FHL3 differentially regulates the expression of MyHC isoforms through interactions with MyoD and pCREB

    FHL3 differentially regulates the expression of MyHC isoforms through interactions with MyoD and pCREB Yunxia Zhang, Wentao Li, Mingfei Zhu, Yuan Li, Zaiyan Xu, Bo Zuo PII: DOI: Reference:

S0898-6568(15)00290-9 doi: 10.1016/j.cellsig.2015.10.008 CLS 8566

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

10 June 2015 9 October 2015 19 October 2015

Please cite this article as: Yunxia Zhang, Wentao Li, Mingfei Zhu, Yuan Li, Zaiyan Xu, Bo Zuo, FHL3 differentially regulates the expression of MyHC isoforms through interactions with MyoD and pCREB, Cellular Signalling (2015), doi: 10.1016/j.cellsig.2015.10.008

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ACCEPTED MANUSCRIPT

FHL3 differentially regulates the expression of MyHC

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isoforms through interactions with MyoD and pCREB

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Yunxia Zhang, Wentao Li, Mingfei Zhu, Yuan Li, Zaiyan Xu*, Bo Zuo *

Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture and Key

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Lab of Agricultural Animal Genetics and Breeding, Ministry of Education, College of

*

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430070, P. R. China.

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Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan,

Correspondence author, E-mail: [email protected];

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[email protected].

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ACCEPTED MANUSCRIPT Abstract In skeletal muscle, muscle fiber types are defined by four adult myosin heavy chain

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(MyHC) isoforms. Four and a half LIM domain protein 3 (FHL3) regulates myoblasts

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differentiation and gene expression by acting as a transcriptional co-activator or co-repressor. However, how FHL3 regulates MyHC expression is currently not clear. In this study, we found that FHL3 down-regulated the expression of MyHC 1/slow and

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up-regulated the expression of MyHC 2a and MyHC 2b, whereas no significant effect

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was found on MyHC 2x expression. MyoD and phosphorylated cAMP response element binding protein (pCREB) played important roles in the regulation of MyHC

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1/slow and MyHC 2a expression by FHL3, respectively. FHL3 could interact with

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MyoD, CREB and pCREB in vivo. pCREB had stronger interaction with the cyclic

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AMP-responsive elements (CRE) of the MyHC 2a promoter compared with CREB, and FHL3 significantly affected the binding capacity of pCREB to CRE. We

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established a model in which FHL3 promotes the expression of MyHC 2a through CREB-mediated transcription and inhibits the expression of MyHC 1/slow by inhibiting MyoD transcription activity during myogenesis. Our data support the notion that FHL3 plays important roles in the regulation of muscle fiber type composition. Keywords: Muscle fiber type; FHL3; MyoD; MyHC; CREB

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ACCEPTED MANUSCRIPT Abbreviations: MyHC, myosin heavy chain;

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FHL, four and half LIM domain protein;

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CRE, cyclic AMP-responsive element; CREB, cAMP-response-element-binding protein;

MyoG, myogenin;

SDS, Sodium Dodecyl Sulfate;

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siRNA, small hairpin RNA;

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cDNA, complementary DNA;

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MyoD, myogenic differentiation 1;

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PKA, cAMP-activited protein kinase A;

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PCR, polymerase chain reaction;

MRFs, myogenic regulatory transcription factors;

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IGFs, insulin-like growth factors; CaN-NFAT, calcineurin-nuclear factor of activated T cells; C/EBP-δ, CCAAT enhancer binding protein δ; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; CDS, coding sequence; bp, base pair; PVD, polyvinylidene fluoride; EMSA, Electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; 3

ACCEPTED MANUSCRIPT 1. Introduction Skeletal muscle comprises different types of muscle fibers. Traditionally, fiber

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types are classified as slow-oxidative (1 type), fast oxidative-glycolytic (2a type) and

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fast-glycolytic (2b type) fibers, according to their contraction and metabolic characteristics [1]. Fiber types are convertible in adult skeletal muscle in response to exercise training [2]. Four postnatal fiber types exist in skeletal muscle, each defined

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by the presence of dominant MyHC isoforms (MyHC 1/slow, MyHC 2a, MyHC 2b,

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MyHC 2x) [3]. In animal production, meat quantity and quality are significantly affected by the composition of the four MyHC isoforms [4]. Skeletal muscle with a

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higher composition of type 2b fibers tends to be larger in diameter. The high

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proportion of MyHC 2b contributes to an increase in muscle mass [5]. However, the

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overall proportion of MyHC 2b has been shown to correlate with the occurrence of pig PSE (pale, soft and exudative) meat, which could lead to a fast pH decline after

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slaughter [6,7]. In human beings, fast-dominant skeletal muscle induced by denervation easily leads to skeletal muscle atrophy compared with type 1 fibers [8]. Oxidative enzymatic activities in human skeletal muscle are correlated with fiber type composition [9]. The fastest, most powerful muscle fiber type (type 2b fibers) tends to be lost in the elderly population [10,11], suggesting that the composition of skeletal muscle is associated with aging-related loss of muscle function and muscle disease. To date, several signal transduction pathways, including myogenic regulatory transcription factors (MRFs), insulin-like growth factors (IGFs), calcineurin-nuclear factor of activated T cells (CaN-NFAT), CCAAT enhancer binding protein δ (C/EBP-δ) 4

ACCEPTED MANUSCRIPT and peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, have been reported in regulating fiber type-specific gene expressions [12-15].

