© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

RESEARCH ARTICLE HMGB2 regulates satellite-cell-mediated skeletal muscle regeneration through IGF2BP2 Xingyu Zhou*, Mingsen Li*, Huaxing Huang, Keren Chen, Zhuning Yuan, Ying Zhang, Yaping Nie, Hu Chen, Xumeng Zhang, Luxi Chen, Yaosheng Chen‡ and Delin Mo‡

ABSTRACT and Rudnicki, 2008). Then, these myoblasts begin to proliferate to Although the mechanism underlying modulation of transcription expand the population of progenitor for repair (Chargé and Rudnicki, factors in myogenesis has been well elucidated, the function of the 2004; Kuang and Rudnicki, 2008). Finally, proliferating myoblasts transcription cofactors involved in this process remains poorly withdraw from the cell cycle, undergo terminal differentiation and understood. Here, we identified HMGB2 as an essential nuclear fuse with each other to form renascent myofibers that replace the transcriptional co-regulator in myogenesis. HMGB2 was highly damaged and dead ones (Apponi et al., 2011; Kang and Krauss, expressed in undifferentiated myoblasts and regenerating muscle. 2010). Sets of transcription factors have been identified that regulate Knockdown of HMGB2 inhibited myoblast proliferation and stimulated the proliferation of satellite cells and terminal differentiation. its differentiation. HMGB2 depletion downregulated Myf5 and cyclin However, more precise molecular mechanism and alternative key A2 at the but not mRNA level. In contrast, overexpression regulators are required to be further elucidated. of HMGB2 promoted Myf5 and cyclin A2 protein upregulation. The high-mobility group (HMG) superfamily consists of three Furthermore, we found that the RNA-binding protein IGF2BP2 is a families, HMGA, HMGB and HMGN, all of which are chromatin- downstream target of HMGB2, as previously shown for HMGA2. binding that share similarities in structural and functional IGF2BP2 binds to mRNAs of Myf5 or cyclin A2, resulting in translation properties (Agresti and Bianchi, 2003; Hock et al., 2007). It has enhancement or mRNA stabilization, respectively. Notably, been well documented that HMG proteins not only act as dynamic overexpression of IGF2BP2 could partially rescue protein levels of modulators of chromatin architecture but also especially influence Myf5 and cyclin A2, in response to HMGB2 decrease. Moreover, DNA replication, recombination, repair and transcriptional depletion of HMGB2 in vivo severely attenuated muscle repair; this regulation (Agresti and Bianchi, 2003; Bianchi and Agresti, 2005; was due to a decrease in satellite cells. Taken together, these results Ueda and Yoshida, 2010). Multiple transcription factors have been highlight the previously undiscovered and crucial role of the HMGB2– verified to interact with HMG proteins at specific during IGF2BP2 axis in myogenesis and muscle regeneration. the process of transcription (Ueda and Yoshida, 2010). Moreover, it has been reported that both HMGA and HMGB families are mainly KEY WORDS: HMGB2, Myogenesis, IGF2BP2, Muscle regeneration expressed in stem cells or progenitors to maintain proliferation and undifferentiated status (Hock et al., 2007). In particularly, HMGA2 INTRODUCTION can induce mouse embryonic stem cells to commit to the myogenic Myogenesis is the term describing the process of myofiber formation, lineage and regulate myoblast proliferation and skeletal muscle which occurs during embryonic development, postnatal growth development (Caron et al., 2005; Li et al., 2012). and muscle regeneration (Chargé and Rudnicki, 2004; Tidball and There are three members of HMGB proteins in mammals, Villalta, 2010). This process is a highly coordinated event and HMGB1, HMGB2 and HMGB3. All of them contain two DNA- implicated in a series of transcriptional networks, among which Pax7, binding domains (basic HMG boxes) followed by a long acidic Myf5, MyoD (also known as MYOD1), and Mrf4 (also C-terminal tail and have more than 80% identity in protein sequences known as MYF6) are crucial and indispensable (Buckingham and (Bustin, 1999; McCauley et al., 2005; Catena et al., 2009). HMGB1 Rigby, 2014; Mok and Sweetman, 2011). Skeletal muscle repair from has been demonstrated to play a key role in multiple biochemical and injury involves in a series of complicated cascade events, which can molecular activities, such as the innate immunity response, necrosis, be divided into the following steps. First, quiescent satellite cells, a arthritis and tumorigenesis (Hock et al., 2007; Yanai et al., 2012). pool of adult muscle stem cells, are activated and become committed Although it has been demonstrated that as a transcriptional co- myoblasts in response to the damage (Kang and Krauss, 2010; Kuang regulator, HMGB2 regulates various differentiation programs, including erythropoiesis, chondrogenesis and spermatogenesis, its role in myogenesis and muscle regeneration remains largely unclear State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, (Laurent et al., 2010; Ronfani et al., 2001; Taniguchi et al., 2011). Guangzhou 510006, China. Post-transcriptional regulation of myogenesis is an important *These authors contributed equally to this work process for muscle development and regeneration (Apponi et al., ‡Authors for correspondence: ([email protected]; 2011). Insulin-like growth factor-II mRNA-binding proteins [email protected]) (IGF2BP2), also known as IMP2, is a member of IMP family that X.Z., 0000-0001-7589-9928; M.L., 0000-0003-3019-3387; H.H., 0000-0001- contains two RNA-binding domains and four K homology motifs 6455-676X; K.C., 0000-0002-3871-7651; Z.Y., 0000-0001-6192-6905; Y.Z., and functions in mRNA localization, turn over and translation 0000-0002-5861-2355; Y.N., 0000-0003-4456-0651; H.C., 0000-0001-8136-7442; modulation (Nielsen et al., 2002, 2003). The involvement of RNA- L.C., 0000-0001-7802-9215; Y.C., 0000-0002-3871-7651; D.M., 0000-0002- 8738-4486 binding proteins, such as IGF2BP2, HuR (also known as ELAVL1), CUGBP1 and Lin-28, in muscle biology has been extensively

Received 18 April 2016; Accepted 17 September 2016 highlighted in recent investigations (Apponi et al., 2011; Li et al., Journal of Cell Science

