pc protein in the same fashion. Together, our results highlight a conserved regulatory mechanism for Pc, the founding member of the PcG family proteins.

RESULTS

Stx Induces Homeotic Transformation in Drosophila

To identify developmental regulators, we performed a Gene Search (GS) based misexpression screen (Toba et al., 1999) for genes located on the X chromosome in Drosophila. Upon expression by an antenna-specific Distal-less (Dll)-Gal4 driver, three independent GS lines that are inserted near the 5’ regulatory sequence of the CG32676 / stuxnet (stx) locus led to defective antennal development (Figure S1). To rule out potential off-target effects of GS lines, we generated transgenic flies expressing stx cDNA. Increased stx expression in antennae induced stereotypical antenna-to-leg transformation (Figure 1B). This phenotype is often associated with reduced Pc activity (Figure 1C), resulting in de-repression of Antennapedia (Antp) (Figure 1E and 1F), a PcG target that represses antennal development (Plaza et al., 2001). Consistently, the expression of one of antennal-related genes, distal antenna (dan), was reduced in antennal discs overexpressing either stx or Pc RNAi (Figure 1H and 1I).

Figure 1. Stx Induces Homeotic Transformation in Drosophila.

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(A–C) Stx induces homeotic transformation in the antenna. Overexpressing stx (B) or Pc RNAi (C) in the antennal disc led to antenna-to-leg transformation. Arrows mark leg bristles, suggesting the transformation of A3 and arista (Ar) antennal segments to a tarsus. Asterisks mark apical bristles, indicating mesothoracic leg identity. Transformation phenotypes are fully penetrant (n>50).

(D–I) Stx alters the expression of Antp and Dan in the antenna. Antp, which was not normally expressed in antenna (D), was de-repressed when stx (E) or Pc RNAi (F) was overexpressed. Accordingly, the expression of an antennal marker Dan was reduced (H and I).

(J–P) stx induces homeotic transformation in the wing. Overexpressing stx in the wing pouch (marked by RFP) led to formation of smaller wings with densely packed shortened hairs (L and M; cf. J and K), and de-repression of Ubx (N). Scale bars: 100 µm.

Intriguingly, we observed ommatidia-like structures on small percentage of transformed antennae (Figure S2B), and misexpression of eye specific genes, eyeless (ey) and eyes absent (eya), in antennal discs (Figure S2G–S2I and S2P–S2R). Antp is known to inhibit eye development in Drosophila; ectopic Antp activity normally transforms antenna exclusively to leg (Plaza et al., 2001). Thus, it is likely that Antp was not the only PcG target regulated by stx. Consistent with this prediction, overexpressed stx was able to induce wing-to-haltere transformation in wing discs (Figure 1J–1M). This is a phenotype associated with ectopic induction of Ubx (Lewis, 1978), a PcG target normally expressed in wildtype haltere but not in wing discs (Figure 1N–1P). The above phenotypes highlight a regulatory role of stx in animal development, most likely participating in homeotic transformation in various tissues.

Stx Genetically Interacts with Pc

To investigate how stx regulates development, we generated two stx alleles; stxd77, a hypomorphic allele created by imprecise P-element excision (Figure S3A), and stx2, a molecularly defined null allele constructed by the FLP-FRT mediated deletion (Parks et al., 2004; Figure S3C). The full-length stx mRNA was undetectable by RT-PCR in stxd77 larvae, but a shorter form without the first exon was still present (Figure S3B). PCR with genomic DNA of stx2 larvae confirmed the deletion of stx locus (Figure S3D), and immunoblotting analyses indicated that Stx protein was not expressed in stx2 larvae (Figure S3E). stx2 flies were homozygous and hemizygous lethal. This lethal phenotype could be rescued by adding back one copy of stx gene but not other genes that were also deleted in stx2 (Figure S3F and S3G), suggesting that stx activity is essential for fly development.

Ubiquitously expressing Pc has little effect on fly development, with the exception of a patterning defect of dorsal abdominal segments known as tergites (Poux et al., 2001). Consistently, we found that increased Pc activity led to disrupted tergite boundary formation (Figure 2B), a phenotype that is linked with disturbance of homeotic genes (Karch et al., 1985; Breen, 1999; Wang et al., 2013; Singh and Mishra, 2014). When stx2 somatic clones were induced, a similar tergite boundary defect was observed (Figure 2C and 2D). Such tergite defect was also found in hemizygotic stxd77 flies albeit with low frequency (Figure S3H). Furthermore, we observed haltere-to-wing as well as metathoracic-to-mesothoracic leg transformations in stx2 homozygous escapers that died as pharate adults (Figure S3I). The stereotypical posterior-to-anterior (i.e. T3-to-T2) transformation led us to hypothesize that stx may act as an epigenetic regulator in development.

Figure 2. Stx Genetically Interacts with Pc.

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(A–D) stx regulates compartmentalization of tergites. Overexpressing Pc disrupted tergite boundaries (B). stx2 mutant clones showed similar tergite malformation phenotype (C and D). Scale bar: 100 µm.

(E) Pupal lethality of stx2 is rescued by loss-of-function PcXT109 mutation.

(F–J) Stx and Pc exhibit reciprocal expression patterns in stx-yfp protein trap embryos. Enlarged boxed area is shown (G–J). Cells (indicated by arrowheads) with dense DAPI staining (I) had higher level of Stx-YFP (G) but lower level of Pc expression (H). Note that stx-yfp mRNA was expressed at a comparable level as endogenous stx (see Figure S4B). Scale bars: 100 µm (F) and 10 µm (G–J).

(K and L) Stx is required for Pc protein fluctuation in cell cycle. Pc expression was reduced while Stx expression was concomitantly increased when S2 cells were blocked at mitotic phase by colchicine treatment (K). Upon releasing from the mitotic blockage, Pc expression was recovered. However, knocking down stx by RNAi inhibited the Pc response to mitotic arrest (L).

Epigenetic silencing of homeotic target genes is mediated through balanced activities between PcG and Trithorax group (TrxG) proteins (Geisler and Paro, 2015). If the malformation of tergite boundaries in stx mutant flies was due to increased Pc activity, we reasoned that disruption of the TrxG function should further enhance the tergite phenotype. As anticipated, the penetrance of tergite phenotype was significantly increased when TrxG activity was compromised (Figure S3H). The genetic relationships between stx and PcG as well as TrxG highlight the involvement of stx in epigenetic regulation in vivo.

Physiological Relevance of Inverse Relationship between Stx and Pc

Given the similarity between stx loss-of-function and Pc gain-of-function phenotypes, we decided to test if stx and Pc genetically interact with each other. Removing one copy of Pc sufficed to rescue pupal lethality caused by stx2, suggesting that stx and Pc may function in opposite directions in epigenetic regulation (Figure 2E). This result fits with classical epistasis model that puts stx as an upstream regulator to repress Pc activity.

To further unveil physiological importance of stx activity on Pc, we carefully examined their protein expression patterns during development. At the subcellular level, fractionation assays with a rabbit antibody that we generated (Figure S3E and S5A) suggested that endogenous Stx was present in the nucleus but with no significant affinity with the chromatin (Figure S4A). As this antibody did not work for immunofluorescence, two homozygous viable YFP protein trap lines (CPTI-004181 and CPTI-004402), in which the yfp cassette is inserted in frame in stx locus (Figure S4B), were employed to analyze Stx protein expression in vivo. Consistent with fractionation data, Stx-YFP and Pc were found ubiquitously expressed in the nucleus in fly embryos and larvae (Figure 2F–2J, S4C–S4J and data not shown), suggesting that Stx may not serve as a cell-type specific Pc regulator. Intriguingly, we observed reciprocal expression pattern of Stx-YFP and Pc in embryos undergoing asynchronous nuclear divisions prior to cellularization (Figure 2F). We found populations of cells expressing higher level of Stx (Figure 2G) but lower level of Pc (Figure 2H) than surrounding cells. As many of those cells displayed condensed DAPI staining (Figure 2I), we believe they might be at the stage of entering mitosis. A reciprocal expression pattern was also observed in late third instar larval wing (Figure S4C–S4J) and leg imaginal discs (data not shown); scattered cells with higher amount of Pc exhibited reduced Stx expression. Interestingly, those cells also appeared to be in a distinct phase of the cell cycle as judged by the DAPI staining.

The expression of Pc protein is known to fluctuate in cell cycle both in vitro (Lanzuolo et al., 2011; Follmer et al., 2012) and in vivo (Buchenau et al., 1998; Steffen et al., 2013), but how this process is regulated is largely unknown. We found that the abundance of Pc protein was reduced in cultured fly S2 cells treated with colchicine to induce mitotic arrest (Rogers et al., 2009; Follmer et al., 2012). This decline of Pc abundance was accompanied by increased Stx expression (Figure 2K), which is consistent with reciprocal expression pattern observed in embryos and larval discs. When Stx function in colchicine-treated S2 cells was compromised by stx RNAi, however, Pc expression was no longer subject to fluctuation (Figure 2L). Thus, Stx might be required for cell cycle coupled regulation of Pc activity.

Stx Physically Interacts with Pc

To provide further evidence in support of a role of stx in Pc regulation, we investigated if Stx functions through physical interaction with Pc. When overexpressed in cl-8 cells, Stx co-immunoprecipitated with Pc (Figure 3A). Furthermore, endogenous Pc was able to form a complex with Stx-YFP (i.e. expressed at a comparable level as endogenous Stx in Figure S4B) in protein lysates extracted from the YFP protein trap flies (Figure 3B). Furthermore, we performed GST pulldown to demonstrate that Stx directly interacted with Pc. More importantly, we located a serine-rich Pc-binding domain (PcB) in Stx required for direct Pc interaction (Figure 3C).

Figure 3. Stx Physically Interacts with Pc.

