Implications for Hspb7 in Muscle Atrophy Stephanie Wales Tobin1,2,3, Dabo Yang4, John Girgis4, Ali Farahzad1,2,3, Alexandre Blais4 and John C

RESEARCH ARTICLE Regulation of Hspb7 by MEF2 and AP-1: implications for Hspb7 in muscle atrophy Stephanie Wales Tobin1,2,3, Dabo Yang4, John Girgis4, Ali Farahzad1,2,3, Alexandre Blais4 and John C. McDermott1,2,3,5,*

ABSTRACT 2000; Prado et al., 2009). In the ubiquitin proteasome pathway, the Mycocyte enhancer factor 2 (MEF2) and activator protein 1 (AP-1) forkhead box protein (FoxO) family of transcription factors transcription complexes have been individually implicated in activates muscle atrophy through induction of two E3 ubiquitin myogenesis, but their genetic interaction has not previously been ligases, MAFbx/atrogin-1 and MuRF1 (also known as TRIM63) addressed. Using MEF2A, c-Jun and Fra-1 chromatin (Sandri et al., 2004). Current treatment programs for muscle immunoprecipitation sequencing (ChIP-seq) data and predicted AP- atrophy include activating the serine/threonine protein kinase (Akt) 1 consensus motifs, we identified putative common MEF2 and AP-1 pathway, which induces muscle hypertrophy by inactivating FoxO target genes, several of which are implicated in regulating the actin proteins (Stitt et al., 2004). However, Akt can be inhibited by β β cytoskeleton. Because muscle atrophy results in remodelling or myostatin, a member of the transforming growth factor (TGF- ) degradation of the actin cytoskeleton, we characterized the superfamily (Trendelenburg et al., 2009), superseding Akt expression of putative MEF2 and AP-1 target genes (Dstn, Flnc, activation as a treatment option. A new antibody recently Hspb7, Lmod3 and Plekhh2) under atrophic conditions using characterized to bind to both members (A and B) of the dexamethasone (Dex) treatment in skeletal myoblasts. Heat shock myostatin/activin type II receptor (ActRII) induces hypertrophy in vivo protein b7 (Hspb7) was induced by Dex treatment and further in a muscle wasting model (Lach-Trifilieff et al., 2014). analyses revealed that loss of MEF2A using siRNA prevented Dex- Additionally, targeting ActRIIB in cancer cachexia models can regulated induction of Hspb7. Conversely, ectopic Fra-2 or c-Jun prevent atrophy, which results in prolonged survival without expression reduced Dex-mediated upregulation of Hspb7 whereas tumour manipulation (Zhou et al., 2010). AP-1 depletion enhanced Hspb7 expression. In vivo, expression of The autophagy pathway is an alternative mechanism of protein Hspb7 and other autophagy-related genes was upregulated in degradation that has also been implicated in muscle wasting. Foxo3, response to atrophic conditions in mice. Manipulation of Hspb7 unlike other members of the FoxO family, is able to regulate – levels in mice also impacted gross muscle mass. Collectively, these autophagy in addition to the ubiquitin proteasome pathway data indicate that MEF2 and AP-1 confer antagonistic regulation of (Mammucari et al., 2007; Zhao et al., 2007). Several possible Hspb7 gene expression in skeletal muscle, with implications for autophagy pathways have been identified in muscle, two of which autophagy and muscle atrophy. are macroautophagy and chaperone-mediated autophagy (CMA). Although both processes ultimately lead to protein degradation in KEY WORDS: Myogenesis, MEF2, AP-1, Autophagy the lysosome, they achieve this through different mechanisms. In CMA, heat shock cognate 70 (Hsc70) targets proteins directly to the INTRODUCTION lysosome (Chiang et al., 1989). Macroautophagy requires de novo Muscle atrophy is a phenomenon associated with reduced muscle synthesis of autophagosomes in a multistep process that involves fibre number and size caused by increased proteolysis and autophagy-related protein (Atg) family members. Autophagy is decreased protein synthesis (Romanick et al., 2013). In the required for muscle homeostasis, as demonstrated by the muscle elderly, muscle wasting is referred to as sarcopenia (Morley atrophy in mouse knockout models that lack proteins involved in et al., 2001); in patients with cancer, AIDS or other chronic autophagosome formation, such as Atg5 and Atg7 (Masiero et al., diseases, muscle atrophy is referred to as cachexia (Kotler, 2000). 2009; Raben et al., 2008). Aged muscle shows decreased autophagy Improving or maintaining muscle mass in these populations has a and therefore build-up of protein aggregates (Demontis and profound impact on the ‘health span’ of individuals. For example, Perrimon, 2010). LC3B (Map1lc3b) is an Atg protein that there is evidence that cachexia in cancer patients directly affects the provides a useful readout for autophagy because it is post- time to tumour progression and disease recurrence (Kadar et al., translationally modified as it becomes part of the autophagosome (Kabeya et al., 2000). First, pro-LC3B is cleaved by Atg4 to form 1Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, cytosolic LC3B-I. Atg7 then lipidates LC3B-I to form LC3B-II, Canada M3J 1P3. 2Muscle Health Research Centre (MHRC), York University, 4700 which can form part of the autophagosome. Using samples from Keele Street, Toronto, Ontario, Canada M3J 1P3. 3Centre for Research in Biomolecular Interactions (CRBI), 4700 Keele Street, Toronto, Ontario, Canada M3J various atrophic mouse models, LC3B was shown to be strongly 1P3. 4Ottawa Institute of Systems Biology, University of Ottawa, Health Sciences upregulated (Lecker et al., 2004). Furthermore, Foxo3 can directly Campus, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. 5Centre for regulate several autophagy-related genes, including that encoding Research in Mass Spectrometry (CRMS), York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3. LC3B (Mammucari et al., 2007; Zhao et al., 2007). A form of autophagy termed chaperone-assisted selective autophagy (CASA) *Author for correspondence ( [email protected]) merges the chaperone-mediated and macroautophagy pathways. In J.C.M., 0000-0001-9696-8929 CASA, Hsc70 forms a complex with Bag3, Hspb8 and E3 ubiquitin ligase CHIP to identify protein aggregates and target them to the

Received 29 March 2016; Accepted 8 September 2016 autophagosome (Arndt et al., 2010). Journal of Cell Science