4305 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

2012). A previous study found that IGF2BP2 functions by binding HMGB2 locates in nuclei of myoblast and functions as a to the 5′ UTR of insulin-like growth factor 2 (IGF2) mRNA and myogenic repressor controlling IGF2 translation (Dai et al., 2011). More recently, In order to further investigate the impact of HMGB2 on myogenesis, IGF2BP2 was also suggested to regulate myoblasts proliferation the C2C12 myoblast differentiation model was studied. The through binding to various target mRNAs and modulating their expression trend of HMGB2 was evaluated during myogenic translation, such as , Sp1, IGF1R, Ccng1 and Nras (Gong et al., differentiation. Similar to the results in vivo, both HMGB2 and 2015; Li et al., 2012). Interestingly, IGF2BP2 has been identified as Pax7 were highly expressed in proliferating C2C12 myoblasts but a direct target of HMGA2 in proliferating myoblasts, NIH/3T3 promptly downregulated after differentiation (Fig. 2A,B). Few fibroblasts and mouse embryos (Brants et al., 2004; Cleynen et al., HMGB2 proteins were detected in mature myotubes (Fig. 2C). Both 2007; Li et al., 2012). However, whether or not HMGB2 is able to in vivo and in vitro analyses showed that HMGB2 proteins were regulate the expression of IGF2BP2 during myogenesis is unknown. mainly located in Pax7-positive muscle stem cells or progenitors but In the present study, we first found that HMGB2 was upregulated rarely in mature myofibers (Figs 1C,D, 2C), indicating that HMGB2 significantly in the early stage of muscle regeneration and highly is mostly expressed in undifferentiated myoblasts. expressed in proliferating myoblasts. Then, we used gain- and loss- As HMGB2 is a chromatin-binding protein and also interacts with of-function approaches to demonstrate that HMGB2 maintains transcription regulators in the nucleus, its location in C2C12 myoblast proliferation and impedes myogenic differentiation by myoblasts and primary satellite cells isolated from adult mice was controlling IGF2BP2, which in return enhanced the protein detected in this work. Primary satellite cells were successfully production of Myf5 and cyclin A2. It was further demonstrated isolated and became activated myoblasts. As expected, that IGF2BP2 could bind either to Myf5 or to cyclin A2 mRNA to immunofluorescence staining showed that HMGB2 proteins were improve the mRNA stability or enhance the translation, only located in the nuclei of C2C12 and primary myoblasts respectively. In vivo data also implied that HMGB2 was required (Fig. 2D,E), which prompted us to speculate that HMGB2 operates for satellite cell proliferation and muscle repair after injury. In with other myogenic regulators in the nucleus. summary, we have identified and characterized the role of the Next, three small interfering RNAs (siRNAs) were designed to HMGB2–IGF2BP2 axis in myogenesis and muscle regeneration. knock down HMGB2 in C2C12 myoblasts (denoted si-HM-1, si- HM-2 and si-HM-3), two of which were efficient (Fig. S2A). Si- RESULTS HMGB2, a mixture of si-HM-2 and si-HM-3, caused a substantial The expression pattern of HMGB2 during muscle knockdown of HMGB2 proteins and was used in all of the following development and generation analysis (Fig. S2B). As shown, depletion of HMGB2 accelerated the To determine the expression pattern of potential in the early initiation of myogenic program, because of a significant increase in stage of muscle repair after injury. RNA sequencing (RNA-seq) was the mRNA expression of early myogenic marker myogenin at 24 h employed to analyze the genome-wide of damaged after differentiation induction (Fig. 3A). Myogenin-positive cells also muscle tissues at various time points after injury. Among sets of increased prominently at 24 h after differentiation when HMGB2 was differentially expressed genes, HMGB2 was focused on because of knocked down (Fig. 3B). A similar increase in the expression of fast its upregulated expression (Fig. 1A). Real-time quantitative PCR myosin skeletal heavy chain (MyHC,alsoknownasMyh1) was also (qPCR) and western blotting also confirmed that the expression of observed in differentiating C2C12 cells after HMGB2 knockdown HMGB2 significantly increased in the early period of muscle repair, (Fig. 3C,E). Consistently, addition of si-HMGB2 in C2C12 cells peaking at day 3 after cardiotoxin (CTX) injection, indicating its caused a significant enhancement of myotube formation (Fig. 3D). potential role in muscle regeneration (Fig. 1B). Furthermore, large However, ectopic expression of HMGB2 led to a remarkable numbers of HMGB2-positive cells were found in the damaged adult concomitant repression of myotube formation (Fig. 3F). Thus, tibialis anterior muscle but not in the uninjured one (Fig. 1C,D). It HMGB2 was identified as a crucial myogenic regulator. has been well documented that activation and proliferation of quiescent satellite cells are required for adult muscle regeneration HMGB2 regulates myoblast proliferation through cell-cycle (Kang and Krauss, 2010). Importantly, immunofluorescence proteins revealed that most of HMGB2-positive cells were simultaneously It has been well established that HMG proteins are mainly specific Pax7-positive, a marker of muscle stem cell or progenitor (Fig. 1D). to stem cells, and they are implicated in the regulation of stem cell Further evidence revealed that both mRNA and protein of HMGB2 proliferation and differentiation (Agresti and Bianchi, 2003; Hock were highly expressed in neonatal muscle but decreased during the et al., 2007). More recently, Zhizhong Li and his colleagues have progress of postnatal development and were not detectable in mature reported that the HMGA2–IGF2BP2 axis affects muscle muscle (Fig. 1E). Collectively, these data support that HMGB2 is development through controlling myoblast proliferation (Li et al., expressed mainly in myoblasts but not in mature myofibers. 2012). Simultaneously, the expression of HMGA2 decreased Intriguingly, the myogenic genes Myf5, myogenin and Mrf4 significantly when HMGB2 was downregulated (Fig. S2C–E). showed a similar expression pattern during muscle development, These results drove us to hypothesize that HMGB2 probably implying that HMGB2 might be expressed during myogenesis suppresses myogenesis by means of regulating myoblast (Fig. S1A). However, MyoD and Pax7 have differing expression proliferation and self-renewal. To test this, the proliferation rate of patterns to HMGB2 in muscle development, which indicates that C2C12 cells and muscle satellite cells transfected with si-HMGB2 they potentially operate through a different mechanism. In addition, and si-NT was examined, respectively. Data from a real-time the expression of HMGB2 in other tissues of adult mice was also monitoring system revealed that the proliferation of C2C12 detected. We found that HMGB2 was widely expressed in various myoblasts and primary satellite cells was reduced after HMGB2 adult tissues (Fig. S1B), raising the possibility that HMGB2 might depletion with siRNAs (Fig. 4A; Fig. S4B). Flow cytometry participate in maintaining the physiological function and analysis followed by propidium iodide staining further revealed that homeostasis of these organs. Taken together, HMGB2 might play an arrest of cell cycle in S phase occurred in response to a HMGB2 a vital role in muscle development and regeneration. decrease in C2C12 myoblasts (Fig. 4B). Journal of Cell Science

4306 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

Fig. 1. HMGB2 expression gradually decreases during postnatal muscle development and is induced in the early of muscle regeneration. (A) Some of the differentially expressed genes identified by RNA-seq during muscle regeneration (n=2 mice per group) represented by a heat map. (B) The tibialis anterior muscle was subjected to cardiotoxin (CTX) injury and then qPCR (left) and western blotting (right) were performed to detect the expression of HMGB2 at day 0, day 1, day 2, day 3 and day 4 after injury, respectively. Data are presented as mean±s.e.m.; n=3 mice per group. (C) Immunofluorescence staining for DAPI (blue), HMGB2 (green) and Pax7 (red) on tibialis anterior muscle sections after 3 days of NaCl (top) or CTX (bottom) injection. White arrows indicate Pax7- and HMGB2-double-positive cells. Scale bar: 100 μm. (D) Immunohistochemistry analysis for HMGB2 on tibialis anterior sections 3 days after CTX or NaCl injection. Brown, HMGB2; blue, nuclei. Scale bar: 100 μm. (E) qPCR (left) and western blotting (right) analysis for HMGB2 expression in tibialis anterior at postnatal day 1, day 14 and day 140. Data are presented as mean±s.e.m.; n=3 mice per group. To address the molecular mechanism underlying the control of Next, a model of CTX-mediated muscle injury concomitant with cell cycle progression through HMGB2, HMGB2 was knocked HMGB2 depletion was used to examine the expression of the above down in myoblasts. The expression of several myogenic-associated regulators (Fig. 4F). Both mRNA and protein levels of HMGB2 factors (Myf5, MyoD, Pax7, and YY1) and cell cycle regulators were successfully downregulated in tibialis anterior at day 3 after [cyclin A2, cyclin E, CDK2, CDK4, Rb, P27 (CDKN1B) and Akt injury by intramuscular injection of si-HMGB2 (Fig. 4G,H), which (Akt1 isoform)] was detected by qPCR. A significant difference in resulted in significant reduction in Myf5, cyclin A2 and Pax7 mRNA level between the si-HMGB2 group and the control group proteins (Fig. 4H). In addition, the number of EdU (5-ethynyl-2′- was not observed for any of the detected genes (Fig. 4C,D). deoxyuridine)-positive cells decreased substantially in tibialis Intriguingly, a pronounced reduction of Myf5 and cyclin A2 anterior when HMGB2 was downregulated (Fig. 4I), implying proteins occurred in response to si-HMGB2 treatment both in that the proliferation of satellite cells was restrained after HMGB2 C2C12 myoblasts and primary muscle satellite cells. However, there was depleted. Based on in vitro and in vivo results, we conclude that was no significant difference for YY1, Pax7 and MyoD proteins HMGB2 maintains myoblast proliferation and self-renewal by (Fig. 4E; Fig. S4C). In addition, overexpression of HMGB2 did not controlling protein expression of Myf5 and cyclin A2. affect the mRNA levels of Myf5 and cyclin A2, but promoted their protein expression (Fig. S3A,B). Consistent with the siRNA IGF2BP2 acts as a downstream target of HMGB2 experiment, overexpression of HMGB2 did not affect either the We found that HMGB2 regulates Myf5 and cyclin A2 at the protein

