1. Volume 57, Issue 2, Pages 361-375 (January 2015)
Locus-Specific Targeting to the X Chromosome Revealed by the RNA Interactome of CTCF 
Johnny T. Kung, Barry Kesner, Jee Young An, Janice Y. Ahn, Catherine Cifuentes-Rojas, David Colognori, Yesu Jeon, Attila Szanto, Brian C. del Rosario, Stefan F. Pinter, Jennifer A. Erwin, Jeannie T. Lee 
Molecular Cell 
Volume 57, Issue 2, Pages (January 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 57, 361-375DOI: (10.1016/j.molcel.2014.12.006)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 The CTCF-RNA Interactome
(A) Percentage of total, sense, and antisense CTCF CLIP peaks from d0 mESC in indicated genomic regions.
(B) Average d0 CLIP peaks profile over a 3 kb metagene (RefSeq genes) ±1 kb flanking region.
(C) Average profile of d0 CLIP peaks within ±4 kb of TSS and TTS.
(D) Scatterplot comparing CLIP-seq and input RNA-seq coverages. d3 data are shown. Input RNAs were assembled from RNA-seq data. For CLIP-seq, coverage under all peaks within each transcript was summed for division over the transcript length. Red diagonal, x = y. Pearson’s correlation r = 0.287, p = 2.94 × 10−178.
(E) Metagene profiles comparing d3 CTCF CLIP (red) and ChIP (blue) peaks.
(F) Average profile of d3 ChIP peaks relative to CLIP peaks. CLIP-seq peak is centered at nt 0 on the x axis.
See also Tables S1 and S2 and Figures S1–S4.
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4. Figure 2 The RNA Interactome and Epigenomic Landscape of CTCF
Normalized CTCF CLIP-seq, ChIP-seq, and RNA-seq signals for (A) Sox2, (B) Sra1, (C) Jpx, (D) Xite and 5′ end of Tsix, and (E) 5′ end of Xist. Below each CLIP and ChIP tracks are corresponding statistically significant “peaks.” Red dashed lines denote repeat motifs within Xist/Tsix. P1 and P2, two Xist promoters. +, Watson strand; −, Crick strand.
See also Tables S1 and S2 and Figures S4 and S6.
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5. Figure 3 Allele-Specific Binding of CTCF on the X Chromosome
(A–C) Day 3 CTCF ChIP peaks, CLIP peaks, and RNA-seq signals over (A) the X chromosome, (B) Kdm6a, and (C) Mid1. Only statistically significant ChIP and CLIP peaks are shown. Composite (comp), sum of all peaks (cas, mus, and neutral). cas, Xa; mus, Xi. Day 3 ES composite Xist CHART, day 7 ES allelic H3K27me3 ChIP (Simon et al., 2013), and mouse embryonic fibroblast allelic H3K4me3 ChIP (Yildirim et al., 2011) data also included for comparison.
(D) qRT-PCR for in vitro RNA pull-down with FLAG-CTCF, FLAG-GFP, and mock. Representative results from four biological replicates shown. Means ± 1 SD. ∗p < 0.05, as determined by unpaired two-tailed Student’s t test comparing each amplicon to Ppia.
(E) UV-RIP qRT-PCR, comparing αCTCF and IgG immunoprecipitation in day 3 female mESC ± UV crosslinking. Means ± 1 SD are shown. Representative results from three biological replicates shown. ∗, significant enrichment (p < 0.05, determined by unpaired two-tailed Student’s t test) of +UV αCTCF pull-down over +UV IgG pull-down; †, significant enrichment of +UV αCTCF pull-down over –UV αCTCF pull-down.
See also Figures S4 and S5.
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6. Figure 4 CTCF Binds RNA Specifically with Very High Affinity
(A) RNA EMSA using 1.5 pmol of purified recombinant FLAG-CTCF or FLAG-GFP and 0.5 pmol of various in-vitro-transcribed, end-labeled RNA probes. Comp, unlabelled competitors at 40× molar excess. ∗, CTCF-RNA shift.
(B) RNA EMSA using 1.5 pmol of CTCF or GFP and 0.5 pmol of Tsix RNA fragments. Map of Xite/Tsix and EMSA probes shown. Comp, unlabelled competitors at 40× molar excess. ∗, CTCF-RNA shift.
(C) RNA EMSA with 0.5 pmol of purified Tsix probed and 1.5 pmol of full-length CTCF (FL), or GST-CTCF fragments: N, N-terminal domain; Zn, zinc-finger domain; C, C-terminal domain; or GST alone. Comp, unlabelled competitors at 40× molar excess.
(D) Double-filter binding assays were used to plot binding isotherms of CTCF at 0.2 nM RNA of indicated species. Serial 2.5-fold dilution from 0 to 30 nM active CTCF. CTCF concentrations corrected using the active fraction calculation (Figure S5B). Bound, nitrocellulose membrane. Free, nylon membrane.
(E) Left: binding isotherms for CTCF-RNA interactions. Right: Kd and R2 values for CTCF binding to RNA species. “≫30 nM” denotes a Kd above measurable range.
(F) Kd values do not correlate with RNA size (p = ).
See also Figures S5 and S6.
