How pluripotency is controlled is one of biology's major puzzles. New research led by Kenneth Kosik of the University of California, Santa Barbara reveals not only a new piece in the puzzle but also how some major pieces fit together.

A set of transcription factors — Klf4, Oct4 and Sox2 — is sufficient to reprogram specialized cells to pluripotency. As pluripotent cells take on a differentiated fate, these transcription factors disappear. Kosik found that one microRNA (miRNA) is able to repress all three of these proteins.

Although these small RNA molecules can silence gene expression in all sorts of cells, their role in pluripotent stem cells is unclear. Kosik screened for miRNAs whose expression increased as embryonic stems cells differentiated.

One of these was miRNA-145, which exists in frogs, fish and mammals and is also enriched in many germline tissues. Computational tools predicted that it would hit mRNA transcripts for Klf4, Oct4 and Sox2.

To make sure that miRNA-145 interacted with the pluripotency proteins, the researchers attached the untranslated regions of Klf4, Oct4 and Sox2 onto the gene for luciferase and found that miRNA-145 repressed luciferase expression in several types of cells, whereas other miRNA constructs did not. Many more experiments, involving mutating the untranslated regions and upregulating and downregulating miRNA-145, all provided evidence that miRNA-145 inhibits these core pluripotency factors.

“[The work] is exciting for many reasons,” says Hannele Ruohola-Baker, who studies stem cells and miRNAs at the University of Washington in Seattle. This knowledge could be exploited not only to understand what keeps cells pluripotent or prompts them to differentiate, but also to make cell therapy safer by providing a mechanism to eliminate unwanted stem cells.

Right now, though, she says the research is at an exploratory stage, with researchers just starting to uncover the miRNAs that control stem cell states. “Let-7, miRNA-21 and now miRNA-145 have all been shown to be miRNAs that are not expressed in stem cells and, at varying levels of certainty, have been shown to make cells differentiate,” she says. “Who knows, maybe this one [miRNA-145] is the most powerful so far.”

And miRNA-145 seems to have a particularly interesting interaction with stem cells' pluripotency machinery. Further study of the genome identified an Oct4 binding site near the part of the genome coding for miRNA-145. Using a luciferase reporter tied to the miRNA-145 region, the researchers found that higher levels of Nanog did not affect the miRNA's expression, but higher levels of Oct4 did. Thus, miRNA-145 represses three powerful pluripotency transcription factors and is itself repressed by one of them — Oct4.

Kosik says that the identification of miRNA-145 and this interesting mechanism opens up many additional questions. “What we need to do now is understand how the double-negative feedback loop is creating a homeostatic state or an altered state. To do that we need a lot more information.”

For instance, he says, the number of copies of the various components could help set the balance between the differentiated state and the pluripotent state. Understanding that kind of stoichiometry will be difficult but could be essential to manipulating miRNAs.

There is a precedent for this sort of feedback loop in murine embryonic stem cells, says Isidore Rigoutsos, manager of the bioinformatics and pattern discovery group at the IBM Thomas J. Watson Research Center in Yorktown Heights, New York. He and others have shown that miRNA-296 targets Nanog and that Nanog and Oct4 regulate miRNA-296 (refs 2,3).

Still, Kosik's work is “particularly exciting,” Rigoutsos says. “It shows that the details of a feedback loop may change fairly drastically from organism to organism even if one confines oneself to studying specific genes.” Understanding such differences could be crucial to manipulating cell states for applications from disease modeling to drug discovery to cell therapy.

Related articles

Versatile microRNAs, two-faced transcription factors and embryonic stem cells

MicroRNAs boost reprogramming, boot out cMyc

A gene used to reprogram differentiated cells blocks microRNA processing

Efficient ES cell differentiation: the right tweak at the right time

Haifan Lin: peeling back layers of stem cell control

MicroRNAs help embryonic stem cells grow fast

MicroRNA reins in tumor-initiating cells

Repressing microRNAs for pluripotency