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(A and B) Stx immunoprecipitates (IP) with Pc. Stx-GFP extracted from cl-8 cells stably expressing stx-gfp co-immunoprecipitated with endogenous Pc (A). Stx-YFP extracted from stx-yfp protein trap larvae co-immunoprecipitated with Pc (B).

(C) Stx binds directly with Pc. Different lengths of MBP-Stx fusions were subject to GST pulldown with recombinant GST-Pc protein. Y and N denote positive and negative interactions between Stx and Pc, respectively. MBP-Stx fusions pulled down by GST-Pc were detected with anti-MBP (upper panel). Inputs of MBP-Stx and GST-Pc were shown in lower panels. The Pc binding domain (PcB) that mediates direct interaction between Stx and Pc was mapped to amino acids (aa.) 210–300.

(D) Nuclear protein extracts of S2 cells stably expressing HA-Pc were fractionated on a Superose 6 column. The protein in fractions 4-–26 (fraction number indicated on top of each lane) was analyzed by immunoblotting as indicated. HA-Pc and Rpd3, which served as a positive control as it co-fractionated with Pc, were present in fractions 4–20. Endogenous Stx was found in fraction 14, while Psc, a component of canonical PRC1, was in fractions 4–12. The column was calibrated with a series of standard proteins, and molecular mass is indicated on the top with arrows. The sizes of protein complexes in fractions 15, 20 and 24 are estimated as 670, 150 and 44 kDa, respectively.

(E) Stx is not a member of the canonical PRC1. Protein lysates extracted from S2 cells overexpressing stx-gfp was subjected to IP with Stx-GFP. Stx interacted with endogenous Pc, but not with other components of the PRC1: Ph, Psc or Sce. β-Tubulin served as a negative control for IP.

(F) Stx does not form a complex with the PRC2. Protein lysates extracted from S2 cells overexpressing stx-FLAG was subject to IP with Stx-FLAG. Stx interacted with endogenous Pc, but not with Rpd3 or the components of the PRC2 including Esc and E(z).

As Stx directly interacts with a core component of the PRC1 complex, we wondered if Stx is a member of the PRC1. Thus, we conducted Superose 6 fractionation in S2 cells (Figure 3D) as well as in wildtype fly embryos (Figure S5C). Stx co-fractionated with Pc, but not with Psc, another core member of the PRC1, suggesting that Stx was not present in canonical PRC1 (Figure 3D and S5B). This conclusion was further strengthened by observation that Stx co-immunoprecipitated only with Pc but not other three core PRC1 members (Figure 3E). We also examined the association of Stx with E(Z) and Esc, two core components of the PRC2, as well as a non-canonical Pc interacting protein Rpd3, but failed to detect any interaction with Stx (Figure 3F). These data collectively demonstrate that Stx directly interacts with Pc to form a complex that is distinct from canonical PRC1.

Stx Promotes Pc Protein Degradation in the Proteasome

We next investigated the consequence of the Stx interaction with Pc. Increased stx activity sufficed to reduce the level of endogenous Pc protein in wing discs (Figure 4D). Removing the Pc-binding domain of Stx (StxΔPcB) abolished this effect (Figure 4G). To delve further into the in vivo control mechanism of the Pc regulation by Stx, we took advantage of an hs-Pc transgene whereby the production of Pc protein was controlled by a heat-shock (hs) promoter (Fauvarque et al., 1995). Stx was still able to reduce the level of hs-induced Pc (Figure 4J). Significantly, hs-Pc protein accumulated in cells lacking stx in mosaic clones (Figure 4M), indicating that the regulation of Pc by Stx likely took place at the post-transcriptional level. This conclusion was also verified by in situ analyses showing the Pc mRNA expression was not affected by stx (Figure S4M).

Figure 4. Stx Promotes Pc Protein Degradation in the Proteasome.

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(A–O) Stx negatively regulates Pc in vivo. Overexpressing stx (D–F and J–L) but not stxΔPcB (G–I) in the posterior compartment of the wing disc (marked by GFP) reduced the abundance of endogenous (D) as well as heat shock-induced Pc (hs-Pc) (J). In contrast, hs-Pc protein accumulated in stx2 homozygous mutant cells (marked by the absence of GFP) (M). Scale bars: 100 µm (A–L) and 50 µm (M–O).

(P–X) Pc is degraded through the proteasome. FLAG-Pc, induced for four hours in S2 cells, degraded rapidly, with a half-life around two hours (P). Proteasome (MG132 and ALLN) but not lysosome (E64 and NH4Cl) inhibitors prevented FLAG-Pc from degradation (Q). Stx facilitated Pc degradation in S2 cells, but this effect was reversed by MG132 treatment (R). Knocking down stx in S2 cells by dsRNA prevented FLAG-Pc from degradation (S). FLAG-Pc degraded much faster when stx was overexpressed (T). When stx expression was reduced by RNAi, the half-life of FLAG-Pc was extended (U). Note that FLAG-Pc shown in panels Q-S was chased for four hours after induction. Similarly, Stx-facilitated hs-Pc degradation (cf. J) was partially prevented when wing discs were incubated with MG132 (V). Scale bar: 100 µm (V–X).

