Fig. 3

Allelic silencing of flanking endogenous genes upon XIST induction. a Allele-discriminating RT-PCR pyrosequencing assay for genes closest to 5 Mb of integration site for each integration site, comparing triplicate cDNAs from untreated cells (No DOX) and following 5-day DOX induction of XIST in duplicate pyrosequencing reactions. cDNA from a different integration was also assessed (additional assays are shown in Additional file 3). P values of significantly silenced genes are listed. b Summary of the silencing observed for individual genes for each of the nine integration sites (color-coded as shown in the legend); plotted by distance from the integration site on the chromosome (from short to long arm). The allelic change is shown as percent silencing, which was calculated as: (allele frequency No DOX – allele frequency 5d DOX)/allele frequency No DOX × 100) for the pyrosequencing assays. For the Xq integration the silencing was determined by q-RT-PCR since the chromosome is hemizygous. Phase was determined for the 3q integration but for all integrations the allelic change is shown as silencing. Integrations on other chromosomes showed no silencing upon DOX induction. c Correlation between extent of silencing and level of XIST RNA. Five different clones (symbols) and cultures show variation in the level of XIST RNA after DOX induction (as measured by qRT-PCR for XIST relative to PGK1), and for 12q this correlates well with the extent of silencing of two genes assayed by allelic pyrosequencing after RT-PCR (OAS3, P <0.0001; POLR3B, P = 0.0004). A similar analysis (d) for the chromosome 8p integration site showed a similar variation in XIST levels, but no correlation with extent of silencing of two loci on 8p. e Removal of XIST expression after 5-day DOX induction resulted in substantial reactivation of endogenous genes in the 8p and Xq integration sites