- Email: [email protected]
Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells
Accepted Manuscript Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells Yue Zhu, Hui-Li Tong, Shu-Feng Li, Yun-Qin Yan PII:
S0006-291X(17)30257-7
DOI:
10.1016/j.bbrc.2017.01.182
Reference:
YBBRC 37244
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 30 January 2017 Accepted Date: 31 January 2017
Please cite this article as: Y. Zhu, H.-L. Tong, S.-F. Li, Y.-Q. Yan, Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.182. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells Zhu Yue, Tong Hui-Li, Li Shu-Feng, Yan Yun-Qin *
Heilongjiang 150030, China
RI PT
The Laboratory of Cell and Development, Northeast Agricultural University, Harbin,
*Corresponding author: Yun Qin Yan, E-mail: [email protected], Laboratory of
M AN U
Fang Strict, Harbin, Heilongjiang 150030, China.
SC
Cell and Development, Northeast Agricultural University, Mucai Street No. 59, Xiang
Abstract: Bovine muscle-derived satellite cells (MDSCs) are important for animal growth. In this study, the effect of transcription elongation factor A3 (TCEA3) on MDSC
differentiation
was
investigated.
Western
blotting,
TE D
bovine
immunofluorescence assays, and cytoplasmic and nuclear protein isolation and purification techniques were used to determine the expression pattern and protein
EP
localization of TCEA3 in bovine MDSCs during in vitro differentiation. TCEA3
AC C
expression was upregulated using the CRISPR/Cas9 technique to study its effects on MDSC differentiation in vitro. TCEA3 expression gradually increased during the in vitro differentiation of bovine MDSCs and peaked on the 5th day of differentiation. TCEA3 was mainly localized in the cytoplasm of bovine MDSCs, and its expression was not detected in the nucleus. The level of TCEA3 was relatively higher in myotubes at a higher degree of differentiation than during early differentiation. After transfection with a TCEA3-activating plasmid vector (TCEA3 overexpression) for 24 1
ACCEPTED MANUSCRIPT hours, the myotube fusion rate, number of myotubes, and expression levels of the muscle differentiation-related loci myogenin (MYOG) and myosin heavy chain 3 (MYH3) increased significantly during the in vitro differentiation of bovine MDSCs.
RI PT
After transfection with a TCEA3-inhibiting plasmid vector for 24 hours, the myotube fusion rate, number of myotubes, and expression levels of MYOG and MYH3 decreased significantly. Our results indicated, for the first time, that TCEA3 promotes
SC
the differentiation of bovine MDSCs and have implications for meat production and
M AN U
animal rearing.
Keywords: TCEA3; transcription elongation factor; muscle-derived satellite cell; differentiation
TE D
1. Introduction
Transcription factor IIS (TFIIS or TCEA) exhibits a wide taxonomic distribution [1]. It not only directly binds to RNA polymerase II, allowing it to read through
EP
various transcription arrest sites, but it also activates the transcriptional activity of
AC C
RNA polymerase II to increase the transcription rate [1-2]. In 1991, Yoo et al. [3] studied the internal structure of the TCEA protein and found that it has three domains, i.e., the Ser/Thr phosphorylation domain, RNA pol II domain, and zinc finger (Znf) domain. The amino acid sequences of the RNA pol II and Znf domains are highly conserved and the precise spacing between the domains plays an important role in protein function [3]. It has three distinct isoforms, which are conserved across frogs, mice, and humans [4]. TCEA1 is ubiquitously expressed [5]. It is highly conserved 2
ACCEPTED MANUSCRIPT among eukaryotes and is essential for definitive hematopoiesis in mice [6]. TCEA2 is a testis-specific isoform. The unique structure of the TCEA2 gene determines the chromosomal localization in testicular tissue [7]. The overexpression of TCEA3 in
RI PT
ovarian cancer cells could induce apoptosis in ovarian cancer cells [5]. Xu et al. [9] studied the early development of myocardial structure in humans and found that TCEA3 expression was elevated, indicating that TCEA3 is related to
SC
myocardial development. However, the mechanism by which TCEA3 promotes cell
M AN U
differentiation is not known. In our previous high-throughput sequencing analysis, we found that TCEA3 expression increases significantly during the differentiation of bovine MDSCs [10]. Therefore, in this study, we examined the expression pattern and localization of TCEA3 in bovine MDSCs at various stages of differentiation. We also
TE D
utilized the CRISPR/Cas9 technique to construct activation and inhibition plasmid vectors [11,12,13] to study the effect of TCEA3 on the differentiation of bovine
development.
EP
MDSCs. The results of this study provide a theoretical basis for improving muscle
AC C
2. Materials and Methods
2.1 Experimental materials Experimental material was collected from the skeletal muscle tissue of newborn
calves after obtaining approval from the Animal Welfare Committee of Northeast Agricultural University, Heilongjiang Province, China. Skeletal muscle tissues were pooled and finely minced. Subsequently, they were digested by treatment with 0.2% collagenase XI (Sigma-Aldrich, St. Louis, MO, USA) for 2 h followed by treatment 3
ACCEPTED MANUSCRIPT with 0.25% trypsin (Sigma) for 30 min. The digestion was halted by adding digestive termination medium, composed of phosphate-buffered saline (PBS) and 10% fetal bovine serum. The resulting muscle cell mixture was filtered through a 400 mesh
RI PT
screen. The cells were collected by centrifugation at 800 × g, washed once with PBS, and aliquots were added to cell culture plates coated with polylysine (Sigma). MDSC culture medium was added to each well of the plate prior to seeding. The isolated
SC
bovine MDSCs were cultured in high-glucose Dulbecco’s Modified Eagle Medium
M AN U
(DMEM) containing 20% fetal bovine serum, 10% horse serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C and 5% CO2 in a humidified atmosphere [10] [14]. 2.2 Cell culture and differentiation
TE D
Bovine MDSCs were seeded in a 6-well plate and cultured to 70–80% confluence in DMEM containing 30% fetal bovine serum. Then, the culture medium was discarded and the cells were washed twice with PBS, followed by the addition of
EP
DMEM containing 2% horse serum to culture the bovine MDSCs for 0, 1, 3, 5, and 7
AC C
days of in vitro differentiation.