RNA or protein levels of Pax7 and MyoD (Fig. S3A,B). but not mRNA level, suggesting that a post-transcriptional Journal of Cell Science

4307 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

Fig. 2. The expression and location of HMGB2 in satellite cells and differentiating C2C12 cells. (A) qPCR analysis for the expression of HMGB2 in C2C12 cells cultured in growth medium (GM) and in the first 3 days after differentiation induction (DM). Data are presented as mean±s.e.m.; n=3. (B) Western blotting analysis for the expression of HMGB2, Pax7 and MyHC in C2C12 cells cultured in growth medium and in the first 3 days after differentiation induction. (C) C2C12 cells were induced to differentiate for 3 days before immunofluorescence staining for DAPI, HMGB2 and MyHC (left). The percentage of HMGB2-positive or HMGB2-negative nuclei in MyHC-positive myofibers was calculated (right). White arrows indicate unfused HMGB2-positive nuclei. Data are presented as mean± s.e.m.; n=3. Scale bar: 50 μm. **P<0.01 (Student’s t-test). (D) Immunofluorescence staining for DAPI and HMGB2 in C2C12 cells cultured in growth medium. Scale bar: 50 μm. (E) Satellite cells were isolated from the extensor digitorum longus (EDL) of adult mice and cultured in proliferating medium. Triple immunofluorescence staining for DAPI, Pax7 and HMGB2 was performed. Scale bar: 50 μm. regulation mechanism needed to be further elucidated. IGF2BP2, a results imply that IGF2BP2 is a downstream target of HMGB2 both RNA-binding protein, can bind to the 5′-UTR of target mRNAs and in vivo and in vitro. enhance their stability and/or translation (Dai et al., 2011; Nielsen et al., 2002). A recent report has also suggested that HMGA2 IGF2BP2 regulates myoblast proliferation via promoting directly regulates IGF2BP2 to affect myoblast proliferation and protein generation of Myf5 and cyclin A2 skeletal muscle development (Li et al., 2012). Intriguingly, the To gain insights into the mechanism by which IGF2BP2 regulates expression of IGF2BP2 was found to decrease in tibialis anterior myoblasts proliferation, IGF2BP2 was knocked down in myoblasts. muscle as well during postnatal development and was at a low level Flow cytometry analysis after propidium iodide staining in adult muscle (Fig. 5A). This expression pattern was similar to that demonstrated that a decrease in IGF2BP2 caused the cell cycle of HMGB2. Whether or not IGF2BP2 is a downstream effector of arrest in S phase, as it is evidenced by a higher proportion of HMGB2 and regulates the protein levels of Myf5 and cyclin A2 in myoblasts in S phase after si-IGF2BP2 treatment (Fig. 6A). These myoblasts needs to be further investigated. observations recapitulate the results of HMGB2 knockdown To verify this possibility, both HMGB2 knockdown and (Fig. 4B). Then, RNA-binding protein immunoprecipitation (RIP) overexpression experiments were first conducted. At 2 days after was carried out to identify target mRNAs of IGF2BP2. It was transfection, qPCR and western blotting analyses were performed observed that, compared to the control group, both Myf5 and cyclin in C2C12 myoblasts cultured in growth medium. Results showed A2 mRNAs, but not Pax7, MyoD, CDK2, smurf1 and CDK4 that depletion of HMGB2 reduced IGF2BP2 expression, whereas mRNAs, were well enriched by the IGF2BP2 antibody, suggesting ectopic expression of HMGB2 caused an increase in IGF2BP2 that IGF2BP2 binds to Myf5 and cyclin A2 mRNAs in myoblasts (Fig. 5B,C). Furthermore, consistent with in vitro results, in vivo (Fig. 6B,C). analysis showed that the expression of IGF2BP2 was upregulated Further analysis indicated that depletion of IGF2BP2 could not significantly in the early stages of muscle injury and affect mRNA levels of Myf5 and cyclin A2 but downregulated both downregulated when HMGB2 was depleted (Fig. 5D,E). Next, protein levels in C2C12 myoblasts and muscle satellite cells three siRNAs targeting IGF2BP2 (denoted si-IG-1, si-IG-2 and si- (Fig. 6D,E; Fig. S4E). However, MyoD was not regulated by IG3) were used to repress IGF2BP2 expression. As shown, all of IGF2BP2 (Fig. 6D,E). In contrast, an increase in Myf5 and cyclin them were efficient (Fig. 5F, left). Si-IGF2BP2, a mixture of these A2 proteins accompanied the overexpression of IGF2BP2 (Fig. 6F). three siRNAs, could also significantly inhibit protein expression Remarkably, overexpressing IGF2BP2 could partially rescue the si- of IGF2BP2 (Fig. 5F, right), and was applied in the following HMGB2-mediated reduction of Myf5 and cyclin A2 proteins experiments. It was shown that there was no significant difference (Fig. 6G). Moreover, immunofluorescence analysis demonstrated in HMGB2 expression when IGF2BP2 was knocked down in that overexpression of IGF2BP2 inhibited the enhanced myoblast myoblasts (Fig. 5G). differentiation caused by HMGB2 knockdown in C2C12 cells To test the role of IGF2BP2 in myogenesis, both C2C12 cells and (Fig. 6H). Taken together, these data indicate that HMGB2 muscle satellite cells were induced to differentiate after IGF2BP2 maintains myoblast proliferation and fate determination through was knocked down. More and longer fused myofibers were regulating IGF2BP2, which binds to Myf5 and cyclin A2 mRNAs observed in IGF2BP2 knockdown group than that in the control and enhances their protein production. one, as it is indicated by the higher fusion index (Fig. 5H; Fig. S4D). To further elucidate the mechanism that IGF2BP2 regulates the Thus, similar to HMGB2 depletion, IGF2BP2 downregulation also protein generation of cyclin A2 and Myf5 after binding to their significantly promotes myogenesis in vitro. Taken together, these mRNAs, mRNA decay analyses of IGF2BP2,cyclinA2andMyf5 Journal of Cell Science

