Synergetic Cooperation of microRNAs with Transcription Factors in iPS Cell Generation

Abstract

Induced pluripotent stem (iPS) cells were first generated by forced expression of transcription factors (TFs) in fibroblasts. Recently, iPS cells have been generated more rapidly and efficiently using miRNAs with or without other transcription factors. However, the specific and collaborative roles of miRNAs and transcription factors in pluripotency acquisition and maintenance remain to be further investigated. Here, based on the miRNA profiling in mouse embryonic fibroblasts (MEFs), MEFs infected with Oct3/4, Sox2, Klf4 and c-Myc (OSKM) for 1, 2, 4, or 8 day, two iPS cell lines and ES cells, representing iPS activation and maintenance steps, we found that two unique miRNA sets are responsible for different steps of iPS generation, and the miRNA expression profiles of iPS cells are very similar to that of ES cells. Furthermore, we searched for transcription factors binding sites at the promoter regions of up-regulated miRNAs, and found that up-regulated miRNAs such as the miR-429-200 and miR-17 clusters are directly activated by exogenous TFs. The GO and pathway enrichment for candidate target gene sets of miRNAs or OSKM provided a clear picture of division and collaboration between miRNAs and OSKM during completion of the iPS process. Compared with the pathways regulated by OSKM, we found that miRNAs play critical roles in regulating iPS-specific pathways, such as the adherens junction and Wnt signaling pathways. Furthermore, we blocked miRNA expression using Dicer knockdown, and found that the level of miRNAs was decreased following this treatment, and the efficiency of iPS generation was significantly repressed. By combining high-throughput analysis, biostatistical analysis and functional experiments, this study provides new ideas for investigating the important roles of miRNAs, the mechanisms of miRNAs and related signaling pathways, and the potential for many more applications of miRNAs in somatic cell reprogramming.

microRNA Control of Mouse and Human Pluripotent Stem Cell Behavior

“…c-Myc directly binds the promoters of the miR-17-92, miR-106a-363, and miR-106b-25 clusters and is largely responsible for their activation (Liao et al 2011). In contrast, the miR-290 cluster has been consistently found to reactivate late in reprogramming, along with other core pluripotency transcription factors (Chen et al 2012, Judson et al 2009, Polo et al 2012). Interestingly, the promoter of this cluster is bound by all four reprogramming factors but is activated only following epigenetic remodeling (Judson et al 2009, Marson et al 2008).…”

“…The let-7 family, miR-10a/b, miR-21, miR-26a/b, miR-29a/b, the miR-30 family, miR-34b-5p, miR-34c, miR-99a/b, miR-100, miR-125a/b-5p, miR-136, miR-145, miR-146a/b, miR-199a-3p, miR-210, miR-301, and miR-434-3p have all been identified by multiple groups as miRNAs downregulated during the reprogramming of mouse embryonic fibroblasts (MEFs) with either Oct4, Sox2, and Klf4 (OSK) or Oct4, Sox2, Klf4, and c-Myc (OSKM) (Chen et al 2012, Polo et al 2012, Yang et al 2011). Fewer miRNAs have been found to be consistently upregulated during reprogramming.…”

“…Fewer miRNAs have been found to be consistently upregulated during reprogramming. Several groups have found that members of the miR-17-92, miR-106a-363, and miR-106b-25 clusters, as well as miR-183, are activated early in OSKM reprogramming of MEFs (Chen et al 2012, Li et al 2011, Polo et al 2012). c-Myc directly binds the promoters of the miR-17-92, miR-106a-363, and miR-106b-25 clusters and is largely responsible for their activation (Liao et al 2011).…”

“…Interestingly, the promoter of this cluster is bound by all four reprogramming factors but is activated only following epigenetic remodeling (Judson et al 2009, Marson et al 2008). Other miRNAs with known function in PSCs, including miR-200, miR-130/301/721, and the miR-302 cluster, have been reported to be activated during reprogramming, but the reported degree and timing of activation have been inconsistent, likely reflecting the stochasticity of the assay (Chen et al 2012, Li et al 2011, Liao et al 2011, Polo et al 2012, Ryul Lee et al 2012, Samavarchi-Tehrani et al 2010, Wang et al 2011). Indeed, miR-302 cluster reactivation during reprogramming reportedly requires not only Oct4 binding but also expression of the histone demethylase Jhdm1b and media supplementation with vitamin C, highlighting the variability introduced by both the reprogramming factors and media conditions on miRNA expression during this assay (Wang et al 2011).…”

Epigenetics and ncRNAs in Brain Function and Disease: Mechanisms and Prospects for Therapy

“…Since miRNAs can function by maintaining terminal differentiation and inducing developmental transitions, there has been a growing interest in miRNAs for stimulating stem cells to adopt specific lineages, reprogramming of differentiated cells to induce pluripotency [18][19][20] and, in general, for improving our understanding of stem cell biology [21][22][23]. As drivers or mediators of pathology, miRNAs are promising therapeutic targets.…”