Identification of Myomaker in Yellowfin Seabream (Acanthopagrus latus) (Hottuyn, 1782) and its Transcriptional Regulation by Two MyoDs

Kecheng Zhu1,2,3, Peiying He1, Baosuo Liu1,2,3, Huayang Guo1,2,3, Nan Zhang1,2,3, Liang Guo1,2,3, Shigui Jiang1,2,3 and Dianchang Zhang1,2,3,* 1Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs; South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 510300, Guangzhou, Guangdong Province, PR China 2Guangdong Provincial Engineer Technology Research Center of Marine Biological Seed Industry, Guangzhou, Guangdong Province, PR China 3Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou, Guangdong Province, PR China Article Information Received 16 January 2020 Revised 14 February 2020 Accepted 04 March 2020 Available online 14 October 2020


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consisting of 221 aa that localizes to intracellular vesicles and the plasma membrane in skeletal myocytes (Millay et al., 2013). The structural features of myomaker span the bilayer seven times with an extracellular N-terminal region and cytosolic C-terminal tail with unknown conserved functional domains (Millay et al., 2016). Myogenin (MyoG) and MyoD, members of myogenic regulatory factors (MRFs), are pivotal transcription factors (TFs) in myogenesis and can control the transcription of most muscle-related genes (Braun et al., 1989;Edmondson and Olson, 1990;Berkes and Tapscott, 2005;Braun and Gautel, 2011). The two MRFs play a critical role in the regulation of myoblast differentiation. The transcription of myomaker is mediated through the activity of musclespecific TFs MyoG and MyoD (Luo et al. 2015;Quinn et al., 2017;Ganassi et al., 2018). Additionally, during the acute and chronic muscle regeneration process, MyoD can induce myomaker expression in muscle satellite cells and accelerate myoblast fusion. Then, the expression of myomaker decreased rapidly after fusion (Millay et al., 2014;Landemaine et al., 2014;Demonbreun et al., 2015;Zhang and Roy, 2017). In the MyoD signalling pathway, a downstream gene, myomixer, is activated by MyoD. Nonmyoblasts can fuse into multicellular cells when myomaker and myomixer are overexpressed (Bi et al., 2017;Quinn et al., 2017;. In avians, the essential role of myomaker is in myoblast fusion and shows that MyoD can regulate myomaker expression (Luo et al., 2015). Two highly conserved e-box mutations in the 5'-regulatory region sequence of the myomaker gene in mice and chickens have been verified, suggesting that the importance of the e-box is in regulating the transcription and expression of myomaker (Millay et al., 2014;Luo et al., 2015). Moreover, when two MyoD binding sites of the myomaker promoter are deleted in chickens, its promoter activity is significantly reduced, indicating that MyoDs have a positive regulatory effect on myomaker (Luo et al., 2015). The C-terminal region of myomaker is essential for the function of three cysteine residues, which are speculated to be palmitoylated (Millay et al., 2016). Myomaker displays N-terminal glycine predicted to sustain myristoylation (Bologna et al., 2004). However, the cellular mechanism of myomaker and the regulatory mechanism of its expression during myogenesis have not been determined in marine fish.
Yellowfin seabream (Acanthopagrus latus) (Hottuyn, 1782), Sparidae, and Perciformes are considered significant aquaculture fish in southern China due to their economic value. Nevertheless, the muscle growth rate is too low in A. latus. Consequently, this fish is known as a specific model for exploring regulatory mechanisms in muscle development in marine fish. In the present study, to investigate the underlying function of Almyomaker and transcriptional regulation of two AlMyoDs, this study focused on illuminating the consequence of MyoD in the activation of Almyomaker expression. A musclespecific membrane protein gene from A. latus, myomaker (Almyomaker), was confirmed. Promoter activity assays via the mutation of potential MyoD binding sites are executed to determine key elements in the Almyomaker candidate sequence. The present study may contribute to further exploration of myomaker function in marine fish and help to elucidate the regulatory mechanism for myoblast fusion.

