The production of healthy cells with full pluripotency is essential for stem cell research and its application in regenerative medicine. However, the low efficiency of iPS cell generation and the reduced pluripotency of these cells have long been obstacles. Therefore, fast and effective methods to adjust the pluripotent properties of cells will greatly accelerate the exploration of reprogramming mechanisms and the use of iPS cells in regenerative medicine.
Here, we identified an imprinted genomic region in mouse that was actively expressed in fully pluripotent stem cell lines, such as ES and 4n-iPS cells, but was repressed in partially pluripotent cells as represented by two 2n-iPS cell lines. Protein-coding genes, a large cluster of miRNAs, and other small RNAs derived from this Dlk1-Dio3 imprinted region consistently exhibited significant expression repression or depletion in the 2n-iPS cells as compared with the ES and 4n-iPS cells. Regardless of the genetic background and origin of the ES and iPS cell lines, the expression differences between the fully and partially pluripotent stem cells were consistently observed. Furthermore, although genes and miRNAs from this region had low overall expression in the 2n-iPS cell lines, their expression was slightly higher in the germline-transmittable 2n-iPS cells than in those without germline transmission ability. These results indicated that the degree of activation of the Dlk1-Dio3 imprinted region is positively correlated with the pluripotency levels of stem cells. The coordinated expression changes of protein-coding genes and miRNAs encoded by this region, measured in independent assays, supports the conclusion that this region is affected by some regulatory mechanism that may be responsible for or respond to the pluripotency status of a cell.
Our analysis revealed that the miRNA cluster encoded by the Dlk1-Dio3 region only presented in mammalian genomes and is highly conserved among mammals, indicating its specific and crucial role in regulating mammalian development. We expect that this miRNA cluster should have conserved functions in human and other mammals. As some other genes within the Dlk1-Dio3 region are also conserved in non-mammal species, it is very likely that the pluripotency-correlated expression of the entire Dlk1-Dio3 region is indeed a functional reflection of the miRNA cluster.
After extensively screening the gene and small RNA expression data used in this study, we were not able to identify any other genomic region exhibiting similar or opposite expression patterns as that of the Dlk1-Dio3 imprinted region. These data indicated that the Dlk1-Dio3 region might be the only long genomic locus that exhibits a clear on-and-off switch in cells with full versus partial pluripotency; thus, the expression state of this region is a strong candidate for a marker of the quality of iPS and stem cells from other sources. Fully pluripotent human stem cells are very difficult to obtain via the iPS technique, so identification of such a marker site may greatly accelerate the selection process for good iPS or ES cells and pave the way for their application in regenerative medicine.
The putative targets of miRNAs from the Dlk1-Dio3
region include three genes in the PRC2 silencing complex. These genes are all expressed at lower levels in the ES and 4n-iPS cells as compared with the 2n-iPS cells, perhaps as a result of higher miRNA expression in the ES and 4n-iPS cells. We hypothesize that the repression of PRC2 complex formation contributes to the lack of methylation and silencing of the Dlk1-Dio3
region, resulting in the activated expression of its encoded genes as observed in our results. The activated miRNAs may act in a feedback loop to further prevent the formation of PRC2 and maintain the active state of the Dlk1-Dio3
region. A recent study showed that PRC2 is not necessary for stem cell pluripotency maintenance (22
). Therefore, down-regulation of PRC2 in the ES and 4n-iPS cell lines should not affect their pluripotency. Many studies have been conducted to investigate the function of the Dlk1-Dio3
imprinted region, but no clear conclusions have been made. Here, we propose that some miRNAs from this region regulate cell pluripotency via gene silencing pathways. It is likely that the activated miRNA clusters are involved in the regulation of many developmentally related biological processes as revealed by gene ontology enrichment analysis.
The connection of the Dlk1-Dio3
region miRNAs with PRC2 also suggests potential applications of these miRNAs in cancer therapy. As one of the PRC2 components, the histone methyltransferase Ezh2 is frequently observed to be overexpressed in various types of cancers (23
). Modulating the formation of PRC2 by manipulating the Dlk1-Dio3
region miRNAs might serve as an approach to prevent the uncontrolled proliferation of some types of cancer cells.
The discovery of iPS technology demonstrated that the developmental status of cells can be reverted by the activity of a few transcription factors, but its underlying mechanisms remain unknown. We hypothesize that changes in the methylation status of an imprinted genomic region might be involved in the pluripotency regulation of cells, providing a testable model toward understanding the mechanism of iPS cell formation.
Scientists have long attempted to identify key components that regulate complex traits but with very little success in protein-coding genes. Our results indicated that cell pluripotency levels might be in partially controlled by a group of miRNAs, suggesting the possibility of miRNAs as master regulators of gene expression networks. The synergetic effects of multiple miRNAs toward the same outcome revealed by our model also furthered our understanding of miRNA function mechanisms and shed light on future studies on the coordinated effects of miRNAs.