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Myocyte-enhancer factor 2 (MEF2) is a member of the MADS- contained 6507. To determine the percentage of shared binding sites box family of transcription factors found in many tissues, including across these datasets we used a MEF2A-centric analysis (Fig. 1A). skeletal and cardiac muscle (Edmondson et al., 1994; Lin et al., From this analysis we observed that the majority of MEF2A binding 1997). MEF2 functions in a homo- or heterodimer complex with sites (69%) were independent of c-Jun or Fra-1 recruitment, yet 17% four different MEF2 isoforms in vertebrates (MEF2A–MEF2D), of MEF2A-bound DNA also contained c-Jun recruitment and 12% which bind to the consensus sequence [C/TTA(A/T)4TAG/A]. contained both c-Jun and Fra-1. Fra-1 and MEF2A alone shared few Previously we have shown that MEF2A can target a shared subset of binding sites (2%). genes in C2C12 myoblasts, an in vitro model of skeletal myogenesis, Functional roles for shared MEF2A and AP-1 binding sites were and in primary cardiomyocytes (Wales et al., 2014). Gene ontology identified using the Genomics Regions Enrichment of Analysis (GO) analysis contained terms enriched for actin cytoskeleton Tool (GREAT), which revealed enriched GO terms for biological organization and actin filament-based processes. In addition, many processes, the top ten of which are depicted in Fig. 1B. The GO of these cytoskeletal MEF2 target genes were enriched for activator terms were ranked by binomial raw P-value; the number of genes protein 1 (AP-1) cis elements, suggesting the possibility of within each GO term is indicated. DNA enriched for MEF2A-alone combinatorial control. AP-1, consisting of Jun homodimer or Jun– was associated with the better-known functions of MEF2 such as Fos family heterodimers, recognizes the consensus sequence TGAG/ actin-filament-based processes and skeletal muscle tissue CTCA (Angel et al., 1987). Global analysis of myoblast development, but also with cell development (blue). There were determination protein (MyoD) target genes in skeletal myoblasts no GO terms identified for MEF2A and Fra-1. However, MEF2A likewise showed that AP-1 motifs are prominent in neighbouring and c-Jun had GO terms for striated muscle development, vascular sequences (Blais et al., 2005; Cao et al., 2010). Neighbouring MEF2 development and heart morphogenesis (purple). The complete list and AP-1 cis elements have recently been shown to be enriched in of GO terms is given in Supplementary Dataset S1. Fewer GO terms macrophages and neurons (Ma and Telese, 2015; Nagy et al., 2013). were enriched for genes associated with all three factors and these Several AP-1 subunits have been implicated in myogenesis. In were related to the actin cytoskeleton and negative regulation of particular, c-Jun antagonizes MyoD transcriptional activity in vitro smooth muscle cell proliferation (black). (Bengal et al., 1992; Li et al., 1992). Using high throughput data, Because the MEF2A dataset was obtained from differentiating Blum et al. (2012) reported that c-Jun and MyoD coordinate muscle myoblasts, and given that several other AP-1 components apart from enhancers, indicating a more complex role for AP-1 in muscle than c-Jun and Fra-1 could be associated with MEF2 target genes, we previously anticipated (Blum et al., 2012). Additionally, the Fos determined the location of AP-1 consensus sequences containing the family member Fra-2 is thought to play a role in maintenance of the sequence TGAGTCA using cisGenome and allowing zero skeletal muscle satellite cell population (Alli et al., 2013). mismatches. From the mm9 genome, this search identified 264,537 Although MEF2 and AP-1 have individually been shown to AP-1 consensus sites. We focused on putative MEF2A and AP-1 function in the myogenic program, their potential interaction has not binding sites within ±10 kb of the transcription start site (TSS) of the been documented. Additionally, although loss of MEF2 and AP-1 single nearest gene and observed that 11 out of these 76 genes are have been implicated in loss of sarcomere integrity during associated with the molecular function ‘cytoskeleton protein binding’ development and satellite cell-mediated muscle regeneration (Table S1). We then assessed the recruitment of MEF2A, c-Jun or (Hinits and Hughes, 2007; Potthoff et al., 2007; Windak et al., Fra-1and the presence of AP-1 consensus sequences near five of these 2013), the combined role of these factors in muscle atrophy has not genes, encoding the proteins destrin (Dstn), filamin C (Flnc), heat been investigated. Here, we document that MEF2 and AP-1 regulate shock protein family, member 7 (Hspb7), leiomodin 3 (Lmod3) and several genes associated with the actin cytoskeleton. Among the pleckstrin homology domain containing family H, member 2 gene products, the small heat shock protein, Hspb7 is implicated in (Plekhh2). Dstn is an actin-depolymerizing protein (Carlier et al., muscle atrophy. 1997) and FlInc, a muscle-specific filamin (Thompson et al., 2000) that promotes the cross-linking of actin. We had also previously RESULTS identified the genes encoding Hspb7 (a small heat shock protein) and Identification of biological processes associated with Lmod3 (tropomodulin family member) as MEF2 target genes in MEF2A and AP-1 recruitment cardiac muscle (Wales et al., 2014). Human nemaline myopathy has MEF2 and AP-1 are transcription complexes involved in myoblast been associated with mutations in Lmod3 (Cenik et al., 2015; Garg proliferation and differentiation, yet whether they regulate common et al., 2014; Yuen et al., 2014). Hspb7 expression is higher in mdx (a or non-overlapping target genes during differentiation has not been genetic model of muscular dystrophy) mice than in normal mice and thoroughly explored. To address this, we utilized a previously is also enhanced in ageing muscle (Doran et al., 2006, 2007). reported MEF2A dataset from chromatin immunoprecipitation Recently, a skeletal muscle-specific conditional Hspb7 knockout combined with exonuclease digestion (ChIP-exo) for model was reported to result in progressive myopathy (Juo et al., differentiating C2C12 myogenic cells (48 h in differentiation 2016). The role of Plekhh2 appears to be to stabilize the cortical actin medium; DM) (Wales et al., 2014) and compared these binding cytoskeleton (Perisic et al., 2012). The recruitment pattern of MEF2A, events with data from c-Jun (Blum et al., 2012) and Fra-1 (Wold c-Jun, Fra-1 and any AP-1 consensus sequences is indicated in images group, ENCODE) ChIP in combination with sequencing (ChIP- from UCSC (Fig. S1). The overall trends demonstrate several points. seq), which was likewise carried out in myogenic cells. Fra-1 is First, MEF2A was performed using ChIP-exo, which involves primarily associated with bone development (Eferl et al., 2004; exonuclease digestion prior to sequencing, therefore MEF2A peaks Fleischmann, 2000) and c-Jun has been implicated in many tissues, are more defined. This indicates one of the advantages of ChIP-exo including muscle (Bengal et al., 1992; Blum et al., 2012; Li et al., over conventional ChIP-seq. Second, the scale of c-Jun and Fra-1 1992). Both the c-Jun and Fra-1 ChIP-seq data were completed in recruitment differ dramatically, which could indicate differential C2C12 myoblasts in growth medium (GM), making these data antibody affinities or distinct DNA binding affinities of AP-1 family comparable with our previously generated MEF2A dataset. The c- members. Third, MEF2A and c-Jun or Fra-1 show similar recruitment

Jun dataset contained 9778 binding events and the Fra-1 dataset patterns to a subset of putative target genes. Of these five MEF2A Journal of Cell Science

4077 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4076-4090 doi:10.1242/jcs.190009

MEF2A (2783) A 2% MEF2A with c-Jun only 12% 17% MEF2A with Fra-1 only

MEF2A alone 69% MEF2A with c-Jun and Fra-1

B GO Biological Processes -log10(Binomial p -value) MEF2A alone acn filament-based process (71) regulaon of myeloid cell differenaon (42) negave regulaon of cysteine-type endopepdase acvity involved in apoptoc process (16) negave regulaon of cysteine-type endopepdase acvity (16) posive regulaon of apoptoc signaling pathway (24) acn filament organizaon (29) regulaon of leukocyte migraon (21) regulaon of B cell apoptoc process (9) posive regulaon of myeloid cell differenaon (25) skeletal muscle ssue development (32) 024681012 MEF2A with c-Jun muscle ssue development (30) posive regulaon of cell death (23) hemopoiesis (30) regulaon of vasculature development (18) striated muscle ssue development (28) Ras protein signal transducon (11) heart morphogenesis (25) response to molecule of bacterial origin (18) small GTPase mediated signal transducon (25) posive regulaon of apoptoc process (22) 0123456 MEF2A with c-Jun and Fra-1 negave regulaon of smooth muscle cell proliferaon (5) acn filament-based process (18) negave regulaon of transport (18) middle ear morphogenesis (5) segment specificaon (4) regulaon of cell growth (15) mesoderm development (11) 01234567

