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NCAPG Dynamically Coordinates the Myogenesis of Fetal Bovine Tissue by Adjusting Chromatin Accessibility

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NCAPG Dynamically Coordinates the Myogenesis of Fetal Bovine Tissue by Adjusting Chromatin Accessibility International Journal of Molecular Sciences Article NCAPG Dynamically Coordinates the Myogenesis of Fetal Bovine Tissue by Adjusting Chromatin Accessibility 1,2, 1, 3 4 1 1 1 Xin Hu y, Yishen Xing y, Xing Fu , Qiyuan Yang , Ling Ren , Yahui Wang , Qian Li , Junya Li 1,* and Lupei Zhang 1,* 1 Key Laboratory of Animal Genetics Breeding and Reproduction, Ministry of Agriculture and Rural Affairs; Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China; [email protected] (X.H.); [email protected] (Y.X.); [email protected] (L.R.); [email protected] (Y.W.); [email protected] (Q.L.) 2 Molecular and Cellular Biology, Gembloux Agro-Bio Tech, University of Liège, 5030 Gembloux, Belgium 3 School of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA; [email protected] 4 Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA; [email protected] * Correspondence: [email protected] (L.Z.); [email protected] (J.L.) These authors contributed equally to this work. y Received: 29 January 2020; Accepted: 11 February 2020; Published: 13 February 2020 Abstract: NCAPG is a subunit of condensin I that plays a crucial role in chromatin condensation during mitosis. NCAPG has been demonstrated to be associated with farm animal growth traits. However, its role in regulating myoblast differentiation is still unclear. We used myoblasts derived from fetal bovine tissue as an in vitro model and found that NCAPG was expressed during myogenic differentiation in the cytoplasm and nucleus. Silencing NCAPG prolonged the mitosis and impaired the differentiation due to increased myoblast apoptosis. After 1.5 days of differentiation, silencing NCAPG enhanced muscle-specific gene expression. An assay for transposase-accessible chromatin- high throughput sequencing (ATAC-seq) revealed that silencing NCAPG altered chromatin accessibility to activating protein 1 (AP-1) and its subunits. Knocking down the expression of the AP-1 subunits fos-related antigen 2 (FOSL2) or junB proto-oncogene (JUNB) enhanced part of the muscle-specific gene expression. In conclusion, our data provide valuable evidence about NCAPG’s function in myogenesis, as well as its potential role in gene expression. Keywords: NCAPG; bovine myoblasts; myogenesis; chromatin accessibility; AP-1 1. Introduction The growth rate is the most important trait in farm animal production. Genome variations regulating animal growth and development have been widely studied in farm animals. NCAPG is a gene well-documented to be associated with body size, average daily gain, feed intake efficiency, fetal growth, carcass trait, and muscle development in beef cattle [1–7]; body weight in sheep [8]; and withers height in horses [9,10]. Moreover, in clinical practice, NCAPG is a crucial gene that is correlated with height [11]. NCAPG is a subunit of the condensin I complex, which is essential for the structural maintenance of chromosomes [12]. The core subunits of condensin are SMC2 and SMC4, both of which are the members of structural maintenance of chromosome (SMC) family of chromosomal ATPases [13]. Condensin I also has unique non-SMC subunits: CAP-G, CAP-H, and CAP-D2 [12]. Previous studies Int. J. Mol. Sci. 2020, 21, 1248; doi:10.3390/ijms21041248 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1248 2 of 15 have revealed that both condensin members play essential but distinct roles in mitosis. Condensin I is sequestered in the cytoplasm during the interphase and gains access to chromosomes only after the nuclear envelope break down (NEBD) in the prometaphase. Condensin II functions in the nucleus from the interphase through the prophase as an early regulator of chromosome condensation. After NEBD, condensinInt. J. I Mol. engages Sci. 2020 in, 21 further, 1248 chromosome compaction to facilitate final mitosis [14–16]. In2 of one 15 study, the abrogation of the CAP-D2 subunit resulted in a significant delay of prometaphase to anaphase the nuclear envelope break down (NEBD) in the prometaphase. Condensin II functions in the nucleus progression compared to the control group [15]. This provided direct evidence for the relationship from the interphase through the prophase as an early regulator of chromosome condensation. After betweenNEBD,NCAPG condensinand growth I engages traits. in further chromosome compaction to facilitate final mitosis [14–16]. In Skeletalone study, muscle the abrogation development of the isCAP-D2 oneof subunit the most resulted important in a significant traits delay in farm of prometaphase animal production to becauseanaphase skeletal progression muscle account compared for aboutto