X-inactivation: quantitative predictions of protein interactions in the Xist network

Abstract

The transcriptional silencing of one of the female X-chromosomes is a finely regulated process that requires accumulation in cis of the long non-coding RNA X-inactive-specific transcript (Xist) followed by a series of epigenetic modifications. Little is known about the molecular machinery regulating initiation and maintenance of chromosomal silencing. Here, we introduce a new version of our algorithm catRAPID to investigate Xist associations with a number of proteins involved in epigenetic regulation, nuclear scaffolding, transcription and splicing processes. Our method correctly identifies binding regions and affinities of protein interactions, providing a powerful theoretical framework for the study of X-chromosome inactivation and other events mediated by ribonucleoprotein associations.

Figure 1.

Xist RepA, 4R, 2R and PcG proteins. We predict that Xist RepA (227–760 nt) binds strongly to (a) SUZ12 (RNA interaction strength = 99%), and (b) EZH2 (RNA interaction strength = 75%), in agreement with experimental evidence; (c) SUZ12 does not bind to repeat 4R (318–521 nt; RNA interaction strength = 22%), while (d) EZH2 shows high interaction propensity (RNA interaction strength = 92%). Neither (e) SUZ12 nor (f) EZH2 are in contact with repeat 2R (401–552 nt; RNA interaction strengths = 0; Supplementary Table S1c) (7). Insets (b, d and f) are secondary structures of RepA (red line), 4R and 2R (blue dots) proposed by Maenner et al. (7).

Figure 2.

Xist and alternative splicing factor SFRS1. The interaction fragments algorithm is used to predict Xist ability to interact with SFRS1. (a) SFRS1 shows high propensity to contact Xist 5′. (b) The region studied by Royce-Tolland et al. (8) is marked in grey (nt 16–1181). In agreement with experimental evidence, strong interaction propensity is predicted between SFRS1 and nt 16–1181 (protein interaction strength = 84%); (c) nucleotides 164–932 nt (marked in red) correspond to an RNA region whose deletion abolishes Xist splicing (8). Strong interaction propensity is predicted between SFRS1 and nt 164–930 (protein interaction strength = 92%), as previously reported (8).

Figure 3.

SFRS1 and Xist 5′-UTR. We predict that SFRS1 interacts with the 5′-UTR exon region of Xist, in agreement with CLIP-seq experiments (18).

Figure 4.

Xist and transcriptional repressor Ying and Yang (YY1). The interaction strength algorithm is used to predict YY1 ability to interact with Xist. (a) High interaction propensity is found between YY1 and Xist Repeat C region (RepC; interaction strength = 77%). (b) No interaction is predicted between YY1 and RepA (interaction strength = 0%), as previously reported (9). (c) Experimental binding levels of AF, B, C, BC and eE1 fragments (red bars) are reproduced by catRAPID (blue bars) with high accuracy (Pearson’s correlation = 92%; P = 0.04 estimated with analysis of variance, two-tailed t-test (9) (Supplementary Table S1b). Interactions strengths and RNA-binding levels are normalized subtracting GFP signals (Supplementary Figure S2b). Errors on catRAPID predictions are evaluated using the second derivative of the cumulative distribution function associated with the interaction strength.

Figure 5.

Xist, scaffold attachment factor SAF-A and special AT-rich sequence-binding protein SATB1. (a) In agreement with experimental evidence, SAF-A is predicted to contact Xist in more than one region (10). Red lines and grey boxes indicate experimentally validated regions involved in Xist localization (6). Stars mark primers of elements studied by Hasegawa et al. (10). (b) SAF-A shows strong propensity to bind to Xist region 4934–5056 nt (protein interaction strength = 99%). (c) Multiple binding sites are predicted between Xist and SATB1. (d) We predict that SATB1 binds strongly to nt 292–698 (RepA; protein interaction propensity = 70%), as previously suggested (6,11).

Figure 6.

Xist first exon. RepA and RepC (yellow lines) encompass nt 227–760 and 3098–4713 (8,15). YY1 interactions investigated by Jeon and Lee (9) correspond to nt 1–2406 (AF), 1898–3083 (B), 3084–4940 (C) and 6990–9467 (eE1). The localization signals identified by Wutz et al. (6) are indicated by grey lines at nt 292–698, 1899–3488 and 4725–6079. The primers used by Hasegawa et al. correspond to nt 2339–2515 and 5125–5227 (10).