2.3 Construction and screening of plasmid vectors The effect of TCEA3 on the differentiation of bovine MDSCs was examined. The
promoter region of TCEA3 (ID: 533803) was subjected to target site prediction using ZiFiT Targeter Version 4.2. Three single-guide RNAs (sgRNAs) targeting different sites
of
the
TCEA3
promoter
CACCGAGGTTCTTTCAGGAAAGTC;
were
obtained
as
follows:
TI, T2, 4
ACCEPTED MANUSCRIPT CACCGGATCCAAAGTTCTCAAAGT;
and
T3,
CACCGAGGTTCTTTCAGGAAAGTC. Each sgRNA contained a 20-nt target sequence and 4-nt Bbs I restriction site at the 5′ ends. The oligonucleotides were
RI PT
synthesized, annealed, and cloned into the pSPgRNA plasmid vector (Addgene, Teddington, UK). The ligation product was transformed into Escherichia coli DH5a (purchased from TransGen Biotech Inc., Beijing, China) and the positively
SC
transformed colonies (transformants) were selected for sequencing. Finally, three
M AN U
sgRNAs, pSPgRNA-T1, pSPgRNA-T2, and pSPgRNA-T3, were successfully obtained.
Bovine MDSCs grown to logarithmic phase were inoculated into a 6-well plate and grown until the cells reached 70–80% confluence prior to transfection using
TE D
polyethylenimine (Sigma). Cells were co-transfected with 2 µg of the sgRNA plasmid vector and 2 µg of the SP-dCas9-VPR plasmid vector (purchased from the Addgene Company), for 24 hours each. TCEA3 expression was detected by western blotting
EP
and the sgRNA plasmid vector with the best activation outcome was selected. Cells
AC C
were then co-transfected with 2 µg of the sgRNA plasmid vector and 2 µg of the dCas9 plasmid vector (Addgene Company) for 24 hours each. TCEA3 expression was detected by western blotting and the sgRNA plasmid vector with the best inhibitory effects was selected.
2.4 Western blot assay Cellular proteins were extracted from bovine MDSCs, separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a PVDF 5
ACCEPTED MANUSCRIPT membrane (Millipore Corporation, Billerica, MA, USA). The PVDF membrane was incubated in blocking solution (PBST containing 5% skim milk powder) at 37°C for 1 hour, followed by incubation with the following specific primary antibodies at 37°C
RI PT
for 1 hour: anti-TFIISH (TCEA3) (Abcam, Cambridge, MA, USA), anti-MYOG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MYH3 (Santa Cruz Biotechnology), and anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA,
SC
USA). The secondary antibodies HRP-labeled goat and rabbit IgG (Beijing
M AN U
Biosynthesis Biotechnology Co., Ltd., Beijing, China) were added at 37°C for 45 minutes. The proteins were visualized using the Super ECL Plus Detection Kit (Applygen
Technologies
Inc.,
Beijing,
China)
using
the
Minichemi
Chemiluminescence imaging instrument (Beijing Sage Creation Science Co., Beijing,
TE D
China) according to the manufacturer's instructions. A densitometry analysis was performed using SageCapture™ software (Beijing Sage Creation Science Co.). 2.5 Extraction of cytoplasmic and nuclear proteins
EP
The cytoplasmic and nuclear proteins were extracted from bovine MDSCs at
AC C
various stages of differentiation using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology Co., Ltd., Haimen, China) according to the user manual.
2.6 Immunofluorescence assay Cells were cultured in a 6-well plate and grown to 70–80% confluence. The medium was discarded and cells were washed twice with PBS and fixed in cold methanol for 20 minutes. Subsequently, the fixative was aspired and the cells were 6
ACCEPTED MANUSCRIPT washed with PBST for 10 minutes, followed by incubation in PBS containing 5% BSA and 0.2% TritonX-100 for 60 minutes. After aspiring the blocking solution, the cells were incubated for 60 minutes with the primary antibodies anti-TFIISH (TCEA3)
RI PT
and anti-Desmin (muscle differentiation-related gene, Desmin, DES) (Santa Cruz Biotechnology), which were diluted in PBST containing 5% BSA, followed by washing 3 times with PBST. Cells were then incubated in the dark with the
SC
FITC-labeled rabbit IgG secondary antibody (purchased from Beijing Biosynthesis
M AN U
Biotechnology Co., Ltd.) for 60 minutes and washed 3 times with PBST. The cells were then subjected to nuclear staining with DAPI (4',6-diamidino-2-phenylindole) for 3 minutes and washed 3 times with PBST. A fluorescence quenching agent was added dropwise and the cells were observed under an inverted fluorescence
2.7 Statistical analysis
TE D
microscope to obtain images.
Data are presented as means ± standard error. Comparisons were performed
EP
using ANOVA followed by Tukey's tests (SPSS Inc., Chicago, IL, USA). A value of P
AC C
< 0.05 was considered significant.
3. Results
3.1 Expression and localization of TCEA3 in bovine MDSCs at various stages of differentiation 3.1.1 TCEA3 expression in bovine MDSCs at various stages of differentiation Western blotting was used to detect changes in TCEA3 expression in bovine 7
ACCEPTED MANUSCRIPT MDSCs at various stages of in vitro differentiation. Differentiated bovine MDSCs at day 0 (0 D) were used as the blank control. TCEA3 protein expression increased gradually as differentiation time increased and peaked on the 5th day of
RI PT
differentiation (Fig. 1A). The expression levels of TCEA3 were 1.4-fold (P < 0.05), 2.8-fold (P < 0.01), 3.3-fold (P < 0.01), and 3.25-fold (P < 0.01), greater than those of the blank control after 1, 3, 5, and 7 days of differentiation, respectively. In the same
M AN U
peaked after 7 days of differentiation (Fig. 1B).
SC
samples, the expression levels of MYOG and MYH3 increased significantly and
The IFA results showed that the cytoplasmic TCEA3 protein level gradually increased as the degree of cell differentiation increased (Fig. 1C). 3.1.2 Localization of TCEA3 in bovine MDSCs at various stages of differentiation
TE D
The distribution of TCEA3 in MDSCs before and after differentiation was determined by IFA. TCEA3 was mainly distributed in the cytoplasm and was not expressed in the nucleus (Fig. 2A).
EP
Cytoplasmic and nuclear proteins were extracted from the differentiated bovine
AC C
MDSCs at 0, 3, and 5 days. Western blotting results showed that TCEA3 was not expressed in the nucleus, and its expression level in the cytoplasm gradually increased as the degree of muscle cell differentiation increased (Fig. 2B). 3.2 Effect of TCEA3 on the differentiation of bovine MDSCs 3.2.1 Activation of TCEA3 expression promotes the differentiation of bovine MDSCs To determine the effect of TCEA3 on the differentiation of bovine MDSCs, the pSPgRNA, pSPgRNA-T1, pSPgRNA-T2, and pSPgRNA-T3 plasmid vectors were 8
ACCEPTED MANUSCRIPT separately co-transfected with the SP-dCas9-VPR plasmid vector into bovine MDSCs for 24 hours. The expression levels of TCEA3 in bovine MDSCs transfected with these plasmid vectors were 1.5-fold (P < 0.05), 2.5-fold (P < 0.01), and 3-fold (P <
RI PT
0.01) higher than those of the control group (pSPgRNA) (Fig. 3A and B). Bovine MDSCs transfected with the pSPgRNA-T3 plasmid vector exhibited the highest expression of TCEA3. Therefore, the pSPgRNA-T3 plasmid vector was selected for
SC
subsequent experiments.