4308 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

Fig. 3. Depletion of HMGB2 promotes myogenesis whereas overexpression of HMGB2 compromises myogenesis in vitro. (A,B) C2C12 cells were transfected with si-NT or si-HMGB2 and maintained in growth medium (GM) for 36 h, and then induced to differentiate for 24 h before being analyzed. Subsequently, myogenin expression was detected by qPCR (A, left), western blotting (A, right) and immunofluorescence (B, left). The percentage of myogenin- positive cells out of the total DAPI-stained cells was calculated in three microscopic fields for each group (B, right). Data are presented as mean±s.e.m.; n=3. ***P<0.001 (Student’s t-test). Scale bar: 100 μm. (C–E) C2C12 cells were maintained in growth medium for 36 h after transfection, and then induced to differentiate (DM) for 2 or 3 days before being analyzed. qPCR (C) and western blotting (E) were used to determine HMGB2 expression at indicated time points, respectively. Immunofluorescence for MyHC was carried out at day 3 after induction (D, left). The fusion index (the percentage of nuclei in fused myotubes out of the total nuclei) was calculated in three microscopic fields for each group (D, right). Data are presented as mean±s.e.m.; n=3. **P<0.01, ***P<0.001 (Student’s t- test). Scale bar: 100 μm. (F) C2C12 cells were transfected with control EGFP or HMGB2 plasmids respectively, and cultured in growth medium for 36 h. Subsequently, C2C12 cells were switched to differentiation medium for 3 days before immunofluorescence staining for MyHC (left). The fusion index was calculated in three microscopic fields for each group (right). Data are presented as mean±s.e.m.; n=3. **P<0.01 (Student’s t-test). Scale bar: 100 μm. were performed. As expected, IGF2BP2 mRNA degraded with a HMGB2 was always inhibited during muscle regeneration, si- half-life of nearly 4.5 h in control cells, but its half-life was reduced to HMGB2 or non-targeting siRNA (si-NT) was injected into tibialis 2.5 h after IGF2BP2 downregulation (Fig. 6I, left). Interestingly, anterior every 4 days and HMGB2 was maintained at a low level there was no significant difference in Myf5 mRNA half-life between (Fig. 7A,B). It was shown that, compared to the control muscle, both the siRNA treatment and the control group (Fig. 6I, middle), whereas the centrally located myonuclei number and the myofiber cross- si-IGF2BP2 treatment led to a nearly two-fold acceleration in the sectional area (CSA) in tibialis anterior administrated with si- decay of cyclin A2 mRNA (Fig. 6I, right). In order to elucidate the HMGB2 were significantly reduced at day 5 and day 10 after injury mechanism by which IGF2BP2 regulates Myf5 protein expression, (Fig. 7C,D). Thus, intramuscular addition of si-HMGB2 impaired we performed a further analysis that indicated that when mRNA muscle regeneration. This result was contrary to the observation transcription and protein degradation were simultaneously inhibited, in vitro that knocking down HMGB2 promoted myogenesis (Fig. 3), Myf5 protein decreased significantly after IGF2BP2 was knocked which might be ascribed to the decrease in activated satellite cells down (Fig. 6J). These findings indicate distinctive mechanisms by caused by perturbation of satellite cell proliferation (Fig. 4). which IGF2BP2 promotes protein production of Myf5 and cyclin A2 Collectively, HMGB2 decrease severely compromises muscle after binding to their mRNAs. It is likely that IGF2BP2 enhances the regeneration, suggesting a general requirement for HMGB2 in translation of Myf5 without altering its mRNA stability. Nevertheless, muscle remodeling and maintenance of muscle integrity. the binding of IGF2BP2 improves cyclin A2 mRNA stability, subsequently increasing its protein production. DISCUSSION Myogenesis is a well-orchestrated gene regulation program, and HMGB2 deficiency blunts muscle regeneration in vivo elucidating the underlying molecular mechanism is quite a To define whether the effect of HMGB2 on myogenesis can be challenging work. In this study, we identified the involvement of recapitulated in an in vivo context, a CTX-mediated muscle HMGB2 in myogenesis and muscle regeneration. Previous reports regeneration model was employed. In order to ensure that have revealed that HMGB2 is required for cell cycle progression and Journal of Cell Science

4309 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

Fig. 4. Knocking down HMGB2 induces cell cycle arrest of myoblasts by reducing Myf5 and Cyclin a2 protein levels both in vitro and in vivo. (A) C2C12 cells were treated with si-NT or si-HMGB2 and then cultured in growth medium. After 24 h, cells were digested and seeded into an E-Plate, which is sensitive for cell-electrode impedance. Cellular impedance, which is in direct proportion to cell number, was measured every 6 h. Data are presented as mean±s.e.m.; n=3. *P<0.05 (Student’s t-test). (B) Flow cytometry analysis following propidium iodide staining for the cell cycle phase of C2C12 cells 48 h after transfection with si-NT or si-HMGB2. Data are presented as mean±s.e.m.; n=3. *P<0.05 (Student’s t-test). (C–E) C2C12 cells were transfected with si-NT or si-HMGB2 and maintained in growth medium for 48 h. Then qPCR was performed to examine the mRNA levels of Myf5, MyoD, Pax7, id2 and YY1 (C) and cell cycle regulating proteins as indicated (D). Western blotting was performed to detect the protein expression of cyclin A2, Myf5, YY1, Pax7 and MyoD (E). Data are presented as mean±s.e.m.; n=3. (F–J) Schematic of experimental design that tibialis anterior muscles of adult mice were injected with si-NT or si-HMGB2 followed by CTX or NaCl the next day, and harvested for analysis 3 days after injury (F). Knockdown efficiency of HMGB2 was detected by qPCR (G). The protein expression of Myf5, Pax7, cyclinA2 and HMGB2 was assayed by western blotting (H). EdU was injected intraperitoneally 4 h before mice were killed. Then, EdU staining on frozen tibialis anterior muscle sections was carried out to label cells in S phase and satellite cells (I). Data are presented as mean±s.e.m.; n=3 mice per group. *P<0.05, **P<0.01, ***P<0.001 (Student’s t-test). Scale bar: 50 μm. is mainly expressed in developing embryo and stem cells, including and G2/M transition. Thus, in our study, the si-HMGB2-mediated S mesenchymal stem cells, embryonic stem cells and spermatocytes and G2/M phase arrest can be explained by the simultaneous (Ronfani et al., 2001; Seyedin and Kistler, 1979). In addition, reduction in Myf5 and cyclin A2 proteins. HMGB2 has been shown to be downregulated significantly within By contrast, cell cycle withdraw is required for the initiation of the first 24 h of myogenic differentiation (Rajan et al., 2012). In myogenic differentiation. We revealed that HMGB2 depletion accordance with these observations, our results suggest that induced withdraw from cell cycle and enhanced myotube formation HMGB2 is highly expressed in activated satellite cells and C2C12 in C2C12 cells. Contradictorily, a decrease of HMGB2 attenuated myoblasts, but decreases significantly during myogenesis and is muscle repair, which might be explained by an insufficient satellite scarcely expressed in mature myofibers (Fig. 2). Therefore, we cell population due to impair of myoblast amplification when injury speculate that HMGB2 functions in myoblasts to maintain occurred. Experiments in vitro showed that both knocking down proliferation and keep stemness. This was well confirmed by our and overexpressing HMGB2 did not influence Pax7 expression in data showing that knockdown of HMGB2 impedes progression of C2C12 myoblasts, implying that HMGB2 does not function by myoblasts cell cycle both in vitro and in vivo. regulating Pax7 (Fig. 4C,E). However, depletion of HMGB2 Myf5 is required for muscle lineage determination in the early in tibialis anterior resulted in the reduction of Pax7 protein and phase of muscle development and is considered as a marker of Pax7-positive satellite cells (Fig. 4H,I), which might result from committed myogenic progenitors (Bryson-Richardson and Currie, the decreased number of proliferating satellite cells (Fig. 4I). 2008). Myf5 is also implicated in myoblast proliferation (Apponi Interestingly, we found that the expression of HMGB2 increased et al., 2011). It is well known that cyclin A2 controls both S phase immediately after injury, which is similar to what occurs with Myf5 Journal of Cell Science