the control half of grou an animal’sp [15]. This body provided weight. direct Muscle evidence development for the is classifiedrelationship into prenatal between and NCAPG postnatal andstages. growth traits. The fetal stage is crucial for skeletal muscle development, as the numberSkeletal of muscle muscle fibers development barely increasesis one of the after most birth important [17–19 ].traits in farm animal production because skeletal muscle account for about half of an animal’s body weight. Muscle development is However,classified the into regulatory prenatal and mechanisms postnatal stages. and biological The fetal functionsstage is crucial of NCAPG for skeletalarestill muscle unknown. In particular,development,NCAPG as ’sthe role number in the of muscle development fibers barely of increases myoblasts after in birth the [17–19]. fetal stage needs exploration. In this study,However, the role the of NCAPGregulatoryin mechanis the myogenicms and dibiologicalfferentiation functions of myoblasts of NCAPG are derived still unknown. from fetal In bovine tissue wasparticular determined, NCAPG using’s role in RNA the development interference of tomyoblasts knock downin the fetalNCAPG stage needsgene exploration. expression. In this Moreover, due to NCAPGstudy, the’s role conserved of NCAPG roles in the in othermyogenic tissues, differentiation our study of myoblasts and its meanings derived from may fetal also bovine be applied tissue was determined using RNA interference to knock down NCAPG gene expression. Moreover, elsewhere in the body. due to NCAPG’s conserved roles in other tissues, our study and its meanings may also be applied 2. Resultselsewhere in the body. 2.1. Myogenic2. Results Diff erentiation of Myoblasts Derived from Fetal Bovine Tissue After2.1. 24Myogenic h of di Differentiationfferentiation, of Myobla somests myotubes Derived from could Fetal Bovine be observed. Tissue After 48 h, a majority of the myoblasts hadAfter fused 24 h of into differentiation, myotubes. some After myotubes an additional could be two observed. days After of di ff48erentiation, h, a majority both of the the size and lengthmyoblasts of the had myotubes fused into increased myotubes. (FigureAfter an 1additionalA). RNA two and days proteins of differentiation, were extracted both the fromsize and the cells on dayslength 0, 2, andof the 4 myotubes to analyze increased the muscle-specific (Figure 1A). RN geneA and expression proteins were profile extracted during from proliferationthe cells on and days 0, 2, and 4 to analyze the muscle-specific gene expression profile during proliferation and differentiation. Myoblast determination protein 1 (MYOD) and myogenic Factor 5 (MYF5) mRNA differentiation. Myoblast determination protein 1 (MYOD) and myogenic Factor 5 (MYF5) mRNA expressionexpression decreased decreased during during proliferation proliferation and and diff differentiation,erentiation, while whileMYOG MYOG expressionexpression increased increased until day 2 ofuntil differentiation, day 2 of differentiation, after which after it which decreased it decreased (Figure (Figure1B). 1B). Figure 1.FigureMyogenic 1. Myogenic differentiation differentiation of of myoblasts myoblasts derivedderived from from fetal fetal bovine bovine tissue. tissue. (A) Microscopic (A) Microscopic images ofimages bovine of bovine myoblasts myoblasts on days on days 0, 2,0, and2, and 4 4 of of didifferentiation.fferentiation. Scale Scale bars bars = 100= µm.100 (µBm.) Transcript (B) Transcript levels oflevelsMYF5, ofMYOD, MYF5, MYOD, MYOG, MYOG, MYH1, MYH1, MYH2, MYH2, MYH3 MYH3and MYH4 and MYH4during during proliferation proliferation (P) and(P) and myogenic differentiationmyogenic (D0, differentiation D2, and D4). (D0, The D2, resultsand D4). are The represented results are represented as the mean as thethe mean SEM ± the from SEM at from least three ± independentat least experiments. three independent (C) experiments. Western blot (C evaluating) Western blot the evaluating protein the levels protein of MYODlevels of andMYOD MHCs and in the MHCs in the cultured cells, as described in (B). cultured cells, as described in (B). Int. J. Mol. Sci. 2020, 21, 1248 3 of 15 As for myosin heavy chain (MYH) isoforms in skeletal muscle, the expression of MYH1, MYH2, MYH3, and MYH4 was relatively low during proliferation and early differentiation, but it increased dramaticallyInt. J. Mol. during Sci. 2020, late 21, 1248 di fferentiation (Figure1B). In particular, MYH2 expression increased3 of 15 nearly 200-fold compared to basal level on day 4. Consistently, western bolt analyses showed that MYH expression increasedAs for myosin gradually heavy chain during (MYH)the isoforms differentiation in skeletal muscle, of myoblasts, the expression while of MYH1, MYOD MYH2, expression decreasedMYH3, during and MYH4 differentiation
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