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The LIM domain has one or more cysteine-rich zinc fingers and regulates gene

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transcription [16]. The LIM domain is a zinc-binding structural motif, of which the consensus amino acid sequence is CX2CX16–23HX2CX2CX2CX16–21CX2-3 (C/H/D) [17]. The LIM domain lacks DNA-binding ability and is involved in gene

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expression and cell differentiation through protein-protein interactions [16]. The four

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and a half LIM domain (FHL) proteins, including FHL1, FHL2, FHL3, FHL4 and ACT, are a family of LIM-only proteins with four-and-a-half LIM domains and can

and interacts with other proteins, including FHL2, CREB, Sox15, MZF-1, BKLF,

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18-20

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act as transcriptional coactivators [18]. Fhl3 is highly expressed in skeletal muscle

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Ang and MT-1X, as a transcriptional co-activator or co-repressor [18, 21-26]. FHL3 has been reported to play important roles in cell growth, development, tumorigenesis

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and cancer [27-30]. In skeletal muscle, FHL3 localizes to the nucleus and focal adhesions and is a significant regulator of actin cytoskeletal dynamics in skeletal muscle [31]. FHL3 contributes to the regulation of MyoD dependent transcription of muscle specific genes during myogenesis [32]. In pig production, a polymorphism within the coding region of pig Fhl3 is also significantly associated with meat mass and quality traits [19,20]. Based on these results, we speculat that FHL3 may regulate the expression of MyHC isoforms of different muscle fiber types, thereby regulating the fiber type composition of skeletal muscle. However, no studies regarding the regulation of MyHC gene expression by FHL3 have been reported thus far. In this 5

ACCEPTED MANUSCRIPT study, we present a novel molecular mechanism for the regulation of MyHC expression by FHL3, which may contribute to meat quality control and may benefit

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muscle disease therapy in the future.

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2. Material and methods 2.1.Plasmid constructs

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The coding sequence (CDS) of the Fhl3 gene (870 bp) was obtained by polymerase chain reaction (PCR) using cDNA (complementary DNA) of C2C12

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myoblasts as a template. The 5’ and 3’ ends of the primers contain BamHⅠor XbaⅠ enzyme sites (Table 1). The amplified CDS was digested with BamHⅠand XbaⅠ and

pcDNA3.1-FHL3.

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was then ligated into pcDNA3.1 using T4 DNA Ligase (Takara, Japan) to generate

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The CDS of Creb (1026 bp) was obtained by PCR using cDNA of C2C12 myoblasts as a template. The 5’ and 3’ ends of the primers contain KpnⅠ or XhoⅠ

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enzyme sites (Table 1). The amplified CDS was digested with KpnⅠ and XhoⅠ and was then ligated into pcDNA3.1 using T4 DNA Ligase to generate pcDNA3.1-CREB. Five deletion fragments of the mouse MyHC 2a promoter spanning the sequence from position -2043 bp to + 206 bp (2249 bp), -1097 bp to + 206 bp (1303 bp), -909 bp to + 206 bp (1115 bp), - 309 bp to + 206 bp (515 bp) -109 bp to + 206 bp (315 bp) (relative to translation start site) were obtained by PCR (Fig. 4A). The PCR products were cloned into the pGL3-basic vector. All primers are listed in Table 1. The mutants of CREB binding sites were generated using the overlapping extension PCR and mutagenic primers. 6

ACCEPTED MANUSCRIPT 2.2.Cell culture and differentiation of C2C12 myoblasts

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Mouse C2C12 (ATCC) myoblasts were cultured in 10% (v/v) fetal bovine

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serum (Gibco, Australia) in DMEM (high-glucose Dulbecco’s modified Eagle’s

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medium) (Hyclone, USA) under humidified air containing 5% CO2 at 37°C, and were differentiated at confluence in DMEM with 2% horse serum (Gibco, USA).

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2.3.Transfection of plasmid DNA or siRNA oligonucleotides

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Transfections were performed using plasmid (4 µg) or siRNA (100 pmol) by Lipofectamine 2000 (9 µl) (Invitrogen, USA) after seeded C2C12 myoblasts had been

RNA

(siRNA)

oligonucleotides

were

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hairpin

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allowed to settle for 12-18 hrs, according to the manufacturer’s instruction. Fhl3 small synthesized

according

to

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Mm_FHL3_2_HP siRNA (SI01002890, Qiagen, Germany) and transfected into C2C12 myoblasts as previously described

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. Creb siRNA oligonucleotides (S:

MyoD

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GGACCUUUACUGCCACAAATT; A: UUUGUGGCAGUAAAGGUCCTT) and siRNA

oligonucleotides

(S:

CCCCAAUGCGAUUUAUCATT;

A:

UGAUAAAUCGCAUUGGGGTT) were designed and synthesized by Sangon (China, Shanghai).

2.4.Quantitative real-time PCR

Total RNA from C2C12 myoblasts was extracted using Trizol reagent (Invitrogen, USA). The concentration and quality of RNA were assessed with a NanoDrop 2000 (Thermo, USA) and agarose gel electrophoresis. One microgram of total RNA was used for reverse transcription with the PrimeScript RTreagent kit with 7

ACCEPTED MANUSCRIPT gDNA Eraser (Takara, Japan). The quantitative real-time PCR reaction was performed in a LightCycler 480 II (Roche, Switzerland) system using the THUNDERBIRDTM

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probe qPCR Mix or SYBR®Green Real-time PCR Master Mix (Toyobo, Japan). The

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sequence of Taqman probes and primers can be found in Table 2 and were determined according to the literature [33]. The Ct (2-ΔΔCt) method was used to analyze the

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relative gene expression data according to the literature [34].

2.5.Western blotting

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Cells were lysed in RIPA buffer according to the manufacturer’s instruction

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(Beyotime, Jiangsu, China). Protein lysates were heated at 95°C for 5 min in 5×

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sodium dodecyl sulfate (SDS) sample buffer and were separated by 10% SDS-PAGE

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(30 µg each lane); then, the gel was transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) using a Mini Trans-Blot Cell system (Bio-Rad, USA). The membrane was blocked with 5% non-fat milk for 1.5 h. The primary antibodies

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were incubated overnight at 4°C. The membranes were washed and incubated with secondary antibodies for 1 h at room temperature. The membranes were visualized by ECL (Bio-RAD, USA). Primary antibodies specific for FHL3 (Santa Cruz, CA, USA, sc-166917; 1:200 dilution), myogenin (Santa Cruz, CA, USA, sc-12732; 1:200 dilution), MyHC 2a (Santa Cruz, CA, USA, sc-53095; 1:1000 dilution), CREB (Santa Cruz, CA, USA, sc-240; 1:200 dilution), β-actin (Boster, China, BM0627; 1:1000 dilution), MyHC 2b (Hybridoma Bank, University of Iowa, 10F5; 1:500 dilution), MyHC 1/slow (Abcam, USA, ab11083; 1:6000 dilution), and MyHC 2x (Abcam, USA, ab127539; 1:500 dilution), p300 (Santa Cruz, CA, USA, sc-585; 1:200 dilution), 8

ACCEPTED MANUSCRIPT phospho-CREB at ser133 rabbit monoclonal (Cell Signaling Technology, USA, 87G3), along with goat anti-mouse IgG-HRP (Santa Cruz, CA, USA, sc-2005; 1:3000

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dilution), goat anti-rabbit IgG-HRP (Santa Cruz, CA, USA, sc-2004; 1:3000 dilution),

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and anti-mouse IgM-HRP (Santa Cruz, CA, USA, sc-2064; 1:3000 dilution) secondary antibody were used to detect protein expression. 30 µg of lysate was analysed by western blotting. For quantitative western blotting analysis, films were

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scanned and the band signal intensities determined using ImageJ software. The

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densitometry values were normalised by the corresponding β-actin densitometry

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2.6. Immunofluorescence

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values obtained from the same sample.