4310 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

Fig. 5. IGF2BP2 is a downstream target of HMGB2. (A) qPCR analysis for IGF2BP2 expression in tibialis anterior at postnatal day 1, day 14 and day 140. Data are presented as mean±s.e.m.; n=3 mice per group. (B) C2C12 cells were treated with si-NT or si-HMGB2 for 48 h in growth medium, and then the expression of IGF2BP2 was determined by qPCR (left) and western blotting (right). Data are presented as mean±s.e.m.; n=3. *P<0.05 (Student’s t-test). (C) C2C12 cells were transfected with EGFP or HMGB2 plasmids and cultured in growth medium for 48 h. Then the expression of IGF2BP2 was determined by qPCR (left) and western blotting (right). Data are presented as mean±s.e.m.; n=3. *P<0.05 (Student’s t-test). (D,E) Adult tibialis anterior muscle was subjected to si-NT or si-HMGB2 administration followed by CTX injury the next day. The mRNA level of IGF2BP2 was assessed by qPCR 3 days after injury (D). The protein levels of IGF2BP2 and HMGB2 were detected by western blotting 3 days after injury (E). Data are presented as mean±s.e.m.; n=3 mice per group. *P<0.05, **P<0.01(Student’s t-test). (F) Three siRNAs against IGF2BP2 (si-IG-1, 2 and 3) were transfected into C2C12 cells cultured in growth medium for 48 h. Knockdown efficiency of IGF2BP2 mRNA was evaluated by qPCR (left). The knockdown of IGF2BP2 protein was detected after transfection for 48 h with the mixture of these three siRNAs (si- IGF2BP2) (right). Data are presented as mean±s.e.m.; n=3. **P<0.01 (Student’s t-test). (G) qPCR (left) and western blotting (right) were used to evaluate the expression of HMGB2 in C2C12 cells at day 2 after transfection with si-NT or si-IGF2BP2. Data are presented as mean±s.e.m.; n=3. (H) C2C12 cells were transfected with si-NT or si-IGF2BP2 and immunofluorescence staining for MyHC was performed at day 3 after differentiation (left). The fusion index was calculated in three microscopic fields for each group (right). Data are presented as mean±s.e.m.; n=3. **P<0.01 (Student’s t-test). and MyoD (Tidball and Villalta, 2010). This further indicated that differentiation (Taniguchi et al., 2011). We found that HMGB2 HMGB2 affects muscle regeneration by stimulating satellite cell specifically transactivates IGF2BP2 in muscle stem cells or expansion mainly in the early stage of injury. progenitors (Fig. 5), implying that a potential cooperation between It has been well-established that HMGB proteins can regulate HMGB2 and specific transcription factors on IGF2BP2 promoter gene-specific transcription through interaction with transcription probably exists. factors, as well as remodeling chromatin structure (Laurent et al., The HMGA2–IGF2BP2 regulatory axis during muscle 2010; Ueda and Yoshida, 2010). Transcription factors identified as development, embryogenesis and tumorigenesis has been well the partner of HMGB protein have been well reviewed (Agresti and characterized in numerous publications (Cleynen et al., 2007; Li Bianchi, 2003). Notably, interactions between Oct4 and HMGB2 et al., 2012). Here, we also showed that HMGB2 mediates IGF2BP2 regulates the Akt pathway and embryonic stem cells pluripotency transcription both in vivo and in vitro during myogenesis (Fig. 5). (Campbell and Rudnicki, 2013). Noboru Taniguchi and his Furthermore, HMGB2 was confirmed to positively regulate the colleagues have reported that HMGB2 coordinates with the expression of HMGA2 (Fig. S2C–E). This implies that HMGB2 Runx2–LEF1–β-catenin complex on the Runx2 proximal promoter might control the expression of IGF2BP2 through a HMGA2- to control Runx2 transcription and influence chondrogenic dependent mechanism. RNA-binding proteins (RBPs) could bind to Journal of Cell Science

4311 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

Fig. 6. IGF2BP2 controls myoblast proliferation by binding to Myf5 and cyclin A2 mRNAs to promote their translation. (A) Flow cytometry analysis after propidium iodide staining was used to determine the cell cycle stage of C2C12 cells transfected with si-IGF2BP2 or si-NT for 48 h. si-NT was used as the control. Data are presented as mean±s.e.m.; n=3. *P<0.05 (Student’s t-test). (B,C) RNP immunoprecipitations were performed in C2C12 myoblasts using control anti- EGFP or anti-IGF2BP2 antibodies to enrich the target mRNAs of IGF2BP2. Then, both qPCR (B) and amplification PCR (C) were used to detect the enrichment of cyclin A2, Myf5, Pax7, MyoD, CDK2, CDK4 and Smurf1. Data are presented as mean±s.e.m.; n=3. ***P<0.001 (Student’s t-test). (D) qPCR analysis for IGF2BP2, cyclin A2, Myf5 and MyoD in C2C12 cells treated with si-NT or si-IGF2BP2 for 48 h. Data are presented as mean±s.e.m.; n=3. **P<0.01 (Student’s t-test). (E) Western blotting analysis for protein levels of cyclin A2, Myf5 and MyoD in C2C12 cells treated with si-NT or si-IGF2BP2 for 48 h. (F) Western blotting analysis for protein levels of cyclin A2, Myf5 and IGF2BP2 in C2C12 cells treated with EGFP or IGF2BP2 plasmids for 48 h. (G) EGFP or IGF2BP2 plasmids were transfected into C2C12 cells simultaneously treated with si-NT or si-HMGB2 for 48 h. Then, the proteins cyclin A2, Myf5, IGF2BP2 and HMGB2 were analyzed by western blotting. (H) C2C12 cells were treated as indicated and then induced to differentiate for 24 h. Immunofluorescence staining for myogenin was performed (left). Myogenin-positive cells were counted in three microscopic fields for each group (right). Data are presented as mean±s.e.m.; n=3. **P<0.01, ***P<0.001 (Student’s t-test). (I) qPCR analyses for the half-life of IGF2BP2, cyclin A2 and Myf5 mRNAs after IGF2BP2 knockdown. C2C12 cells were treated with actinomycin D (Act D) after transfection with si-NT or si-IGF2BP2 for 48 h. Then, cells were harvested at indicated time points. Data are presented as mean± s.e.m.; n=3. (J) After being transfected with si-NT or si- IGF2BP2 for 48 h, C2C12 myoblasts were treated with both Act D and MG132 for 6 h. Then, protein levels of Myf5 and IGF2BP2 were detected by western blotting. Journal of Cell Science

4312 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

Fig. 7. Decrease in HMGB2 impairs muscle regeneration. (A) Schematic diagram of si-HMGB2-mediated HMGB2 knockdown in the CTX injury muscle model. (B) The tibialis anterior muscle was treated with si-NT or si-HMGB2 and then injured with CTX. Western blotting analysis for HMGB2 was performed at day 5orday 10. (C) At day 5 and day 10 after injury, tibialis anterior muscles treated with si-NT or si-HMGB2 were stained by hematoxylin and eosin (H&E). (D) The mean myonuclei number per 100 fibers and mean myofiber cross-sectional area (CSA) were counted (top). The percentages of myofibers in the indicated myofiber CSA were calculated (bottom). Data are presented as mean±s.e.m.; n=3. *P<0.05 (Student’s t-test). Scale bar: 100 μm. (E) Schematic diagram of mechanism by which the HMGB2–IGF2BP2 axis regulates myoblast proliferation and muscle regeneration. HMGB2 promotes IGF2BP2 transcription in the nuclei of myoblasts. Then, IGF2BP2 binds to the 5′-UTR of Myf5 and cyclin A2 mRNAs to enhance translation and improve stability, respectively, which leads to increased protein production. With the increase in Myf5 and cyclin A2 proteins, myoblasts are maintained at a proliferating and undifferentiated stage, which facilitates muscle regeneration in response to injury through the expansion of satellite cells. specific sequences of target mRNAs (RNA-binding domains), (Fig. 6H). Further observation indicated that the increased Myf5 allowing the control of localization, stability, degradation and protein generation after IGF2BP2 binding to its mRNA can likely be translation of mRNAs (Kelly and Corbett, 2009; Wilusz and Wilusz, attributed to the enhanced translation (Fig. 6I). Taken together, the 2010). In this regard, IGF2BP2 is a key RBP and targets various specific binding to both mRNAs results in increased protein levels mRNAs (Dai et al., 2011; Gong et al., 2015; Li et al., 2012). and proliferation maintenance in myoblasts. Importantly, strong evidence has been recently provided to prove The canonical Wnt/β-catenin pathway is required for muscle that IGF2BP2 binds to proliferation-relevant mRNAs, including development and satellite cell proliferation, which is responsible for Myc, Sp1, Igf1r, Ccng1 and Nras, and then controls their stability muscle repair (Otto et al., 2008; von Maltzahn et al., 2012). Strong and/or translation (Gong et al., 2015; Li et al., 2012). Similarly, we evidence has confirmed the interactions between HMGB2 and Wnt/ identified Myf5 and cyclin A2 as two new mRNA targets of β-catenin pathway in mesenchymal stem cells and the superficial IGF2BP2 in myoblasts (Fig. 6C), and also demonstrated that zone of articular cartilage (Taniguchi et al., 2011, 2009). This