Animals and tissues collection
Fish (body weight: 289 ± 18.5 g) were collected from Yangjiang Marine Fish Farm in Guangdong Province, China. For the present study, three healthy fish were used, and 13 tissues (heart, male gonad, eye, skin, brain, fin, spleen, small intestine, gill, white muscle, kidney, liver, and stomach) were isolated to analyze the myomaker expression level in different tissues. Then, the flash was frozen in liquid nitrogen and stored at -80°C until use.

Gene cloning and bioinformatics of myomaker
Total RNA (1 μg) was extracted from A. latus white muscle by TRIzol Reagent (Takara, Japan). To synthesize cDNA, the Prime ScriptTM RT reagent Kit (Takara) was used according to the manufacturer's instructions. Putative myomaker, MyoD1 and MyoD2 sequences were acquired according to genomic data of A. latus (Sequence Read Archive under BioProject PRJNA566024). To verify the accuracy of the derived sequence, gene-specific primers were designed (Table I). The PCR system (volume) and procedure employed were described previously (Zhu et al., 2014). The amplified products, linked into the pEASY-T1 vector (TransGen Biotech, China), were purified by a DNA Purification Kit (Tiangen, China) and then sequenced (Invitrogen, China). Confirmed recombinants were transformed into competent Trans1-T1 cells (TransGen Biotech, China). A BLAST search on the supposed myomaker open reading frame (ORF) sequence further confirmed the accuracy and validity of the analysis.
The derived aa sequence from the cloned Almyomaker ORF was aligned with other myomaker orthologue proteins from the NCBI and Ensembl databases (Fig. 2). Multiple sequence alignment was performed by ClustalX version 2.0 (Larkin et al., 2007) with default parameters. Phylogenetic analyses for all myomaker aa sequences were achieved using MEGA 6.0 (Tamura et al., 2013) with maximum likelihood (ML) methods (LG + G

Real-time quantitative PCR analysis
Specific primers for real-time quantitative PCR (qRT-PCR) were designed by Primer Premier 5.0 (Premier Biosoft, USA) based on cloned nucleotide sequences (Table I) (Zhu et al., 2020). The myomaker, MyoDs and elongation factor 1-alpha (EF-1α) were tested and used as a target and reference gene, respectively. qRT-PCR was implemented in a quantitative thermal cycle (Mastercycler® eprealplex; Eppendorf, Germany). The PCR system (volume) was described above. The qRT-PCR program was conducted as follows: 95°C for 2 min followed by 40 cycles of 95°C for 10 s, 56°C for 10 s, and 72°C for 20 s. Gene expression was determined using the 2 −ΔΔCt method (Livak et al., 2001).

