The repeat A region of XIST is necessary and sufficient for silencing of flanking reporter genes. (A) Approximate location of genes analyzed on chromosome 3 relative to the schematic of full-length XIST cDNA construct showing regions included in shorter XIST constructs and location of qRT-PCR primer pairs p1 to p4 and p5 (vector primer pair used to amplify all XIST constructs). (B) Enhanced Green Fluorescent Protein gene (EGFP) expression following one to five days (d1 to d5) induction of full-length XIST or 5’A, measured by flow cytometry and shown relative to d0. (C) qRT-PCR analysis of expression within full length XIST transgene (p2) and upstream (p1) and downstream (p3, p4) of XIST sequence. Genomic DNA was used to normalize for amplification efficiency. Location of qPCR amplicon positions is shown in Figure 1A. (D) Expression of the reporter genes (Hygromycin gene (Hyg) and EGFP) and endogenous genes CLDN16 and IL1RAP following five days of transgene induction measured by qRT-PCR, relative to expression in uninduced cells (d0) and normalized to ACTB expression. Transgene constructs were full XIST, 5’A only, full XIST lacking the 5’A region or vector with no XIST as indicated. Error bars indicate ± 1 S.D. of four to six biological replicates. Significance (P-value <0.05) was calculated using a Mann–Whitney test comparing each transgene construct with the vector alone construct. (E) Allele-specific silencing of flanking endogenous genes following five days of transgene induction. The percent change in allelic ratio upon DOX induction relative to the ratio without DOX was measured by pyrosequencing for expressed polymorphisms in five genes up to 20 Mb from the integration site (see A). Transgene constructs were full XIST, 5’A only, full XIST lacking the 5’A region or vector with no XIST as indicated. Two technical replicates of three biological replicates were averaged for each datapoint.