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After transfection with plasmid (1 µg) or siRNA (25 pmol) by Lipofectamine 2000 (2 µl) (Invitrogen, USA) and differentiation for 3 days, C2C12 myoblasts cultured in 24-well plate were rinsed with phosphate-buffer saline (PBS) two times

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and fixed with 4% paraformaldehyde for 20 min. Next, the cells were washed twice with PBS, and treated with 0.25% Triton X-100 at room temperature for 10 min and washed twice. They were then incubated in blocking solution (5% bovine serum albumin in PBS) to block nonspecific binding at room temperature for 2 h. The samples were incubated with mouse monoclonal anti-MyHC 2a (Santa Cruz, CA, USA, sc-53095; 1:50 dilution), anti-MyHC 2b (Hybridoma Bank, University of Iowa, 10F5; 1:50 dilution) or anti-MyHC 1/slow (Abcam, USA, ab11083; 1:1000 dilution) and rabbit polyclonal anti-FHL3 (Santa Cruz, CA, USA, sc-28692; 1:50 dilution) antibody at 4°C overnight. The next day, the cells were washed three times, and 9

ACCEPTED MANUSCRIPT incubated with anti-rabbit-FITC (Beyotime, Jiangsu, China, A0562; 1:500 dilution) at room temperature for 1 h in the dark room. The anti-rabbit-FITC antibody was

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removed, and then, samples were washed three times and incubated with

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anti-mouse-Alexa Fluor 555 antibody (Beyotime, Jiangsu, China, A0460; 1:500 dilution). After the secondary antibody was removed, the cells were washed three times and the cell nuclei was stained with 4’, 6-diamidino-2-phenylindole (DAPI) in

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the dark room. After washing three times, images were captured with a Olympus

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IX51-A21PH fluorescence microscope (Olympus, Japan).

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2.7.Dual luciferase assay

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After transfection with MyHC 2a promoter constructs (0.8 µg) and

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pcDNA3.1-FHL3 vector (0.8 µg) by Lipofectamine 2000 (2 µl) (Invitrogen, USA), C2C12 myoblasts were differentiated for 2 days, cultured in a 24-well plate, washed with PBS, lysed in 100 µl of lysis buffer, and then, the cells were assayed for

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promoter activity using a dual luciferase reporter assay system (Promega, USA). The enzymatic activity of luciferase was measured using a PerkinElmer 2030 Multilabel Reader (PerkinElmer). To normalize the transfection efficiency, the cells were transfected with 0.04 µg of the Renilla luciferase reporter plasmid (pRL-TK, Promega, USA).

2.8.Immunoprecipitation

C2C12 myoblasts were seeded in 10-cm dishes and differentiated for 3 days. Cells were harvested and lysed in 1ml lysis buffer (Sangon, Shanghai, China) and protease 10

ACCEPTED MANUSCRIPT inhibitor (Sangon, Shanghai, China). The lysate was centrifuged to remove insoluble components and incubated with either anti-FHL3 monoclonal antibody (Santa Cruz,

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CA, USA sc-166917), phospho-CREB at ser133 rabbit monoclonal (Cell Signaling

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Technology, USA, 87G3), anti-MyoD monoclonal antibody (Santa Cruz, CA, USA sc-166917X) or IgG antibody (Beyotime, Jiangsu, China) overnight at 4°C in the presence of protein A/G-Sepharose beads (Beyotime, Jiangsu, China) after removing

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25 µl lysates for the input control. The beads were washed four times using lysis

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buffer. The proteins were analyzed by western blotting as described above.

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2.9.Electrophoretic mobility shift assay (EMSA)

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C2C12 myoblasts cultured in 15-cm dishes and differentiated for 3 days were

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extracted using a Nuclear Extraction Kit (Active Motif, CA, USA) for EMSA. Double-stranded oligonucleotides (Sangon, Shanghai, China) corresponding to the CRE within the MyHC2a promoter were synthesized and annealed into double strands.

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The DNA binding activity of CREB protein was detected by a chemiluminescent EMSA Kit (Beyotime, Jiangsu, China). The reactions were analyzed by electrophoresis in 6.0% polyacrylamide gels at 100 V for 1 h and were then transferred to a nylon membrane. The dried nylon was visualized using ECL (Bio-Rad, USA). The antibodies were specific for CREB rabbit monoclonal (Cell Signaling Technology, USA, 48H2), phospho-CREB at ser133 rabbit monoclonal (Cell Signaling Technology, USA, 87G3). The DNA-binding reaction system and double-stranded oligonucleotides are listed in Table 3 and Fig 5D, respectively.

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ACCEPTED MANUSCRIPT 2.10. Chromatin immunoprecipitation (ChIP) assay

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C2C12 myoblasts were seeded in 10-cm dishes. ChIP assays were performed

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following the transfection of the pcDNA3.1-FHL3 plasmid into C2C12 myoblasts and

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then differentiation for 3 days using the ChIP Assay Kit (Beyotime, Jiangsu, China). Briefly, after crosslinking the chromatin with 1% formaldehyde at 37°C for 20 min

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and neutralizing with glycine for 5 min at room temperature, C2C12 myoblasts were washed with cold PBS, scraped and collected. Nuclear lysates were sonicated 15

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times for 10 s with 10 s intervals on ice water using a Sonics VCX 130 (Sonics, USA). The chromatin complex was immunoprecipitated at 4°C overnight with antibodies

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against CREB rabbit monoclonal (Cell Signaling Technology, USA, 48H2), phospho-CREB at ser133 rabbit monoclonal (Cell Signaling Technology, USA, 87G3)

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and normal mouse IgG (Beyotime, Jiangsu, China). Immunoprecipitated products were collected after incubation with Protein A + G coated magnetic beads. The beads

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were washed, and the bound chromatin was eluted in ChIP Elution Buffer. Then the proteins were digested with Proteinase K for 4 h at 45°C. The DNA was purified using phenol/chloroform. PCR was performed to analyze the DNA fragments using 2.0% Agarose gel. The primers are listed in Table 1.