M AN U
After co-transfection with the pSPgRNA-T3 plasmid vector and SP-dCas9-VPR plasmid vector for 24 hours, changes in the myotube fusion rate and the number of myotubes in bovine MDSCs during in vitro differentiation were observed by Desmin immunofluorescence staining. The myotube fusion rate and the number of myotubes
TE D
in bovine MDSCs both increased significantly. The myotube fusion rate and the number of myotubes were 1.58-fold (P < 0.01) and 1.76-fold (P < 0.01) greater than those of the control group (pSPgRNA), respectively (Fig. 3C, D, and E).
EP
The expression levels of muscle cell differentiation-related proteins (MYOG and
AC C
MYH3) were determined by western blotting. In addition to the increase in TCEA3 expression, the expression levels of MYOG and MYH3 were 1.9-fold (P < 0.05) and 1.89-fold (P < 0.05) higher than those of the control group (pSPgRNA), respectively (Fig. 3F and G).
These results showed that TCEA3 activation could promote the differentiation of bovine MDSCs. 3.2.2 Interference of TCEA3 inhibits the differentiation of bovine MDSCs 9
ACCEPTED MANUSCRIPT The pSPgRNA, pSPgRNA-T1, pSPgRNA-T2, and pSPgRNA-T3 plasmid vectors were separately co-transfected with the dCas9 plasmid vector into bovine MDSCs for 24 hours. The expression levels of the TCEA3 protein in bovine MDSCs
RI PT
transfected with these plasmid vectors were 0.41-fold (P < 0.01), 0.3-fold (P < 0.01), and 0.25-fold (P < 0.01) those of the control group (pSPgRNA) (Fig. 4A and B). Bovine MDSCs transfected with the pSPgRNA-T3 plasmid vector had the lowest
SC
TCEA3 expression levels. Therefore, pSPgRNA-T3 was selected for subsequent
M AN U
experiments.
After co-transfection with the pSPgRNA-T3 plasmid vector and dCas9 plasmid vector for 24 hours, changes in the myotube fusion rate and the number of myotubes in bovine MDSCs during in vitro differentiation were determined by Desmin IFA to
TE D
study the effects of TCEA3 inhibition on the differentiation of bovine MDSCs. During the in vitro differentiation of bovine MDSCs, the myotube fusion rate and the number of myotubes were significantly lower in co-transfected cells than in control
EP
cells. The myotube fusion rate and the number of myotubes were 0.58-fold (P < 0.05)
AC C
and 0.70-fold (P < 0.05) those of the control group (pSPgRNA), respectively (Fig. 4C, D, and E).
Based on western blotting, the decline in TCEA3 expression was accompanied
by changes in the expression levels of MYOG and MYH3, which were 0.5-fold (P < 0.05) and 0.15-fold (P < 0.01) those in the control group (pSPgRNA), respectively (Fig. 4F and G). These results showed that the inhibition of TCEA3 expression could inhibit the 10
ACCEPTED MANUSCRIPT in vitro differentiation ability of bovine MDSCs. 4. Discussion Little is known about functions of the transcription elongation factor TCEA3,
RI PT
also known as TFIIS type 3. The overexpression of TCEA3 inhibits the proliferation and colony formation of gastric cancer cells [8]. Additionally, TCEA3 overexpression in ovarian cancer cells could induce apoptosis [5]. In 2009, Xu et al. [9] studied the
SC
early development of myocardial structure in humans and found that TCEA3
M AN U
expression was upregulated, but the function of TCEA3 in cardiac muscle cells (cardiomyocytes) has not been studied. Our results indicated that the expression of TCEA3 in bovine MDSCs was upregulated during in vitro differentiation. IFA results showed that more highly differentiated myotubes express higher levels of TCEA3
TE D
compared with the expression in less-differentiated myotubes, consistent with our previous high-throughput sequencing results showing that the mRNA expression of TCEA3 in bovine MDSCs is upregulated during in vitro differentiation.
EP
We observed the localization of TCEA3 in bovine MDSCs by IFA and detected
AC C
TCEA3 expression in the cytoplasm of bovine MDSCs, but not in nuclei. To further study the role of TCEA3 in the cytoplasm, we will detect TCEA3-interacting proteins in the cytoplasm by co-immunoprecipitation and fluorescence resonance energy transfer. We will also examine the location of TCEA3 in tissues of different species to evaluate species-specificity in the future. Park et al. found that the overexpression of TCEA3 could inhibit the differentiation of mouse embryonic stem cells under in vitro differentiation conditions. 11
ACCEPTED MANUSCRIPT The inhibition of TCEA3 expression promotes the development of mouse embryonic stem cells into mesoderm and ectoderm lineages [15]. Another study confirmed that vascular endothelial growth factor A (VEGFA) and VEGFC, major transcription
RI PT
factors that regulate vasculogenesis, are activated in Tcea3-knockdown mouse embryonic stem cells [16].
Our results showed that TCEA3 overexpression results in a large number of
SC
elongated myotubes and increases in the myotube fusion rate and number of myotubes,
M AN U
as well as significant increases in the expression levels of MYOG and MYH3. The inhibition of TCEA3 expression resulted in the shrinkage of myotubes, decreases in the myotubes fusion rate and number of myotubes, as well as significant decreases in the expression levels of MYOG and MYH3. These results confirmed that TCEA3
TE D
plays an important role in promoting the differentiation of bovine MDSCs, different from the results obtained by Park et al. [15] regarding the role of TCEA3 in the in vitro differentiation of mouse embryonic stem cells.
EP
The results of our study demonstrated, for the first time, that TCEA3 plays an
AC C
important role in promoting the differentiation of bovine MDSCs. We also characterized the mechanisms underlying the association between TCEA3 and cell differentiation. These results provide an important basis for improving meat yield and domestic animal breeding. Acknowledgments This work was supported by the breeding program for new high-quality varieties of genetically modified bovine from the National Major Transgenic Project [grant 12
ACCEPTED MANUSCRIPT number 2014ZX08007-002]. References [1] M. Wind, D. Reines, Transcription elongation factor SII, BioEssays 22 (2000)
RI PT
327–336. [2] D. Reines, J.W. Conaway, R.C. Conaway, The RNA polymerase II general elongation factors, Trends Biochem. Sci. 22 (1996) 351–355.
SC
[3] O.J. Yoo, H.S. Yoon, K.H. Baek, et al., Cloning, expression and characterization of
M AN U
the human transcription elongation factor, TFIIS, Nucleic Acids Res. 19 (1991) 1073–1079.
