by Monya Baker
An analysis of when and where pluripotency factors bind indicate that c-Myc shuts down specialization and the remaining three turn on pluripotency
Scientists are still shocked that only a handful of introduced genes can turn a specialized cell's clock back so far that it behaves like an embryonic stem cell. Even with current established techniques, such reprogramming is a rare event, and researchers around the world are trying to figure out exactly how reprogramming can be triggered by genes for the pluripotency factors, which encode the transcription factors Oct4, Sox2, Klf4 and c-Myc. Publishing in Cell, researchers led by Kathrin Plath of the University of California, Los Angeles, show which genes are bound by the transcription factors in mouse embryonic stem cells, fully reprogrammed cells (induced pluripotent stem cells) and partially reprogrammed cells. The results, she believes, will help researchers find small molecules to replace the viral vectors currently used in the reprogramming process, which could result in more homogenous cells that are better suited for clinical applications.
To find out where the transcription factors were physically binding on DNA, Plath and colleagues used a technique known as chromatin immunoprecipitation (ChIP) assays. They examined two lines for each cell type and found that results were so similar within each cell type that the values could be averaged together.
The patterns were striking: c-Myc binding was very similar between partially reprogrammed, fully reprogrammed and embryonic stem (ES) cells. Binding for the other three factors was very similar only between fully reprogrammed and ES cells. Not surprisingly, genes co-occupied by the three factors were the ones that had the most dramatic expression changes between reprogrammed cells and fibroblasts.
This could not be because of the concentrations of Oct4, Sox2 and c-Myc. As cells are fully reprogrammed, they shut down the extra viral copies of the pluripotency factor genes and activate the endogenous genes — that means all of the pluripotency factors' levels are threefold to eightfold higher in partially reprogrammed cells than in fully reprogrammed cells. So why do Oct4, Sox2 and Klf4 behave so differently in partially reprogrammed cells?
Plath says the analysis suggests two reasons. The first is that the state of the chromatin in partially reprogrammed cells does not allow the factors to bind their sites. Some differences in binding, but not most, could be because the partially reprogrammed cells lack activating histone modifications nearby. The second is that Oct4, Sox2 and Klf4 must bind cooperatively with yet other pluripotency factors, particularly Nanog, that are not present in the partially reprogrammed cells.
The work suggests two approaches for finding ways to replace the viral vectors used for the four reprogramming factors. One is to hunt for proteins or small molecules that might activate the genes that code for Oct4, Sox2 and Klf4. Another is to figure out when and how to encourage the earlier process, in which c-Myc expression starts to reset fibroblast expression. Other work has shown that c-Myc can potentially be replaced by, say, valproic acid or Wnt3a ligands, she says. "It will be interesting if they have a similar mechanism."
Source: Nature