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RNA Interference Screen IDs New Human Embryonic Stem Cell Regulatory Genes

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – A team of researchers led by investigators in Singapore reported in the early, online version of Nature yesterday that they have used a genome-wide RNA interference screen to uncover genes involved in pluripotency and self-renewal in human embryonic stem cells (hESC).

"Our study helps to build a better understanding of hESCs and this will help in the development of technologies to further the utilities of these cells such as their potential to be used for clinical and therapeutic applications," senior author Huck-Hui Ng, stem cell and development biology group leader at the Genome Institute of Singapore and associate director of biology at the institute, said in a statement.

"The unprecedented scale of this screen has added considerable new information to our understanding of pluripotency and will help efforts to improve reprogramming of adult cells," Janet Rossant, a developmental and stem cell biology researcher at Toronto's Hospital for Sick Children, who was not involved in the study, added.

In particular, the team's RNAi screen and follow-up experiments highlighted the importance of certain chromatin remodeling and transcription factor-related genes in human stem cell function.

By characterizing one such transcription factor gene, PRDM14, the team uncovered clues to improving the efficiency with which differentiated human cells are reprogrammed to produce induced pluripotent cells.

"Altogether, our study uncovers a wealth of novel hESC regulators wherein PRDM14 exemplifies a key transcription factor required for the maintenance of hESC identity and the reacquisition of pluripotency in human somatic cells," Ng and his co-authors wrote.

In an effort to uncover regulatory networks in human stem cells, the researchers first did a genome-wide RNAi screen in human embryonic stem cells containing a green fluorescent protein reporter that could be used to distinguish between undifferentiated and differentiated cells.

Using a small interfering RNA library targeting more than 21,100 genes, they found 566 genes with potential roles in stem cell function, including a handful of genes identified in previous studies of mouse embryonic stem cells and several genes involved in transcription and translation.

In addition, protein-protein interaction predictions for hundreds of the genes pointed to stem cell networks containing chromatin remodeling, spliceosome, and other complexes, the researchers noted.

Using a second RNAi screen focusing in on 200 of the genes detected in the initial screen, combined with additional stem cell marker and screening data, the team further narrowed in on genes that seemed to be promising candidates for functional studies.

Of these, the researchers began characterizing one transcription factor, PRDM14, which seems to influence the expression of at least one gene previously implicated in pluripotency: the transcription factor gene POU5F1.

Their subsequent experiments suggest PRDM14 also improves the efficiency with which the transcription factors Oct4, Sox2, Klf4, and c-Myc can prompt human fibroblasts to become stem cells.

Nevertheless, they explained, PRDM14 does not seem to have the same sorts of stem cell related functions in mouse embryonic stem cells as it does in human embryonic stem cells. Instead, past studies suggest the gene is involved in germ cell establishment in mice.

And while they noted that more research is needed to better understand the role of some of the other transcription factor, chromatin remodeling, and signaling genes identified in their screen, those involved in the study say that their findings so far underscore the promise of the approach for finding key players in stem cell processes.

"Our study shows that these regulators may hold the potential in advancing the methodology and understanding the mechanisms of human somatic cell reprogramming," they concluded.

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