[4] P. Labhart, G.T. Morgan, Identification of novel genes encoding transcription elongation factor TFIIS (TCEA) in vertebrates: conservation of three distinct TFIIS
TE D
isoforms in frog, mouse, and human, Genomics 52 (1998) 278–288. [5] Y. Cha, D.K. Kim, J. Hyun, et al., TCEA3 binds to TGF-beta receptor I and induces Smad-independent, JNK-dependent apoptosis in ovarian cancer cells, Cell.
EP
Signal. 25 (2013) 1245–1251.
AC C
[6] T. Ito, K. Doi, N. Matsumoto, et al., Lack of polymorphisms in the coding region of the highly conserved gene encoding transcription elongation factor S-II (TCEA1), Drug Discov. Ther. 1 (2007) 9–11. [7] Z.A. Weaver, C.M. Kane. Genomic characterization of a testis-specific TFIIS (TCEA2) gene, Genomics 46 (1997) 516–519. [8] L. Jia, J. Yin, P. Shuang, et al., TCEA3 attenuates gastric cancer growth by apoptosis induction, Med.Sci. Monit. 21 (2014) 3241–3246. [9] X.Q. Xu, S.Y. Soo, W. Sun, et al., Global expression profile of highly enriched 13
ACCEPTED MANUSCRIPT cardiomyocytes derived from human embryonic stem cells, Stem Cells 27 (2009) 2163–2174. [10] H.L. Tong, H.Y. Yin, W.W. Zhang, et al., Transcriptional profiling of bovine
sequencing, Cell. Mol. Biol. Lett. 20 (2015) 351–373.
RI PT
muscle-derived satellite cells during differentiation in vitro by high throughput RNA
[11] L.S. Qi, M.H. Larson, L.A. Gilbert, et al. Repurposing CRISPR as an
SC
RNA-guided platform for sequence-specific control of gene expression, Cell 2013,
M AN U
152 (2013) 1173–1183.
[12] D. Bikard, W. Jiang, P. Samai, et al., Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system, Nucleic Acids Res. 41 (2013) 7429–7437.
TE D
[13] M.L. Maeder, S.J. Linder, V.M. Cascio, et al., CRISPR RNA-guided activation of endogenous human genes, Nature Meth. 10 (2013) 977–979. [14] S. Lee, H.S. Shin, P.K. Shireman, et al., Glutathione-peroxidase-1 null muscle
EP
progenitor cells are globally defective, Free Rad. Biol. Med. 41 (2006) 1174–1184.
AC C
[15] K.S. Park, Y. Cha, C.H. Kim, et al., Transcription elongation factor Tcea3 regulates the pluripotent differentiation potential of mouse embryonic stem cells via the Lefty1-Nodal-Smad2 pathway, Stem Cells 31 (2013) 282–292. [16] Y. Cha, S.H. Heo, H.J. Ahn, et al., Tcea3 regulates the vascular differentiation potential of mouse embryonic stem cells, Gene Expr. 16 (2013) 25–30.
14
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Figure 1
15
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
Figure 2
16
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Figure 3
17 Figure.4
ACCEPTED MANUSCRIPT
Fig. 1. Expression of TCEA3 during MDSC differentiation. (A) Protein expression of TCEA3, MYOG, and MYH3 in MDSCs at days 0 (0D), 3 (3D), 5 (5D), and 7.(7D)
RI PT
of differentiation. (B) Statistical analysis of the protein expression levels of TCEA3, MYOG, and MYH3. (NS: no significant difference, *p < 0.05, **p < 0.01). (C) Immunofluorescence assay of TCEA3 in MDSCs at days 0 (0D), 3 (3D), 5 (5D), and
M AN U
SC
7 (7D) of differentiation (200×).
Fig. 2. Localization of TCEA3 during MDSC differentiation. (A) Localization of TCEA3 in MDSCs at days 0 (0D), 3 (3D), and 5 (5D) of differentiation. TCEA3 (green). Cell nuclei (blue). Magnification, 200×. (B) Protein expression of TCEA3 in
TE D
the cytoplasm and nucleus at various stages (0D, 3D, and 5D).
Fig. 3. Activation of TCEA3 to promote MDSC differentiation. (A) Protein
EP
expression of TCEA3 in MDSCs after co-transfection with the SP-dCas9-VPR
AC C
expression plasmid and pSPgRNA-Tn. (B) Statistical analysis of TCEA3 in MDSCs after co-transfection with the SP-dCas9-VPR expression plasmid and pSPgRNA-Tn. pSPgRNA was used as the control group. (NS: no significant difference, *p < 0.05, **p < 0.01). (C) Immunofluorescence detection of DES in MDSCs. DES (green). Cell nuclei (blue). Magnification, 200×. Cells were co-transfected with the SP-dCas9-VPR and pSPgRNA-T3 vectors (200×). (D) Myotube fusion rate according to DES staining presented in C. (E) Number of myotubes according to DES staining presented in C. (F) 18
ACCEPTED MANUSCRIPT Western blot results for the expression of TCEA3, MYOG, and MYH3 after co-transfection with SP-dCas9-VPR and pSPgRNA-T3. (G) Statistical analysis of the expression of TCEA3, MYOG, and MYH3 after co-transfection with SP-dCas9-VPR
RI PT
and pSPgRNA-T3.
Fig. 4. Inhibition of TCEA3 to inhibit MDSC differentiation. (A) Protein
SC
expression of TCEA3 in MDSCs cells after co-transfection with the dCas9 expression
M AN U
plasmid and pSPgRNA-Tn. (B) Statistical analysis of TCEA3 in MDSCs cells after co-transfection with the dCas9 expression plasmid and pSPgRNA-Tn. pSPgRNA was used as the control group. (NS: no significant difference, *p < 0.05, **p < 0.01). (C) Immunofluorescence detection of DES in MDSCs. DES (green). Cell nuclei (blue).
TE D
Magnification, 200×. Cells were co-transfected with dCas9 and pSPgRNA-T3. (D) Myotube fusion rate according to DES staining presented in C. (E) Number of myotubes according to DES staining presented in C. (F) Western blot results for the
EP
expression of TCEA3, MYOG, and MYH3 after co-transfection with dCas9 and
AC C
pSPgRNA-T3. (G) Statistical analysis of the expression of TCEA3, MYOG, and MYH3 after co-transfection with dCas9 and pSPgRNA-T3.
19
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights
EP
TE D
M AN U
SC
RI PT
Muscle-derived satellite cell differentiation is promoted by TCEA3. TCEA3 protein was localized in the cytoplasm, but not nuclei of bovine MDSCs. TCEA3 levels increased as myotube differentiation increased. TCEA3 affected myotube fusion, myotube counts, and MYOG and MYH3 levels.
AC C
• • • •
S0006-291X(17)30257-7
DOI:
10.1016/j.bbrc.2017.01.182
Reference:
YBBRC 37244
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 30 January 2017 Accepted Date: 31 January 2017
Please cite this article as: Y. Zhu, H.-L. Tong, S.-F. Li, Y.-Q. Yan, Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.182. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells Zhu Yue, Tong Hui-Li, Li Shu-Feng, Yan Yun-Qin *
Heilongjiang 150030, China
RI PT
The Laboratory of Cell and Development, Northeast Agricultural University, Harbin,
*Corresponding author: Yun Qin Yan, E-mail: [email protected], Laboratory of
M AN U
Fang Strict, Harbin, Heilongjiang 150030, China.
SC
Cell and Development, Northeast Agricultural University, Mucai Street No. 59, Xiang
Abstract: Bovine muscle-derived satellite cells (MDSCs) are important for animal growth. In this study, the effect of transcription elongation factor A3 (TCEA3) on MDSC
differentiation
was
investigated.
Western
blotting,
TE D
bovine
immunofluorescence assays, and cytoplasmic and nuclear protein isolation and purification techniques were used to determine the expression pattern and protein
EP
localization of TCEA3 in bovine MDSCs during in vitro differentiation. TCEA3
AC C
expression was upregulated using the CRISPR/Cas9 technique to study its effects on MDSC differentiation in vitro. TCEA3 expression gradually increased during the in vitro differentiation of bovine MDSCs and peaked on the 5th day of differentiation. TCEA3 was mainly localized in the cytoplasm of bovine MDSCs, and its expression was not detected in the nucleus. The level of TCEA3 was relatively higher in myotubes at a higher degree of differentiation than during early differentiation. After transfection with a TCEA3-activating plasmid vector (TCEA3 overexpression) for 24 1
ACCEPTED MANUSCRIPT hours, the myotube fusion rate, number of myotubes, and expression levels of the muscle differentiation-related loci myogenin (MYOG) and myosin heavy chain 3 (MYH3) increased significantly during the in vitro differentiation of bovine MDSCs.
RI PT
After transfection with a TCEA3-inhibiting plasmid vector for 24 hours, the myotube fusion rate, number of myotubes, and expression levels of MYOG and MYH3 decreased significantly. Our results indicated, for the first time, that TCEA3 promotes
SC
the differentiation of bovine MDSCs and have implications for meat production and
M AN U
animal rearing.
Keywords: TCEA3; transcription elongation factor; muscle-derived satellite cell; differentiation
TE D
1. Introduction
Transcription factor IIS (TFIIS or TCEA) exhibits a wide taxonomic distribution [1]. It not only directly binds to RNA polymerase II, allowing it to read through
EP
various transcription arrest sites, but it also activates the transcriptional activity of
AC C
RNA polymerase II to increase the transcription rate [1-2]. In 1991, Yoo et al. [3] studied the internal structure of the TCEA protein and found that it has three domains, i.e., the Ser/Thr phosphorylation domain, RNA pol II domain, and zinc finger (Znf) domain. The amino acid sequences of the RNA pol II and Znf domains are highly conserved and the precise spacing between the domains plays an important role in protein function [3]. It has three distinct isoforms, which are conserved across frogs, mice, and humans [4]. TCEA1 is ubiquitously expressed [5]. It is highly conserved 2
ACCEPTED MANUSCRIPT among eukaryotes and is essential for definitive hematopoiesis in mice [6]. TCEA2 is a testis-specific isoform. The unique structure of the TCEA2 gene determines the chromosomal localization in testicular tissue [7]. The overexpression of TCEA3 in
RI PT
ovarian cancer cells could induce apoptosis in ovarian cancer cells [5]. Xu et al. [9] studied the early development of myocardial structure in humans and found that TCEA3 expression was elevated, indicating that TCEA3 is related to
SC
myocardial development. However, the mechanism by which TCEA3 promotes cell
M AN U
differentiation is not known. In our previous high-throughput sequencing analysis, we found that TCEA3 expression increases significantly during the differentiation of bovine MDSCs [10]. Therefore, in this study, we examined the expression pattern and localization of TCEA3 in bovine MDSCs at various stages of differentiation. We also
TE D
utilized the CRISPR/Cas9 technique to construct activation and inhibition plasmid vectors [11,12,13] to study the effect of TCEA3 on the differentiation of bovine
development.
EP
MDSCs. The results of this study provide a theoretical basis for improving muscle
AC C
2. Materials and Methods
2.1 Experimental materials Experimental material was collected from the skeletal muscle tissue of newborn
calves after obtaining approval from the Animal Welfare Committee of Northeast Agricultural University, Heilongjiang Province, China. Skeletal muscle tissues were pooled and finely minced. Subsequently, they were digested by treatment with 0.2% collagenase XI (Sigma-Aldrich, St. Louis, MO, USA) for 2 h followed by treatment 3
ACCEPTED MANUSCRIPT with 0.25% trypsin (Sigma) for 30 min. The digestion was halted by adding digestive termination medium, composed of phosphate-buffered saline (PBS) and 10% fetal bovine serum. The resulting muscle cell mixture was filtered through a 400 mesh
RI PT
screen. The cells were collected by centrifugation at 800 × g, washed once with PBS, and aliquots were added to cell culture plates coated with polylysine (Sigma). MDSC culture medium was added to each well of the plate prior to seeding. The isolated
SC
bovine MDSCs were cultured in high-glucose Dulbecco’s Modified Eagle Medium
M AN U
(DMEM) containing 20% fetal bovine serum, 10% horse serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C and 5% CO2 in a humidified atmosphere [10] [14]. 2.2 Cell culture and differentiation
TE D
Bovine MDSCs were seeded in a 6-well plate and cultured to 70–80% confluence in DMEM containing 30% fetal bovine serum. Then, the culture medium was discarded and the cells were washed twice with PBS, followed by the addition of
EP
DMEM containing 2% horse serum to culture the bovine MDSCs for 0, 1, 3, 5, and 7
AC C
days of in vitro differentiation.
2.3 Construction and screening of plasmid vectors The effect of TCEA3 on the differentiation of bovine MDSCs was examined. The
promoter region of TCEA3 (ID: 533803) was subjected to target site prediction using ZiFiT Targeter Version 4.2. Three single-guide RNAs (sgRNAs) targeting different sites
of
the
TCEA3
promoter
CACCGAGGTTCTTTCAGGAAAGTC;
were
obtained
as
follows:
TI, T2, 4
ACCEPTED MANUSCRIPT CACCGGATCCAAAGTTCTCAAAGT;
and
T3,
CACCGAGGTTCTTTCAGGAAAGTC. Each sgRNA contained a 20-nt target sequence and 4-nt Bbs I restriction site at the 5′ ends. The oligonucleotides were
RI PT
synthesized, annealed, and cloned into the pSPgRNA plasmid vector (Addgene, Teddington, UK). The ligation product was transformed into Escherichia coli DH5a (purchased from TransGen Biotech Inc., Beijing, China) and the positively
SC
transformed colonies (transformants) were selected for sequencing. Finally, three
M AN U
sgRNAs, pSPgRNA-T1, pSPgRNA-T2, and pSPgRNA-T3, were successfully obtained.