As the canonical PRC1 is comprised of Pc, Ph, Psc and Sce, we asked if stx regulates the expression of other PRC1 components. stx had little effect on Sce (Figure S6A). Similar to its role on Pc, increased stx activity led to reduced Ph expression (Figure S6D). However, the Stx regulation on Ph was distinct from that on Pc as the production of hs-induced Ph (Fauvarque et al., 1995) was not affected by stx (Figure S6G). Interestingly, we found that the amount of ph mRNA was reduced in response to overexpressed stx (Figure S4Q) or Pc RNAi (Figure S4R). These observations are consistent with a previous report that ph transcription is negatively regulated by Ph itself but positively regulated by Pc (Fauvarque and Dura, 1993). Thus, we reasoned that the regulation of ph by Stx may be mediated through its effect on Pc. In contrast to its role on Pc and ph, increased stx activity led to upregulation of Psc (Figure S6J). We suspected this effect could also be a consequence of reduced Pc expression in stx overexpressing cells as Psc is a bona fide PcG target (Park et al., 2012). Indeed, we found that the Psc level was increased in Pc mutant cells (Figure S6M). These data together with genetic interactions between stx and Pc led us to conclude that the developmental role of stx is mediated mainly through Pc. Thus, we focused our study on revealing the underlying mechanism of how stx regulates Pc.

To date, not much is known about homeostatic regulation of PcG proteins. The genetic and physical interaction between Stx and Pc implies that Stx may be required for maintenance of Pc protein stability. To test this hypothesis, we performed pulse-chase experiments in S2 cells and found that Pc protein had a short half-life around two hours (Figure 4P). This rapid Pc turnover was due to regulated protein degradation in the proteasome; treating cells with specific proteasome, but not lysosome inhibitors, protected Pc protein from degradation (Figure 4Q). We then asked if Stx participated in proteasomal degradation of Pc. Overexpressed stx promoted Pc degradation in the proteasome (Figure 4R) whilst stx RNAi increased Pc abundance in S2 cells (Figure 4S). Consistently, heightened Stx activity resulted in shortened half-life whilst stx RNAi led to prolonged half-life of Pc protein (Figure 4T and 4U).

These in vitro results were validated by ex vivo analyses as Stx-induced proteasomal degradation of hs-Pc (Figure 4V) as well as endogenous Pc protein (Figure S6S) were partially prevented when wing discs were incubated with a proteasomal inhibitor. Together, our data support a direct role of Stx in promoting Pc protein degradation in the proteasome. Based on its activity on Pc protein stability, we named it Stuxnet after an infamous PC computer virus.

The UBL Domain of Stx Is Required for Regulated Pc Degradation

It is generally believed that most proteins destined for degradation in the proteosome are marked with ubiquitin (Ub) conjugated on specific lysine residues. Indeed, Pc was covalently modified by Ub (Figure 5A). Pc protein contains 35 lysine residues. In order to identify crucial lysines as Ub conjugation sites responsible for Pc degradation, we performed arginine scan on Pc in which lysine residues with high probability for Ub conjugation were replaced by arginines either individually or by combination. However, none of these mutant forms of Pc exhibited increased protein stability (data not shown). More surprisingly, the Pc mutant where all 35 lysines were replaced by arginines (PcKR) was still subject to Stx regulation in the proteasome (Figure 5B and 5C). To confirm this result, we expressed in vivo the yeast deubiquitinase UBP2 that can remove Ub from conjugated substrates in flies (DiAntonio et al., 2001). Elevated deubiquitination in the dorsal compartment of the wing disc protected Cubitus interuptus (Ci) protein (Figure 5D; cf. Figure S7C), a known ubiquitinated substrate subject to proteasomal degradation (Du et al., 2011), but not Pc protein (Figure 5E) from degradation. These findings together indicate that Pc may be degraded through an Ub-independent mechanism in the proteasome (Erales and Coffino, 2014).

Figure 5. Pc Protein Degradation in the Proteasome Is Independent of Ubiquitination Modification.

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(A–F) FLAG-PcKR, which was not modified by ubiquitination (A), was still subject to Stx-mediated proteasomal degradation (B and C). Overexpressing yeast ubp2 in the dorsal compartment (d) of the wing disc protected Ci protein from degradation (D), but had little effect on Pc (E). Scale bar: 100 µm (D–F).

(G–K) Stx facilitates Pc degradation through interacting with the proteasome. Shown is the alignment of UBL domains of Stx orthologs with experimentally verified PIM in Parkin and Rad23 proteins (G). “E” represents a β-strand and “H” an β-helix. The core PIM residues are highlighted in yellow (positively charged) and in blue (hydrophobic). Full-length Stx but not StxΔUBL co-immunoprecipitated with Rpn10 in S2 cell nuclear extracts (H). Overexpressing stxΔUBL led to accumulation of endogenous Pc in the wing disc (I). Scale bar: 100 µm (I–K).