IGF2BP2 maintains the stability of cyclin A2 but not Myf5 mRNA guides us to hypothesize that HMGB2 also influences myoblasts Journal of Cell Science

4313 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944 proliferation and muscle regeneration through Wnt/β-catenin Western blotting pathway. Cells or tibialis anterior muscle were incubated in cell lysis buffer on ice to In summary, the data present here highlights the crucial role of completely release total protein. Total protein was separated by SDS-PAGE the HMGB2–IGF2BP2 axis in myogenesis and early muscle and transferred onto PVDF member (Bio-Rad, Shanghai, China). Then, regeneration (Fig. 7E). HMGB2 induces proliferation of muscle immunoblotting for target proteins were carried out by specific antibodies as described in Table S4. α-Tubulin was used as the internal control. stem cells or progenitors through IGF2BP2, and is necessary for proper muscle regeneration. The insights into these distinct CTX injury mechanisms provide potential therapeutic approaches to enhance In adult mice, right tibialis anterior muscles were administrated with 100 μl the regenerative ability of muscle stem cells, thereby possibly of 10 mM CTX and an equal volume of NaCl was injected into left tibialis treating muscle diseases such as muscular dystrophies. anterior muscles as a control.

MATERIALS AND METHODS Intramuscular transfection of siRNAs Animals Reagent A was prepared by mixing 12.5 μg si-NT or the target siRNA with 8-week-old male Mus musculus (C57BL/6) were housed in a specific- 12.5 μl physiological saline solution. Reagent B was prepared by mixing pathogen-free (SPF) facility with a 12-h dark and 12-h light cycle. All animal 6.25 μl Entranster-in vivo (Engreen, Beijing, China) with 18.75 μl experiments were approved by the Animal Care and Use Committee of physiological saline solution. Then, reagent B was added to reagent A Guangdong Province and carried out in accordance with ethical standards. and mixed completely. The mixture was incubated at room temperature for 15 min before injecting into tibialis anterior muscles with a syringe. Cell culture and differentiation C2C12 cells, provided by ATCC, were cultured in in Dulbecco’s modified RNA-binding protein immunoprecipitation Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) (GIBICO, The RIP experiment was performed as described previously (Keene et al., Shanghai, China) (growth medium) until confluence. When cells had 2006). In brief, first, prepare the following solutions: polysome lysis buffer, reached 100% confluence (day 0), C2C12 cells were switched into DMEM 100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.0), 0.5% NP40, 1 mM with 2% horse serum (GIBICO) (induction medium). All cells are DTT, 100 units/ml RNase, 400 µM vanadyl ribonucleoside complexes maintained at 37°C in a humidified incubator with 5% CO2. (VRC; added before use), protease inhibitor cocktail (added before use); and NT2 buffer, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2 and Satellite cell isolation and culture 0.05% NP40. C2C12 cells in growth medium were collected and broken Satellite cells were isolated from the extensor digitorum longus (EDL) of down in polysome lysis buffer on ice for 5 min. To coat antibody on Protein- adult mice. The EDL was isolated from 8-week-old mice and digested with A/G Magnetic beads (Millipore, Shanghai, China), IgG or IGF2BP2 Ab was 0.2% Collagenase type I (Sigma, Shanghai, China) solution at 37°C until added into the bead slurry and incubated for 18 h followed by washing four sufficient myofibers were released. Dead myofibers were removed by or five times with ice-cold NT2 buffer. Then, messenger ribonucleoprotein transferring the digestion products into new prewarmed DMEM (10% horse (mRNP) lysate was added into antibody–Protein-A/G bead mixture and serum) for at least three times. Single myofibers were maintained in DMEM incubated for 4 h at 4°C. After washing the beads with ice-cold NT2 buffer, (10% horse serum, 0.5% chick embryo extract) for 1 day and then switched proteinase K was used for 30 min at 55°C to release the RNP components. to proliferation medium (20% FBS, 10% horse serum, 2% chick embryo Finally, RNA was isolated from the immunoprecipitated pellet by TRIzol® extract in DMEM). When primary myoblasts grew and migrated out from Reagent and reverse-transcribed as described above. The enrichment of the basal lamina, cells were cultured in F10 (GIBICO) medium [20% horse target mRNAs was detected by qPCR. serum, 2.5 ng/ml bFGF (Invitrogen, Shanghai, China), 1% Gluta-Max (GIBCO), 1% penicillin-streptomycin (GIBICO)]. Immunohistochemistry and H&E staining Tibialis anterior muscles were isolated from adult mice and fixed in 4% RNA interference and overexpression paraformaldehyde for 12 h at 4°C. Then, samples were subjected to A set of three Stealth RNAi™ siRNAs against mouse HMGB2 and against dehydration using graded ethanol and paraffin embedding. Paraffin- mouse IGF2BP2 (Invitrogen, Shanghai, China) were purchased from Life embedded samples were cut into 4-μm sections. Paraffin sections were Technologies. Their sequences are provided in Table S2 and Table S3, then analyzed by H&E staining or immunostaining with HMGB2 antibody respectively. Stealth siRNA Negative Control containing medium GC content using a cell and tissue staining kit (rabbit kit HRP-DAB system; R&D, (Invitrogen, Shanghai, China) was used as the negative control. The CDS Shanghai, China) as per the manufacturer’s instruction. sequences encoding mouse HMGB2 or IGF2BP2 were inserted into a PCDNA3.1 plasmid backbone (Invitrogen). C2C12 cells were seeded in the 6- Immunofluorescence and EdU analysis well or 12-well plates 1 day before treatment. The expression plasmids or Tibialis anterior muscles were immediately frozen in liquid nitrogen upon siRNAs were transfected into cells with Lipofectamine® 2000 (Invitrogen, being isolated from adult mice and embed in O.C.T. compound. Cryostat Shanghai, China) as per the manufacturer’s instruction. The medium with sections (10 μm) were prepared on a cryostat microtome. Cells cultured in transfection mixture was replaced with fresh growth medium at 6 h after 12-well plates or on cryostat slides were fixed on ice for 10 min using 4% transfection. paraformaldehyde, followed by permeabilization with PBST solution (0.5% Triton X-100 in PBS) for 15–20 min. After blocking with goat serum, qPCR and mRNA decay analyses immunostaining with specific antibodies (mentioned in Table S4) was Total RNA was extracted from cells or tibialis anterior muscle by using performed, followed by counterstaining with DAPI. TRIzol® Reagent (Invitrogen, Shanghai, China), and then cDNA was Intraperitoneal injection of EdU (Sigma, Shanghai, China) in PBS was synthesized using a reverse transcription kit (Promega, Beijing, China). The performed 4 h before killing the mice (50 mg/kg). Tibialis anterior muscles real-time quantitative PCR was performed using a SYBR Green qPCR Kit were then isolated, frozen and sectioned at 10 µm. Cryostat slides were fixed (Genestar, Beijing, China) and detected in the LightCycler 480 II system and permeabilized as described above. Finally, EdU detection was (Roche, Basel, Switzerland). All results were normalized to that of GAPDH. performed using an in vivo EdU Click Kit 488 (Sigma, Shanghai, China) The primers used for qPCR were given in Table S1. as per the manufacturer’s instruction. Following transfected with si-IGF2BP2 or si-NT for 24 h, C2C12 cells were harvested directly or treated with 5 μM actinomycin D (Act D) and then Flow cytometry analysis harvested at the indicated time points. Total RNA extraction, reverse transcription C2C12 cells were digested with tripsin and fixed in 70% ethanol overnight. and qPCR were performed as above indicted to detect mRNA decay. After three washes in PBS, fixed cells were treated with propidium iodide Journal of Cell Science