Fig. 1. Comparison of MEF2A and AP-1 target genes in skeletal muscle. (A) Percentage overlap between MEF2A, c-Jun, and Fra-1. The total number of MEF2A ChIP-seq binding events is 2783. All datasets were converted to the mm9 genome and then overlapping peaks were identified from individual bed files for MEF2A, c-Jun and Fra-1 using the UCSC intersect function. (B) Functional roles for MEF2A alone, MEF2A and c-Jun, and MEF2A with c-Jun and Fra-1. Using the datasets from 1A, GREAT analysis revealed GO terms for biological processes. The total number of genes per GO term are indicated to the right of each GO term. target genes, c-Jun was recruited to an overlapping or neighbouring others. We assessed the expression pattern of MEF2A and AP-1 binding event near Flnc, Hspb7 and Plekhh2 (Fig. S1). Fra-1 and family members during C2C12 differentiation (Fig. 2A). As MEF2A recruitment only overlapped at the promoter of Flnc,andFra- myogenin and MEF2A levels increased during myogenesis, only 1 was detected within the second intron of Hspb7, where c-Jun also one AP-1 subunit (Fra-2) also increased. c-Jun, JunD and, most showed recruitment (Fig. S1). This in silico analysis identifies a dramatically, Fra-1 levels were reduced in DM. To move forward in potential interplay between AP-1 and MEF2 in the transcriptional determining a role for MEF2 and AP-1 in myogenesis, we focused control of target genes involved in muscle stability. on Fra-2 and c-Jun because they are expressed in myoblasts and have been shown to have a role in myogenesis (Alli et al., 2013; Actin cytoskeletal genes are regulated by MEF2A and AP-1 Bengal et al., 1992; Blum et al., 2012; Li et al., 1992). Additionally, Aside from c-Jun and Fra-1, AP-1 has many family members that the Fra-2–c-Jun heterodimer is one of the main AP-1 binding are expressed in muscle, including Fra-2, JunD, c-Fos and several complexes present in C2C12 differentiation (Andreucci et al., Journal of Cell Science

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ABGM 24 hr 48 hr 120 * MEF2A

100 c-Jun

80 Fra-2

60 0 hr Fra-1 48 hr Fold Change Fold 40 *

JunD * 20 * MyoG 0 Dstn Flnc Hspb7 Lmod3 Plekhh2

Acn

C D 1.8

2 1.6

1.4 scr siMEF2A sic-Jun siFra- 1.2 MEF2A 1.0 siMEF2A siFra-2 0.8 *

Fra-2 Change Fold sic-Jun 0.6 ** * ** c-Jun 0.4

0.2 Acn 0.0 48 hr DM Dstn Flnc Hspb7 Lmod3 Plekhh2 MEF2A Fra-2 c-Jun

Flnc E Dstn Hspb7 1.6 120 500 1.4 1.2 70 300 1.0 0.8 100 0.6 20 Fold Change Fold

Fold Change Fold 4.0 0.4 5 0.2 2.0 0.0 0 0.0 0 hr DM 24 hr DM 72 hr DM 0 hr DM 24 hr DM 72 hr DM 0 hr DM 24 hr DM 72 hr DM Lmod3 Plekhh2 45 600 40 F pcDNA3 GFP-Tam67 35 0 hr 24 hr 72 hr 0 hr 24 hr 72 hr 30 25 GFP 100 Control 20 4.0 Tam67 Fold Change Fold 15 10 Acn 2.0 5 0.0 0 0 hr DM 24 hr DM 72 hr DM 0 hr DM 24 hr DM 72 hr DM

Fig. 2. See next page for legend. Journal of Cell Science

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Fig. 2. MEF2A and AP-1 regulation of actin cytoskeletal genes. (A) MEF2A marginal effects on Dstn, Flnc, Hspb7, Lmod3 and Plekhh2 and AP-1 protein expression during myogenesis. C2C12 cells were allowed to expression at 0 and 24 h in DM (Fig. 2E). However, after 72 h in differentiate from myoblasts (GM) to 48 h DM. (B) Expression pattern of MyoG, DM, all five genes were robustly upregulated by Tam67 expression. Dstn, Flnc, Hspb7, Lmod3 and Plekhh2 during C2C12 differentiation in GM – and 48 h DM. Values were calculated using the ΔΔCt method and normalized Expression of GFP Tam67 is depicted in Fig. 2F. Together these to β-actin (n=3, mean±s.e.m., *P<0.05, **P<0.01). (C) Efficiency of knockdown data indicate that a subset of MEF2A target genes are of MEF2A, Fra-2 and c-Jun in C2C12 myocytes (48 h DM) using siRNA- transcriptionally regulated by AP-1. mediated gene silencing. C2C12 cells were transfected with siRNA and allowed to differentiate for 48 h before western blot analysis. scr indicates a Atrophy induced by age or dexamethasone modulates scrambled siRNA control. (D) siRNA-mediated knockdown of MEF2A, Fra-2 expression of MEF2A and AP-1 cytoskeletal target genes and c-Jun at 48 h DM was carried out to assess changes in target gene The cytoskeleton is integrally linked to the contractile unit of the expression. Data were analysed as in B. (E) C2C12 cells were transfected with α GFP–Tam67 using calcium phosphate. RNA was isolated at the indicated time myofibril, the sarcomere, which is mainly comprised of -actin and points and analysed via qRT-PCR as in B. (F) Western blot depicting myosin. During muscle atrophy (a phenomenon observed in expression of GFP-tagged Tam67. sarcopenia, cachexia and various genetic diseases) the cytoskeleton and components of the sarcomere become degraded, 2002). Fra-2 is present in different isoforms and subject to post- resulting in overall muscle loss and weakness. Because these five translational modification by ERK (Alli et al., 2013; Andreucci proteins (Dstn, Flnc, Hspb7, Lmod3 and Plekhh2) are involved in et al., 2002). To date, Fra-2 ChIP-seq data is not available. the actin cytoskeleton, to varying degrees, we next determined The expression of cytoskeletal genes during myogenesis was whether expression of the corresponding MEF2 and AP-1 target determined using quantitative reverse transcription (qRT)-PCR in genes changes under atrophic conditions. To determine whether GM (myoblasts) and at 48 h in DM (myocytes) (Fig. 2B). During these genes were differentially expressed in growing postnatal C2C12 differentiation, the expression of each gene except Dstn muscle compared with mature adult muscle we isolated RNA from increased. To confirm MEF2A recruitment to Dstn, Flnc, Hspb7, the gastrocnemius and quadriceps of 8- and 63-week-old mice Lmod3 and Plekhh2, ChIP-qPCR was performed in growth (Fig. 3A). In the gastrocnemius, Hspb7 was upregulated with age. In conditions (GM) and during differentiation (48 h DM). During the quadriceps, Dstn, Flnc and Hspb7 were upregulated. differentiation, MEF2A was recruited to each gene, compared with a In cell culture, muscle atrophy can be replicated by treatment with control region upstream of SMA (Fig. S2). MEF2A recruitment to Dex, a synthetic glucocorticoid. To model glucocorticoid-induced Lmod3 and Hspb7 was the most significant and reflects the ChIP- atrophy, C2C12 myoblasts were allowed to differentiate for 72 h in seq data (Fig. S1). Fra-2 and c-Jun recruitment were also assessed at DM, and then treated with Dex (Fig. 3B). In this analysis we the same regions as MEF2A (Figs S1 and S2). c-Jun was moderately included two E3 ubiquitin ligases, MAFbx and MuRF1, which are recruited to Flnc, Hspb7 and Plekhh2 in both GM and 48 h DM associated with muscle atrophy and serve as positive controls for the equally but was not detected near Dstn or Lmod3. Fra-2 enrichment process. These E3 ligases promote atrophy and ubiquitinate proteins was detected for all genes at 48 h DM, with the strongest recruitment for degradation. In muscle, MuRF1 directly targets myosin and observed on Flnc (Fig. S2). myosin binding proteins for degradation, contributing to sarcomere To determine whether actin cytoskeletal genes are sensitive to the loss (Clarke et al., 2007; Cohen et al., 2009). After 6 or 24 h of loss of AP-1 and MEF2 we utilized siRNA-mediated gene treatment with Dex, MAFbx and MuRF1 were upregulated. silencing. MEF2A is the predominant MEF2 subunit in Treatment with Dex for 6 h increased Dstn and Hspb7 expression differentiating myogenic cells and our previous ChIP-exo data and decreased Flnc. Expression of Lmod3 and Plekhh2 was was completed using a MEF2A antibody; therefore, we initially unchanged. After 24 h treatment with Dex these trends were similar; used siRNA targeting MEF2A. Because AP-1 could function in however, Hspb7 was upregulated fivefold, equivalent to the degree Jun–Fos or Jun–Jun homodimers and because in muscle c-Jun and of MAFbx and MuRF1 induction. Fra-2 have been documented to be crucial factors in regulating myoblast proliferation (Alli et al., 2013; Bengal et al., 1992), we MEF2A and c-Jun and Fra-2 regulate atrophy-induced Hspb7 used siRNA targeting c-Jun and Fra-2 (Fig. 2C). Depletion of expression MEF2A resulted in downregulation of Hspb7 and Lmod3 (Fig. 2D). Hspb7 was the most dynamically regulated gene during ageing in Dstn, Flnc and Plekhh2 showed MEF2A recruitment (Fig. S2) but skeletal muscle and was also highly inducible in response to Dex no differential expression with MEF2A knockdown, possibly treatment; therefore, we pursued the regulation of this gene by reflecting some level of redundancy in the MEF2 family. A MEF2A, c-Jun and Fra-2 in more detail. A previous study had reduction in c-Jun did not significantly affect target gene expression, identified target genes of the glucocorticoid receptor (GR) in Dex- although decreases in Flnc, Plekhh2 and Fra-2 were observed. treated C2C12 myotubes (Kuo et al., 2012). Interestingly, this study Similarly, although Fra-2 expression was partially reduced, this did found GR recruitment to an intron of Hspb7. Using the UCSC not result in dramatic changes in gene expression, potentially browser we plotted the MEF2A, c-Jun, Fra-1 and AP-1 peaks and indicating AP-1 functional redundancy. compared them with GR recruitment reported by Kuo et al. (2012) To account for partial knockdown with siRNA technology or the within the Hspb7 gene (Fig. 4A). GR was recruited following Dex involvement of other AP-1 family members in expression of these treatment to within the second intron of Hspb7, which contained c- genes, we exogenously expressed GFP–Tam67, a potent dominant Jun and Fra-1 enrichment peaks. We utilized Dex treatment to negative form of c-Jun that lacks the transactivation domain but further determine how MEF2A, AP-1 and GR might contribute to contains the DNA binding and dimerization domains. Previously, Hspb7 expression. Tam67 has been used as a dominant negative of the AP-1 complex At the protein level, Dex treatment upregulated Hspb7 expression and, as such, is a useful tool to use when redundancy of the complex in the late stages of differentiation but not under growth conditions AP-1 subunits is suspected (Hennigan and Stambrook, 2001). We (Fig. 4B). Next, the effect of exogenous expression of MEF2A, Fra- assessed expression of the five target genes in growth conditions 2 or c-Jun in combination with Dex treatment was assessed in