In order to understand how Stx facilitates Pc protein degradation, we searched for known motifs present in Stx protein. Stx contains an Ub-like (UBL) domain at its N-terminus (Figure 3C). Increasing evidence suggest that a subset of N-terminal UBL domain-containing proteins, such as Rad23 and Parkin, interacts with the 19S regulatory particle subunit Rpn10 (Hiyama et al., 1999; Sakata et al., 2003). The proteasome-interacting motif (PIM) present in the UBL forms an interacting surface with the 26S proteasome, bridging the substrates to degradation machinery (Upadhya and Hegde, 2003). The presence of a conserved PIM in Stx (Figure 5G) hints a possibility that Stx may directly interact with the proteasome. Indeed, full-length, but not UBL domain-deleted Stx (StxΔUBL) co-immunoprecipitated with Rpn10 in nuclear extracts of S2 cells (Figure 5H). The physiological importance of this UBL domain is revealed in wing discs, where ectopically expressed stxΔUBL was sufficient to enrich Pc expression in vivo (Figure 5I). The opposite effects between stx and stxΔUBL were not due to differential transgene expression as they were targeted to attP2 landing site using the ΦC31 integrase (Markstein et al., 2008). Given that stxΔUBL was designed to disrupt the ability of Stx in proteasomal interaction, it was perhaps not surprising that StxΔUBL acted in a dominant negative fashion. Based on these results, we conclude that Stx utilizes the UBL domain to facilitate Pc protein degradation in the proteasome.

Stx Negatively Regulates PcG Activity

In Drosophila, Pc is recruited to the Polycomb response elements (PRE) on target chromatins upon H3K27me3 modification, thereby helping maintain chromatin compaction and target gene repression (Müller and Verrijzer, 2009). To investigate if Stx could modulate PcG activity in vivo, we examined the effect of stx on Pc recruitment to polytene chromosomes in fly salivary gland cells (Dietzel et al., 1999). We observed a stereotypical chromatin recruitment pattern of Pc in wildtype cells (Figure 6B and S7F). Overproduced Stx colocalized with Pc in the nucleus, but not on chromatin (Figure 6E–6H). Consequently, a general decrease in the amount and intensity of Pc binding on polytene chromosomes was observed in cells overexpressing stx (Figure 6F and S7J). This effect relies on the PcB domain of Stx as Pc recruitment to chromatin was not affected in cells overexpressing stxΔPcB (Figure 6J and S7N).

Figure 6. Stx Regulates PcG Activity In Vivo.

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(A–L) Stx regulates the recruitment of Pc to chromatin. In the fly salivary gland cells, Pc was recruited to polytene chromosomes to form a stereotypical banding pattern (B). Overexpressed stx disrupted the polytene chromosome localization of Pc-GFP; the majority of Pc-GFP co-localized with Stx in the nucleus (E–H). In contrast, overexpressing stxΔPcB did not alter chromatin recruitment of Pc-GFP (J). Scale bar: 10 µm.

(M) Stx regulates Pc occupancy on PcG targets. The amount of Pc bound to PREs was significantly reduced in wing discs overexpressing stx when analyzed by qChIP. The iab-7PRE and PGRP-LE served as positive and negative controls, respectively. The intensity of ChIP signal is represented as percentage of the input chromatin. Means ± SD from four independent experiments were shown. *p < 0.05, **p < 0.01 (unpaired, two-tailed Student’s t test).

(N–V) Stx regulates PRE activity. Reducing stx expression by stx RNAi in the posterior compartment of the wing disc (marked by GFP) led to decreased expression of ph-pPRE-lacZ (S). Conversely, overexpressing stx enhanced the expression of ph-pPRE-lacZ (V). Also see Figure S4Q for the role of elevated stx expression on ph-p transcription. Scale bar: 100 µm.

(W) A model showing that Stx epigenetically regulates target gene expression through its ability to facilitate regulated Pc protein degradation in the proteasome.

To quantitatively validate these results, we performed chromatin immunoprecipitation (ChIP) to compare, in wildtype and stx-overexpressing wing discs, the Pc recruitment on a well-characterized iab-7PRE in the bithorax complex (Mihaly et al., 1997) as well as several known PcG target genes (Martinez et al., 2009; Gonzalez et al., 2014), including Antp, bithoraxoid (bxd), engrailed (en), hedgehog (hh), Serrate (Ser) and reaper (rpr). We found that Pc did not bind to PGRP-LE, a non-PcG target, however, its recruitment to known PcG target chromatins was obviously reduced in stx overexpressed wing discs (Figure 6M). It has been reported recently that several proteasome subunits associate with the chromatin at the sites of stalled RNA Polymerase II (Pol II) (Gillette et al., 2004), and PcG components are known to regulate the distribution of stalled Pol II (Chopra et al., 2011; Schaaf et al., 2013). As Stx interacts with both the proteasome and Pc, we wondered if Stx had any preference towards PcG targets close to Pol II binding on the chromatin. However, Stx similarly affected those PcG targets despite the appearance of Pol II nearby (Figure S7Q).

