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    • Introduction Self renewal and the potential

    2018-11-06

    Introduction Self-renewal and the potential to differentiate into any cell type are the defining features of pluripotent stem cells (PSCs). Maintaining the ddr1 in this unique state is a carefully regulated process that requires the coordinated interaction of multiple transcription factors (TFs) and epigenetic regulators (Jaenisch and Young, 2008). A subset of core pluripotency factors, including OCT4, SOX2, and NANOG, have been the focus of many studies, and their target loci and protein interaction partners have been reported (Apostolou et al., 2013; de Wit et al., 2013; Jaenisch and Young, 2008; Kim et al., 2008). Several other genes have been linked to the regulation of pluripotency, including Sall4, Esrrb, Tbx3, Tcl1, and Utf1 (Ng and Surani, 2011). Undifferentiated embryonic cell transcription factor 1 (UTF1) was one of the initial 24 factors selected by Takahashi and Yamanaka (2006) to screen for cellular reprogramming to pluripotency, and was later shown to increase reprogramming efficiency when included with the four core factors (Zhao et al., 2008). Utf1 was first classified as a transcriptional coregulator expressed in embryonic stem cells (ESCs), embryonic carcinoma cells, cells of the germline, and teratocarcinoma cells (Okuda et al., 1998). Subsequent knockdown studies of Utf1 suggested that it plays a regulatory role in cellular differentiation by controlling the chromatin organization at its associated target sites, many of which are cobound by the core pluripotency factors OCT4, SOX2, and NANOG (Kooistra et al., 2010). More recent reprogramming studies have shown that Utf1 undergoes early chromatin dynamics that precede its transcriptional activation (Koche et al., 2011), and based on single-cell, time-course expression analysis, it appears that Utf1 is expressed in a restricted population of reprogramming cells, making it a potential marker to detect early, successful reprogramming events (Buganim et al., 2012). Another recent study reported that UTF1 and PRC2 compete for the same target sites, thereby balancing the deposition of H3K27me3 at bivalent genes, which in turn may be essential to control the induction of the respective genes upon exit of pluripotency (Jia et al., 2012). In line with that, Utf1 knockout cells showed less effective differentiation. As a complementary mechanism, UTF1 recruits the DCp1a subunit, a component of the mRNA-decapping machinery, to these same bivalent genes (Jia et al., 2012). Therefore, UTF1 has been proposed to serve a dual role in preventing excessive binding of the PRC2 complex to bivalent genes while simultaneously facilitating the tagging of mRNA from leaky repression for degradation. Interestingly, Marks et al. (2012) showed that Utf1 is one of the most downregulated genes upon culture under the inhibitor-based, serum-free 2i/leukemia inhibitory factor (LIF) condition (i.e., dual inhibition of MEK1 and GSK3 by small-molecule inhibitors), which maintain pluripotent cells in a more homogeneous undifferentiated state (Ying et al., 2008). Utf1’s tightly regulated expression during reprogramming and within pluripotent cells under conventional serum/LIF conditions, in addition to its potential role as a transient suppressor of lineage-specifying genes, support the growing interest in this factor. Here we report the generation and characterization of Utf1 reporter cell lines, and highlight their utility and faithful expression with several relevant examples.
    Results
    Discussion Defining and characterizing key regulators of the pluripotency network remains a major task in the stem cell field. Utf1 is tightly linked to pluripotency, but despite several recent studies, its exact role remains incompletely understood. Here, we report the generation and characterization of two Utf1 reporter lines and demonstrate their utility for efficient tracking, mapping, and purification of the endogenous UTF1 protein. Our results confirm the dynamic expression of Utf1 in vivo and in vitro, and show the power of these reporter systems for live-cell imaging. Two findings of particular relevance to the stem cell community are the rapid and reversible downregulation of Utf1 when ESCs were switched from serum/LIF to 2i, and the late but highly specific upregulation of this factor during reprogramming. The dynamic regulation observed during the switch to 2i not only establishes Utf1 as a very sensitive marker that distinguishes two LIF-regulated pluripotent states but also suggests that it may be a possible regulator of downstream transcriptional and epigenetic changes (Marks et al., 2012). The reprogramming experiments highlight the utility of Utf1 as a specific marker for the isolation of fully reprogrammed cells, and suggest that it may facilitate screens for small molecules or additional factors that specifically enhance late-stage transitions from intermediate to fully reprogrammed cells. In summary, the multipurpose design and high specificity of the endogenous Utf1 reporter system make it a valuable resource that can be specifically applied to address numerous questions in stem cell and developmental biology.