PUS7 replaced exogenous OCT4 in the Yamanaka cocktail for reprogramming human cells into stem cells: the resulting cells more closely resembled embryonic stem cells on several measures than those produced with the standard cocktail
PUS7 replaced exogenous OCT4 in the Yamanaka cocktail for reprogramming human cells into stem cells: the resulting cells more closely resembled embryonic stem cells on several measures than those produced with the standard cocktail
On October 12, Nature Chemical Biology published a study from the laboratories of Yanhong Shi (City of Hope) and Chuan He (University of Chicago). Cells from people with a mutation in PUS7 were much less efficiently reprogrammed into iPS cells (stem cells capable of becoming any cell type in the body). The authors traced the defect to a single RNA modification and used the enzyme to replace exogenous OCT4.
The Yamanaka cocktail consists of four proteins, OCT4, SOX2, KLF4 and MYC, introduced into a cell to reprogram it into a stem cell. Reprogramming human cells without exogenous OCT4 has rarely succeeded, although it has worked in mice: in 2019, Hans Schöler’s group used SOX2, KLF4 and MYC alone to generate mouse iPS cells that could develop into animals. The group suggested that excess OCT4 activates genes that are not needed for reprogramming.
PUS7 converts uridine in RNA into its isomer pseudouridine, creating a chemical modification. Mutations in PUS7 cause intellectual disability. The researchers used the Yamanaka cocktail to reprogram cells from four people with these mutations and four healthy people. Reprogramming involves two waves of gene activation: through day 6, cells carrying a mutation progressed through the first wave much like healthy cells, but they failed to initiate the second wave, when the cells’ own stem cell genes are activated.
Mapping pseudouridine modifications in iPS cells with and without PUS7 identified position 250 in 7SK RNA. This RNA had been linked to chromatin accessibility, and PUS7 had already been shown to modify it in colon cancer cells. ARID1A, a protein in the BAF complex that loosens DNA packaging, binds more strongly to 7SK carrying the modification. Without PUS7, less BAF associates with chromatin, chromatin becomes less accessible, and OCT4 binds DNA less frequently on day 16. Targeting PUS7 to 7SK with the dCas13b system restored the modification and allowed cells carrying the mutation to form colonies again.
Based on this mechanism, the authors tested whether PUS7 could replace exogenous OCT4, which works with BAF. They removed OCT4 from the cocktail along with an RNA encoded in the same construct that suppresses p53, a protein that limits the division of damaged cells, and added PUS7. The PSKM cocktail produced iPS cell colonies, and the cells formed tumors containing tissues from all three germ layers when injected into mice. SOX2, KLF4 and MYC produced almost no colonies without functional PUS7, but yielded iPS cells when PUS7 was targeted to 7SK. PSKM cells activated their own OCT4; disabling BRG1, a gene encoding a component of the BAF complex, reduced reprogramming efficiency. The authors write:
“To our knowledge, PUS7 is the first RNA-modifying enzyme capable of replacing OCT4 in the reprogramming of human cells into iPS cells.”
The researchers reprogrammed the same starting cells with the standard cocktail and with PSKM, then compared the resulting cells against embryonic stem cells as a reference. In the DLK1–DIO3 region of the genome, only the gene copy inherited from one parent is active, and previous studies have linked disruptions in this region to reduced developmental potential in iPS cells. Two genes in this region showed elevated activity in cells produced with the standard cocktail, whereas their activity in PSKM cells matched that in embryonic stem cells. Neural progenitors derived from PSKM cells activated brain gene programs, while those derived from cells produced with the standard cocktail shifted toward mesodermal programs. The overall gene expression profile of PSKM cells was slightly closer to that of embryonic stem cells. The authors attribute the difference to the composition of the cocktail, probably the absence of exogenous OCT4, while allowing for a contribution from the modification of 7SK.
Chromatin accessibility was nearly identical in cells produced with the two cocktails, although endogenous OCT4 bound to different DNA sites. The authors suggest that different reprogramming routes may lead to a similar stem cell state.