What is the relationship between induction of cell differentiation and DNA methylation status?

Question

This may sound like a broad question to ask, but I am working on interpreting a review article for my epigenetics course and I'm having trouble reconciling two seemingly contradictory things this review is saying.

Ehrlich, M., & Lacey, M. (2013). DNA methylation and differentiation: Silencing, upregulation and modulation of gene expression. Epigenomics, 5(5), 553-568. doi:10.2217/epi.13.43

On page 554,

Constantinides et al. found that treatment of an embryonic fibroblast cell line with 5-azacytidine (5azaCR), an inhibitor of DNA methylation, induces the formation of [myotubes] [35].

and

Treatment with DNA demethylating agents can not only convert non-myogenic progenitor cells to Mt, but also can induce other cell- type interconversions in progenitor cells. For example, with 5azaCR treatment, the C2C12 Mb cell line can be induced to express genes for key osteogenic transcription factors as well as adipocyte markers [40]. The outcome of DNA demethylation treatment is dependent upon the cell type as well as the treatment and growth conditions [41].

and

The conversion of a multipotent adult stem cell to dissimilar differentiation products by treatment with DNA demethylating agents can be explained by the hypothesis that some genomic methylation restricts the differentiation potential of progenitor cells [40].

These three quotes, all on the same page, seem to indicate that at the very least, induction of differentiation is contingent on demethylation. More specifically, DNA methylation restricts the possible differentiation activity that a progenitor cell can undergo. However, just a little further down,

Terminal differentiation predominantly led to increases in DNA methylation and both increases and decreases in H3K27me3, depending on the gene involved.

and

[...] to hypothesize that changing the DNA methylation status of pluripotency genes in vivo is critical to their function [43]. DNA methylation is considered a more stable repressive mark than repression-associated histone modifications [42]. The association of differentiation and the loss of pluripotency with DNA methylation at previously unmethylated sites (de novo methylation)is consistent with the inability of ESCs to differentiate when Dnmt1, the most abundantly expressed DNA methyltransferase gene, is homozygously knocked out [44].

I'm working on a presentation on DNA methylation and gene silencing, and I want to make an accurate portrayal of the role of DNA methylation in cell differentiation, and this is only a portion of the whole presentation, so I am not including a lot of data on this topic. However, I want whatever I do present to be self-consistent and accurate. How do I reconcile these two phenomena? From what I can tell, there is no explanation in the intervening text between these passages about how these two phenomena are similar but distinct. Am I accurate in determining that this is a difference between induction of differentiation and terminal methylation status? If so, how is it that ESCs can be differentiated with demethylating agents, but also ESCs have trouble differentiating when Dmnt1, which promotes methylation, is knocked out?

Answer 1

This is a good question. I'm not as steeped in this literature as I'd like, but here is my understanding of the process:

Methylation is one of the key methods by which cell fate is restricted. The review you're reading is giving specific examples of cell lines that are at least partially restricted into cells that are of an entirely different type. Thus de-methylation is a method for de-differentiation, which then allows differentiation along a different line.

Your three examples that it sounds like you're interpreting as induction of differentiation, are actually examples of that effect -- de-differentiation that then allows for differentiation to a different fate. Constantinides work, for example, showed the conversion of a non-myoblast precursor into functional striated muscle cells. Not only can fibroblasts be turned into muscle cells, myoblast cell lines can be made to look like bone cells or fat cells. None of these things would happen without re-programming the cell fate by de-differentiating the cell lines to form dis-similar differentiation products.

This is consistent with other findings about de-methylation in vivo. An important example is the global de-methylation of primordial germ cells, as demonstrated in this figure from the linked review, summarizing the methylation state of the germ line in mice. Pay particular attention to the blue line. The pronucleus of the highly differentiated spermatazoon (along with its highly methylated DNA and histones) undergoes global de-methylation. Its products include the primordial germ cells of the developing mouse, the most de-differentiated state, which, in the case that it is a male, then become methylated as they become primed to differentiate, and then differentiate.