Plasmid construction, cell culture and dual-luciferase reporter assays
Briefly, to clone the target fragment of Almyomaker, genomic DNA was isolated from the white muscle of A. latus, as previously described in other marine fish (Sun et al., 2013). The ORF sequence upstream of the myomaker gene was obtained from genomic data of A. latus. Moreover, to obtain the recombinants of MyoD1 and MyoD2, the integrated sequences of MyoD1 and MyoD2 ORFs were inserted into the pCDNA3.1-Flag vector (Invitrogen, USA) using specific primers (Table I) (Zhu et al., 2020). To confirm the effect of AlMyoD1 and AlMyoD2 on Almyomaker expression, five different truncated regions from Almyomaker were amplified by peculiar primers with KpnI and XhoI restriction sites ( Table I). The forward primers (Myomaker-pF1, Myomaker-pF2, Myomaker-pF3, Myomaker-pF4, and Myomaker-pF5) were designed with a 5′ KpnI site, and the common reverse primer (Myomaker-pR) was designed with a 3′ XhoI site (Table I). These primers were used to acquire the target region (Myomaker-pF1, 2,090 bp) and four truncated fragments ((i) Myomaker-pF2, 1,483 bp; (ii) Myomaker-pF3, 1,211 bp; (iii) Myomaker-pF4, 436 bp and (iv) Myomaker-pF5, 302 bp) (Fig. 5). PrimeSTAR Master Mix (Takara, Japan) was used to amplify the five truncated mutants. The PCR program consisted of 95°C for 4 min, followed by 30 cycles of 95°C for 40 s, 56°C for 40 s, and 72°C for 1 min. Subsequently, the PCR products were purified by the general DNA Purification Kit (Tiangen, China). A pGL3basic (Promega, USA) vector and purified PCR products were digested with KpnI and XhoI and linked by T4 DNA ligase (Takara, Japan) overnight at 16°C. Recombined plasmids were extracted using the EndoFree Plasmid Giga Kit (Tiangen, China), and recombinants were validated by sequencing, as described above. Furthermore, to investigate the possible function of MyoD1 or MyoD2 binding sites on the core myomaker promoter and 5'UTR sequences, three truncated mutations of recombinant plasmids were established. The TF binding site prediction (TFBS)-JASPAR database (http://jaspar. genereg.net/), TRANSFAC ® , and MatInspector ® were used to search for potential binding sites in the myomaker sequence with MyoD1 and MyoD2, respectively. Then, according to the manufacturer's protocol, site-directed mutagenesis was conducted with a QuickChange II Site-Directed Mutagenesis Kit (Vazyme, USA) from the deletion mutant pGL3-basic-myomaker-p5, which was regarded as the wild-type plasmid. The prediction of three binding sites (M1, M2, and M3) was directly deleted, and the schematic diagram and sequences of homologous

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TF binding sites are shown in Figure 6A and Table II, respectively. The role of TF binding site mutations on the promoter activity of Almyomaker was explained by a dual luciferase assay, as described below (Genecreate, China). Human embryonic kidney (HEK293T) cells were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS) (Invitrogen, USA) accompanied by 100 μg/ mL streptomycin and 100 U/mL penicillin (Thermo Fisher Scientific, USA) at 37°C in a humidified incubator under 5% CO 2 . The procedure of transfection and dual luciferase reporter assays were described by Li et al. (2017). Relative luciferase activities (the ratio of firefly and ranilla luciferase activities) were measured and calculated using the VICTOR TM X2 Multi-label Plate Reader (PerkinElmer, Inc., Waltham, MA, USA).

Statistical analysis
All trials were executed in triplicate in the present study. All values are shown with three replicates ± SE. Significant differences were calculated by one way ANOVA tests. P values <0.05 were considered to be significant.

Sequence characterization of Almyomaker
The genomic sequence of Almyomaker is 3,383 bp, including 6 exons and 5 introns ( Supplementary Fig. S1). The full-length ORF of Almyomaker is 855 bp (Fig. 1A), encoding peptides of 285 amino acids with a predicted molecular weight of 32.14 kDa and a theoretical isoelectric point of 9.44 (Accession No. MN266854). A conserved domain (DUF3522 superfamily starting from the 3 rd amino acid and ending at the 185th amino acid) was detected using BLAST (Huang et al., 2019). Hydrophobicity profiles show that the Almyomaker protein includes more hydrophobic amino acids than hydrophilic amino acids (Fig. 1B). The results of TMHMM indicate that there are 6 obvious transmembrane domains in the Almyomaker protein (Fig. 1C), which is consistent with the analysis of goose Anser cygnoides (He et al., 2017). The myomaker amino acids in A. latus reveal sequence homology with those in the other metazoan species ( Fig. 2A) and are most highly homologous with Xiphophorus maculatus, Oryzias latipes, Poecilia formosa, and Tetraodon nigroviridis. Moreover, a BLAST analysis indicated that the Almyomaker protein sequence shares high sequence identity with myomaker sequences from other teleosts containing tilapia (O. niloticus, 92 %), platyfish (X. maculatus, 87.1%), medaka (O. latipes, 86.7%), and molly (P. formosa, 86.3%) and low sequence identity with humans (Homo sapiens, 56.5%) and mice (Mus musculus, 54.7%) (Fig. 2B).