Bovine MDSCs grown to logarithmic phase were inoculated into a 6-well plate and grown until the cells reached 70–80% confluence prior to transfection using
TE D
polyethylenimine (Sigma). Cells were co-transfected with 2 µg of the sgRNA plasmid vector and 2 µg of the SP-dCas9-VPR plasmid vector (purchased from the Addgene Company), for 24 hours each. TCEA3 expression was detected by western blotting
EP
and the sgRNA plasmid vector with the best activation outcome was selected. Cells
AC C
were then co-transfected with 2 µg of the sgRNA plasmid vector and 2 µg of the dCas9 plasmid vector (Addgene Company) for 24 hours each. TCEA3 expression was detected by western blotting and the sgRNA plasmid vector with the best inhibitory effects was selected.
2.4 Western blot assay Cellular proteins were extracted from bovine MDSCs, separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a PVDF 5
ACCEPTED MANUSCRIPT membrane (Millipore Corporation, Billerica, MA, USA). The PVDF membrane was incubated in blocking solution (PBST containing 5% skim milk powder) at 37°C for 1 hour, followed by incubation with the following specific primary antibodies at 37°C
RI PT
for 1 hour: anti-TFIISH (TCEA3) (Abcam, Cambridge, MA, USA), anti-MYOG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MYH3 (Santa Cruz Biotechnology), and anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA,
SC
USA). The secondary antibodies HRP-labeled goat and rabbit IgG (Beijing
M AN U
Biosynthesis Biotechnology Co., Ltd., Beijing, China) were added at 37°C for 45 minutes. The proteins were visualized using the Super ECL Plus Detection Kit (Applygen
Technologies
Inc.,
Beijing,
China)
using
the
Minichemi
Chemiluminescence imaging instrument (Beijing Sage Creation Science Co., Beijing,
TE D
China) according to the manufacturer's instructions. A densitometry analysis was performed using SageCapture™ software (Beijing Sage Creation Science Co.). 2.5 Extraction of cytoplasmic and nuclear proteins
EP
The cytoplasmic and nuclear proteins were extracted from bovine MDSCs at
AC C
various stages of differentiation using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology Co., Ltd., Haimen, China) according to the user manual.
2.6 Immunofluorescence assay Cells were cultured in a 6-well plate and grown to 70–80% confluence. The medium was discarded and cells were washed twice with PBS and fixed in cold methanol for 20 minutes. Subsequently, the fixative was aspired and the cells were 6
ACCEPTED MANUSCRIPT washed with PBST for 10 minutes, followed by incubation in PBS containing 5% BSA and 0.2% TritonX-100 for 60 minutes. After aspiring the blocking solution, the cells were incubated for 60 minutes with the primary antibodies anti-TFIISH (TCEA3)
RI PT
and anti-Desmin (muscle differentiation-related gene, Desmin, DES) (Santa Cruz Biotechnology), which were diluted in PBST containing 5% BSA, followed by washing 3 times with PBST. Cells were then incubated in the dark with the
SC
FITC-labeled rabbit IgG secondary antibody (purchased from Beijing Biosynthesis
M AN U
Biotechnology Co., Ltd.) for 60 minutes and washed 3 times with PBST. The cells were then subjected to nuclear staining with DAPI (4',6-diamidino-2-phenylindole) for 3 minutes and washed 3 times with PBST. A fluorescence quenching agent was added dropwise and the cells were observed under an inverted fluorescence
2.7 Statistical analysis
TE D
microscope to obtain images.
Data are presented as means ± standard error. Comparisons were performed
EP
using ANOVA followed by Tukey's tests (SPSS Inc., Chicago, IL, USA). A value of P
AC C
< 0.05 was considered significant.
3. Results
3.1 Expression and localization of TCEA3 in bovine MDSCs at various stages of differentiation 3.1.1 TCEA3 expression in bovine MDSCs at various stages of differentiation Western blotting was used to detect changes in TCEA3 expression in bovine 7
ACCEPTED MANUSCRIPT MDSCs at various stages of in vitro differentiation. Differentiated bovine MDSCs at day 0 (0 D) were used as the blank control. TCEA3 protein expression increased gradually as differentiation time increased and peaked on the 5th day of
RI PT
differentiation (Fig. 1A). The expression levels of TCEA3 were 1.4-fold (P < 0.05), 2.8-fold (P < 0.01), 3.3-fold (P < 0.01), and 3.25-fold (P < 0.01), greater than those of the blank control after 1, 3, 5, and 7 days of differentiation, respectively. In the same
M AN U
peaked after 7 days of differentiation (Fig. 1B).
SC
samples, the expression levels of MYOG and MYH3 increased significantly and
The IFA results showed that the cytoplasmic TCEA3 protein level gradually increased as the degree of cell differentiation increased (Fig. 1C). 3.1.2 Localization of TCEA3 in bovine MDSCs at various stages of differentiation
TE D
The distribution of TCEA3 in MDSCs before and after differentiation was determined by IFA. TCEA3 was mainly distributed in the cytoplasm and was not expressed in the nucleus (Fig. 2A).
EP
Cytoplasmic and nuclear proteins were extracted from the differentiated bovine
AC C
MDSCs at 0, 3, and 5 days. Western blotting results showed that TCEA3 was not expressed in the nucleus, and its expression level in the cytoplasm gradually increased as the degree of muscle cell differentiation increased (Fig. 2B). 3.2 Effect of TCEA3 on the differentiation of bovine MDSCs 3.2.1 Activation of TCEA3 expression promotes the differentiation of bovine MDSCs To determine the effect of TCEA3 on the differentiation of bovine MDSCs, the pSPgRNA, pSPgRNA-T1, pSPgRNA-T2, and pSPgRNA-T3 plasmid vectors were 8
ACCEPTED MANUSCRIPT separately co-transfected with the SP-dCas9-VPR plasmid vector into bovine MDSCs for 24 hours. The expression levels of TCEA3 in bovine MDSCs transfected with these plasmid vectors were 1.5-fold (P < 0.05), 2.5-fold (P < 0.01), and 3-fold (P <
RI PT
0.01) higher than those of the control group (pSPgRNA) (Fig. 3A and B). Bovine MDSCs transfected with the pSPgRNA-T3 plasmid vector exhibited the highest expression of TCEA3. Therefore, the pSPgRNA-T3 plasmid vector was selected for
SC
subsequent experiments.
