Those picking the lock of pluripotency tend to focus on transcription factors, particularly the combination of Nanog, Sox2, Oct4 and others, which allow cells to proliferate without differentiating. More recently, researchers have found pluripotency depends not just on how transcription factors bind DNA, but in how DNA is packaged up into chromatin.

To explore this function, researchers led by Barbara Panning at the University of California studied over a thousand genes associated with chromatin regulation. Using RNAi, they knocked down the genes one by one to see how that would change mouse embryonic stem cells. Sixty-eight genes affected the cells' appearance or growth; seven of these encoded proteins that were part of a huge 17-member conglomerate called the Tip60-p400 histone acetyltransferase and nucleosome remodeling complex, and that's where Panning decided to focus her efforts.

Left, normal embryonic stem cells. Right, embryonic stem cells lacking Tip60-p400 Credit: Panning laboratory

“We knew it was going to be interesting because of the unusual change in the appearance and because it didn't alter expression of the known pluripotency transcription factors,” says Panning. “It's like we hamstrung the transcription factors.” Even though these factors were present, the cells didn't look like ES cells: rather than growing in dense spheres, these cells grew in a flat layer with little cell-cell contact. They also couldn't form teratomas or embryoid bodies.

Tip60-p400 is an unusual suspect, says Panning; the protein complex is expressed in nearly all cell types and performs a range of housekeeping functions like DNA repair. It was not expected to have a special role in embryonic stem cells. But further work showed that while the complex generally has an activating role on genes in normal cells, it has a repressive role in ES cells.

The team used chromatin immunoprecipitation to learn where in the genome the complex binds and found that it likely binds to the regulatory regions of most genes, and in particular areas that have been chemically marked with a chromatin modification known as H3K4me3. This mark is often found near genes that trigger differentiation and that are repressed in a sort of spring-loaded fashion. Proteins known as polycomb factors sit on these genes, physically blocking their expression in a way that can be quickly reversed.

But Panning suspects another mechanism is at work as well: many of the genes that are misregulated in the absence of Tip60-p400 are also misregulated in the absence of Nanog. However, the two do not seem to interact directly. Plus, she says, the components of Tip60-p400 that weren't identified in the initial screen have an interesting story to tell.

But there are also plenty of stories in those genes that were identified in the screen, says Rick Young, who studies ES cells and gene regulation at the Whitehead Institute in Cambridge, Massachusetts. He calls the publication a landmark study. “With this broad range of genes she's identified, this protein complex is just the tip of the iceberg.”