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Dual AID for Reprogramming and DNA Methylation

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The main aim of this work was to study the process of DNA demethylation in human cells, a relatively unknown epigenetic process, but of key importance in gene regulation and therefore the reprogramming process. Initial analysis of heterokaryon cells showed the rapid upregulation of human OCT4 and NANOG transcripts, which are utilised as surrogate indictors of the reprogramming process, and rapid demethylation of DNA at their respective promoter regions. These changes were observed over three days, compared to over two weeks which is usually required in iPSC generation, and took place without cell division or DNA replication as measured by Ki67 staining and BrdU incorporation analysis. This is an important finding, suggesting that an active, replication-independent mechanism is involved in DNA demethylation, rather than a passive, replication-dependent dilution of this mark. This is the first time this phenomenon has been demonstrated and is an important finding in the study of DNA demethylation in humans.

 

The next part of the study concentrated on a factor implicated in DNA demethylation in Zebrafish (Rai et al, Cell, 2008), which has also been observed in mammalian pluripotent germ cells where DNA demethylation is known to occur (Morgan, JBC, 2004). This factor, AID (or AICDA) (Activation induced cytidine deaminase), is thought to deaminate methylated cytosines (DNA methylation) creating a thymine and therefore a G-T base pair mismatch. It is then suggested that this mismatch can be repaired by DNA repair mechanisms, thereby allowing active DNA demethylation. siRNA of AID inhibited OCT4 and NANOG expression in the heterokaryons and also inhibited DNA demethylation at their promoter sequences. However, over-expression studies showed no increase in the speed of reprogramming or amount of DNA demethylation, and indeed over-expression of AID seemed to retard these processes, perhaps suggesting that other unknown factors are limiting for this process. A final experiment exhibited AID binding to promoter sequences of genes which are required to undergo demethylation for expression (OCT4 and NANOG in human fibroblasts and Cdx2 in mESC), giving further credence to the role of AID in the reprogramming process, although, the authors note, this does again suggest that other factors may be required to initiate DNA demethylation at promoter sequences.

This model system represents an attractive tool in which to study the reprogramming process and identify other factors involved alongside the reprogramming effectors, such as early epigenetic changes, other important regulators of cell fate (perhaps by altering the differentiated cell type) and also the study of other interesting cell types where reprogramming occurs, for example, primordial germ cells (PGCs). However this system is reliant on species-specific expression and chromatin immunoprecipitation PCR analysis and a large degree of homology between species for the desired pathways or systems to be interrogated.

In another related study, Popp and colleagues investigated genome-wide DNA methylation and demethylation in relation to AID, with a particular emphasis on primordial germ cells (PGCs) in mouse. This was established using unbiased sequencing of bisulphite-treated DNA by next generation sequencing, linked to analysis by Illumina Solexa arrays. This analysis revealed that DNA from male and particularly female PGCs was highly unmethylated (approximately 15 and 7% methylation of total DNA respectively) compared to methylated DNA levels in sperm (~85%), mESC (~80%), the foetus (~75%) and placenta (~45%). Further, this investigation was repeated with AID-deficient tissues. In most tissues examined (sperm, foetus and placenta) AID deficiency did not alter the level of DNA methylation, however in male and particularly female PGCs an increase in methylation was observed (~5% and ~10%), indicating a loss of DNA demethylation in these cells. This loss of DNA demethylation suggests that AID is important for this process but, in line with the findings of Bhutani and colleagues, that other factors are involved. Results from more detailed genomic analysis (promoters, exons, introns, intergenic sequences and retrotransposable elements) further showed that levels of DNA methylation are at their lowest in PGCs and that this cell type displays the greatest increase in DNA methylation under AID deficiency.

This study suggests that DNA methylation is lost during normal development in PGCs and thus, as the authors note, may limit the scope for potential trans-generational epigenetic inheritance. Importantly, he authors also note that this study suggests that trans-generational epigenetic inheritance may be under genetic control.

Both papers clearly implicate the role of AID in DNA demethylation, but also that other mechanisms are likely to contribute to this process. Suggested candidates are the APOBECs which have deaminase activity, the TET family of genes, which may act to remove 5-methyl-cytosine by oxidation, or glycolases. Continued investigation into the factors involved in DNA repair mechanisms, such as base-excision repair, would also be of great interest for further study of the reprogramming processes involved in PGC development and specification, SCNT, and iPSC generation alongside other normal developmental processes.

 

References and Further Suggested Reading

 

Original Articles

Reprogramming towards pluripotency requires AID-dependent DNA demethylation.

Bhutani N, Brady JJ, Damian M, Sacco A, Corbel SY, Blau HM.

Nature. 2010 Feb 25;463(7284):1042-7.

 

Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency.

Popp C, Dean W, Feng S, Cokus SJ, Andrews S, Pellegrini M, Jacobsen SE, Reik W.

Nature. 2010 Feb 25;463(7284):1101-5.

 

Editorials

AID in reprogramming: Quick and efficient: identification of a key enzyme called AID, and its activity in DNA demethylation, may help to overcome a pivotal epigenetic barrier in reprogramming somatic cells toward pluripotency.

Deng W.

Bioessays. 2010 Apr 9;32(5):385-387.

 

AID for reprogramming.

Agarwal S, Daley GQ.

Cell Res. 2010 Mar;20(3):253-5.

 

Other Related References

 

DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45.

Rai K, Huggins IJ, James SR, Karpf AR, Jones DA, Cairns BR.

Cell. 2008 Dec 26;135(7):1201-12.

 

Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues: implications for epigenetic reprogramming.

Morgan HD, Dean W, Coker HA, Reik W, Petersen-Mahrt SK.

J Biol Chem. 2004 Dec 10;279(50):52353-60.