M AN U
After co-transfection with the pSPgRNA-T3 plasmid vector and SP-dCas9-VPR plasmid vector for 24 hours, changes in the myotube fusion rate and the number of myotubes in bovine MDSCs during in vitro differentiation were observed by Desmin immunofluorescence staining. The myotube fusion rate and the number of myotubes
TE D
in bovine MDSCs both increased significantly. The myotube fusion rate and the number of myotubes were 1.58-fold (P < 0.01) and 1.76-fold (P < 0.01) greater than those of the control group (pSPgRNA), respectively (Fig. 3C, D, and E).
EP
The expression levels of muscle cell differentiation-related proteins (MYOG and
AC C
MYH3) were determined by western blotting. In addition to the increase in TCEA3 expression, the expression levels of MYOG and MYH3 were 1.9-fold (P < 0.05) and 1.89-fold (P < 0.05) higher than those of the control group (pSPgRNA), respectively (Fig. 3F and G).
These results showed that TCEA3 activation could promote the differentiation of bovine MDSCs. 3.2.2 Interference of TCEA3 inhibits the differentiation of bovine MDSCs 9
ACCEPTED MANUSCRIPT The pSPgRNA, pSPgRNA-T1, pSPgRNA-T2, and pSPgRNA-T3 plasmid vectors were separately co-transfected with the dCas9 plasmid vector into bovine MDSCs for 24 hours. The expression levels of the TCEA3 protein in bovine MDSCs
RI PT
transfected with these plasmid vectors were 0.41-fold (P < 0.01), 0.3-fold (P < 0.01), and 0.25-fold (P < 0.01) those of the control group (pSPgRNA) (Fig. 4A and B). Bovine MDSCs transfected with the pSPgRNA-T3 plasmid vector had the lowest
SC
TCEA3 expression levels. Therefore, pSPgRNA-T3 was selected for subsequent
M AN U
experiments.
After co-transfection with the pSPgRNA-T3 plasmid vector and dCas9 plasmid vector for 24 hours, changes in the myotube fusion rate and the number of myotubes in bovine MDSCs during in vitro differentiation were determined by Desmin IFA to
TE D
study the effects of TCEA3 inhibition on the differentiation of bovine MDSCs. During the in vitro differentiation of bovine MDSCs, the myotube fusion rate and the number of myotubes were significantly lower in co-transfected cells than in control
EP
cells. The myotube fusion rate and the number of myotubes were 0.58-fold (P < 0.05)
AC C
and 0.70-fold (P < 0.05) those of the control group (pSPgRNA), respectively (Fig. 4C, D, and E).
Based on western blotting, the decline in TCEA3 expression was accompanied
by changes in the expression levels of MYOG and MYH3, which were 0.5-fold (P < 0.05) and 0.15-fold (P < 0.01) those in the control group (pSPgRNA), respectively (Fig. 4F and G). These results showed that the inhibition of TCEA3 expression could inhibit the 10
ACCEPTED MANUSCRIPT in vitro differentiation ability of bovine MDSCs. 4. Discussion Little is known about functions of the transcription elongation factor TCEA3,
RI PT
also known as TFIIS type 3. The overexpression of TCEA3 inhibits the proliferation and colony formation of gastric cancer cells [8]. Additionally, TCEA3 overexpression in ovarian cancer cells could induce apoptosis [5]. In 2009, Xu et al. [9] studied the
SC
early development of myocardial structure in humans and found that TCEA3
M AN U
expression was upregulated, but the function of TCEA3 in cardiac muscle cells (cardiomyocytes) has not been studied. Our results indicated that the expression of TCEA3 in bovine MDSCs was upregulated during in vitro differentiation. IFA results showed that more highly differentiated myotubes express higher levels of TCEA3
TE D
compared with the expression in less-differentiated myotubes, consistent with our previous high-throughput sequencing results showing that the mRNA expression of TCEA3 in bovine MDSCs is upregulated during in vitro differentiation.
EP
We observed the localization of TCEA3 in bovine MDSCs by IFA and detected
AC C
TCEA3 expression in the cytoplasm of bovine MDSCs, but not in nuclei. To further study the role of TCEA3 in the cytoplasm, we will detect TCEA3-interacting proteins in the cytoplasm by co-immunoprecipitation and fluorescence resonance energy transfer. We will also examine the location of TCEA3 in tissues of different species to evaluate species-specificity in the future. Park et al. found that the overexpression of TCEA3 could inhibit the differentiation of mouse embryonic stem cells under in vitro differentiation conditions. 11
ACCEPTED MANUSCRIPT The inhibition of TCEA3 expression promotes the development of mouse embryonic stem cells into mesoderm and ectoderm lineages [15]. Another study confirmed that vascular endothelial growth factor A (VEGFA) and VEGFC, major transcription
RI PT
factors that regulate vasculogenesis, are activated in Tcea3-knockdown mouse embryonic stem cells [16].
Our results showed that TCEA3 overexpression results in a large number of
SC
elongated myotubes and increases in the myotube fusion rate and number of myotubes,
M AN U
as well as significant increases in the expression levels of MYOG and MYH3. The inhibition of TCEA3 expression resulted in the shrinkage of myotubes, decreases in the myotubes fusion rate and number of myotubes, as well as significant decreases in the expression levels of MYOG and MYH3. These results confirmed that TCEA3
TE D
plays an important role in promoting the differentiation of bovine MDSCs, different from the results obtained by Park et al. [15] regarding the role of TCEA3 in the in vitro differentiation of mouse embryonic stem cells.
EP
The results of our study demonstrated, for the first time, that TCEA3 plays an
AC C
important role in promoting the differentiation of bovine MDSCs. We also characterized the mechanisms underlying the association between TCEA3 and cell differentiation. These results provide an important basis for improving meat yield and domestic animal breeding. Acknowledgments This work was supported by the breeding program for new high-quality varieties of genetically modified bovine from the National Major Transgenic Project [grant 12
ACCEPTED MANUSCRIPT number 2014ZX08007-002]. References [1] M. Wind, D. Reines, Transcription elongation factor SII, BioEssays 22 (2000)
RI PT
327–336. [2] D. Reines, J.W. Conaway, R.C. Conaway, The RNA polymerase II general elongation factors, Trends Biochem. Sci. 22 (1996) 351–355.
SC
[3] O.J. Yoo, H.S. Yoon, K.H. Baek, et al., Cloning, expression and characterization of
M AN U
the human transcription elongation factor, TFIIS, Nucleic Acids Res. 19 (1991) 1073–1079.