2.11. Statistical analysis

Statistical analysis was performed using one-way ANOVA. The data are presented as the means ± S.D., and the level for statistical significance was set at p < 0.05. 12

ACCEPTED MANUSCRIPT 3.

Results

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3.1.FHL3 up-regulates the expression of MyHC 2a and MyHC 2b, and

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down-regulates the expression of MyHC 1/slow

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To investigate the effects of FHL3 on the expression of four MyHC isoforms during C2C12 myoblast differentiation, total RNA and protein were extracted from

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C2C12 myoblasts that were transfected with pcDNA3.1-FHL3 or empty plasmid

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(pcDNA3.1) and then differentiated at 0 day (D0), day 2 (D2), day 4 (D4), or day 6 (D6) in DMEM with 2% horse serum. Real-time PCR, western blotting and

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immunofluorescence were performed to detect gene expression of Fhl3 and MyHC

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isoforms. The expression level of Fhl3 was increased with overexpression by at least

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50% compared with the control (Fig. 1A and B). The expression of MyHC 2a and MyHC 2b were up-regulated, and that of MyHC 1/slow was down-regulated after

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overexpression of Fhl3, while no significant change was found for MyHC 2x expression (Fig. 1A-E). The mRNA level of the marker gene myogenin (MyoG) decreased (Fig. 1A), which was in agreement with the previous study [32]. The knockdown of Fhl3 decreased the expression of MyHC 2a and MyHC 2b but increased the expression of MyHC 1/slow at both the mRNA and protein levels (Fig. 2A-E). The results of immunofluorescence confirmed those of real-time PCR and western blotting. In summary, FHL3 up-regulated the expression of MyHC 2a and MyHC 2b and down-regulated the expression of MyHC 1/slow, whereas no significant effects on MyHC 2x expression were found.

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ACCEPTED MANUSCRIPT 3.2.FHL3 represses the expression of MyHC 1/slow through functional interaction

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with MyoD, but promotes the MyHC 2a expression by other mechanism

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MyoD is a bHLH transcription factor and binds to E-box sites of promoters of

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muscle-specific genes [35]. The promoters of MyHC 2a, MyHC 2b and MyHC 1/slow all contain several E-box elements analyzed using the TFsearch software

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(http://www.cbrc.jp/research/db/TFSEARCH.html). The previous study showed that FHL3 could interact with MyoD and thereby inhibited MyoD transcriptional

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activation [32]. To determine whether FHL3 regulated the expression of MyHC isoforms through MyoD, MyoD was knocked down and Fhl3 was overexpressed in

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C2C12 cells. The results showed that MyoD could increase the expression of MyHC 2a, MyHC 2b and MyHC 1/slow (Fig. 3A and B). MyHC 2a and MyHC 2b expression

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was also increased by Fhl3 overexpression after knockdown of MyoD, but there was no effect of Fhl3 overexpression on the MyHC 1/slow expression after MyoD

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knockdown (Fig. 3A and B). To further confirm this, Fhl3 was knocked down and MyoD was overexpressed in C2C12 cells. MyoD overexpression stimulated MyHC 1/slow expression, and knockdown of Fhl3 strengthened the promoting function of MyoD (Fig. 3C and D), suggesting that FHL3 represses the expression of MyHC 1/slow through inhibition of MyoD transcriptional activity. We also performed a co-expression of Fhl3 and MyoD in C2C12 myoblasts. Although MyoD or Fhl3 overexpression increased MyHC 2a and MyHC 2b gene expression, respectively, the MyHC 2a expression levels after co-expression of Fhl3 and MyoD did not significantly changed compared with the control (Fig. 3E and F). This phenomenon 14

ACCEPTED MANUSCRIPT may be caused by the following reasons: the exogenous expressed FHL3 and MyoD may interact with each other which resulted in no excess FHL3 and MyoD to regulate

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MyHC 2a expression, thereby the activating effects of two proteins were abolished.

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Based on the above results, we concluded that FHL3 repressed the expression of MyHC 1/slow through MyoD, but regulates the expression of MyHC 2a and MyHC 2b

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through other transcription factors instead of MyoD.

3.3.FHL3 up-regulates MyHC 2a expression primarily through improving CREB

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transcription activity

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To elucidate how FHL3 regulated the expression of MyHC 2a and MyHC 2b, we

reporter

gene

(Fig.

4A).

Co-transfection

with

pcDNA3.1

or

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luciferase

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generated a series of MyHC 2a promoter deletions driving the transcription of the

pcDNA3.1-FHL3 and luciferase reporter indicated that the transcription activity of the

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region between nucleotides -909 bp and + 206 bp (1115 bp) containing cAMP response elements (CRE) was improved by Fhl3 overexpression (Fig. 4A). To confirm whether CREB participated in the regulation of MyHC 2a expression, Creb overexpression and knockdown was performed in C2C12 myoblasts. The results showed that CREB also promoted the protein expression of MyHC 2a (Fig. 4B and C). Based on the previous study that FHL3 could strongly interact with CREB and function as a coactivator of CREB protein [18], we hypothesize that FHL3 may regulate MyHC 2a through CREB mediated transcription. To confirm this, Fhl3 and Creb were co-expressed in C2C12 myoblasts. Overexpression of Fhl3 or Creb

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ACCEPTED MANUSCRIPT promoted MyHC 2a expression, and co-expression of two proteins further enhanced MyHC 2a expression (Fig. 4D and E). By contrast, after transfection of Creb siRNA

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fragments, overexpression of Fhl3 did not increase the expression of MyHC 2a

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compared with the control (Fig. 4F and G), whereas no significant difference for MyHC 1/slow expression was observed after overexpression or knockdown of Creb (Fig. 4D-G), suggesting that CREB plays an important role in the regulation of MyHC

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2a expression by FHL3. To further determine whether FHL3 increased the promoter

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transcriptional activity of MyHC 2a through CREB binding sites, we analyzed the transcriptional activity of MyHC 2a promoter with the CRE mutation after

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overexpression of Fhl3 and Creb. The transcriptional activity of the MyHC 2a

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promoter was enhanced by Fhl3 or Creb overexpression, whereas no significant

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difference was found for the activity of the MyHC 2a promoter with the mutation at CRE (Fig. 4H), indicating that FHL3 was involved in the CREB-mediated gene

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expression.

3.4.FHL3 forms a complex with pCREB to regulate MyHC 2a expression

CREB is an important transcriptional factor activated by phosphorylation at ser-133 [36]. cAMP-activated protein kinase A (PKA) can lead to activation of the transcriptional factor CREB [37]. To detect whether FHL3 was involved in CREB phosphorylation-mediated MyHC 2a expression, we used the PKA inhibitor H-89 to decrease the CREB phosphorylation level [38]. We treated the C2C12 cells with 10 µM , 20 µM, 30 µM, 40 µM, 50 µM H-89, and selected 20 µM as the optimal

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ACCEPTED MANUSCRIPT working concentration. CREB phosphorylation levels were decreased by H-89 but were not changed by Fhl3 overexpression (Fig. 5A). Thus we inferred that FHL3

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might interact with pCREB to regulate MyHC 2a expression. Fhl3 overexpression

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up-regulated the expression level of MyHC 2a, which was abolished by the PKA inhibitor H-89 (Fig. 5A). These results showed that the effect of FHL3 on MyHC 2a expression was dependent on CREB protein phosphorylation by PKA. To investigate

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whether FHL3 could form a complex with CREB to regulate MyHC 2a expression,

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we explored the interaction of FHL3 with other proteins in vivo. CREB and p300 were all co-immunoprecipitated by the antibody against FHL3 but were not

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co-immunoprecipitated by control IgG, pCREB also was co-immunoprecipitated by

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FHL3 antibody and there was weak non-specific binding with IgG (Fig. 5B),

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indicating that FHL3 participated in the complex with pCREB and p300. Co-immunoprecipitation experiments were also performed with anti-pCREB and

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anti-MyoD. The results confirmed that FHL3 could interact with both pCREB and MyoD (Fig. 5C).

3.5.FHL3 affects the binding capacity of pCREB to the CRE within the MyHC 2a promoter

The binding capacity of CREB with promoter sequences was analyzed by EMSA and ChIP assay. As shown in Fig. 5D, the incubation of nuclear extracts of C2C12 myoblasts with CRE probes formed a DNA-protein complex (Lane 2). The complex became weaker with 1× cold probes in the mixture (Lane 3). However, the

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ACCEPTED MANUSCRIPT complex did not change in the mutation cold probe reaction (Lane 4). No super-shift band was detected after the addition of anti-CREB to the mixture (Lane 5), while the

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addition of anti-pCREB to the binding reaction caused a super-shift band (Lane 6).

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These results suggested that the CRE of the MyHC 2a promoter was capable of binding to pCREB in vitro. ChIP was also performed on C2C12 myoblasts to determine whether FHL3 regulated the binding capacity of CREB to the CRE of the

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MyHC 2a promoter in vivo. The CRE of the MyHC 2a promoter was capable of

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binding to both CREB protein and pCREB in vivo, and pCREB had a stronger binding capacity compared with CREB (Fig. 5E), which was in agreement with the EMSA

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result. Knockdown of Fhl3 decreased the binding capacity of pCREB to the CRE of

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4. Discussion

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the MyHC 2a promoter (Fig. 5F).

MyHC has been classified into 9 to 11 classes [39]. Class II is also called

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“conventional” MyHCs and includes two developmental isoforms (MyHC-embryonic and MyHC-perinatal) and four adult skeletal muscle isoforms (MyHC 2a, MyHC 2b, MyHC 2x, and MyHC 1/slow) [40]. No reports have been published regarding the effects of FHL3 on the expression of any individual member of the MyHC family thus far. In this study, we observed that FHL3 differentially regulates the expression of MyHC isoforms through interactions with MyoD and pCREB, suggesting that FHL3 increases fast fiber specific gene expression and decreases slow fiber specific gene expression. Adult MyHC isoforms are highly expressed in C2C12 cells differentiated for day 4 and day 6, and hardly expressed in C2C12 myoblasts [41, 42]. In this study, 18

ACCEPTED MANUSCRIPT the Fhl3 overexpression peak occurred at 1-2 days of transfection, and then began to decline afterward. Therefore, we infer that there may be the hysteresis effects of

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FHL3 change at early differentiation on the MyHC proteins at later differentiation. In

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general, FHL3 inhibits expression of total MyHC protein and myotube formation [32]. Our study showed that FHL3 promoted MyHC 2a and MyHC 2b expression, and inhibited MyHC 1/slow expression after C2C12 myoblasts differentiation. It can be

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inferred that the other members of MyHC family such as MyHC-embryonic and

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MyHC-perinatal may be inhibited by FHL3, which resulted in a decreased expression in total MyHC protein. This observation points to a novel mechanism by which FHL3

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differentially regulates muscle fiber specific gene expression.

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Several signaling pathways, including calcineurin [43], PGC-1α [15], and

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Ras/ERK-1/2 signalling [44], have been implicated in skeletal muscle fast-to-slow fiber-type shift and slow fiber gene expression. The MyoG gene is highly expressed in

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slow oxidative muscles [45]. The expression of MyHC 1/slow and MyoG shows the same upward trend after Ca2+ and continuous mild heat stress stimulation which induces a fast-to-slow fiber-type shift, but this observation was different for other myogenic factors [46]. The present results also showed a similar expression trend of both MyHC 1/slow and MyoG after Fhl3 overexpression and knockdown, and synergistic effects for MyHC 1/slow expression were detected after co-transfection of FHL3 siRNA and MyoD overexpression vector. As MyoD also positively regulates the expression of the MyHC 1/slow gene by binding to E-box in the promoter [47], we concluded that the regulation mechanism of MyHC 1/slow expression by FHL3 19

ACCEPTED MANUSCRIPT agreed with the previous results that FHL3 negatively regulates MyoG expression by inhibiting MyoD transcriptional activity [32].

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The studies on the regulation of MyHC 2a, MyHC 2x and MyHC 2b expression

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were primarily focused on the specific signaling pathways, such as p38 MAPK, IGF-1 and intracellular calcium [13, 48, 49]. CREB is a transcription factor that can be activated by growth factor and kinases [50, 51] and that can regulate gene expression

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important to the development of cardiovascular diseases [52, 53], immune responses

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[54], and memory formation [55]. CREB promotes the transcription of target genes via the recruitment of the p300/CBP coactivators [56], and the binding of CREB to

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p300 requires the phosphorylation of CREB at Ser-133 [57]. Emerging evidence has

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also revealed functions of CREB in myogenesis, muscle degeneration and

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regeneration in muscular dystrophy [58-60]. However, the role of CREB protein in the expression regulation of different MyHC isoforms has not been studied. Our

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results showed that pCREB played a more important role in the regulation of MyHC 2a expression than unphosphorylated CREB, and FHL3 regulated MyHC 2a expression through its interaction with pCREB and possible interaction with p300. This conclusion was different from the previous report in which FHL3 did not require phosphorylation of CREB at ser-133 to stimulate transcriptional activity [18]. The present data also showed that MyoD and FHL3 promoted MyHC 2a expression with different molecular mechanisms. FHL3 played the predominant role in the up-regulation of MyHC 2a expression by improving pCREB transcriptional activity rather than changing MyoD transcriptional activity. In addition, MyoD also could 20

ACCEPTED MANUSCRIPT bind to pCREB and FHL3 [32, 61], a tertiary complex of FHL3/MyoD/pCREB may exist and play the roles in the gene regulation by another mechanism. As the promoter

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of MyHC 2b also contains CRE, we inferred that FHL3 promoted the MyHC 2b

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expression with the same mechanism as that of MyHC 2a expression regulation, but the molecular mechanism remains to be determined. 5. Conclusions

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We propose a model whereby FHL3 forms a complex with CREB or MyoD to

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regulate the expression of MyHCs in the nucleus of myoblasts (Fig. 6). Both FHL3 and MyoD participate in the up-regulation of the MyHC 2a expression, and FHL3

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plays the leading role in the up-regulation of MyHC 2a expression, primarily through

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its direct interaction with pCREB. p300 may also participate in the complexes by a

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possible interaction with FHL3 (Fig. 6A), as FHL3 does not directly bind to p300 [29]. In contrast, FHL3 down-regulates the expression of MyHC 1/slow possibly through

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decreasing the binding capacity of MyoD to the E-box of its promoter (Fig. 6B). Regulating muscle fiber composition is possible by altering Fhl3 expression levels, thereby improving meat quality and providing therapy for muscle disease. Our results elucidated a novel molecular mechanism regarding the regulation of MyHCs expression by FHL3. FHL3 might be required for repression of MyHC 1/slow expression during C2C12 myoblasts differentiation. FHL3 promoted the expression of fast fiber genes (MyHC 2a and MyHC 2b) through its interaction with pCREB but inhibited the expression of slow fiber genes (MyHC 1/slow) by inhibiting MyoD transcriptional activity. 21

ACCEPTED MANUSCRIPT Acknowledgments

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This study was financially supported by the Key National High Technology Development Project

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of China (Project No. 2011AA100301), the National Natural Science Foundation of P.R. China

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(Grants No. 31001043), the Agricultural Innovation Fund of Hubei Province (Grant No. 2007-620), the Fundamental Research Funds for the Central Universities (Grant No. 2014PY038).

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We also thank Prof. Guoquan Liu and Prof. Chunyan Mu for their kind assistance in manuscript

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writing and Dr. Xinyu Wu for technical help in the experiment.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Fhl3 overexpression alters the expression levels of MyHC isoforms during C2C12

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differentiation. (A and B) The mRNA and protein expression levels of Fhl3 and four MyHC

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isoforms after Fhl3 overexpression. C2C12 myoblasts were transfected with pcDNA3.1-FHL3 vector or empty vector when reaching confluence and then differentiated at D0, D2, D4 and D6 in DMEM with 2% horse serum. (A) Quantitative RT-PCR results of Fhl3, MyHC isoform and

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MyoG mRNA levels after Fhl3 overexpression at D0, D2, D4 and D6. The data are presented as

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mean ± S.D. (n=3). *P<0.05, **P<0.01, N.S., no significance between the two groups (the same below). (B) Western blotting results of Fhl3 and MyHC isoform protein levels after FHL3

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overexpression at D0, D2, D4 and D6. (C) Relative protein expression levels represented by ratio

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of detected protein to β-actin protein expression level after Fhl3 overexpression at D0, D2, D4 and

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D6. The quantifications of Western blotting data are presented as the mean ± S.D. (n=3) (the same below). (D) Immunofluorescence results of Fhl3 and MyHC isoform protein expression levels

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after Fhl3 overexpression. C2C12 myoblasts overexpressing pcDNA3.1-FHL3 or vector pcDNA3.1 were differentiated for 3 days, and stained with anti-FHL3 antibodies (green), anti-MyHC 2a antibodies (red), anti-MyHC 2b antibodies (red), anti-MyHC1/slow antibodies (red), and DAPI (blue) and then imaged by fluorescence microscopy. Bars, 200 µm. (E) The protein expression level of MyHC 2a, MyHC 2b and MyHC 1/slow observed in D was determined by ratio of the number of nuclei within MyHC isoforms positive myotubes to the total number of nuclei. Values are shown as means ±SD of three independent experiments. Fig. 2. Fhl3 knockdown alters the levels of MyHC isoforms during C2C12 differentiation. (A and B) The mRNA and protein expression levels of Fhl3 and MyHC isoforms after Fhl3 knockdown. 27

ACCEPTED MANUSCRIPT C2C12 myoblasts were transfected with siRNA oligonucleotides (siFHL3) or a negative control (NC) and were then differentiated at D0, D2, D4 and D6 in DMEM with 2% horse serum. (A)

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Quantitative RT-PCR results of Fhl3, MyHC isoforms and MyoG mRNA levels after Fhl3

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knockdown at D0, D2, D4 and D6. (B) Western blotting results of Fhl3, MyHC isoforms and MyoG protein levels after Fhl3 knockdown at D0, D2, D4 and D6. (C) Relative protein levels of Fhl3, MyHC 2a, MyHC 2b, MyHC1/slow and MyHC2x after Fhl3 knockdown at D0, D2, D4 and

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D6. (D) Immunofluorescence results of Fhl3 and MyHC isoform protein expression level after

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Fhl3 knockdown. Transfected C2C12 myoblasts were differentiated for 3days, and stained with anti-FHL3 antibodies (green), anti-MyHC 2a antibodies (red), anti-MyHC 2b antibodies (red),

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anti-MyHC1/slow antibodies (red), and DAPI (blue) and were imaged by fluorescence microscopy.