(0 h DM) and after 24 and 72 h in DM. Tam67 expression had growth conditions and in 72 h myotubes. Under growth conditions, Journal of Cell Science

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A B 6 hr Gastrocnemius 3.5 4 * 3.0 2.5 3 0 μM 2.0 2 Young 1.5 1 μM

Adult Change Fold 1.0 10 μM Fold Change Fold 1 0.5 100 μM 0 0.0 Dstn Flnc Hspb7 Lmod3 Plekhh2

Quadriceps 24 hr 6 * 6 5 5 4 * 4 0 μM * Young 3 3 1 μM 2 Adult

Fold Change Fold 2 1 Change Fold 10 μM 1 0 100 μM Dstn Flnc Hspb7 Lmod3 Plekhh2 0

Fig. 3. Atrophy induced by aging or dexamethasone causes changes in MEF2A and AP-1 cytoskeletal target genes. (A) RNA from the gastrocnemius and quadriceps of 8- and 63-week-old C57BL/6 mice was isolated for qRT-PCR analysis. Values were calculated using the ΔΔCt method and normalized to Gapdh (n=3 except for Flnc expression in the gastrocnemius sample where n=2, mean±s.e.m., *P<0.05). (B) Dex treatment of myotubes induces atrophy and modulates the expression of MEF2A and AP-1 target genes. C2C12 cells were allowed to differentiate for 72 h and then treated with Dex for 6 h (upper graph)or 24 h (lower graph) at the indicated concentrations. Values were calculated using the ΔΔCt method and normalized to Gapdh.

Hspb7 expression was only enhanced by the combined Wisdon and Verma, 1993). Together, these data indicate the overexpression of MEF2A and Dex treatment. Dex treatment possibility of antagonistic regulation of Hspb7 by AP-1 and MEF2. alone could not induce induction of Hspb7 (Fig. 4C). In myotubes, Also, MEF2 could function in combination with GR recruitment for Dex treatment consistently upregulated Hspb7 expression, except upregulation of Hspb7. To address the possible involvement of GR, when c-Jun was ectopically expressed. This further indicated the we performed ChIP-qPCR and assessed GR recruitment to the repressive role of AP-1 at this locus (Fig. 4D). By 48 h in DM, second intron of Hspb7 after Dex treatment (48 h DM plus 24 h endogenous expression of MEF2A was significantly higher 10 μM Dex). Indeed, we observed a substantial recruitment of GR to (Fig. 2A). However, premature expression of MEF2A under the the Hspb7 locus after Dex treatment, in agreement with ChIP-seq conditions for exogenous expression contributed to upregulation of data generated by Kuo et al. (2012), but no change in MEF2A Hspb7 at this time point, and this was enhanced by Dex treatment. binding at the 3′-end (Fig. 4F). Finally, to determine whether MEF2 and AP-1 are necessary for Dex-induced upregulation of Hspb7, C2C12 myoblasts were Characterization of the role of Hspb7 in muscle atrophy transfected with siRNA targeting MEF2A, Fra-2 or c-Jun, Small heat shock proteins have a documented role in protecting the allowed to differentiate for 48 h and then treated with Dex to cytoskeleton during stress (Garrido et al., 2012). Hspb7 (also known determine whether Hspb7 expression was affected (Fig. 4E). Under as cvHsp) is highly expressed in skeletal and cardiac muscle (Krief Dex treatment, loss of MEF2A prevented Dex-dependent induction et al., 1999) and has been linked to cardiac morphogenesis, of Hspb7 expression; however, loss of Fra-2 or c-Jun upregulated cardiomyopathies and skeletal muscle integrity in the adult (Chiu Hspb7 and this was enhanced by Dex treatment. Of note, when et al., 2012; Juo et al., 2016; Rosenfeld et al., 2013). In other cell types, expressed exogenously, c-Jun was able to block Hspb7 induction Hspb7 has been shown to prevent protein aggregation (Eenjes et al., whereas Fra-2 could not, yet loss of either of these proteins led to the 2016; Minoia et al., 2014) and to localize to nuclear speckles (Vos upregulation of Hspb7 expression (Fig. 4D,E). As we previously et al., 2009), a sub-nuclear location in which pre-mRNA is spliced. showed, c-Jun and Fra2 function as a heterodimer in skeletal muscle Using an HA-tagged Hspb7 expression construct we observed (Andreucci et al., 2002), therefore this disparity could be related to that exogenous expression of Hspb7 did not influence myogenic the relative transcriptional potency of these transcription factors: c- induction in vitro, as shown by the lack of change in MyoG Jun is a robust transcriptional regulator and can form a homodimer expression (Fig. 5A), in agreement with Juo et al. (2016) who in the absence of Fra-2, whereas Fra-2 is a weaker transcriptional showed that Hspb7 does not contribute directly to myogenesis. activator that has no intrinsic transactivation domain and relies Hspb7 has been associated with autophagy but the mechanism is on recruitment of the Jun heterodimeric partner for bringing unclear (Vos et al., 2010). Bag3 is a component of CMA that shows transactivation properties to the AP-1 complex (Suzuki et al., 1991; enriched expression in striated muscle, and Bag3-null mice develop Journal of Cell Science

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A ChIP-seq read densies within the Hspb7 gene

GN 48 hr DM 72 hr DM Control MEF2A Fra-2 c-Jun B Dex + 6 + 24 + 6 + 24 + 6 + 24 C Dex ++ + + Hspb7 Hspb7 Acn MEF2A D Control MEF2A Fra-2 c-Jun Dex ++ + + Fra-2 Hspb7 c-Jun

