Reprogramming towards pluripotency requires AID-dependent DNA demethylation

Abstract

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 the first evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.Reprogramming of somatic cell nuclei towards pluripotency has been achieved by nuclear transfer into enucleated oocytes 1,2 and the introduction of four defined factors to generate iPS cells [3][4][5][6] . These remarkable advances enable the generation of patient-specific cells for tissue replacement, modelling human diseases in tissue culture, and drug discovery. However, an understanding of the molecular mechanisms underlying nuclear reprogramming has been elusive, largely due to the technical challenges associated with nuclear transfer and the low efficiency of iPS cell generation.

Mechanisms of nuclear reprogramming by eggs and oocytes: a deterministic process?

“…Typically, reactivation of previously silent genes is detected between 24 and 48 hours after nuclear transplantation 10,11,16,43 . Similar kinetics are observed following mouse ES cell fusion to human lympho cytes 37 or fibroblasts 45 . The oocyte exerts its transcriptional reprogramming activity on a very large scale, not only reactivating pluripotency genes, such as OCT4, SOX2 and NANOG, but also activating, to a certain extent, many different genes that are not actively transcribed in the oocyte 49 .…”

“…Two examples of enzymes that are thought to play a part in active DNA demethylation are growth arrest and DNA damage-inducible 45α (GADD45α) 42 and activation-induced deaminase (AID) 45 . More recently, in mouse eggs and early embryos, the TET enzymes have been shown to help active DNA demethylation by hydroxylating of methylated cytosine [46][47][48] .…”

“…Eggs and oocytes also possess active DNA demethylation activity, which is a requirement for the transcriptional reactivation of silenced genes (for example, OCT4); this activity is observed after nuclear transfer [41][42][43][44] , as well as in cell fusion to ES cells 45 . Two examples of enzymes that are thought to play a part in active DNA demethylation are growth arrest and DNA damage-inducible 45α (GADD45α) 42 and activation-induced deaminase (AID) 45 .…”

AID in reprogramming: Quick and efficient

“…In a report published online in Nature on December 21, 2009, Bhutani et al [1] developed a strategy in which reprogramming was initiated by fusing mouse ESCs with human skin cells to create hybrids called heterokaryons, resulting in astonishingly quick and efficient reprogramming in just 1 day and with extremely high efficiency ($70%). Key advantages of this heterokaryon-based approach included rapid access to the earliest reprogramming events and a more efficient and synchronous environment through which specific pluripotency mechanisms could be uncovered.…”

“…Key advantages of this heterokaryon-based approach included rapid access to the earliest reprogramming events and a more efficient and synchronous environment through which specific pluripotency mechanisms could be uncovered. Moreover, the researchers showed that reprogramming toward pluripotency in single heterokaryons was initiated without cell division or DNA replication [1]. The use of interspecies (between mouse and human) heterokaryons allowed for a distinction of transcripts derived from the two fused cell types, and reprogramming was assessed immediately after fusion without the need for cell division or DNA replication.…”