Almyomaker structural and phylogenetic analyses
The genomic structural features and phylogenetic relationship of myomaker were determined and constructed in metazoans (Fig. 3). The lengths and distributions of the exons and introns of metazoan myomaker genes are displayed in Supplementary Table I Furthermore, the sizes of the exon sequences reveal that there is nearly no diversity among species. Additionally, the phylogenetic tree was constructed with the full-length myomaker aa of various fish, mammalia, aves, and amphibia species.
Almyomaker is grouped together with other Perciformes, such as O. niloticus. The phylogenetic tree clearly shows that fish evolve into one branch followed by Amphibians, Aves and then Mammals (Huang et al., 2019).

Tissue expression of Almyomaker
The mRNA levels of Almyomaker are determined by qRT-PCR in various kinds of tissues. qRT-PCR shows that Almyomaker is broadly distributed in various tissues, and the abundance of mRNA varies among tissues. The highest Almyomaker transcriptions were detected in the white muscle followed by the brain and spleen, whereas the lowest Almyomaker expression levels were found in the liver, skin and stomach (Fig. 4).   in the Almyomaker sequence, a full-length target fragment and 4 truncated mutants were inserted with a promoterless luciferase reporter vector, pGL3-basic (Promega, USA). The result of promoter activity analysis shows that the construct of myomaker-p5 (-34 bp to +267 bp) is higher than that of other constructs with cotransfection of MyoD1 or MyoD2, suggesting that the core binding region is located at -34 bp to +267 bp, which may include several MyoD1 or MyoD2 binding sites (Fig. 5). Therefore, the sequence of construct myomaker-p5 (-34 bp to +267 bp) was used for further functional analysis.
To understand the MyoD1 and MyoD2 binding sites in Almyomaker, the presumptive binding sites are mutated ( Fig. 6A; Table II). The effects on promoter activity were investigated in 293T cells, together transfected with each mutant and MyoD1 or MyoD2. The results reveal that mutation of the M1 binding site (GGACAGCTGGGT, -14 bp to -3 bp) generates a marked reduction in promoter activity (Fig. 6B), showing that M1 is the MyoD1 binding site on the Almyomaker promoter. Moreover, mutation of the M2 binding site (GGACATCTACTT, +92 bp to +103 bp) resulted in a significant reduction in promoter activity (Fig. 6C), suggesting that M2 is the potential MyoD2 binding site in the Almyomaker gene 5'-UTR region. Notably, one predicted binding site (M3) does not activate luciferase activity with MyoD1 or MyoD2, suggesting that this site is not required for triggering Almyomaker expression with two MyoDs.

DISCUSSION
The present study investigated the potential mechanisms of the transcriptional regulation of myomaker by MyoD1 and MyoD2 in A. latus. The sequence and functional characterization, tissue expression and regulation relationship between two AlMyoDs and Almyomaker were determined. The Almyomaker aa sequence was 54.7%-92.0% identical to myomaker proteins from other teleosts.
In general, the expression of several genes depends on RNA polymerases and TFs binding to specific sequences on the promoters of target genes in eukaryotic organisms (O'Malley, 1977;Xie et al., 2018;Zhu et al., 2019). Furthermore, the integrity and activity of a promoter could regulate gene transcription. The TF MyoDs monitored skeletal muscle growth in mammals and altered the transcription of muscle-related genes in teleosts and avians (Millay et al., 2014;Luo et al., 2015). Notably, evidence has demonstrated that overexpression of MyoD could increase downstream gene myomaker expression in zebrafish (Bi et al., 2017;Quinn et al., 2017;. It was unclear whether MyoD could upregulate Almyomaker in marine fishes. In the present study, dualluciferase reporter assays were conducted to realize regulatory mechanisms whereby MyoD1 and MyoD2 were believed to accommodate Almyomaker expression. The results of truncated mutants indicated that Almyomaker reporter activity was activated by the overexpression of two AlMyoDs. The core binding region in the Almyomaker promoter was -34 to +267 bp (Fig. 6). This evidence was the first to show that the expression of Almyomaker could be upregulated by both AlMyoD1 and AlMyoD2 in marine fishes.
MyoDs possess a highly conserved bHLH domain, which is necessary for heterodimerization with characteristic DNA binding to the e-box motif (consensus CANNTG) detected in the regulatory regions of their target genes (Murre et al., 1989;Lassar et al., 1989Lassar et al., , 1991Davis and Weintraub, 1992). Consequently, to investigate the two MyoD binding sites on the Almyomaker promoter, three mutant vectors were constructed. Mutation of the MyoD1 M1 binding site (GGACAGCTGGGT) and MyoD2 M2 binding site (GGACATCTACTT) resulted in observably decreased promoter activity (Fig. 6B, C), suggesting that the MyoD1 binding M1 site and MyoD2 binding M2 site were essential for Almyomaker promoter activity, respectively. The sequences of the above two binding sites were representative e-box motifs (Davis and Weintraub, 1992). In brief, MyoD1 and MyoD2 could mediate myomaker promoter expression by combining e-box motifs (M1 and M2 binding sites) in fish, respectively, which was consistent with the findings in M. musculus and G. gallus (Millay et al., 2014;Luo et al., 2015). Moreover, the MyoD2 M2 binding site was located in the 5'UTR, which was analogous to previous studies (Wu et al., 2018;Khan et al., 2019).
Phylogenetic analysis of myomakers in tetrapods and teleosts showed that a protein of uniform length (221 aa) was observed in ancestral nonteleost fish, and teleost myomaker sequences were classified into three groups according to myomaker protein length (Landemaine et al., 2019). In the present study, phylogenetic analysis showed that a typical phylogeny revealing the amino acid sequence of Almyomaker was closely matched to myomakers of O. niloticus, X. maculatus, O. latipes, P. formosa, and T. nigroviridis, with analogous lengths. This result was similar to that reported in Landemaine et al. (2019). A genome structure analysis showed that all myomakers contained 6 exons and 5 introns in metazoans, except for D. rerio, T. nigroviridis, P. sinensis, L. chalumnae, G. gallus, H. sapiens and M. musculus myomaker, which only possessed 5 exons and 4 introns, suggesting that the last exon might be lost during evolution.
Previous studies have focused on the role of myomaker O n l i n e

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Identification of Myomaker in Yellowfin Seabream during embryonic development and have determined that myomaker was indispensable for the facilitation of myoblast fusion in such species as D. rerio, G. gallus, and M. musculus (Millay et al., 2013;Landemaine et al., 2014;Luo et al., 2015;He et al., 2017;Zhang and Roy, 2017). The mRNA expression patterns of the myomaker gene during 8 different postnatal developmental stages in the Japanese flounder (Paralichthys olivaceus) showed that the expression of myomaker at 180 dph was higher than that at other periods (Huang et al., 2019). Moreover, myomaker is expressed only in skeletal muscle in M. musculus, G. gallus and Oncorhynchus mykiss (Millay et al., 2013;Landemaine et al., 2019). In the present study, the tissue-specific expression pattern revealed that the highest Almyomaker mRNA expression was detected in white muscle, showing that Almyomaker played an important role in muscle development. Almyomaker is also broadly expressed in other tissues; however, in mammals, chicks and fish, Myomaker is nearly undetectable in other organs (Millay et al., 2013;Landemaine et al., 2019). This discrepancy may be attributable to species diversity.

CONCLUSION
In conclusion, the full-length Almyomaker genome sequence was cloned. The tissue expression profile indicated that the mRNA level of Almyomaker was highest in the heart and gonad among the detected tissues. The luciferase activity analysis showed that the region from -34 bp to +267 bp includes the core binding region. Mutation analyses indicated that the activity of the Almyomaker promoter significantly decreased after the targeted mutation of the M1 and M2 binding sites with MyoD1 and MyoD2, respectively. This study mainly focused on the transcription of Almyomaker by MyoDs in heterologous cells. In the future, more attention should be paid to the mRNA or protein levels of Almyomaker after overexpression of MyoDs in endogenous cells.