[4] P. Labhart, G.T. Morgan, Identification of novel genes encoding transcription elongation factor TFIIS (TCEA) in vertebrates: conservation of three distinct TFIIS
TE D
isoforms in frog, mouse, and human, Genomics 52 (1998) 278–288. [5] Y. Cha, D.K. Kim, J. Hyun, et al., TCEA3 binds to TGF-beta receptor I and induces Smad-independent, JNK-dependent apoptosis in ovarian cancer cells, Cell.
EP
Signal. 25 (2013) 1245–1251.
AC C
[6] T. Ito, K. Doi, N. Matsumoto, et al., Lack of polymorphisms in the coding region of the highly conserved gene encoding transcription elongation factor S-II (TCEA1), Drug Discov. Ther. 1 (2007) 9–11. [7] Z.A. Weaver, C.M. Kane. Genomic characterization of a testis-specific TFIIS (TCEA2) gene, Genomics 46 (1997) 516–519. [8] L. Jia, J. Yin, P. Shuang, et al., TCEA3 attenuates gastric cancer growth by apoptosis induction, Med.Sci. Monit. 21 (2014) 3241–3246. [9] X.Q. Xu, S.Y. Soo, W. Sun, et al., Global expression profile of highly enriched 13
ACCEPTED MANUSCRIPT cardiomyocytes derived from human embryonic stem cells, Stem Cells 27 (2009) 2163–2174. [10] H.L. Tong, H.Y. Yin, W.W. Zhang, et al., Transcriptional profiling of bovine
sequencing, Cell. Mol. Biol. Lett. 20 (2015) 351–373.
RI PT
muscle-derived satellite cells during differentiation in vitro by high throughput RNA
[11] L.S. Qi, M.H. Larson, L.A. Gilbert, et al. Repurposing CRISPR as an
SC
RNA-guided platform for sequence-specific control of gene expression, Cell 2013,
M AN U
152 (2013) 1173–1183.
[12] D. Bikard, W. Jiang, P. Samai, et al., Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system, Nucleic Acids Res. 41 (2013) 7429–7437.
TE D
[13] M.L. Maeder, S.J. Linder, V.M. Cascio, et al., CRISPR RNA-guided activation of endogenous human genes, Nature Meth. 10 (2013) 977–979. [14] S. Lee, H.S. Shin, P.K. Shireman, et al., Glutathione-peroxidase-1 null muscle
EP
progenitor cells are globally defective, Free Rad. Biol. Med. 41 (2006) 1174–1184.
AC C
[15] K.S. Park, Y. Cha, C.H. Kim, et al., Transcription elongation factor Tcea3 regulates the pluripotent differentiation potential of mouse embryonic stem cells via the Lefty1-Nodal-Smad2 pathway, Stem Cells 31 (2013) 282–292. [16] Y. Cha, S.H. Heo, H.J. Ahn, et al., Tcea3 regulates the vascular differentiation potential of mouse embryonic stem cells, Gene Expr. 16 (2013) 25–30.
14
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Figure 1
15
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
Figure 2
16
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
Figure 3
17 Figure.4
ACCEPTED MANUSCRIPT
Fig. 1. Expression of TCEA3 during MDSC differentiation. (A) Protein expression of TCEA3, MYOG, and MYH3 in MDSCs at days 0 (0D), 3 (3D), 5 (5D), and 7.(7D)
RI PT
of differentiation. (B) Statistical analysis of the protein expression levels of TCEA3, MYOG, and MYH3. (NS: no significant difference, *p < 0.05, **p < 0.01). (C) Immunofluorescence assay of TCEA3 in MDSCs at days 0 (0D), 3 (3D), 5 (5D), and
M AN U
SC
7 (7D) of differentiation (200×).
Fig. 2. Localization of TCEA3 during MDSC differentiation. (A) Localization of TCEA3 in MDSCs at days 0 (0D), 3 (3D), and 5 (5D) of differentiation. TCEA3 (green). Cell nuclei (blue). Magnification, 200×. (B) Protein expression of TCEA3 in
TE D
the cytoplasm and nucleus at various stages (0D, 3D, and 5D).
Fig. 3. Activation of TCEA3 to promote MDSC differentiation. (A) Protein
EP
expression of TCEA3 in MDSCs after co-transfection with the SP-dCas9-VPR
AC C
expression plasmid and pSPgRNA-Tn. (B) Statistical analysis of TCEA3 in MDSCs after co-transfection with the SP-dCas9-VPR expression plasmid and pSPgRNA-Tn. pSPgRNA was used as the control group. (NS: no significant difference, *p < 0.05, **p < 0.01). (C) Immunofluorescence detection of DES in MDSCs. DES (green). Cell nuclei (blue). Magnification, 200×. Cells were co-transfected with the SP-dCas9-VPR and pSPgRNA-T3 vectors (200×). (D) Myotube fusion rate according to DES staining presented in C. (E) Number of myotubes according to DES staining presented in C. (F) 18
ACCEPTED MANUSCRIPT Western blot results for the expression of TCEA3, MYOG, and MYH3 after co-transfection with SP-dCas9-VPR and pSPgRNA-T3. (G) Statistical analysis of the expression of TCEA3, MYOG, and MYH3 after co-transfection with SP-dCas9-VPR
RI PT
and pSPgRNA-T3.
Fig. 4. Inhibition of TCEA3 to inhibit MDSC differentiation. (A) Protein
SC
expression of TCEA3 in MDSCs cells after co-transfection with the dCas9 expression
M AN U
plasmid and pSPgRNA-Tn. (B) Statistical analysis of TCEA3 in MDSCs cells after co-transfection with the dCas9 expression plasmid and pSPgRNA-Tn. pSPgRNA was used as the control group. (NS: no significant difference, *p < 0.05, **p < 0.01). (C) Immunofluorescence detection of DES in MDSCs. DES (green). Cell nuclei (blue).
TE D
Magnification, 200×. Cells were co-transfected with dCas9 and pSPgRNA-T3. (D) Myotube fusion rate according to DES staining presented in C. (E) Number of myotubes according to DES staining presented in C. (F) Western blot results for the
EP
expression of TCEA3, MYOG, and MYH3 after co-transfection with dCas9 and
AC C
pSPgRNA-T3. (G) Statistical analysis of the expression of TCEA3, MYOG, and MYH3 after co-transfection with dCas9 and pSPgRNA-T3.
19
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Highlights
EP
TE D
M AN U
SC
RI PT
Muscle-derived satellite cell differentiation is promoted by TCEA3. TCEA3 protein was localized in the cytoplasm, but not nuclei of bovine MDSCs. TCEA3 levels increased as myotube differentiation increased. TCEA3 affected myotube fusion, myotube counts, and MYOG and MYH3 levels.
AC C
• • • •