Extended Data Fig. 3: PTBP1, MATR3, CELF1 and TDP-43 directly bind the Xist E-repeat, comprising a tandem array of 20–25nt C/U/G-rich elements.
From: A protein assembly mediates Xist localization and gene silencing
a (i), Top, diagram of the Xist genomic locus. The IVT E-repeat RNA used in d is indicated. Bottom, PTBP1, MATR3 and CELF1 i/eCLIP–seq profiles across the Xist locus in male tetO-Xist ES cells after 6 h of dox induction. CELF1 input profile is shown, read counts indicated on left. (ii), PTBP1 ChIP–seq profiles across the Xist locus before or after 20 h of dox treatment in male tetO-Xist ES cells. (iii), PTBP1, PTBP2 and TDP-43 iCLIP–seq profiles across the Xist locus in the female mouse brain. b, Table of mapping statistics for PTBP1, MATR3 and CELF1 i/eCLIP–seq data in a. Note that Xist is overexpressed in this experiment, which influences the number of reads mapping to the locus. c (i), The first 1,500 nt of exon 7 of Xist are shown, capturing the E-repeat. The sequence remaining after splicing of the XistΔE transcript is underlined and italicized. The C/U/G tandem repeats within the 5′ half of the E-repeat are indicated (pink-full and blue-truncated repeats) as are the CU-tracts (green) in the 3′ half. Potential TDP-43 sites are indicated in orange. (ii), Alignment of the 25 full C/U/G-tandem repeats (pink) from (i). Brown tracts encode putative PTBP1/MATR3 binding sites, red tracts putative CELF1/TDP-43 binding sites. (iii), Alignment of the nine truncated C/U/G-tandem repeats (blue) from (i). Orange coloured nucleotides are variable within each truncated repeat unit. d, Left: EMSA of IVT E-repeat RNA (see a) and either none, or increasing amounts of rPTBP1 (0, 1.95 nM, 3.9 nM, 7.8 nM, 15.6 nM, 31.3 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1 μM and 2 μM). Right, quantification of the bound RNA fraction (dissociation constant, Kd ≈ 200 nM, from two independent experiments, with s.e.m shown). For source data see Supplementary Fig. 1.
