by Monya Baker

An RNAi screen finds a protein complex that keeps pluripotency genes open for transcription

In efforts to piece together the network that maintains pluripotency, one strategy is to remove potential components and see if pluripotency is disrupted. Researchers led by Frank Buchholz of the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, knocked down thousands of genes one by one and found evidence that embryonic stem cells have specialized gene-transcription machinery. Buchholz's team set up a screen so that they could identify whether levels of a key pluripotency protein, Oct4, dropped in the presence of a series of over 25,000 short RNA molecules (which could be mapped to approximately 15,000 annotated genes). This screen identified hundreds of genes, so the researchers focused on the ones that reproducibly made Oct4 levels drop the most dramatically, and they decreased this number to a set of 16 genes. (Previous to this process, they also ascertained that several known pluripotency genes showed up as hits in their screens, whereas 'housekeeping' genes not specifically required for stem cell maintanance did not.) Identified genes fwere associated with functions like transcription regulation (Nfya, Ptbp1, Ctr9, Rtf1, Wdr61, Cpsf3, Fip1l1, Iws1 and Thoc2), chromatin modulation (Rnf2, Cxxc1, Cnot1, Rtf1, Ctr9, Wdr61 and Ncl), cell signalling (Apc) and protein degradation/DNA repair (Ube2m and Shfdg1).

Two of the genes with the strongest effect coded for a protein complex, Paf1C, that guides RNA polymerase II on its gene-transcribing missions. Silencing these and other members of this six-protein complex lowered levels of Oct4, Nanog and several other pluripotency genes. When levels of Paf1C were increased, embryonic stem cells were less responsive to differentiation cues.

Further work determined where Paf1C interacts with the genome: it binds the promoters of several pluripotency genes, spots where the complex could keep chromatin unwound and open for gene expression. More specifically, Paf1C maintains the H3K4me3 markers on histones that are associated with active genes.

Interestingly, other researchers conducting a separate RNA interference (RNAi) screen that focused on genes involved in chromatin regulation identified another protein complex. Seven hits out of the approximately thousand genes studied were part of the 17-member conglomerate called the Tip60-p400 histone acetyltransferase and nucleosome remodelling complex, which was not previously thought to play a special role in embryonic stem cells.

Paf1C, however, seems to be more directly involved in the expression of other pluripotency genes. The authors believe it is probably an integral part of the Nanog-Oct4 regulatory circuit. "Understanding the endogenous regulation of Oct4 may reveal alternative ways to activate Oct4 expression in somatic cells," they write in the Cell Stem Cell paper. Not only could this help dissect how pluripotency is regulated, but also it has practical applications in terms of new ways to make induced pluripotent stem cells.