Reprogramming towards pluripotency requires AID-dependent DNA demethylation.
Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.
Bhutani
Nidhi
N
Baxter Laboratory for Stem Cell Biology, Institute for Stem Cell Biology and Regenerative Medicine, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5175, USA.
Brady
Jennifer J
JJ
Damian
Mara
M
Sacco
Alessandra
A
Corbel
Stéphane Y
SY
Blau
Helen M
HM
D000818:Animals; D002455:Cell Division; D002459:Cell Fusion; D002460:Cell Line; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics / Q000502:physiology*; D047369:Chromatin Immunoprecipitation; D003564:Cytidine Deaminase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004261:DNA Replication; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D008168:Lung / Q000166:cytology / Q000196:embryology; D051379:Mice; D008954:Models, Biological; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D013997:Time Factors; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D004247:DNA; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase