Nuclear reprogramming of human somatic cells by xenopus egg extract requires BRG1.
Animal cloning by nuclear transplantation in amphibia was demonstrated almost half a century ago and raised the question of the mechanisms and genes involved in nuclear reprogramming. Here, we demonstrate nuclear reprogramming of permeabilized human cells using extracts from Xenopus laevis eggs and early embryos. We show upregulation of pluripotency markers Oct-4 and germ cell alkaline phosphatase (GCAP) in 293T cells and human primary leukocytes. Reprogrammed leukocytes had a limited life span and did not express surface antigens characteristic of pluripotent cells, indicating that reprogramming was incomplete. Reprogramming activity was detected in egg and early embryo extracts until early blastula stage. Late blastula-stage extracts were not only inactive but also inhibitory to reprogramming. Screening for factors required for reprogramming identified the chromatin remodeling ATPase BRG1. Antibody depletion of BRG1 protein or expression of dominant-negative BRG1 abolished the reprogramming ability of amphibian extracts. Conversely, overexpression of BRG1 in Xenopus animal caps extended their competence from blastula to gastrula stage to respond to basic fibroblast growth factor (bFGF) treatment with induction of the mesodermal marker Xbra. Dissection of the molecular machinery using a simplified assay system may aid in achieving complete nuclear reprogramming of somatic cells for regenerative medicine.
Hansis
Christoph
C
Division of Molecular Embryology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Barreto
Guillermo
G
Maltry
Nicole
N
Niehrs
Christof
C
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002457:Cell Extracts / Q000235:genetics / Q000494:pharmacology*; D002467:Cell Nucleus / Q000502:physiology; D002478:Cells, Cultured; D004265:DNA Helicases; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects* / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008845:Microinjections; D009687:Nuclear Proteins / Q000378:metabolism* / Q000494:pharmacology; D050814:Octamer Transcription Factor-3; D010063:Ovum / Q000378:metabolism*; D044968:Regenerative Medicine / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism* / Q000494:pharmacology; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000378:metabolism*
D002457:Cell Extracts; D017931:DNA Primers; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D020825:T-Box Domain Proteins; C116765:TBXT protein, Xenopus; D014157:Transcription Factors; D029867:Xenopus Proteins; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase; C084108:SMARCA4 protein, human; D004265:DNA Helicases