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DNA methylation in stem cells

Final Report Summary - METSTEM (DNA methylation in stem cells)

Establishing methylation patterns in embryonic stem cells
In animals, the genome-wide DNA methylation pattern is initially erased in the early embryo and then re-established in each individual at about the time of implantation. This is carried out by a process in which almost all the DNA is subject to de novo methylation while CpG island-like regions are protected on the basis of underlying sequence motifs, but the biological logic and molecular mechanism of this process were still unknown. Analysis of these CpG-rich sites indicated that they are highly enriched for transcription start sites and characterized by the presence of binding motifs [Straussman, 2009 #4934] for many transcription factors. This close correlation suggested the possibility that protection from de novo methylation may actually be dictated by the binding of transcription complexes at recognized sites in the pre-implantation embryo. In this project, we used bioinformatic tools as well as genetic techniques to test this idea both in vitro, as well as in vivo. The results of these experiments have led to a completely new concept for how DNA methylation functions during development.

We have also identified a new pathway in embryonic stem cells that specifically carries out CpG-island demethylation. Using genetics, we have demonstrated that this involves hydroxymethylation (Tet1), deamination (Aid), glycosylation (Mbd4) and excision repair (Gadd45a) genes. Surprisingly, this biochemical route is not required for “setting up” the overall bimodal DNA modification pattern, but it does play a key role in reprogramming somatic-cell DNA methylation profiles by selectively resetting all CpG island-like sequences, including the Oct-3/4 and Nanog pluripotency gene promoters, as well as those that have become aberrantly modified through aging or cancer. These studies provide new insights into how stem cells are programmed for maintaining epigenetic plasticity.

Aberrant methylation of stem cells in culture
Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we have shown that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicated that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. This modification, which is very stable, occurs on key developmental genes and may thus cause inhibition of normal differentiation. It may also predispose to cancer if cells are used for replacement therapy. The aberrant methylation pattern observed in differentiating ES cells is very similar to that seen in placenta, suggesting that this process is governed by an inherent program that may be used in vivo during normal extra-embryonic development. It is hoped that by deciphering the rules of this aberrant methylation it will be possible to prevent this event, thereby generating physiologically-normal stem cells.

Programming asynchronous replication timing in stem cells
Many regions of the genome have been found to replicate asynchronously. These regions are associated with monoallelically expressed genes and it is thought that the differential replication pattern in each cell may be involved in choosing one allele over the other. In this paper, we have explored the developmental aspects and molecular mechanisms of this process and showed that, as opposed to somatic cells that maintain replication timing in a clonal manner, embryonic and adult stem cells appear to undergo switching such that daughter cells with an early paternal allele are derived from mother cells which have a late paternal allele. This novel phenomenon is carried out according to a predetermined molecular program aimed at maintaining allelic diversity. In the early embryo, gametic patterns of asynchronous replication are erased and all of these regions replicate synchronously. Using ground-state ES cells as a model system, we have demonstrated that in the initial transition to asynchronous replication, it is always the paternal allele that is chosen to be early, suggesting that the primary act of allelic choice is directed by pre-set markers derived from gametic DNA. Using a genetic approach, we have determined that the mechanisms of both “switching” and “choosing” are controlled by DNA methylation.