Simultaneous overexpression of Oct4 and Nanog abrogates terminal myogenesis.
Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.
Lang
Kuan Chih
KC
Dept. of Life Sciences, National Central University, 300 Jhongda Rd., Jhongli 32054, Taiwan, ROC.
Lin
I Hsuan
IH
Teng
Han Fang
HF
Huang
Yi Cheng
YC
Li
Chung Leung
CL
Tang
Kam Tsun
KT
Chen
Shen Liang
SL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D019070:Cell Lineage; D025461:Feedback, Physiological; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D064326:MEF2 Transcription Factors; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008969:Molecular Sequence Data; D024510:Muscle Development / Q000235:genetics; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D032448:Myoblasts, Skeletal / Q000378:metabolism*; D018006:Myogenic Regulatory Factors / Q000235:genetics / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D051763:PAX7 Transcription Factor / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D013724:Teratoma / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D015854:Up-Regulation
D018398:Homeodomain Proteins; D064326:MEF2 Transcription Factors; C578041:Mef2c protein, mouse; D018006:Myogenic Regulatory Factors; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; D051763:PAX7 Transcription Factor; C497322:Pax7 protein, mouse; C494174:Pou5f1 protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse