Kenneth Zaret is the associate director of the Penn Institute for Regenerative Medicine and is also Professor of Cell and Developmental Biology at the University of Pennsylvania. Zaret’s laboratory has examined the process by which adult cells are reprogrammed to make induced pluripotent stem cells (IPSCs). Shinya Yamanaka won the Nobel Prize this year for the discovery of iPSCs, and iPSCs seem to offer many of the advantages of embryonic stem without the moral messiness of destroying human embryos to make them.
The production of iPSCs required genetic engineering techniques that introduce genes into cells. Introducing four genes – Oct4, Sox2, Klf4, and c-Myc – into adult cells drives them to de-differentiate at become iPSCs, but this process is very slow – it can take up to a month – and is quite inefficient – one in 1,000 cells becomes an iPSC. Also, even though iPSCs share many characteristics with embryonic stem cells, they are not exactly the same and differ in some ways.
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This study, which was published in the journal Cell, attempted to define the reasons why iPSC production takes so long and is so inefficient. Zaret and his group examined the genomes of adult cells, 48 hours after the introduction of the four genes, and compared them to the genomes of the starting adult cells, fully reprogrammed iPSCs, cells near the end of the re-programming process (pre-iPSCs), and embryonic stem cells.
At 48 hours, Zaret and others found that of the transcription factors that had been introduced and overexpressed in the adult cells, three of them, Oct4, Klf4, and Sox2, tended to bind to the “distal enhancer elements” of genes. The phrase “distal enhancer elements” refers to regions of genes that help control when the gene is turned on. Most genes consist of a sequence of DNA known as the “coding region” that contains the sequences that are used to make the messenger RNA that will be translated into protein. However, genes also have other sequences that tell the cell when and where to make the messenger RNA. These control sequences are called “enhancers.” The Oct4, Klf4, and Sox2 proteins bind to enhancers in target genes and influence their expression. Because these proteins do this early heavy lifting, Zaret called them “pioneer factors.”
Now, there is a problem. The DNA of the cell is not an open book, but is bound up into a compact structure called chromatin. DNA tightly wound into chromatin does not allow access to proteins. Therefore, the pioneer factors for cell reprogramming are ready to bind to enhancers, but the DNA of the genome is too tightly wound to permit their binding. What is the cell to do? This is the job of the c-Myc protein, which enhances the binding of the other pluripotency factors to chromatin.
There is another problem, however, and this is the genuinely remarkable finding of Zaret’s lab: 48 hours after the initiation of reprogramming, large sections of the genome of the cell are refractory to the binding of the pioneering factors. In Zaret’s own words: “Basically, large chunks of the human genome were physically resisting these factors from entering. That provided some understanding that you’ve got to overcome the binding requirement to get these factors to their final destination.”
What caused these chunks of the genome to be off-limits to the pioneer factors? Chromatin results from the assembly of DNA with very positively-charged proteins called histones. Histones act as miniature spools around which the DNA is wound and packaged. Chemical modification of the histones can influence the tightness of the chromatin. For example, the attachment of acetate groups tends to make chromating rather loose, and gene expression can readily occur, but the attachment of methyl (CH3-) groups tends to cinch the chromatin down so tightly that little gene expression can occur.
In the case of the off-limits portions of the genome in adult cells undergoing reprogramming, a histone modification called “H3K9me3,” which is a short hand for saying lysine residue number 9 on histone #3 had three methyl groups attached to it, blocks the pioneer factors from accessing the DNA under its structural compaction. However, if Zaret and his workers treated cells with an inhibitor that prevents the enzymes from modifying histones in this manner, they found that the reprogramming process was significantly accelerated.
Zaret thinks that these findings might not only tell us about the roadblocks to reprogramming, but also give us clues as to a way to work around the difficulties to reprogramming. His lab has uncovered a normal mechanism by which cells protect themselves from being reprogrammed under normal circumstances. In his own words: “We went into this thinking that we were going to learn something about the mechanism of conversion to pluripotency, but at the end of the day we ended up discovering new ways that cells control gene expression by shutting down parts of their genome.”
The importance of this work is difficult to overstate. In the words of Susan Haynes, from the National Institutes of Health General Medical Sciences division, which funded Zaret’s work, “These studies provide detailed insights into how reprogramming factors interact with the chromatin of differentiated cells and start them down the path toward becoming stem cells. Dr. Zaret’s work also identified a major structural roadblock in the chromatin that the factors must overcome in order to bind DNA. This knowledge will help improve the efficiency of reprogramming, which is important for any future therapeutic applications.”