Development of B cells, white blood cells that make antibodies, follows a progression of stages: common lymphoid progenitors, pre-pro-B cells, pro-B cells, pre-B cells, immature B cells, and then more mature and specialized B cells. By the time the development hits the pro-B stage, the cell is fated to stay a B cell rather than another type of cell.
But researchers from the School of Veterinary Medicine and Perelman School of Medicine have found that knockout of YY1in pro-B cells impairs this lineage commitment, enabling unusual plasticity in blood cell formation. YY1 is a ubiquitous transcription factor that is capable of both activation and repression functions and plays significant roles in cell proliferation and replication, DNA repair, and the development of embryos.
They found that YY1 knockout pro-B cells can generate T lineage cells—which help B cells produce antibodies—in vitro and in a mouse model. Their findings are published in the journal Genes & Development .
"The data has come out better than my wildest fantasy," says senior author Michael Atchison , professor of biomedical sciences at Penn Vet. He says of YY1, "Since it's expressed everywhere and it's involved with so many lineages, the potential for regenerative medicine is quite high."
He envisions the ability to temporarily knock down YY1, push cells in another direction, and then remove the block so now there is a new lineage. Muscle cells, for example, could be reimplanted into a patient with a muscle disease.
"Although B cell lineage plasticity has been observed following knockout of several lineage-specific transcription factors," the authors write, "YY1 is unique in being a ubiquitous transcription factor expressed in all cell types suggesting a potentially universal mechanism of lineage commitment."
Atchison says that, just after he came to Penn Vet as an assistant professor in 1989, one of his graduate students cloned YY1. It was a time when it was shocking that a transcription factor could activate some genes while repressing the expression of others, he says. Several labs published on this simultaneously more than 30 years ago, and another researcher named the transcription factor Yin Yang 1 because of this dual function.
Atchison says the basis for this paper began nearly a decade ago, with a former postdoctoral researcher who, looking at RNA sequencing data, had the idea that YY1 knockout pro-B cells could turn into T cells. Sarmistha Banerjee, first author on the paper, took over this work as a senior research investigator at Penn.
The researchers eliminated YY1 with Mb1-driven CRE. CRE is a protein that can delete DNA and Mb1 is a promoter that drives expression of CRE in pro-B cells.
"We saw this transition happening from B to T and thought it would be a gradual transition, but there were a few weird-looking genes that were expressed in the mature T cells that developed," Atchison says. Co-author Joshua Rhoades, a bioinformatician, suggested doing single-cell RNA sequencing, which Atchison says "turned out to be really transformative. Then we saw all these other cell types coming up."
The sequencing data showed that as YY1 knockout pro-B cells developed in culture, 85% identified as monocytic or dendritic cells, which are involved in the presentation of antigens to B and T cells to mount an immune response, while only 3% identified as T cells. But incubation for three weeks resulted in the downregulation of most of these alternative lineage genes and increased expression of T lineage genes.
"A very useful, insightful, and unexpected finding was that while we were pushing these B cells to T cells, in the appropriate T cell environment, we observed that during this transition (midway) they were making a lot of other cell types," says co-author Sarah Naiyer, a research associate in immunology at Penn. She says, "Because we showed that Notch, a receptor in a highly conserved cell signaling pathway, was involved in the process, this could mean that it's the gradient of Notch signaling that is required to make these other cell types during the early phase while also inhibiting their differentiation during later stages, pushing them to T cells."
Atchison says it was satisfying to then see these results replicated in vivo. "It's coming out pretty close to what we fantasized about how the genome has changed when you knock out YY1 and why that allows the cells to be less committed to one lineage and able to be pushed into another direction," he says.
Looking forward, the mountain of data the team amassed "can be mined for a long time to look at what's happening when you knock out YY1," Atchison says. "We've looked at a small handful of genes, but there's 20,000 genes in the genome, and there's a lot we can do just with the data we have."
Another avenue of research would be to see how well YY1 knockout works in other cell types and tissues, and another would be to see if the T cells could yet still move to another lineage.
Michael Atchison is a professor in the Department of Biomedical Sciences, director of the VMD-Ph.D. Program, and director of the NIH/Boehringer-Ingelheim Summer Research Program at the University of Pennsylvania School of Veterinary Medicine.
Sarmistha Banerjee is a former researcher in the Atchison Lab at Penn Vet.
Sarah Naiyer is a research associate in immunology at Penn Vet.
Joshua Rhoades is a bioinformatician at Penn Vet.
The other co-authors are Penn Vet's Nasreen Bano, Dawei Dong, Suchita Hodawadekar, and Sulagna Sanyal; and David Allman and Anupam Banerjee of the Department of Pathology and Laboratory Medicine at the Perelman School of Medicine.
This work was supported by the National Institutes of Health (grants R01AI162879, R01AI155540, R01AI139123, and R01AI175185) and an Aspire award from the Mark Foundation for Cancer Research.