MEF2A GFP Acn Fra-2 GM + 24 hr treatment

E Control siMEF2A siFra-2 sic-Jun c-Jun Dex ++ + +

GFP Hspb7

Acn MEF2A 48 hr DM + 24 hr treatment

F 0.08 0.10 Fra-2 0.06 0.08 0.06 0.04 c-Jun 0.04 % Input % Input 0.02 0.02 0 0.00 IgG GR IgG GR IgG Mef2a IgG Mef2a Acn DMSO Dex DMSO Dex 48 hr DM + 24 hr treatment

Fig. 4. Changes in Hspb7 expression induced by dexamethasone are modulated by MEF2A and AP-1. (A) UCSC genome browser image depicting recruitment of MEF2A (blue), Fra-1 (brown; C2 FOSL1), c-Jun (green) and glucocorticoid receptor (GR, red) to Hspb7. AP-1 consensus sequences are indicated by vertical black lines. (B) Dex treatment of myotubes strongly upregulated Hspb7 protein expression. C2C12 cells were treated with 10 μM Dex for the indicated times. Actin was used as a loading control. (C,D) Exogenous expression of MEF2A, Fra-2 or c-Jun with Dex treatment in growth conditions (C) or 72 h DM (D). C2C12 cells were transfected with the indicated construct using calcium phosphate and allowed to recover. Cells were then treated with 10 μM Dex for 24 h after 24 h in GM (C) or after 48 h in DM (D). (E) Loss of MEF2A, Fra-2 or c-Jun affects the induction of Hspb7 expression by Dex treatment. Cells were transfected with the indicated siRNA and allowed to differentiate for 48 h, after which they were treated with 10 μM Dex for 24 h. (F) At 48 h, DM C2C12 cells were treated with 10 μM Dex for 24 h and prepared for ChIP-qPCR analysis. Enrichment of GR or MEF2A was detected at the 2nd intron or 3′-end, respectively. Data were calculated using the percentage input method. Journal of Cell Science

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B 6 hr 24 hr A Rapa ++ GM 24 hr DM 48 hr DM Hspb7 pCAGGSnHC ++ + Hspb7-HA ++ + I I LC3B LC3B II II Acn Hspb7 C LC3B EYFP Merge HA

MyoG DMSO

GFP

Acn Rapa

D E F DMSOBafA BafA+Rapa 3.0 pCAGGSnHC Hspb7-HA pCAGGSnHC Hspb7-HA ++pCAGGSnHC Rapa +++ 2.5 I Hspb7-HA +++ LC3B II I 2.0 LC3B II 1.5 HA HA 1.0 Hspb7 LC3BII/AcnUnits) (Arbitrary Hspb7 0.5 Acn 0.0 Acn DMSO BafA BafA+Rapa

Fig. 5. Role of Hspb7 in skeletal muscle atrophy. (A) Overexpression of Hspb7 in differentiating C2C12 myoblasts. Cells were transfected with Hspb7–HA and allowed to differentiate for the indicated times. Extracts were prepared for western blot. (B) Rapamycin treatment decreases Hspb7 expression. Myotubes (72 h) were treated with rapamycin (10 μg/ml) for the indicated times. Protein extracts were then prepared for western blot analysis. (C) Myoblasts transfected with EYFP–Hspb7 were treated with rapamycin (10 μg/ml) for 6 h and then assessed for LC3B co-localization (red). Hoechst stain was used to visualize nuclei. (D) C2C12 cells were transfected with Hspb7–HA or control and treated with rapamycin (10 μg/ml) for 6 h in GM, 48 h post-transfection. Protein extracts were then prepared for western blot. (E) C2C12 cells were prepared as in D. Cells were treated with either BafA alone (200 nM) or BafA plus rapamaycin (10 μg/ml) for 6 h. (F) Quantification of LC3B-II expression was relative to actin. Values were quantified in ImageJ. myopathies (Homma et al., 2006). Interestingly, Hspb7 and Bag3 To investigate whether Hspb7 associates with autophagosomes, single nucleotide polymorphisms (SNPs) have been associated with we generated an EYFP–Hspb7 construct and determined the co- heart failure (Garnier et al., 2015). Hspb7 has also been shown to localization of Hspb7 with LC3B under rapamycin treatment in interact with Hspb8 (Sun et al., 2004), an autophagy-related protein myoblasts (Fig. 5C). After 6 h of rapamycin treatment, LC3B was that interacts with Bag3 (Carra et al., 2008). Therefore, we detected in puncta throughout the cell and in large aggregates, where investigated whether Hspb7 could affect autophagy by using the EYFP–Hspb7 was also observed. conversion of LC3B-I to LC3B-II as a molecular readout for this Because rapamycin affected endogenous Hspb7 protein levels process. We observed that LC3B-II levels were not significantly and Hspb7 and LC3B co-localize, we hypothesized that Hspb7 affected by Hspb7 overexpression; however, exogenous Hspb7 could be directly involved in autophagic flux. Related family protein was rapidly degraded within 24 h whereas GFP protein member Hspb8 was able to induce LC3B-II accumulation in levels were maintained (Fig. 5A). We hypothesized that the turnover previous studies, but Hspb7 had no effect (Vos et al., 2010) even of Hspb7 protein could indicate its involvement in autophagy and though these studies were carried out without pharmacological subsequent degradation in the autolysosome. To confirm whether activation of autophagy and without lysosome inhibitors, which are the loss of exogenous Hspb7 demonstrated in Fig. 5A could be via probably necessary to interpret autophagic flux. Therefore, we autophagy we monitored endogenous Hspb7 proteins levels in treated cells with rapamycin in the presence or absence of Hspb7– response to treatment with rapamycin, an mTOR inhibitor. HA (Fig. 5D). It was observed that rapamycin alone induced LC3B- Endogenous Hspb7 was reduced after 24 h of rapamycin II levels but this was reduced with Hspb7–HA expression. Changes treatment (Fig. 5B), indicating that Hspb7 could be degraded in LC3B-II levels without lysosomal blockade are invoked by either within the autophagosome. increased or decreased autophagic flux; therefore, we next used Journal of Cell Science

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BafA in combination with rapamycin and Hspb7 exogenous To determine whether Hspb7 has a protective role during expression to determine whether the rate of autophagy was altered muscle atrophy induced by starvation we exogenously expressed by Hspb7 expression. Interestingly, we saw reduced LC3B-II levels an HA-tagged Hspb7 construct into the TA muscle using (Fig. 5E, quantified in Fig. 5F), indicating the possibility that Hspb7 electroporation. These were ‘paired’ experiments whereby the reduces autophagy. TA muscle of one leg received the Hspb7–HA expression vector while the contralateral leg TA received the control vector, thus A role for Hspb7 in skeletal muscle autophagy in vivo serving as a control in the same animal (Fig. 7A). Interestingly, Based on the above data, we suspected that Hspb7 is induced under total mass of the TA was reduced with Hspb7–HA expression in muscle atrophy and has a potential role in autophagy. Although we the colchicine plus fasting condition (Fig. 7B). Total RNA was propose that Hspb7 is involved in mature muscle homeostasis, our isolated for qRT-PCR analysis of autophagy-related genes in vitro experiments were limited to early growth conditions (Fig. 7C). Under control conditions (saline), Hspb7–HA did not because exogenous expression of Hspb7 or siRNA was lost in alter expression of Map1lc3b, p62, Foxo1 or Foxo3. These genes cultured myotubes (>72 h DM). Therefore, to determine whether were upregulated in response to colchicine and fasting. However, Hspb7 is associated with autophagy in mature muscle we used a in Hspb7–HA transfected limbs, induction of Map1lc3b, p62 and previously characterized in vivo model of autophagy via 24 h fasting Foxo1 was weaker, although expression was not significantly of mice (Ju et al., 2010). Twenty-four hours prior to fasting, different from the paired leg control (Fig. 7C). At the protein level, colchicine (0.4 mg/kg/day) was injected to serve as an autophagic we noticed that mice from the colchicine plus fasting condition block and this treatment was repeated every 24 h (Fig. 6A). Under that did not receive Hspb7–HA showed an upregulation of total colchicine plus fasting conditions, Hspb7 expression was increased LC3B (note LC3B-I in lanes 7–9) compared with non-fasted, in the quadriceps and gastrocnemius muscles but not the heart saline conditions (Fig. 7D). This change in protein level correlates (Fig. 6B). We also assessed expression of Hspb7 and autophagy- with increased mRNA expression of Map1lc3b (Fig. 7C). Hspb7– related genes in the tibialis anterior (TA) muscle under these HA expression showed weaker, though not significantly different, conditions and observed an upregulation of Map1lc3b, p62, MyoG expression of total LC3B compared with the paired leg controls Atg7, Foxo1, Foxo3 and Hspb7 (Fig. 6C). from the same mice (Fig. 7D).

