Zbed6基因敲除对小鼠骨骼肌生长发育的影响及其分子作用机制研究

doi:10.16431/j.cnki.1671-7236.2023.09.020

Abstract

Abstract: 【Objective】 The purpose of this study was to investigate the effect of zinc finger BED domain containing protein 6 (ZBED6) gene knockout on skeletal muscle growth and development in adolescent mice and its molecular mechanism.【Method】 Wild type (WT) and Zbed6 gene knockout (Zbed6^-/-) C57BL/6 mice at 8 weeks old were used as experimental subjects.Blood was collected and serum testosterone content was determined by ELISA method.Skeletal muscle tissue was isolated and muscle weight and muscle ratio were recorded.Skeletal muscle fiber area and IGF2 protein expression levels were determined by histological analysis and immunohistochemical analysis.Transcriptome sequencing was performed on WT and Zbed6^-/- male and female mice by Illumina Novaseq^TM 6000 high-throughput sequencing.Combined with ZBED6 target gene data of mice,the downstream target genes that played a role in Zbed6 gene knockout mouse muscle tissue were screened.Real-time quantitative PCR was used to verify the reliability of differentially expressed genes in transcriptome sequencing.【Result】 Compared with WT mice,muscle weight and muscle ratio of Zbed6^-/- male and female mice were extremely significantly increased (P<0.01).After Zbed6 gene knockout,the mRNA expression of Zbed6 gene in skeletal muscle of female and male mice was significantly decreased (P<0.05),and the mRNA and protein expression of IGF2 were significantly or extremely significantly increased (P<0.05 or P<0.01),serum testosterone content was extremely significantly increased (P<0.01).Transcriptome sequencing showed that 38 differentially expressed genes were detected in the skeletal muscle of male mice in Zbed6^-/- group,of which 22 genes were up-regulated and 16 genes were down-regulated.49 differentially expressed genes were detected in skeletal muscle of female mice,among which 19 genes were up-regulated and 30 genes were down-regulated.A total of 10 differentially expressed genes were co-expressed in female and male mice,among which Dock3,Lhx2,Brsk2,Caskin1 and Gbe1 were ZBED6 target genes.Real-time quantitative PCR showed that the expression of the 4 differentially expressed genes was consistent with the transcriptome sequencing analysis,which confirmed the accuracy and reliability of the sequencing results.【Conclusion】 Zbed6^-/- promoted skeletal muscle growth and increased serum testosterone content in adolescent mice.Five ZBED6 target genes including Dock3, Lhx2,Brsk2,Caskin1 and Gbe1 were screened.The results provided new gene targets and research directions for further exploring the function of ZBED6,contribute to improving the functional map of ZBED6,and provided an important theoretical basis for the improvement of large animal varieties and the genetic breeding of lean meat traits of livestock and poultry.

Key words: zinc finger BED domain containing protein 6; skeletal muscle; growth and development; transcriptome sequencing

CLC Number:

LIU Ling, WANG Shengnan, WANG Dandan, MA Yuehui, JIANG Lin, CUI Kai. Effect and Molecular Mechanism of Zbed6 Gene Knockout on the Growth and Development of Skeletal Muscle in Mice[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3641-3651.