PcG proteins interact with the PRE to confer Hox gene silencing in Drosophila (Lewis, 1978; Zink and Paro, 1989). To test if Stx-regulated chromatin recruitment of Pc had any impact on PcG target gene expression, we visualized directly the effect of stx on PcG activity in wing discs using a classical PRE reporter ph-pPRE-lacZ, which includes a 2.9 kbp P{418} fragment located upstream of ph-p transcription start site (Bloyer et al., 2003). Elevated stx expression in the posterior compartment of the wing disc led to de-repression of ph-pPRE-lacZ (Figure 6V). Reduced stx expression, on the other hand, enhanced PcG activity to further repress ph-pPRE-lacZ (Figure 6S). Together, these data led us to conclude that Stx functions as a general PcG regulator in development.

Stx Is a Conserved PcG Regulator

Orthologous Stx proteins have been identified in vertebrates, but their function in development is still unknown (Tsukahara et al., 2000; Zielak et al., 2008; Hofmeister-Brix et al., 2013). The fact that these orthologs contain an UBL domain led us to hypothesize that vertebrate Stx might act in a similar fashion on Pc protein homeostasis. There are five orthologous Pc proteins in vertebrates, named Cbx2, 4, 6, 7 and 8 (Bernstein et al., 2006). Intriguingly, mouse Stx (mStx) specifically destabilized Cbx4, but not other four Cbx proteins in 293T cells (Figure 7A). Cbx4 protein was also subject to proteasomal degradation, resulting in a relatively short half-life around three hours (Figure 7B and 7C). Similar to fly Stx, only full-length but not the UBL domain-deleted mStx promoted Cbx4 degradation in the proteasome (Figure 7D and 7E).

Figure 7. Stx Function Is Conserved from Drosophila to Vertebrates.

Figure 7

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(A–E) mStx facilitates proteasomal degradation of vertebrate Pc protein. mStx reduced the expression of Cbx4 when co-expressed in HEK293T cells (A). Cbx4 protein was subject to degradation in the proteasome (B), with a half-life around four hours (C). Proteasome inhibitors (MG132 and ALLN) protected Cbx4 from mStx-mediated degradation (D). Similar to fly Stx, the conserved UBL domain was required for mStx-facilitated Cbx4 degradation (E).

(F–I) mStx is functionally conserved in Drosophila. Overexpressing mStx in wildtype antennae induced antenna-to-leg transformation (F). This phenotype was resulted from reduced expression of Pc (G) and Dan (H), and de-repression of Antp in antennal discs (I). Arrow and asterisk indicate tarsal and apical bristles, respectively. Arrowhead marks ommatidia-like structure.

To demonstrate functional conservation between fly and vertebrate Stx in vivo, we generated transgenic flies expressing mstx. Importantly, ectopic expression of mstx in antenna was sufficient to induce antenna-to-leg transformation (Figure 7F–7I). Similar to that caused by fly stx, mstx induced ommatidia-like structures on transformed antennae and de-repression of eye-specific genes (Figure S2C, S2J–S2L and S2S–S2U). Therefore, the Stx activity on Pc protein homeostasis is conserved from flies to vertebrates.

DISCUSSION

Taking advantage of genetic tools available in Drosophila, we explored the function of an UBL domain-containing protein Stx, and uncovered its unexpected role of regulated Pc protein degradation in epigenetic repression. Our analyses on classical PcG targets demonstrate that Stx functions as a Pc-specific regulator that negatively modulates the PcG activity. Importantly, we find that this mode of regulation is conserved from flies to vertebrates.

Stx Regulation of PcG Activity

stx activity is essential for Drosophila development. The pupal lethal phenotype associated with loss-of-function stx mutations can be rescued by removing 50% of Pc activity strongly supports that modulating the Pc expression is the major developmental process regulated by stx. Stx might not be a constitutive component of the canonical PRC1 (Figure 3D–3F). However, the ability of Stx to reduce Pc recruitment to target gene loci argues that Stx may act as a gatekeeper to control Pc availability to form highly dynamic PRC complexes on target chromatin (Ficz et al., 2005). As stx activity is necessary for PcG target expression, Stx could function in an intrinsic machinery to regulate Pc protein homeostasis. Stx directly binds Pc through a serine-rich PcB domain and interacts with the proteasome through the UBL domain. As Pc protein degradation does not rely on ubiquitination, the UBL domain in Stx, upon interaction with Pc, could serve as a recognition signal that marks Pc protein for degradation in the proteasome. Thus, we propose a model in which Stx acts first as an adapter and then a chaperon-like protein to facilitate proteasomal degradation of Pc, resulting in altered PcG activity in animal development (Figure 6W). Intriguingly, upon inspection of modENCODE database (Contrino et al., 2012), we find in stx locus multiple binding sites for PcG components, including Pc, Psc, Sce and Pho, and Ubx, which is itself a PcG target, thus pointing to the existence of a potential feedback loop between Stx and PcG activity.