AB Hspb7 6 * ** d0 d1 d2 5 4 Fasng Control Colchicine 3 Colchicine 2 Fold Change Fold Colchicine+Fast 1 0 Heart Quad Gastroc

C TA 30 ** 25

15 Saline ** Colchicine

Fold Change ** 10 ** ** Colchicine+Fast ** 5 ** 0

Fig. 6. Hspb7 expression is associated with autophagy. (A) Experimental design for in vivo experiments. 6- to 8-week-old C57BL/6 mice were treated with saline, colchicine (0.4 mg/kg/day) or colchicine plus fasting (24 h). (B) Hspb7 expression in the heart, quadriceps (Quad) and gastrocnemius (Gastroc). Values were calculated using the ΔΔCt method and normalized to Gapdh (n=3, mean±s.e.m., *P<0.05, **P<0.01). (C) Changes in autophagy genes in response to fasting. RNA was isolated from the TA for qRT-PCR analysis. Data were analysed as in B. Journal of Cell Science

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45 A B 40 35 d0 d3 d4 d5 ** 30 Fasng 25 20 Hspb7-HA pCAGGSnHC Colchicine 15 weight (mg) Electroporaon 10 of TA 5 0 Control Hspb7-HA Control Hspb7-HA ** ** Saline Colchicine+Fast 3.0 C * * 2.5 Saline Control 2.0 Saline Hspb7-HA 1.5 Colchicine+Fast Control

Fold Change Fold 1.0 Colchicine+Fast Hspb7-HA 0.5

0.0 Map1lc3 p62 Foxo1 Foxo3

D GFP RFP Merge Saline Colchicine+Fast E Hspb7-HA +++ +++

I Control LC3B II

HA Saline Gapdh

d0 d2 d4 F Control Hspb7

si-Hspb7 scr Colchicine Electroporaon Hspb7 of mouse TA with siRNA Colchicine+Fast

G H 50 * Saline Colchicine 40 si-Hspb7 +++ +++ 30 20

Hspb7 weight (mg) 10 0 Gapdh scr siHspb7 scr siHspb7 Saline Colchicine

Fig. 7. Hspb7 regulates gross muscle mass and LC3B expression. (A) Experimental design for in vivo experiments. Hspb7–HA was electroporated into the TA muscle while the contralateral TA muscle received the control plasmid (pCAGGSnHC). Mice were then treated with saline or colchicine plus fasting for the indicated times (colchicine, 0.4 mg/kg/day; fasting, 24 h). (B) Total TA muscle mass under the indicated experimental conditions (n=6; mean±s.e.m., **P<0.01). (C) In vivo gene expression during colchicine plus fasting with exogenous expression of Hspb7–HA or control in paired leg experiment. Saline, n=6; colchicine plus fasting, n=5; mean±s.e.m.; *P<0.05, **P<0.01 compared with saline control. Values were calculated using the ΔΔCt method and normalized to the geometric mean of β-actin, Gapdh, Tbp and Rps26. (D) Samples from C were analysed using western blot to determine level of exogenous expression of Hspb7–HA and LC3B. (E) TA muscles were electroporated with tandem LC3 and Hspb7–HA or tandem LC3 and control plasmid and allowed to recover for 3 days before being treated with saline or colchicine plus fasting, as depicted in A. Transverse sections of the muscle were then prepared for analysis of tandem LC3 localization. (F) Experimental design of siRNA experiments. Three independent siRNAs targeting Hspb7 were pooled into a final concentration of 450 pmol (150 pmol of each) and electroporated into the TA muscle. Control siRNA (scr) was electroporated into the contralateral control TA muscle. At 2 days post-electroporation, colchicine was administered daily for a further 2 days. (G) Expression of Hspb7 protein levels in the TA muscle after siRNA knockdown. (H) Total TA muscle mass under the indicated experimental conditions (n=3; mean±s.e.m., *P<0.05).

To further define the role of Hspb7, we utilized a tandemly tagged and RFP+) or red (GFP− and RFP+), respectively. We LC3 plasmid containing both GFP and mRFP (Kimura et al., 2007). electroporated this plasmid into the TA muscle of mice with GFP becomes degraded in the acidic lysosomal compartment Hspb7–HA and initiated a protocol of colchicine plus fasting, as and, therefore, the difference in fluorescence corresponds to depicted in Fig. 6A. Compared with saline, mice treated with autophagosomes or autolysosomes, which appear yellow (GFP+ colchicine plus fasting showed upregulation of LC3 puncta with the Journal of Cell Science

