A team of researchers from the University of Cambridge examined the early stages of somatic cell reprogramming using nuclear transfer (NT) in Xenopus oocytes to understand the mechanism that makes the method work. Cellular reprogramming could provide researchers with the required quantities of customized pluripotent stem cells to advance disease research and cell-based therapies. The research, published in the Proceedings of the National Academy of Science (PNAS), identified a linker histone in frog oocytes called B4, which binds to the somatic cell nucleus’ chromatin and turns the pluripotency genes back on.
“The challenge for the team was to figure out one of the very early events, which has to take place for the nuclei to be reprogrammed,” said John Gurdon, an emeritus professor in the zoology department at the University of Cambridge and one of the authors of the study.
In NT, researchers remove a somatic cell’s nucleus and transfer it into an enucleated egg cell. Xenopus oocytes—precursors to mature frog eggs—are often used as the host egg cell because they are larger, easier to culture, and more copious than human cells. After the transfer, the host egg cell reprograms the somatic cell nucleus. Exactly how that happens is a mystery that researchers are trying to decrypt.
Gurdon and his colleagues extracted nuclei from a human bone cancer cell line with a reporter gene attached and injected them directly into the transparent nuclei of oocytes, isolated from the opaque eggs. This allowed the researchers to monitor the reprogramming in real time under a confocal microscope. Less than 3 hours after the transfer, B4 from the oocyte had almost completely replaced another histone, H1, in the ES nuclei.
After confirming that B4 was a key player, the team further explored the mechanism. To determine whether the nuclei had to remove H1 before the addition of B4, the researchers tested whether histone replacement would still occur if the ES nuclei were saturated with H1. The team injected oocytes with mRNA that increased the production of H1 five-fold. Surprisingly, B4 replacement occurred during nuclear transfer just as before.
To take a closer look at B4, the researchers compared nuclei that had available B4-binding sites with control nuclei injected with an anti-B4 antibody that rendered the B4-binding sites unavailable. The team found that B4 accumulated near promoter sites for three pluripotency genes—sox2, nanog, and oct4—within six hours. After 24 hours, sox2 was reactivated and expressed at 100%, oct2 was moderately reactivated, and nanog was expressed in only some of the oocytes. Since the promoters for the other two genes are methylated, the researchers hypothesize that the results are somehow related to sox2’s unmethylated promoter. In the control nuclei injected with anti-B4 antibody, the genes remained off.
Gurdon said that it is likely that B4 is only one of many proteins that are involved in nuclear reprogramming, and has plans to look for others. “If we knew all these molecules, what they are, and how they reprogram, we might be able to introduce them into somatic cells and achieve reprogramming without having to use eggs.”
The paper, “Characterization of somatic cell nuclear reprogramming by oocytes in which a linker histone is required for pluripotency gene reactivation,” was published online ahead of print in the Mar. 8 issue of PNAS.