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Bars, 200 µm. (E) The protein expression level of MyHC 2a, MyHC 2b and MyHC 1/slow

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observed in D was determined by ratio of the number of nuclei within MyHC isoforms positive myotubes to the total number of nuclei . Values are shown as means ±SD of three independent

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experiments.

Fig. 3. The co-expression of Fhl3 and MyoD affects the protein expression levels of MyHC 2a, MyHC 2b and MyHC 1/slow. (A) Western blotting results of MyHC 2a, MyHC 2b and MyHC 1/slow after Fhl3 overexpression and MyoD knockdown. C2C12 myoblasts were co-transfected with pcDNA3.1-FHL3 and siRNA oligonucleotides and (indicated at the bottom) and differentiated for 3 days. The cell lysates were subject to western blotting analysis with anti-FHL3, anti-MyoD, anti-MyHC 2a, anti-MyHC 2b and anti-MyHC 1/slow antibodies (indicated at the left). (B) Relative protein expression levels of MyHC 2a, MyHC 2b and MyHC 1/slow after Fhl3 overexpression and MyoD knockdown. (C) Western blotting results of MyHC 1/slow after MyoD 28

ACCEPTED MANUSCRIPT overexpression

and

Fhl3

knockdown.

C2C12

myoblasts

were

co-transfected

with

pcDNA3.1-MyoD and siRNA oligonucleotides and (indicated at the bottom) and differentiated for

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3 days. The cell lysates were subject to western blotting analysis with anti-FHL3, anti-MyoD and

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anti-MyHC1/slow antibodies (indicated at the left). (D) Relative protein expression levels of MyHC1/slow after MyoD overexpression and Fhl3 knockdown. (E) Western blotting results of MyHC 2a and MyHC 2b protein expression levels after Fhl3 and MyoD overexpression. C2C12

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myoblasts were co-transfected with pcDNA3.1-FHL3 and pcDNA3.1-MyoD (indicated at the

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bottom) and were then differentiated for 3 days. Cell lysates were subject to western blot analysis with anti-FHL3, anti-MyoD, anti-MyHC 2a, and anti-MyHC 2b antibodies (indicated to the left).

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(F) Relative protein expression levels of MyHC 2a and MyHC 2b after Fhl3 and MyoD

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overexpression.

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Fig. 4. FHL3 up-regulates MyHC 2a expression primarily through improving CREB transcription activity. (A) Promoter activities of a series of deleted constructs determined by luciferase assay

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after Fhl3 overexpression and differentiation for 2 days. Left panel, schematic representation of the deleted fragments linked with the luciferase gene in the pGL3 vector. The nucleotides are numbered relative to the translation start site that was assigned as + 1. Schematic diagram of MyHC 2a promoter deletion containing the CRE and E-box are displayed. Right panel, the relative activities of a series of deleted constructs of the pGL3-MyHC 2a construct determined by luciferase assay. (B) Western blotting results of MyHC 2a protein expression levels after Creb overexpression. C2C12 myoblasts were transfected with pcDNA3.1-CREB and empty vector and were then differentiated at D2 and D4; Relative protein levels of MyHC 2a was also represented. (C) Western blotting results of MyHC 2a protein expression levels after Creb knockdown. C2C12 29

ACCEPTED MANUSCRIPT myoblasts were transfected with Creb siRNA (siCREB) and NC and were then differentiated at D2 and D4; Relative protein levels of MyHC 2a was also represented. (D) Western blotting results of

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MyHC 2a protein levels after Fhl3 and CREB co-expression. C2C12 myoblasts were

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co-transfected with pcDNA3.1-FHL3 and pcDNA3.1-CREB (indicated at the bottom) and were then differentiated for 3 days. Cell lysates were subject to western blotting with anti-FHL3, anti-CREB, anti-pCREB anti-MyHC 2a, and anti-MyHC 1/slow antibodies (indicated at the left).

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(E) Relative protein levels of MyHC 2a and MyHC 1/slow after Fhl3 and CREB co-expression. (F)

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Western blotting results of MyHC 2a and MyHC 1/slow protein expression levels after co-transfection of pcDNA3.1-FHL3 and siCREB and differentiation for 3 days in C2C12

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myoblasts. (G) Relative protein expression levels of MyHC 2a and MyHC 1/slow after

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co-transfection of pcDNA3.1-FHL3 and siCREB. (H) Promoter activities of MyHC 2a (-909/+206)

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or MyHC 2a with a mutation at CRE (MutCRE -909/+206) was determined by a luciferase assay after overexpression of Fhl3 or Creb and differentiation for 2 days. The data are presented as mean

AC

± S.D. (n = 6).

Fig. 5. FHL3 promotes MyHC 2a expression through its interaction with pCREB and p300 in C2C12 myoblasts. (A) The effects of FHL3 on MyHC 2a expression protein levels through pCREB. After Fhl3 overexpression, C2C12 myoblasts were differentiated for 3 days and then treated or not with 20 µM H-89 for 1 h before harvest, and the control cells were treated with 0.1% DMSO (indicated at the bottom). Protein expression levels of pCREB and MyHC 2a were detected by western blotting; Relative protein expression levels of pCREB and MyHC 2a were also represented (B) The immunoprecipitation results showing the interaction among FHL3, p300 and pCREB in vivo. C2C12 myoblasts were differentiated for 3 days, harvested and pull-downed 30

ACCEPTED MANUSCRIPT by antibodies. Lane 1: the analysis of total cell lysates before immunoprecipitation to verify expression of CREB and pCREB. Lane 2: immunoprecipitation results with anti IgG monoclonal

IP

T

antibody as a negative control; Lane 3: immunoprecipitation results with anti-FHL3 monoclonal

SC R

antibody. (C) The immunoprecipitation results showing the interaction of FHL3 with pCREB and MyoD. Lane 3: immunoprecipitation results with anti-pCREB monoclonal antibody; Lane 4: immunoprecipitation results with anti-MyoD monoclonal antibody. (D) EMSA results showing the

NU

binding of CREB and pCREB to the MyHC2a promoter in C2C12 myoblasts differentiated for 3

MA

days. The probes were incubated with nuclear extract in the absence or presence of 1-fold excess of various competitor probes (mutant or non-labeled probe) or antibodies (anti-CREB or pCREB).