4314 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944 solution (10 mg propidium iodide, 0.5 ml Triton X-100, 200 mg sodium Bianchi, M. E. and Agresti, A. (2005). HMG proteins: dynamic players in gene citrate and 129.6 ml PBS in 200 ml, pH 7.2–7.6) for 30 min in the dark. regulation and differentiation. Curr. Opin. Genet. Dev. 15, 496-506. Then, cells were dissociated by pipetting up and down gently, and analyzed Brants, J. R., Ayoubi, T. A., Chada, K., Marchal, K., Van de Ven, W. J. M. and Petit, M. M. R. (2004). Differential regulation of the insulin-like growth factor II using a BD FACSCalibur system (BD Biosciences, Franklin Lakes, USA). mRNA-binding protein genes by architectural HMGA2. FEBS Lett. 569, 277-283. xCELLigence cell proliferation assay Bryson-Richardson, R. J. and Currie, P. D. (2008). The genetics of vertebrate Cell proliferation was evaluated by an impedance-based RTCA xCELLigence myogenesis. Nat. Rev. Genet. 9, 632-646. DP system (ACEA Biosciences, CA) that can monitor real-time cell Buckingham, M. and Rigby, P. W. J. (2014). Gene regulatory networks and transcriptional mechanisms that control myogenesis. Dev. Cell 28, 225-238. proliferation. Cells were seeded into an E-Plate 16 (ACEA Biosciences) μ Bustin, M. (1999). Regulation of DNA-dependent activities by the functional motifs with 200 l growth medium and allowed to grow for 62 h at 37°C in a 5% of the high-mobility-group chromosomal proteins. Mol. Cell. Biol. 19, 5237-5246. CO2 atmosphere. Cellular impedance was detected every 6 h. The data Campbell, P. A. and Rudnicki, M. A. (2013). Oct4 interaction with Hmgb2 regulates collected from cell-electrode impedance reflects the cell proliferation index. Akt signaling and pluripotency. Stem Cells 31, 1107-1120. Caron, L., Bost, F. M., Prot, M., Hofman, P. and Binétruy, B. (2005). A new role for the oncogenic high-mobility group A2 transcription factor in myogenesis of RNA-seq analysis embryonic stem cells. Oncogene 24, 6281-6291. Total RNA was extracted from CTX-injured tibialis anterior muscles using Catena, R., Escoffier, E., Caron, C., Khochbin, S., Martianov, I. and Davidson, I. ® TRIzol reagent as per the manufacturer’s instruction. Sequencing analysis (2009). HMGB4, a novel member of the HMGB family, is preferentially expressed was performed with a Illumina Genome Analyzer (Illumina, CA) according in the mouse testis and localizes to the basal pole of elongating spermatids. Biol. to the manufacturer’s instructions. Reprod. 80, 358-366. Chargé, S. B. P. and Rudnicki, M. A. (2004). Cellular and molecular regulation of muscle regeneration. Physiol. Rev. 84, 209-238. Antibodies Cleynen, I., Brants, J. R., Peeters, K., Deckers, R., Debiec-Rychter, M., Sciot, R., All antibodies used in current study are provided in Table S4. Previous studies Van de Ven, W. J. M. and Petit, M. M. R. (2007). HMGA2 regulates transcription have demonstrated that all of them are validated for use (Aguilera et al., 2011; of the Imp2 gene via an intronic regulatory element in cooperation with nuclear Averous et al., 2012; Della Pietra et al., 2015; Dixon et al., 2011; Gong et al., factor-κB. Mol. Cancer Res. 5, 363-372. 2015; Kim et al., 2011; Li et al., 2012; Murphy et al., 2011; Nissar et al., 2012; Dai, N., Rapley, J., Angel, M., Yanik, M. F., Blower, M. D. and Avruch, J. (2011). Rao et al., 2014; Sarkar and Zohn, 2012; Takeda et al., 2014; Tan et al., 2011; mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry. Genes Dev. 25, 1159-1172. Taniguchi et al., 2011; Wu et al., 2010; Zhang et al., 2011). Della Pietra, E., Simonella, F., Bonavida, B., Xodo, L. E. and Rapozzi, V. (2015). Repeated sub-optimal photodynamic treatments with pheophorbide a induce an Statistical analysis epithelial mesenchymal transition in prostate cancer cells via nitric oxide. Nitric All data are presented as the mean±s.e.m.; significance of differences in Oxide 45, 43-53. comparisons were determined by a Student’s t-test. Values of P<0.05 were Dixon, A. S., Pendley, S. S., Bruno, B. J., Woessner, D. W., Shimpi, A. A., Cheatham, T. E., III and Lim, C. S. (2011). Disruption of Bcr-Abl coiled coil considered as statistically significant. oligomerization by design. J. Biol. Chem. 286, 27751-27760. Gong, C., Li, Z., Ramanujan, K., Clay, I., Zhang, Y., Lemire-Brachat, S. and Acknowledgements Glass, D. J. (2015). A long non-coding RNA, LncMyoD, regulates skeletal muscle Tong Jiang is acknowledged for qPCR experiments and vector construction. differentiation by blocking IMP2-mediated mRNA translation. Dev. Cell 34, 181-191. Competing interests Hock, R., Furusawa, T., Ueda, T. and Bustin, M. (2007). HMG chromosomal The authors declare no competing or financial interests. proteins in development and disease. Trends Cell Biol. 17, 72-79. Kang, J.-S. and Krauss, R. S. (2010). Muscle stem cells in developmental and regenerative myogenesis. Curr. Opin Clin. Nutr. Metab. Care 13, 243-248. Author contributions Keene, J. D., Komisarow, J. M. and Friedersdorf, M. B. (2006). RIP-Chip: the X.Z. and M.L. performed most of the experiments, data analysis and manuscript isolation and identification of mRNAs, microRNAs and protein components of writing. H.H. helped western blotting assay. Z.Y. and Y.N. carried out some of the ribonucleoprotein complexes from cell extracts. Nat. Protoc. 1, 302-307. experiments on animals. Y.Z., H.C., X.Z. and L.C. helped analyze experimental data Kelly, S. M. and Corbett, A. H. (2009). Messenger RNA export from the nucleus: a and gave advice. D.M. and Y.C. designed the study and helped revise the series of molecular wardrobe changes. Traffic 10, 1199-1208. manuscript. Kim, S., Zaghloul, N. A., Bubenshchikova, E., Oh, E. C., Rankin, S., Katsanis, N., Obara, T. and Tsiokas, L. (2011). Nde1-mediated inhibition of ciliogenesis Funding affects cell cycle re-entry. Nat. Cell Biol. 13, 351-360. This work was supported by the National Natural Science Foundation of China Kuang, S. and Rudnicki, M. A. (2008). The emerging biology of satellite cells and (NSFC)-Guangdong Joint Fund (U1201213); the National Natural Science their therapeutic potential. Trends Mol. Med. 14, 82-91. Foundation of China (31272417); and the Ministry of Agriculture of the People’s Laurent, B., Randrianarison-Huetz, V., Marechal, V., Mayeux, P., Dusanter- Republic of China (CASR-36). Fourt, I. and Dumenil, D. (2010). High-mobility group protein HMGB2 regulates human erythroid differentiation through trans-activation of GFI1B transcription. Data availability Blood 115, 687-695. Li, Z., Gilbert, J. A., Zhang, Y., Zhang, M., Qiu, Q., Ramanujan, K., Shavlakadze, The RNA-Seq data have been deposited with links to BioProject accession number T., Eash, J. K., Scaramozza, A., Goddeeris, M. M. et al. (2012). An HMGA2- PRJNA348136 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/ IGF2BP2 axis regulates myoblast proliferation and myogenesis. Dev. Cell 23, bioproject/PRJNA348136). 1176-1188. McCauley, M., Hardwidge, P. R., Maher, L. J., III and Williams, M. C. (2005). Dual Supplementary information binding modes for an HMG domain from human HMGB2 on DNA. Biophys. J. 89, Supplementary information available online at 353-364. http://jcs.biologists.org/lookup/doi/10.1242/jcs.189944.supplemental Mok, G. F. and Sweetman, D. (2011). Many routes to the same destination: lessons from skeletal muscle development. Reproduction 141, 301-312. Reference Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A. and Kardon, G. Agresti, A. and Bianchi, M. E. (2003). HMGB proteins and gene expression. Curr. (2011). Satellite cells, connective tissue fibroblasts and their interactions are Opin. Genet. Dev. 13, 170-178. crucial for muscle regeneration. Development 138, 3625-3637. Aguilera, C., Nakagawa, K., Sancho, R., Chakraborty, A., Hendrich, B. and Nielsen, F. C., Nielsen, J., Kristensen, M. A., Koch, G. and Christiansen, J. Behrens, A. (2011). c-Jun N-terminal phosphorylation antagonises recruitment of (2002). Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH the Mbd3/NuRD repressor complex. Nature 469, 231-235. domains. J. Cell Sci. 115, 2087-2097. Apponi, L. H., Corbett, A. H. and Pavlath, G. K. (2011). RNA-binding proteins and Nielsen, J., Adolph, S., Rajpert-DeMEYTS, E., Lykke-Andersen, J., Koch, G., gene regulation in myogenesis. Trends Pharmacol. Sci. 32, 652-658. Christiansen, J. and Nielsen, F. (2003). Nuclear transit of human zipcode- Averous, J., Gabillard, J. C., Seiliez, I. and Dardevet, D. (2012). Leucine limitation binding protein IMP1. Biochem. J. 376, 383-391. regulates and myoD expression and inhibits myoblast differentiation. Exp. Nissar, A. A., Zemanek, B., Labatia, R., Atkinson, D. J., van der Ven, P. F. M.,

