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. 2014 Aug 18;111(35):12591–12598. doi: 10.1073/pnas.1413620111

Fig. 3.

Fig. 3.

XCIFs function by promoting Xist expression and/or localization, and DNMT1 is a transcriptional activator of Xist on the Xi. (A) qRT-PCR analysis monitoring Xist expression in the 13 XCIF KD ES cell lines following differentiation. Expression in differentiated ES cells was normalized to that obtained with the NS shRNA, which was set to 1. Error bars indicate SE. (B) RNA FISH monitoring localization of Xist in the 13 XCIF KD ES cell lines following differentiation. Cells were categorized as having either a typical Xist cloud or “other” pattern, which includes either the lack of a detectable Xist signal or presence of two small Xist signals, as in undifferentiated ES cells. (C) RNA FISH monitoring expression of Xist (Upper) and Mecp2 (Lower) in BMSL2 cells treated with an Xist locked nucleic acid antisense oligonucleotide (LNA ASO) or a control LNA ASO. (D) ChIP analysis monitoring binding of DNMT1 and POL2 to the Xist promoter and exon 2 in BMSL2 cells expressing a NS or Dnmt1 shRNA. Error bars indicate SD. (E) Nuclear run-on assay monitoring transcription of Xist, Hprt, and Tbp in BMSL2 cells expressing a NS or DNMT1 shRNA. (F) qRT-PCR analysis monitoring Xist levels in BMSL2 cells expressing a NS or Dnmt1 shRNA following treatment with actinomycin D. Actin mRNA was used as a normalization control. Error bars indicate SD. (G) qRT-PCR analysis monitoring Xist expression in MEFs isolated from female Dnmt1+/+ and Dnmt1−/− embryos. Four different litters were analyzed (n = 4 mice total per genotype), and the results were averaged. Expression was normalized to that observed in Dnmt1+/+ MEFs, which was set to 1. Error bars indicate SD. *P < 0.001 (Student t test). (H) qRT-PCR monitoring levels of Xist and Tsix in H4SV cells expressing a NS or DNMT1 shRNA. Expression was normalized to that obtained with the NS shRNA, which was set to 1. Error bars indicate SD. (I) qRT-PCR analysis monitoring Hprt and Xist expression in BMSL2 cells treated in the absence or presence of 5-azacytidine (5-AZA). Expression was normalized to that observed in the absence of 5-AZA, which was set to 1. Error bars indicate SD.