Altered Pc protein abundance has been noted in several biological processes. In the Sce mutant fly embryos, the bulk level of Pc protein is significantly reduced, but Ph and Psc are not affected (Pengelly et al., 2015). Similar results have been reported in mouse ES cells for RING1B and Cbx4, mammalian orthologs of Sce and Pc (Leeb and Wutz, 2007). However, the significance of such regulation was not understood. We suspect that binding with Sce might stabilize Pc, which is crucial for PRC1 assembly. It is interesting to note that the level of Pc changes rapidly in cell cycle (Lanzuolo et al., 2011; Follmer et al., 2012). The oscillation of Pc protein during cell cycle is thought to be important for establishment and maintenance of cellular epigenetic memory (Follmer et al., 2012). Our observation of the reciprocal expression pattern of Pc and Stx as well as the ability of Stx to control Pc abundance in cell cycle are in favor of a notion that regulated Pc protein stability may be one way to dynamically control Pc activity under physiological contexts. How Stx participates in such regulation is an interesting question that awaits further exploration.

The PRC1 is composed of four core subunits, each of which has unique molecular activities and is non-exchangeable for each other (Schwartz and Pirrotta, 2013). However, the loss-of-function phenotypes of individual PRC1 subunits in Drosophila only partially overlap, revealing the complexity of PRC1 regulation in various cellular processes (Classen et al., 2009; Feng et al., 2011; Martinez et al., 2009). The differential requirement of PRC1 subunits in development might be due to the presence of distinct PRC complexes in a temporal and tissue specific manner (Kang et al., 2015). This view is further complicated in vertebrates by partially redundant orthologous PRC1 proteins and formation of multiple non-canonical complexes (Strübbe et al., 2011). Thus, it is required to explore the regulatory machineries utilized by individual PRC1 components in order to better understand how PRC complexes exert versatile functions in vivo. We have shown that Stx targets Pc for proteasomal degradation, but whether parallel regulators existed for other PRC1 components are still unknown.

Regulated Protein Degradation Plays a General Role in Modulating PcG Activity

Our study of Stx regulation on Pc stability reveals that the activity of Pc protein, the founding member of the PRC complexes, can be controlled through regulated protein degradation. Surprisingly, we found that fly Pc protein is largely regulated by ubiquitination-independent degradation (UID). The list of substrates that undergo UID expands rapidly in recent years. Intriguingly, many UID substrates are localized to nucleus, including transcription factors and chromatin remodeling factors (Erales and Coffino, 2014). The addition of Pc, a key epigenetic regulator, to this list leads us to believe that UID in the nucleus may participate in the control of gene expression.

Consistent with a role of Stx on Pc stability in Drosophila development, proteasomal degradation has been reported to affect stability of several PcG components in cultured vertebrate cells, including three PRC1 proteins BMI1, RING1B, PHC and one PRC2 protein EZH2 (Ben-Saadon et al., 2006; Maertens et al., 2010; Wu et al., 2010; Zaaroor-Regev et al., 2010; Sahasrabuddhe et a., 2011; Wu and Zhang, 2011; Yu et al., 2013;). It is thus highly likely that protein degradation may play a general role in regulating PcG activity.

Appropriate PcG activity is essential for stem cell maintenance and lineage specification in vertebrates (Gil and O'Loghlen, 2014). Altered PcG activity is associated with malignant human diseases, including cancer (Rajasekhar and Begemann, 2007). Furthermore, dysregulated stx expression and Stx mutations are reported in several forms of cancer in the COSMIC database (Forbes et al., 2015). Consistently, genes co-expressed with stx shown in COXPRESdb (Okamura et al., 2015) are clustered into pathways in cancer as well as Notch and MAPK signaling pathways. Very recently, Stx mutations are found in patients with autism spectrum disorders (ASD) by whole exome sequencing (Butler et al., 2015). Given the strong connection between PcG and ASD (Gao et al., 2014; Kumari and Usdin, 2014), Stx may play a role in ASD through its regulation on PcG activity. Thus, the identification of regulators of PcG activity, such as Stx, may provide additional therapeutic targets for relevant diseases.

EXPERIMENTAL PROCEDURES

Fly Genetics

Details of fly strains and crosses used in this study are listed in Supplemental Experimental Procedures.

Molecular Biology

Fly stx (CG32676) and Pc, mouse stx (gene ID: 59090) and Cbx were cloned into pUAST, pMT or pcDNA3 vectors containing Myc, FLAG or GFP tags. The StxΔUBL, StxΔPcB and mStxΔUBL mutations were created by PCR. FLAG-PcKR was generated through multiple rounds of site-directed mutagenesis (Stratagene). GST and MBP fusions were cloned in pGST-parallel2.1 or pMBP-parallel2.1. All plasmids were verified by sequencing. Primers used for generating various mutants are listed in Supplemental Experimental Procedures.