4085 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4076-4090 doi:10.1242/jcs.190009 control plasmid (Fig. 7E). True LC3 puncta corresponding to A striking feature of Fra-1 and c-Jun recruitment is that the anti- autophagosomes or autolysosomes can be difficult to determine inflammatory GR also binds within the same location of the second using this method because exogenous LC3 is prone to aggregation intron of Hspb7 (Fig. 4A). GR and AP-1 competitive regulation is (Kuma et al., 2007) and, indeed, we did see minimal aggregation at not a new phenomenon and was observed several decades ago on the periphery of myofibres with saline and control plasmids. Hspb7 the gene encoding collagenase I (Jonat, 1990). Subsequently, many has been shown to prevent protein aggregation in vitro (Eenjes et al., other genes have been shown to be regulated by GR and AP-1. 2016), therefore we predicted that Hspb7 could also reduce LC3 Interestingly, GR cooperates with a Jun homodimer but inhibits aggregation. Interestingly, we observed an overall trend whereby co- Jun–Fos heterodimers (Adcock and Caramori, 2001; Herrlich, expression of Hspb7–HA with tandem-LC3 resulted in enhanced 2001). Additionally, AP-1 has been shown to potentiate GR aggregation of LC3 (Fig. 7E). recruitment by promoting accessible chromatin in epithelial cells We next utilized siRNA to knockdown Hspb7 in vivo,in (Biddie et al., 2011); however, in the case of Hspb7 expression, AP- combination with 2 days of colchicine treatment (Fig. 7F). Loss of 1 and GR do not synergize. In the case of Hspb7, we observed that Hspb7 protein was detected most clearly with colchicine treatment loss of c-Jun or Fra-2 induces Hspb7 expression upon Dex (Fig. 7G). Interestingly, conditions with a significant reduction of treatment, indicating that a Jun–Fos dimer is involved in Hspb7 protein levels were associated with an increase in muscle regulation of this gene. Moreover, our studies implicate a mass (Fig. 7H). Collectively, these data suggest a possible widespread level of cooperativity between AP-1 and MEF2 that involvement of Hspb7 in regulation of overall muscle mass and warrants further investigation. autophagic flux. Further studies are required to fully characterize the In striated muscle, the actin cytoskeleton stabilizes the sarcomere complex function of Hspb7 in muscle in vivo. in concert with the costamere, which tethers the Z-line of the sarcomere to the sarcolemma (Ervasti and Campbell, 1993). This DISCUSSION facilitates sarcomere stabilization and connects the actin From our previous study, in which we identified novel MEF2A cytoskeleton to the extracellular matrix via the costamere, but it target genes in skeletal and cardiac muscle (Wales et al., 2014), we also has roles in other cell processes including migration, adhesion observed that AP-1 consensus cis elements were enriched by and gene expression (Zheng et al., 2009). Therefore, identifying MEF2A binding events and, based on GO term analysis, there was pathways that mediate actin cytoskeletal gene expression has indication that MEF2A regulates the actin cytoskeleton (Potthoff implications for different types of muscle disease. MEF2 has a et al., 2007; Wales et al., 2014). In the work presented here, we show well-established role in sarcomere organization, because it regulates through bioinformatic and biochemical analyses that MEF2 and AP- key target genes associated with the costamere and sarcomeric 1 share a number of putative common target genes related to the proteins (Ewen et al., 2011; Hinits and Hughes, 2007; Potthoff et al., establishment and maintenance of the actin cytoskeleton, some of 2007). There are fewer studies that have investigated the role of AP-1 which was validated using ChIP-qPCR and knockdown analysis. In in sarcomere integrity; however, in cardiomyocytes c-Jun has been particular, we focused on Hspb7 under conditions of muscle atrophy shown to have an important role in promoting sarcomere gene and observed that AP-1 and MEF2 antagonistically regulate expression and sarcomere integrity (Windak et al., 2013). expression of this gene. Moreover, experiments in mice and in Interestingly, destabilization of the actin cytoskeleton triggers c- cultured muscle cells suggest a role for Hspb7 in autophagy, having Jun activity in vitro and represses glucocorticoid receptor activity possible implications for muscle atrophy. (Oren et al., 1999). The myofibres in mouse models of cachexia are associated with a defective sarcolemma, similar to that seen in MEF2 and AP-1 regulation of the actin cytoskeleton and muscular dystrophies (Acharyya et al., 2005); therefore, common muscle atrophy structural and cytoskeletal defects could contribute to various MEF2 and AP-1 are ubiquitous transcription factors yet their pathologies. In cancer cachexia, expression of a dominant negative potential interaction at the transcriptional level has not been AP-1 factor Tam67 or AP-1 and NF-κB double inhibitor can prevent studied. Based on our results, MEF2 and AP-1 appear to inversely loss of muscle mass (Moore-Carrasco et al., 2006, 2007). By regulate Hspb7, wherein MEF2 promotes expression and AP-1 contrast, JunB was found to be universally downregulated in models represses it. We also identified several other potential common of atrophy (Lecker et al., 2004) and loss of AP-1 factors in target genes that MEF2 and AP-1 might co-operatively or denervation-induced muscle atrophy prevents upregulation of competitively regulate. Based on GO term analysis, Fra-1 and MAFbx and MURF1 (Choi et al., 2012). Dysregulation of AP-1 in MEF2A do not share a significant number of target genes cancer could therefore not only modulate proliferation and metastasis compared with MEF2A and c-Jun (Fig. 1). This could indicate (Eferl and Wagner, 2003) but also impact muscle health, which is that Fra-1 and MEF2 have fundamentally different functions and known to be modulated in cancer cachexia (Dodson et al., 2011). also that Fra-1 associates with another Jun family member such as JunB or JunD to target differential AP-1 target genes under A role for Hspb7 in muscle disease and sarcopenia proliferative conditions. Interestingly, MEF2 and c-Jun were To date, it has been thought that the role of small heat shock enriched for several muscle-related GO terms. This could reflect proteins, including Hspb7, is stabilization of the cytoskeleton under the role of c-Jun in priming muscle-specific enhancers with MyoD stress conditions. Hspb7 can interact with α-filamin (Krief et al., (Blum et al., 2012) and possibly MEF2. Fra-1 and c-Jun exclusive 1999), filamin C (Juo et al., 2016), the cytoskeleton in tachypaced targets were mainly associated with angiogenesis and the response cardiomyocytes (Ke et al., 2011) and myofibrils in response to to bacterium, the latter being associated with inflammation and cardiac ischemia (Golenhofen et al., 2004). However, based on cytokine production, one of the traditional roles of AP-1 (Hess studies by Vos and colleagues, it appears that Hspb7 could have a et al., 2004; Shaulian and Karin, 2002). The bioinformatic analysis role in autophagy and protein degradation, making its role more demonstrates that analysis of differential transcription factors can extensive than previously thought (Vos et al., 2009, 2010). reveal distinct roles of cooperative and exclusive biological Recently, Hspb7 has been shown to prevent aggregate formation, functions. and not to contribute to aggregate clearance (Eenjes et al., 2016). Journal of Cell Science

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Our study confirms that Hspb7 is upregulated with age, and also in Transfections the context of GR-mediated atrophy and fasting conditions in vivo. C2C12 myoblasts were transfected using the calcium phosphate These observations agree with the previously stated idea that Hspb7 precipitation method. Cells were then harvested at 48 h post-transfection has a protective role (Doran et al., 2007); however, our data indicate or the medium was changed to DM. that after rapamycin treatment in vitro the exogenous expression of Plasmids Hspb7 localizes with LC3B and decreases autophagic flux. The rate – – of autophagy is considered to be a delicate balance because too Expression plasmids for pMT2 MEF2A, pCMV c-Jun, pcDNA3.1-Fra-2, pCMV–dsRed2, pcDNA–GFP, GFP–Tam67 have been described much or too little results in different molecular deficiencies; previously (Alli et al., 2013; Hennigan and Stambrook, 2001; Perry et al., therefore, the ability of Hspb7 to modulate this process in muscle 2009). pCAGGSnHC–HSPB7-HA was described by Lin et al. (2014). could have pivotal consequences in the context of sarcopenia and EYFP–Hspb7 was sub-cloned from Hspb7–HA into EYFP–pcDNA3 using other forms of muscle atrophy. XhoI and EcoRI. Because impairment of autophagy is believed to be associated with sarcopenia (Rubinsztein et al., 2011), our findings could have Antibodies and reagents important implications in future therapies that seek to target Rabbit polyclonal MEF2A antibody has been previously described (Cox autophagy in muscle wasting. Deficits in satellite cell numbers et al., 2003). The following antibodies were purchased from Santa Cruz has also recently been associated with decreased autophagy (García- Biotechnology: actin (sc-1616), dsRed (sc-33354), MEF2A (sc-313X; used Prat et al., 2016), which could implicate a role for Hspb7 in satellite in ChIP), donkey anti-goat IgG-HRP (sc-2020), Fra-2 (sc-604), c-Jun (sc- cell biology. Although Hspb7 is primarily associated with 1694), GFP (sc-9996), MCK (sc-365046) and MyoD (sc-304). Anti-LC3B was from Cell Signaling (2775). Myogenin and HA monoclonal antibodies autophagy (Vos et al., 2010) it could have uncharacterized roles were obtained from the Developmental Studies Hybridoma Bank. The in proteasomal degradation pathways in other tissues. Additionally, remaining antibodies were as follows: Hspb7 (Abcam, ab150390), the induction of Hspb7 via glucocorticoids could result in a glucocorticoid receptor (Abcam, ab3579), rabbit IgG (Millipore, 12-370). subsequent maladaptive role for Hspb7 if signalling is prolonged. Dexamethasone (sc-29059) was used at a concentration of 10 μM, unless Skeletal muscles deficient in Hspb7 do not show defects in otherwise indicated. DMSO was used as a volume control. Rapamycin myogenesis but still develop myopathies postnatally, indicating that (10 μg/ml) and BafA1 (200 nM) were purchased from Santa Cruz Hspb7 has a potentially crucial role in skeletal muscle integrity in Biotechnology (sc-3504 and sc-201550). adults (Juo et al., 2016). It would be interesting to further investigate this in the context of ageing or fasting. Hspb7 is highly expressed in siRNA transfection of C2C12 myoblasts the heart, and a crucial role for Hspb7 in heart development has been Knockdown of target genes was carried out using siRNAs obtained from Sigma-Aldrich; targets are listed in Table S2. In C2C12 myoblasts, siRNA shown in zebrafish (Rosenfeld et al., 2013). Mutations in Hspb7 are MEF2A c-Jun correlated with cardiomyopathies and, co-incidentally, Hspb7 SNPs was transfected at the following concentrations: (30 nM), (50 nM) and Fra-2 (50 nm). within the second intron (where GR and AP-1 recruitment was observed) are associated with cardiomyopathies (Garnier et al., Immunoblots 2015; Matkovich et al., 2010). In our studies, although Hspb7 was Cells were washed with 1× PBS and lysed in NP-40 lysis buffer (50 mM induced in skeletal muscles in response to fasting it was not Tris, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA, 100 mM NaF and 10 mM upregulated in the heart (Fig. 7). This could indicate that Hspb7 is Na pyrophosphate) containing protease inhibitor cocktail (Sigma-Aldrich), induced to protect tissues in response to different forms of stress. 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) and 1 mM sodium The importance of MEF2, AP-1 and GR in Hspb7 regulation in the orthovanadate (Bioshop). Protein concentrations were determined by heart could also be related to cardiac development and it would be Bradford assay (Bio-Rad). Twenty micrograms of total protein were interesting to determine whether Hspb7, and the GR–MEF2–AP-1 resolved on 10% SDS–PAGE and then transferred onto Immobilon-FL signaling axis as highlighted here, has a role in cardiac hypertrophy PVDF membrane (Millipore) for 1 h or overnight. Nonspecific binding sites or other cardiomyopathies. were blocked using 5% milk in PBS or TBST. Membranes were incubated with primary antibodies overnight at 4°C in 5% milk in PBS or 5% BSA in In conclusion, MEF2 and AP-1 have historically been assigned TBST. Horseradish peroxidase-conjugated secondary antibody was added fairly distinct functions in tissue-specific gene expression and for 1 h at room temperature. Protein was detected with ECL growth control, respectively. Here, we implicate them together in chemiluminescence reagent (Pierce). For cyto-nuclear fractionation, the the antagonistic or cooperative control of cytoskeletal genes such as Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit Hspb7 in skeletal muscle. These studies also highlight the role of the (78833) was used. actin cytoskeleton and its regulation by MEF2 and AP-1 transcriptional regulators, which may be of particular importance Chromatin immunoprecipitation for muscle function and pathology. Methods were carried out as described previously described (Wales et al., 2014) with the addition of a third immunoprecipitation wash buffer (IP wash MATERIALS AND METHODS buffer III; 20 mM Tris pH 8.1, 250 mM LiCl, 1% NP-40, 1% deoxycholate, Cell culture 1 mM EDTA). C2C12 myoblasts were obtained from American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle’s medium RNA extraction (DMEM) with high glucose and L-glutamine (Hyclone) supplemented with Total RNA was extracted from C2C12 myoblasts using the RNeasy Plus kit 10% foetal bovine serum (HyClone) and 1% penicillin/streptomycin (Qiagen) and Qiashredder (Qiagen). RNA isolated from tissue was extracted (Invitrogen). C2C12 myoblasts were induced to differentiate in using Trizol (Invitrogen). RNA was converted to cDNA using Superscript differentiation medium containing DMEM/high glucose/L-glutamine III (Invitrogen) according to the manufacturer’s instructions. supplemented with 2% horse serum (Hyclone) and 1% penicillin/ streptomycin for the indicated times. Cells were maintained in an Quantitative PCR humidified, 37°C incubator at 5% CO2 and replenished with fresh SybrGreen (BioRad or ABM) was combined with 2.5 μl gDNA or cDNA medium every 24 h. Pharmacological drug treatments were completed for and 500 nM primers in a final volume of 20 μl. cDNA was diluted 1:10 prior the indicated times. to use. Each sample was prepared in triplicate and analysed using Rotor- Journal of Cell Science