References

[1] MARKLJUNG E,JIANG L,JAFFE J D,et al.ZBED6,a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth[J].PLoS Biology,2009,7(12):e1000256.
[2] JEON J T,CARLBORG O,TÖRNSTEN A,et al.A paternally expressed QTL affecting skeletal and cardiac muscle mass in pigs maps to the IGF2 locus[J].Nature Genetics,1999,21(2):157-158.
[3] NEZER C,MOREAU L,BROUWERS B,et al.An imprinted QTL with major effect on muscle mass and fat deposition maps to the IGF2 locus in pigs[J].Nature Genetics,1999,21(2):155-156.
[4] YOUNIS S,SCHÖNKE M,MASSART J,et al.The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals[J].Proceedings of the National Academy of Sciences of the United States of America,2018,115(9):E2048-E2057.
[5] ANDERSSON L,ANDERSSON G,HJÄLM G,et al.ZBED6:The birth of a new transcription factor in the common ancestor of placental mammals[J].Transcription,2010,1(3):144-148.
[6] VAN LAERE A S,NGUYEN M,BRAUNSCHWEIG M,et al.A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig[J].Nature,2003,425(6960):832-836.
[7] HUANG Y Z,HE H,ZHAN Z Y,et al.Relationship of polymorphisms within ZBED6 gene and growth traits in beef cattle[J].Gene,2013,526(2):107-111.
[8] HUANG Y Z,ZHAN Z Y,SUN Y J,et al.Comparative analysis of the IGF2 and ZBED6 gene variants and haplotypes reveals significant effect of growth traits in cattle[J].Genome,2013,56(6):327-334.
[9] XIANG G,REN J,HAI T,et al.Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs[J].Cellular Molecular Life Sciences,2018,75(24):4619-4628.
[10] WANG D,PAN D,XIE B,et al.Porcine ZBED6 regulates growth of skeletal muscle and internal organs via multiple targets[J].PLoS Genetics,2021,17(10):e1009862.
[11] WANG S,TIAN W,PAN D,et al.A comprehensive analysis of the myocardial transcriptome in ZBED6-knockout Bama Xiang pigs[J].Genes,2022,13(8):1382.
[12] LIU X,LIU H,WANG M,et al.Disruption of the ZBED6 binding site in intron 3 of IGF2 by CRISPR/Cas9 leads to enhanced muscle development in Liang Guang Small Spotted pigs[J].Transgenic Research,2019,28(1):141-150.
[13] JIANG L,WALLERMAN O,YOUNIS S,et al.ZBED6 modulates the transcription of myogenic genes in mouse myoblast cells[J].PLoS One,2014,9(4):e94187.
[14] YOUNIS S,NABOULSI R,WANG X,et al.The importance of the ZBED6-IGF2 axis for metabolic regulation in mouse myoblast cells[J].FASEB Journal,2020,34(8):10250-10266.
[15] WANG X,JIANG L,WALLERMAN O,et al.ZBED6 negatively regulates insulin production,neuronal differentiation,and cell aggregation in MIN6 cells[J].FASEB Journal,2019,33(1):88-100.
[16] WANG X,JIANG L,WALLERMAN O,et al.Transcription factor ZBED6 affects gene expression,proliferation,and cell death in pancreatic beta cells[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(40):15997-16002.
[17] ZHANG W,CLINE M A,LIU D,et al.Knockdown of ZBED6 is not associated with changes in murine preadipocyte proliferation or differentiation[J].Adipocyte,2013,2(4):251-255.
[18] AKHTAR ALI M,YOUNIS S,WALLERMAN O,et al.Transcriptional modulator ZBED6 affects cell cycle and growth of human colorectal cancer cells[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(25):7743-7748.
[19] NABOULSI R,LARSSON M,ANDERSSON L,et al.ZBED6 regulates Igf2 expression partially through its regulation of miR483 expression[J].Scientific Reports,2021,11(1):19484.
[20] ZOU H,YU D,YAO S,et al.Efficient editing of the ZBED6-binding site in intron 3 of IGF2 in a bovine model using the CRISPR/Cas9 system[J].Genes,2022,13(7):1132.
[21] NEIRIJNCK Y,CALVEL P,KILCOYNE K R,et al.Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells[J].FASEB Journal,2018,32(6):3321-3335.
[22] CHARALAMBOUS M,SMITH F M,BENNETT W R,et al.Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(14):8292-8297.
[23] WILTROUT K,FERRER A,VAN DE LAAR I,et al.Variants in DOCK3 cause developmental delay and hypotonia[J].European Journal Human Genetics,2019,27(8):1225-1234.
[24] HELBIG K L,MROSKE C,MOORTHY D,et al.Biallelic loss-of-function variants in DOCK3 cause muscle hypotonia,ataxia,and intellectual disability[J].Clinical Genetics,2017,92(4):430-433.
[25] REID A L,WANG Y,SAMANI A,et al.DOCK3 is a dosage-sensitive regulator of skeletal muscle and Duchenne muscular dystrophy-associated pathologies[J].Human Molecular Genetics,2020,29(17):2855-2871.
[26] TARNOPOLSKY M A.Myopathies related to glycogen metabolism disorders[J].Neurotherapeutics,2018,15(4):915-927.
[27] URUNO A,YAGISHITA Y,KATSUOKA F,et al.Nrf2-mediated regulation of skeletal muscle glycogen metabolism[J].Molecular Cellular Biology,2016,36(11):1655-1672.

Effect and Molecular Mechanism of Zbed6 Gene Knockout on the Growth and Development of Skeletal Muscle in Mice

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