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  • Molecular ESC analyses and the discovery of


    Molecular ESC analyses and the discovery of iPSC reprogramming attributed pluripotency and self-renewal functions to the transcriptional regulators OCT4, SOX2, NANOG, and others (Ivanova et al., 2006; Loh et al., 2006; Takahashi and Yamanaka, 2006). Since then, numerous PGRN versions have been proposed (Festuccia et al., 2013; Yang et al., 2010), and the list of factors continues to grow. However, the hierarchy of the PGRN, the order of regulatory links, and the principles of PRGN function remain largely elusive. In our studies (see flowchart in Figure S1), hundreds of single mESCs grown either under serum + LIF or serum-free 2i + LIF conditions have been collected, and their expression signatures with respect to 46 pluripotency genes were retrieved using high-throughput microfluidic single-cell qRT-PCR (White et al., 2011). Clustering individual Baricitinib based on their gene expression profiles revealed the presence of two major cell subpopulations in cells grown under the serum + LIF condition. In contrast, under 2i conditions, the two populations collapsed into one, which is in agreement with recent data suggesting a reduction in gene expression heterogeneity in 2i versus LIF alone (Marks et al., 2012). Comparison of our single-cell data with published single-cell data (Kumar et al., 2014; Tang et al., 2010) established that one subpopulation detected under the LIF condition has a gene expression signature similar to that of the inner cell mass (ICM) (Boroviak et al., 2014), whereas the other subpopulation resembles more mature epiblast cells from the embryo. Detection of subpopulations became possible here because of the large number of analyzed cells (96 cells on each chip, seven chips in total). We integrated the single-cell data obtained in this study with the data available for knockdowns of major pluripotency transcription factors (Feng et al., 2009; Ivanova et al., 2006; Loh et al., 2006; Lu et al., 2009; Martello et al., 2012). PGRNs reconstructed based on the integrated data revealed network motifs such as incoherent feedforward loops (iFFL) (Goentoro et al., 2009; Milo et al., 2002; Papatsenko and Levine, 2011), linking OCT4 and NANOG with their target genes and suggesting an antagonistic interaction between OCT4 and NANOG. Certain genes alternatively regulated by OCT4 and NANOG (Sall4 and Zscan10) appear to feed back to Oct4 and Sox2. We discuss how these loops may stabilize OCT4 concentrations required for self-renewal.
    Experimental Procedures
    Author contributions
    Introduction Myotonic dystrophy type 1 (DM1) is an autosomal dominant muscular dystrophy that affects a wide range of body systems (DM1 [OMIM: 160900]). It results from a trinucleotide CTG repeat expansion (50–4,000 copies) in the 3′ UTR of the dystrophia myotonica protein kinase gene (DMPK) (Aslanidis et al., 1992; Brook et al., 1992). The CTG repeat region, which resides within a CpG island (CGI), commonly results in hypermethylation and the spread of heterochromatin when expanded (Cho et al., 2005; Filippova et al., 2001; Steinbach et al., 1998). Hypermethylation is largely age- and tissue-specific and does not necessarily correlate with expansion size in somatic cells of patients (López Castel et al., 2011; Spits et al., 2010). In addition, when the CTG repeats expand, they commonly result in a reduction in the expression of a neighboring gene, SIX5 (Klesert et al., 1997, 2000; Korade-Mirnics et al., 1999; Sarkar et al., 2000, 2004; Thornton et al., 1997). The contribution of hypermethylation to disease pathogenesis is still not fully understood, nor is the precise mechanism by which CTG expansion leads to SIX5 reduction in cis. Using a wide range of DM1-affected human embryonic stem cell (hESC) lines, we aimed to uncover the mechanistic relationship between CTG expansion, aberrant methylation, and reduced expression of SIX5 in DM1.