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Gene Q (Qiagen). Parameters for qRT-PCR using BioRadwere were 30 s positive charged slides and fixed with 4% paraformaldehyde for 10 min at 95°C, [5 s 95°C, 30 s 60°C]×40 cycles. Parameters for qRT-PCR using room temperature. Sections were rinsed with PBS and mounted with ABM were 10 m 95°C, [3 s 95°C, 30 s 60°C]×35 cycles. Parameters for Prolong Gold anti-fade with DAPI. Images were acquired using a Zeiss ChIP-qPCR were 5 min 95°C, [5 s 95°C, 15 s 60°C]×40 cycles. Fold Observer Z1 microscope. enrichment (ChIP-qPCR) and fold change (qRT-PCR) were quantified using the ΔΔCt method. Primers used in qRT-PCR and ChIP-qPCR are Statistics listed in Tables S3 and S4, respectively. Data are presented as mean±s.e.m. Statistical analysis was carried out using two-tailed unpaired Student’s t-test. Bioinformatics AP-1 consensus sequences were mapped using cisGenome. GREAT Acknowledgements (default settings) identified GO terms based on DNA sequences obtained We would like to thank G. Sweeney (York University, Toronto, Canada) for providing from available datasets: GSE61207 for MEF2A; ENCSR000AIK for Fra-1; the tandem LC3 plasmid (tflc3) and Y. Nakamura (University of Chicago, Chicago, USA) for providing Hspb7–HA. and GSE37525 for c-Jun. All datasets were converted to mm9 using UCSC LiftOver. Overlapping binding sites were determined using the UCSC Competing interests Table Browser Intersect function using default settings. The authors declare no competing or financial interests.

Animal care Author contributions For ageing experiments, 63- and 8-week-old C57BL/6 male mice were S.W.T. completed most of the data acquisition, analysis and interpretation. D.Y. and obtained from Jackson Lab or Charles River, respectively. Mice were J.G. completed in vivo experiments, which were then analysed by S.W.T. A.B. sacrificed using cervical dislocation in accordance with the Institutional assisted in bioinformatic analysis. A.F. assisted with in vitro immunofluorescence and imaging, ChIP-qPCR and Tam67 experiments. S.W.T., A.B. and J.C.M. Animal Care and Use Committee of York University. For autophagy designed experiments and wrote the manuscript. experiments, 6- to 8-week-old C57BL/6 male mice were sacrificed using cervical dislocation in accordance with University of Ottawa Animal Care Funding and Use Committee. Autophagic flux was induced using a combination of This work was supported by a grant from the Canadian Institutes of Health Research daily intraperitoneal colchicine administration (0.4 mg/kg/day) and fasting to J.C.M. as described by Ju et al. (2010) with the following changes: (1) mice were placed on a 24 h fast; (2) 3 days of recovery were allowed after ptfLC3, Supplementary information Hspb7–HA or pCAGSSnHC electroporation. Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.190009.supplemental Electroporation of plasmid DNA into mouse tissue Plasmid DNA was prepared as follows: twenty-five micrograms of Hspb7– References HA or empty vector (pCAGGSnHC) in sterile half saline were injected into Acharyya, S., Butchbach, M. E. R., Sahenk, Z., Wang, H., Saji, M., Carathers, M., Ringel, M. D., Skipworth, R. J. E., Fearon, K. C. H., Hollingsworth, M. A. et al. the right and left TA muscles of 6- to 8-week-old C57BL/6 male mice, (2005). Dystrophin glycoprotein complex dysfunction: a regulatory link between respectively. Co-electroporation of tfLC3 and Hspb7–HA or empty vector muscular dystrophy and cancer cachexia. Cancer Cell 8, 421-432. was carried out using equal amounts of 12.5 μg. After injection, muscles Adcock, I. M. and Caramori, G. (2001). Cross-talk between pro-inflammatory were subject to electrical stimulation using an electroporation system (ECM transcription factors and glucocorticoids. Immunol. Cell Biol. 79, 376-384. 830, BTX) with electroporation array needle (model no.533, BTX) Alli, N. S., Yang, E. C., Miyake, T., Aziz, A., Collins-Hooper, H., Patel, K. and McDermott, J. C. (2013). Signal-dependent fra-2 regulation in skeletal muscle programmed at 80 V, 6 pulses, 50 ms/pulse and 500 ms interval between reserve and satellite cells. Cell Death Dis. 4, e692. pulses. Tissues were harvested immediately after cervical dislocation and Andreucci, J. J., Grant, D., Cox, D. M., Tomc, L. 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