D

The specific DNA-protein complex and the super-shift complex (DNA-protein-antibody complex)

TE

bands are indicated by arrows. The sequences of various probes are shown under the panel. *,

CE P

non-specific binding. (E) ChIP assay results showing the interaction of CREB and pCREB with the MyHC 2a promoter in vivo in C2C12 myoblasts differentiated for 3 days. Immunoprecipitated

AC

DNA was amplified by PCR for 35 cycles. Total chromatin was used as the input, and normal mouse IgG was used as the negative control (the same as below). (F) ChIP assay results showing the effect of the Fhl3 knockdown on the binding capacity of pCREB to the MyHC 2a promoter. Immunoprecipitated DNA was amplified by PCR for 30 cycles. Fig. 6. Model for the regulation of MyHC 2a and MyHC 1/show expression by FHL3 in the cell nucleus. (A) FHL3 forms a complex with CREB and p300 to positively regulate MyHC 2a expression. MyoD also up-regulates MyHC 2a expression. (B) FHL3 physically interacts with MyoD to inhibit MyHC 1/slow expression.

31

ACCEPTED MANUSCRIPT Table 1 Primers used in the expression vector construction Gene

S:CGGGATCCGTCATGAGCGAGGCATTTGA A: GCTCTAGATCAGGGGCCTGCTTGGCTG S:GGGGTACCTTTAAAAAGCTCCAAGGTA S:GGGGTACCAGGTGACAAACAGAAACACA S:GGGGTACCAAACATTTTCCCAAATATCA S:GGGGTACCTCAGTGGTTAAAAGTAATTG S:GGGGTACCTACAAGTCAAATCGTGATAA A:GGCTCGAGACCGCTCCTGCTTCTGTTTT S:GGGTACCGTCATGACCATGGAATCTGG A:GCTCGAGTTAATCTGATTTGTGGCA S: CTCCTAATGTTGCTACCCTG A: AAGTACACTTATGAAGTAGC S:TCCTCATGTTGTGGGTACCTCATGACAGAA A:TTCTGTCATGAGGTACCCACAACATGAGGA

870 bp

T

Fhl3

2249 bp 1303 bp 1115 bp 515 bp 315 bp

CREB

MA

MyHC 2a-ChIP

NU

SC R

IP

pMyHC 2a-delection

Mut-CRE

Product length

Primer sequence（5’-3’）

1026 bp 89 bp

AC

CE P

TE

D

S, sense; A, antisense; six red base pairs, enzyme digested sites; two red base pairs, mutant sites; underlined, translation start site.

32

ACCEPTED MANUSCRIPT Table 2 Primers and TaqMan probes used in the Quantitative real-time PCR Gene.

Ann. temp(AT)

Primer sequence（5’-3’）

Product length

S: CCATGAGCGAGGCATTTGAC Fhl3

A: CAGTAGGGGCCACTGTCTGTC

60°C

P: CGTTTGAGAATCCAAGGCTCA S: CAGCTGCACCTTCTCGTTTG

SC R

A: CTTCTCAGACTTCCGCAGGAA

IP

S: GCCTGGGCTTACCTCTCTATCAC MyHC1/ slow

T

P:AATGCAACGAGTCCTTGTACGGCCGCA

85 bp

60°C

116 bp

60°C

81 bp

A: CCCGAAAACGGCCATCT

MyHC 2a

P: TGAGTTCAGCAGTCATGAG S: ATGAGCTCCGACGCCGAG A: TCTGTTAGCATGAACTGGTAGGCG S: GGACCCACGGTCGAAGTTG

62°C

506 bp

MyHC 2x

A: GGCTGCGGGCTATTGGTT

60°C

70 bp

60°C

80 bp

60°C

72 bp

59°C

184 bp

60°C

241 bp

MA

NU

MyHC 2a

P:CTAAAGGCAGGCTCTCTCACTGGGCTG S: CAATCAGGAACCTTCGGAACAC MyHC 2b

A: GTCCTGGCCTCTGAGAGCAT

D

P:TGCTGAAGGACACACAGCTGCACCT

TE

β-actin

S: CGTGAAAAGATGACCCAGATCA A: CACAGCCTGGATGGCTACGT P: TTGAGACCTTCAACACCCCAGCCATG S:GCCTCACTGTCCACCTTCCA A:AGCCATGCCAATGTTGTCTCTT

CE P

β-actin

S: CCATCCAGTACATTGAGCGCCTACA A: ACGATGGACGTAAGGGAGTGCAGAT

MyoG

AC

S, sense; A, antisense; P, TaqMan probe. All probes were 5’-labelled with FAM and 3’-labelled with TAMRA.

33

ACCEPTED MANUSCRIPT Table 3 The EMSA reaction system Cold competitor

Mutant cold competitor

Super-shift (CREB antibody)

Up to 10 µl 2 µl

Up to 10 µl 2 µl

Up to 10 µl 2 µl

Up to 10 µl

Up to 10 µl

0 µl 1 µl 0 µl 0 µl

2 µl 1 µl 0 µl 0 µl

2 µl 1 µl 1 µl 0 µl

2 µl 1 µl 0 µl 1 µl

2 µl 1 µl 0 µl 0 µl

2 µl 1 µl 0 µl 0 µl

0 µl 10 µl

0 µl 10 µl

0 µl 10 µl

0 µl 10 µl

10 µg 10 µl

10 µg 10 µl

IP 2 µl

MA

NU

SC R

2 µl

T

Sample reaction

AC

CE P

TE

D

Nuclease Free water 5×binding buffer Extract Probe Competitor Mutant Competitor Antibody Total

NC reaction

34

Super-shift (pCREB antibody) Up to 10 µl 2 µl

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 1

35

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 2 36

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 3

37

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 4 38

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 5 39

AC

Fig. 6

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

40

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Hightlights 1. FHL3 regulates the expression of MyHC isform genes. 2. FHL3 inhibits the expression of MyHC 1/slow by MyoD. 3. CREB enhances MyHC 2a prompter activity and increases the expression of MyHC 2a. 4. FHL3 promotes the expression of MyHC 2a by interaction with pCREB

41