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7. Figure 5 CTCF Prefers RNA over DNA
(A) DNA EMSAs using 0.2 nM probe of the indicated species, titrated against 2-fold serial dilutions of active CTCF protein up to 300 nM. ∗, CTCF-DNA shift.
(B) Kd and R2 values for CTCF binding to various DNA species. “≫300 nM” denotes a Kd above measurable range.
(C) Map of competition EMSA probes. RNA probe corresponds to a CLIP-seq fragment of Tsix RNA as shown. DNA probes are derived from ChIP-seq peaks within Xite and Tsix, as shown.
(D) Competition EMSA using 0.2 nM Tsix RNA CLIP probe and 5 nM purified CTCF, in the presence of 0, 0.2, 2.0, 20, and 200 nM of cold DNA competitor (comp) indicated. ∗, CTCF-Tsix RNA shift. “Tsix CLIP DNA” refers to DNA probe bearing the sequence of the Tsix CLIP-seq fragment, serving as negative control.
(E and F) Reciprocal competition EMSA using 0.2 nM ChIP1 (E) or ChIP2 (F) DNA probe and 200 nM purified CTCF, in the presence of 0, 0.2, 2.0, 20, and 200 nM of the cold RNA competitor (comp) indicated. ∗, CTCF-DNA shift. ChIP1 and ChIP2 RNA refer to RNA probe bearing the sequence of ChIP1 and ChIP2, serving as negative control.
See also Figure S5.
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8. Figure 6 Tsix and Xite RNAs Are Required for X-X Pairing
(A) Map of Xic and pairing center, with positions for RIP-qPCR primers and EMSA probes (arrowheads). TsixKD positions (asterisks): blue, shRNA; green, LNA; red, LNA. The Tsix major promoter accounts for 90% of Tsix transcripts. Xite enhancer expresses an eRNA.
(B) RIP qRT-PCR, ±UV, on d3 female mESC at various domains with Tsix and Xite, with Jpx as positive control and U1 snRNA as negative control. qPCR positions shown in (A). Means ± 1 SD are shown from two biological replicates. All values normalized to 1% of input RNA. p, determined by unpaired two-tailed Student’s t tests comparing CTCF to IgG pulldowns in the +UV samples.
(C) Effect of TsixKD on pairing in female clones stably expressing shTsix versus shScr. DNA FISH using a two-probe combination of RP24 (centromeric) and pSx9 (Xist/Tsix) was performed. To exclude XO artifacts, we scored only nuclei with two RP24 signals. Cumulative frequency curves shown for decile with closest X-X distances. Whole distributions are shown in Figure S7. The significance of the difference, p, in pairwise comparisons between ScrKD and TsixKD on various differentiation days is determined using unpaired two-tailed Student’s t tests. Representative results are shown for two independent biological replicates. Sample sizes, n: ScrKD: 261 (d0), 297 (d2), 295 (d4), 254 (d6); TsixKD: 263 (d0), 332 (d2), 282 (d4), 246 (d6).
(D and E) Quantitation of Tsix RNA after ScrKD versus TsixKD using two LNAs. Pairing analysis performed as in (C). Whole distributions are shown in Figure S7. Representative results shown from two to three independent biological replicates. (D) Sample sizes: ScrKD: 295 (d0), 289 (d3), 294 (d6); TsixKD: 277 (d0), 303 (d3), 310 (d6). (E) Sample sizes: ScrKD: 212 (d0), 186 (d3), 171 (d6); TsixKD, 205 (d0), 202 (d3), 186 (d6).
(F) EB outgrowth of shTsix KD mESC was severely compromised. Scale bar, 100 μm.
(G) Pluripotency markers are appropriately downregulated in female shTsix KD cells, suggesting proper cell differentiation. Means ± 1 SD shown.
(H) Xist RNA FISH in shRNA TsixKD versus ScrKD during differentiation. p, determined by χ2 test comparing the distribution of Xist+ cells for ScrKD versus TsixKD from d0, d2, d4, and d6; ([observed-expected]2/expected), degrees of freedom = 3.
See also Figure S7.
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9. Figure 7 Tsix and Xite RNAs Target CTCF In cis to the Pairing Center
(A) Map of Xic and pairing center, with positions for ChIP primers. TsixKD positions, asterisks.
(B) ChIP-qPCR in stable shTsix or shScr KD clones. Means ± 1 SD shown from four biological replicates. p determined by unpaired two-tailed t tests.
(C) Control ChIP experiments. Top: ChIP-seq analysis of CTCF and various control epitopes (OCT4, SMC3, and H3K27me3) at other positions along the X chromosome. Bottom: ChIP-qPCR in shScr and shTsix at indicated sites of X chromosome shows that CTCF binding is not significantly affected by the Tsix/Xite knockdown. Means ± 1 SD shown for three independent biological replicates.
(D) Site-specific action of Tsix RNA facilitates locus-specific targeting of CTCF. POL-II transcribes Tsix RNA, which remains tethered to the site of synthesis as the RNA binds CTCF. A rapid turnover of Tsix RNA (t1/2, 30–60 min) enables its site-specific action. Targeting of CTCF in turn mediates X-X pairing.
Molecular Cell  , DOI: ( /j.molcel )
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