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    • br Results br Discussion Here we demonstrate that

    2018-11-06


    Results
    Discussion Here, we demonstrate that the status of the X chromosome is dynamic during human female somatic cell reprogramming. Extending previous studies that examined X chromosome status in iPSCs after completion of reprogramming, we determined the change of X chromosome status in KN-93 at different stages of reprogramming and after they were established as iPSC clones. We found that strong ectopic expression of reprogramming factors markedly suppresses XIST and RNF12, and mediates the reactivation of the inactive X chromosome (reprogramming XCR [rXCR] in Figure S5D). Although it is transient, the reactivated X chromosome at rXCR is transcriptionally active (Figure 3). When reprogramming was completed, the nascent iPSC clones were shown to be composed mostly of cells possessing the inactive X chromosome marker H3K27me3 foci. These results suggest that the reactivated X chromosome state in cells under active reprogramming is transient, and the timing of inactivation of the X chromosome is well correlated with the silencing of ectopic reprogramming factors (Chan et al., 2009). In murine ESCs, the reprogramming factors Oct4 and Nanog bind to intron 1 of Xist and suppress its transcription, whereas Myc and Klf4 bind to DXPas34 of Tsix to activate the transcription, resulting in two active X chromosomes (Deuve and Avner, 2011). Likewise, the high expression of the reprogramming cocktail in the current study may suppress XIST in cells undergoing reprogramming and reactivate the inactive X chromosome. When cells become bona fide iPSCs, the retroviral silencing machinery becomes activated (Chan et al., 2009; Matsui et al., 2010) and reduces the ectopic expression of reprogramming factors and thus the suppression of XIST. Remarkably, the formation of monoallelic iPSC clones composed of the same inactive X chromosome indicates that out of many cells undergoing reprogramming, only one cell becomes an iPSC clone and thus has one allele of the inactive X chromosome. Tracing of the nascent iPSC clones that were composed mostly of H3K27me3 foci-positive cells showed that cells with no H3K27me3 markers became dominant in some clones during very early passages. There are two possible explanations for this: either H3K27me3 foci-positive class II iPSCs become H3K27me3 foci-negative class I or class III cells, or some existing H3K27me3 foci-negative iPSCs that have a growth advantage become dominant during passaging. In a detailed analysis of the X chromosome state, we found that class I iPSCs with two active X chromosomes, as well as class III iPSCs with one active and one eroded X chromosome, arose in early passages (Figure 4F). Although a previous report by the Eggan lab suggested that the long-term culture of iPSCs results in X chromosome erosion (Mekhoubad et al., 2012), our data show that X chromosome erosion occurs even in very early passages. Currently, it is unknown how class I iPSC clones arise from class II nascent iPSCs. Perhaps the neighboring nonreprogrammed cells suppress the XCR in nascent iPSCs via paracrine factors (Bendall et al., 2007; Xu et al., 2005), and when the iPSCs are picked and placed in a new culture plate without the influence of these factors, XCR may occur. During development, the X chromosome shows dynamic changes in state. The X chromosome becomes activated in the ICM in mouse (Lessing et al., 2013). Preimplantation human embryos also show two X chromosomes in the pre-XCI state (Lengner et al., 2010; Okamoto et al., 2011). Following random inactivation in the epiblast stage, the X chromosome becomes reactivated in PGC development (Sugimoto and Abe, 2007). The reactivation of the X chromosome during reprogramming shown by our results suggests that reprogramming mimics either preimplantation embryo development or PGC formation where XCR occurs. iPSC clones with different X chromosome status have been isolated by several groups (Ananiev et al., 2011; Cheung et al., 2011; Pomp et al., 2011; Tchieu et al., 2010). Some groups isolated iPSCs with one active X chromosome and others isolated two active X chromosomes. The medium used for reprogramming does not seem to be responsible for the different results, because all of these groups used a standard medium composed of knockout serum replacement and basic fibroblast growth factor (Amit et al., 2000). The reprogramming methods used by each group may not result in iPSCs with different X chromosome status. The Plath group used retro- or lentiviral polycistronic vectors that express four reprogramming factors in one backbone (Tchieu et al., 2010). The Ellis, Colman, Chang, and Eggan groups all used retrovirus for reprogramming (Ananiev et al., 2011; Mekhoubad et al., 2012; Pomp et al., 2011). We used a lentiviral STEMCCA vector that was used by the Plath group (Figure 3A). Although the Plath group did not report XCR, we found that the STEMCCA vector gives rise to iPSCs with XCR. Thus, the vectors used for reprogramming do not unambiguously explain the differential X chromosome status in iPSCs. Although isogenic iPSC clones can be isolated from reprogramming of female fibroblasts, some of the above-cited papers reported that monoallelic iPSC clones with only one inactive X chromosome, but no other X chromosomes, were isolated from some lines (Cheung et al., 2011; Pomp et al., 2011), whereas we readily isolated iPSC clones with two different X chromosome states. OCT4, SOX2, MYC, and KLF4 were shown to play critical roles in reactivation of the inactive X chromosome (Deuve and Avner, 2011; Lessing et al., 2013; Navarro et al., 2010). Differences in viral infectivity or the amount of virus added may have influenced the expression of reprogramming factors and thus X chromosome state during reprogramming. Indeed, the analysis of provirus integration in our iPSC clones showed many more integrations compared with those derived by the Eggan group (Figure S3G; Boulting et al., 2011; Mekhoubad et al., 2012). Another possibility is the difference in the fibroblast line of resistance to reprogramming, for which the epigenetic barriers may prevent the XCR during reprogramming. The different feeder conditions used cannot be ruled out as a possible cause, since the Yamanaka group showed that feeder cells that produce high LIF support the derivation of iPSCs with two active X chromosomes (Tomoda et al., 2012). However, the feeder we used does not express high LIF and is less likely to be a cause of X reactivation.