Generation of Stx Antibody

A rabbit polyclonal antibody was raised against a synthetic peptide (CHKTGNNRITRTKHRHYHGQ) corresponding to aa. 585–603 of fly Stx (YenZym Antibodies, LLC). Its specificity was confirmed by immunoblotting (1:10,000) (Figure S3E and S5A). This antibody was not suitable for immunofluorescence even after affinity purification with Bio-Rad Affi-Gel 10 gel.

Antibody Staining and In Situ Hybridization

Standard procedures were used for antibody staining and in situ hybridization (Su et al., 2011). A full list of antibodies and antisense RNA probes used is provided in Supplemental Experimental Procedures. Images were acquired with a Leica TCS SP8 Confocal system or a Zeiss Axio Imager Z1 microscope equipped with an ApoTome. The figures were assembled in Adobe Photoshop CS5. Minor image adjustments (brightness and/or contrast) were performed in AxioVision 4.8.1 or Adobe Photoshop.

Mounting of Adult Fly Structures

Adult flies were fixed overnight in isopropanol before dissection. Adult antennae, halters, legs and wings were mounted in Euparal mounting media (BioQuip). Images were acquired with a Leica DMIL inverted microscope or a Leica MZ16F stereomicroscope equipped with a QImaging digital camera.

Cell Culture and Drug Treatments

Drosophila Schneider 2 (S2) cells and S2 cells stably expressing HA-Pc (Tie et al., 2007) were cultured in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% FBS and transfected using calcium phosphate method. Fly clone 8 (cl-8) cells were cultured in M3 insect medium (Sigma) supplemented with 2.5% FBS and 2.5% fly extract. A cell line stably expressing copper inducible stx-gfp was generated by transfecting pMT-stx-gfp in cl-8 cells using Effectene (Qiagen). HEK293T cells were maintained in DMEM with 10% FBS and transfected using Lipofectamine 2000 (Invitrogen). In some experiments, MG132 (50 µM; Sigma) or ALLN (50 µM; Sigma) was added to cultured cells or wing discs for six to eight hours to inhibit proteasome activity, while E64 (50 µM; Sigma), leupeptin (50 µM; Sigma) or NH4Cl (10 mM) was used to disrupt lysosome function. Cycloheximide (CHX; 50 µg/ml; Sigma) was added to S2 cells for six hours to inhibit nascent protein synthesis (Zhang et al., 2014), while colchicine (30 µM; Sigma) was added to S2 cells for 12 hours to induce mitotic arrest (Rogers et al., 2009).

Biochemistry

Cultured cells or fly larval tissues were collected for immunoblotting. Immunoprecipitation was performed using either agarose anti-GFP (Vector Lab.) or anti-FLAG M2 affinity gel (Sigma). Further details for ubiquitination and GST pulldown are provided in Supplemental Experimental Procedures.

Quantitative Chromatin Immunoprecipitation (qChIP)

qChIP assays for wing imaginal discs were performed using protocols described previously (Classen et al., 2009; Martinez et al., 2009; Gonzalez et al., 2014). Rabbit anti-Pc (gift of Giacomo Cavalli) was used in ChIP. All ChIP analyses were repeated three times with independent biological replicates. The primer pairs used for qPCR are listed in Supplemental Experimental Procedures.

Supplementary Material

1
2

Highlights.

  • -

    Stuxnet (Stx) and Polycomb (Pc) proteins display reciprocal expression patterns.

  • -

    Stx facilitates Pc protein degradation through a ubiquitin-independent pathway.

  • -

    Stx modulates PRC1 activity to epigenetically control target gene transcription.

  • -

    Stx effect on Pc is conserved from flies to vertebrates.

Acknowledgments

We thank David Allis, Sharon Bickel, Giacomo Cavalli, Stephen Cohen, Renjie Jiao, Shunsuke Ishii, Florence Maschat, Jocelyn McDonald, Jürg Müller, Jessica Treismn, Rongwen Xi, Bloomington, DGRC and VDRC stock centers, and DSHB for fly stocks and antibodies. We thank Zhu lab members for help on wing disc dissection for ChIP analyses. This work was supported by the Ministry of Sciences and Technology of China (2014CB942804 to A.J.Z.), National Natural Science Foundation of China (31371410 to A.J.Z., 31401241 to M.L. and 31471382 to J.Z.), NIH (R01GM39255 to P.J.H. and R01GM85175 to A.J.Z.), Peking-Tsinghua Center for Life Sciences and State Key Laboratory of Membrane Biology to A.J.Z.

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

J.D., J.Z. and A.J.Z. designed the study and wrote the paper. J.D., J.Z., T.H., Y.L., Y.S., F.T., M.L. and P.J.H. preformed experiments, data analyses and interpretation.

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NIHMS790141-supplement-1.pdf (14MB, pdf)
2
NIHMS790141-supplement-2.doc (22.5KB, doc)

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