Fig. 1: Schematic, overlaid on a colony of human embryonic stem cells (hESCs), showing how the transcription factors PBX1, KLF4, SOX2 and OCT4 work together to activate the NANOG gene, which forms an auto-regulatory network with SOX2 and OCT4 and controls numerous functions related to hESC maintenance.
© 2009 A*STAR
Embryonic stem cells (ESCs) are remarkable for their ability to develop into literally any cell type in the body. This so-called pluripotency is not a passive state, however, and requires the active, coordinated effort of several genes encoding maintenance factors that help ESCs to preserve their ‘stem-ness’ and essentially prevent them from maturing. These same genes can also be used to reprogram adult cells into induced pluripotent stem cells (iPSCs), which are seemingly identical to naturally derived ESCs.
The NANOG gene and its upstream activators OCT4 and SOX2 are among the most essential contributors, regulating transcription of numerous important stem cell genes. “These transcription factors are a high-level ‘master switch’ for pluripotency in stem cells,” says Ken Kwok-Keung Chan of the Bioprocessing Technology Institute of A*STAR in Singapore, “and they are even more important in iPSC reprogramming.” To help fill the gap in our knowledge about how NANOG’s involvement in stem cell maintenance is controlled, Chan and co-workers developed an assay that enabled them to identify two additional factors that contribute to human ESC (hESC) pluripotency via direct regulation of NANOG.
The researchers found that these factors, KLF4 and PBX1, stimulate NANOG gene activity and protein production; importantly, the genes encoding these two factors are highly activated in hESCs, with expression levels more than double those observed in differentiated cells.
The NANOG gene is controlled by an upstream regulatory region called the promoter, which contains four stretches of DNA whose sequence has been highly conserved throughout evolution. They also revealed that two of these sequences (CR1 and CR2) are binding sites for PBX1 and KLF4, and appear to facilitate NANOG’s role in maintaining hESC pluripotency.
Chan and co-workers then showed that PBX1 and KLF4 work cooperatively in stimulating NANOG activity, but they observed the highest levels of activation when these factors were accompanied by OCT4 and SOX2, indicating that all four act synergistically to maintain pluripotency (Fig. 1). Interestingly, KLF4 and PBX1 could each be replaced by other, closely related transcription factors, such as KLF2 and PBX2, suggesting that these other proteins may act as redundant ‘failsafe’ regulators for hESCs.
Having uncovered another component of the pluripotency maintenance system, Chan and co-workers now plan to delve deeper into this process. “We are still interested in understanding the molecular mechanisms controlling hESC self-renewal and differentiation,” he says. “Our team’s future plan is to investigate the epigenetic factors and other complexes involved in controlling these functions.”
The A*STAR-affiliated authors in this highlight are from the Bioprocessing Technology Institute.