Cell Res. 318, 217-227. Furst, D. O. and Hawke, T. J. (2012). Skeletal muscle regeneration is delayed by Journal of Cell Science

4315 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

reduction in Xin expression: consequence of impaired satellite cell activation? Tan, K. Y., Eminli, S., Hettmer, S., Hochedlinger, K. and Wagers, A. J. (2011). Am. J. Physiol. Cell Physiol. 302, C220-C227. Efficient generation of iPS cells from skeletal muscle stem cells. PLoS ONE 6, Otto, A., Schmidt, C., Luke, G., Allen, S., Valasek, P., Muntoni, F., Lawrence- e26406. Watt, D. and Patel, K. (2008). Canonical Wnt signalling induces satellite-cell Taniguchi, N., Carames, B., Kawakami, Y., Amendt, B. A., Komiya, S. and Lotz, proliferation during adult skeletal muscle regeneration. J. Cell Sci. 121, M. (2009). Chromatin protein HMGB2 regulates articular cartilage surface 2939-2950. maintenance via beta-catenin pathway. Proc. Natl. Acad. Sci. USA 106, Rajan, S., Chu Pham Dang, H., Djambazian, H., Zuzan, H., Fedyshyn, Y., Ketela, 16817-16822. T., Moffat, J., Hudson, T. J. and Sladek, R. (2012). Analysis of early C2C12 Taniguchi, N., Carames, B., Hsu, E., Cherqui, S., Kawakami, Y. and Lotz, M. myogenesis identifies stably and differentially expressed transcriptional regulators (2011). Expression patterns and function of chromatin protein HMGB2 during whose knock-down inhibits myoblast differentiation. Physiol. Genomics 44, mesenchymal stem cell differentiation. J. Biol. Chem. 286, 41489-41498. 183-197. Tidball, J. G. and Villalta, S. A. (2010). Regulatory interactions between muscle Rao, T., Ranger, J. J., Smith, H. W., Lam, S. H., Chodosh, L. and Muller, W. J. and the immune system during muscle regeneration. Am. J. Physiol. Regul. Integr. (2014). Inducible and coupled expression of the polyomavirus middle T antigen Comp. Physiol. 298, R1173-R1187. and Cre recombinase in transgenic mice: an in vivo model for synthetic viability in Ueda, T. and Yoshida, M. (2010). HMGB proteins and transcriptional regulation. mammary tumour progression. Breast Cancer Res. 16, R11. Biochim. Biophys. Acta 1799, 114-118. Ronfani, L., Ferraguti, M., Croci, L., Ovitt, C., Scholer, H., Consalez, G. and von Maltzahn, J., Chang, N. C., Bentzinger, C. F. and Rudnicki, M. A. (2012). Wnt Bianchi, M. (2001). Reduced fertility and spermatogenesis defects in mice signaling in myogenesis. Trends Cell Biol. 22, 602-609. lacking chromosomal protein Hmgb2. Development 128, 1265-1273. Wilusz, C. J. and Wilusz, J. (2010). Consequences of mRNA wardrobe Sarkar, A. A. and Zohn, I. E. (2012). Hectd1 regulates intracellular localization and malfunctions. Cell 143, 863-865. secretion of Hsp90 to control cellular behavior of the cranial mesenchyme. J. Cell Wu, M., Liu, H., Fannin, J., Katta, A., Wang, Y., Arvapalli, R. K., Paturi, S., Biol. 196, 789-800. Karkala, S. K., Rice, K. M. and Blough, E. R. (2010). Acetaminophen improves Seyedin, S. M. and Kistler, W. (1979). Levels of chromosomal protein high mobility protein translational signaling in aged skeletal muscle. Rejuvenation Res. 13, group 2 parallel the proliferative activity of testis, skeletal muscle, and other 571-579. organs. J. Biol. Chem. 254, 11264-11271. Yanai, H., Ban, T. and Taniguchi, T. (2012). High-mobility group box family of Takeda, A.-N., Oberoi-Khanuja, T. K., Glatz, G., Schulenburg, K., Scholz, R.-P., proteins: ligand and sensor for innate immunity. Trends Immunol. 33, 633-640. Carpy, A., Macek, B., Remenyi, A. and Rajalingam, K. (2014). Ubiquitin- Zhang, S. J., Ye, F., Xie, R. F., Hu, F., Wang, B. F., Wan, F., Guo, D. S. and Lei, T. dependent regulation of MEKK2/3-MEK5-ERK5 signaling module by XIAP and (2011). Comparative study on the stem cell phenotypes of C6 cells under different cIAP1. EMBO J. 33, 1784-1801. culture conditions. Chin. Med. J. 124, 3118-3126. Journal of Cell Science

4316