TY - JOUR
AU - Calabrese, J, Mauro
AB - Abstract Xist requires Repeat-A, a protein-binding module in its first two kilobases (2kb), to repress transcription. We report that when expressed as a standalone transcript in mouse embryonic stem cells (ESCs), the first 2kb of Xist (Xist-2kb) does not induce transcriptional silencing. Instead, Xist-2kb sequesters RNA produced from adjacent genes on chromatin. Sequestration does not spread beyond adjacent genes, requires the same sequence elements in Repeat-A that full-length Xist requires to repress transcription and can be induced by lncRNAs with similar sequence composition to Xist-2kb. We did not detect sequestration by full-length Xist, but we did detect it by mutant forms of Xist with attenuated transcriptional silencing capability. Xist-2kb associated with SPEN, a Repeat-A binding protein required for Xist-induced transcriptional silencing, but SPEN was not necessary for sequestration. Thus, when expressed in mouse ESCs, a 5′ fragment of Xist that contains Repeat-A sequesters RNA from adjacent genes on chromatin and associates with the silencing factor SPEN, but it does not induce transcriptional silencing. Instead, Xist-induced transcriptional silencing requires synergy between Repeat-A and additional sequence elements in Xist. We propose that sequestration is mechanistically related to the Repeat-A dependent stabilization and tethering of Xist near actively transcribed regions of chromatin. INTRODUCTION Long noncoding RNAs (lncRNAs) play essential roles in development and homeostasis by regulating gene expression (1). Emerging data suggest that lncRNAs encode regulatory function in a modular fashion via discrete domains that each recruit effector proteins to carry out specific actions (2–10). An example of this modularity comes from studies of the lncRNA Xist, which functions to silence nearly all genes along the 165 megabase (Mb) X chromosome as part of X-chromosome inactivation (XCI), the dosage compensation process that occurs early during the development of eutherian mammals. At the level of sequence composition, Xist is notable for the presence of several internal domains of tandem repeats (11–14). These repeats have been shown to recruit different subsets of RNA-binding proteins that help Xist achieve repressive function, and in this regard, they can be considered as functional modules (7–10). One such repeat is found in the first thousand nucleotides of Xist and is called ‘Repeat-A’. Repeat-A consists of eight to nine tandemly arrayed, 50 nucleotide long repeating elements that each harbor a degenerate U-rich region followed by a GC-rich region that is highly conserved among eutherians (11,12). Overexpressed Xist cDNA transgenes lacking Repeat-A accumulate around the mouse embryonic stem cell (ESC) inactive X chromosome in wild-type-like patterns, but they are incapable of silencing gene expression. Repressive function of the mutant transgenes can be restored by appending Repeat-A or synthetic, Repeat-A-like sequences to their 3′ ends, demonstrating that Repeat-A functions as a self-contained module necessary to confer gene silencing activity to Xist (15–17). Repeat-A is thought to induce gene silencing by recruiting the protein SPEN. Knockout or knockdown of SPEN in multiple experimental contexts results in failure of XCI, establishing SPEN as an essential Xist cofactor (7,18–20). In transgenic Xist cDNA overexpression experiments, Repeat-A is necessary to recruit SPEN to Xist (7). Concordantly, in vivo, iCLIP demonstrates robust association between SPEN and Repeat-A, and in vitro, SPEN associates with single-stranded, U-rich regions of Repeat-A that are located directly adjacent to its structured, GC-rich segments (3,21). In contexts outside of XCI, SPEN has been shown to repress transcription of target genes through its association with SMRT/NCoR and NuRD co-repressor complexes and several histone deacetylases (HDACs; (22–24)). Transient knockdown of SMRT and HDAC3 in male and in female ESCs reduces the efficacy of Xist-induced gene silencing, providing support for the notion that during XCI, SPEN silences gene expression by recruiting co-repressors to Xist (18). Despite clear links between SPEN and Repeat-A, questions remain regarding the mechanism through which Repeat-A functions. Most notably, while SPEN is necessary for XCI, it is not known if SPEN binding to Repeat-A is sufficient to induce transcriptional silencing (18). Moreover, outside of transgenic contexts, deletion of Repeat-A from the endogenous Xist locus causes not only a failure of XCI, but the destabilization of Xist itself (25,26). The mechanisms by which Repeat-A promotes Xist stability are unclear. Lastly, in transgenic Xist cDNA overexpression experiments, Repeat-A appears to be involved in targeting Xist to actively transcribed regions (27,28). How Repeat-A is involved in this targeting is unclear. It has been noted that the mechanism is difficult to disentangle from Repeat-A-mediated stabilization of Xist (29). We recently developed a transgenic assay that recapitulates Repeat-A-dependent gene silencing, which we called TETRIS (Transposable Element to Test RNA’s effect on transcription in cis; (30)). In the assay, expression of the first 2 kilobases (kb) of Xist (Xist-2kb), which contains Repeat-A, is driven by a doxycycline-inducible promoter positioned adjacent to a constitutively expressed luciferase reporter gene. The linked Xist-2kb and luciferase genes are inserted into the genomes of ESCs using the piggyBac transposase. Addition of doxycycline to the media induces expression of Xist-2kb and results in an 80–90% reduction of luciferase activity relative to cells that did not receive doxycycline. We demonstrated that repression of luciferase by Xist-2kb in TETRIS requires the same sequence motifs within Repeat-A that are required for transcriptional repression by full-length Xist—the GC-rich portion of Repeat-A but not its U-rich spacer sequences, as well as three adjacent structured elements and their intervening sequences (6,15,30,31). Given questions surrounding the mechanisms through which Repeat-A functions in Xist, we sought to investigate the mechanism of repression induced by Xist-2kb in TETRIS as well as in a transgenic, single-copy insertion assay that is analogous to transgenic assays previously employed in mouse ESCs to identify seminal aspects of Xist biology (7,8,15–20,27). We found, quite surprisingly, that Xist-2kb represses gene expression not at the transcriptional level, but by sequestering the mRNA of neighboring genes on chromatin. To our knowledge, our study is the first to demonstrate that expression of a lncRNA can block the nuclear export of RNA produced from an adjacent gene. We demonstrate that the ability to sequester RNA on chromatin is not unique to Xist-2kb but can also be induced by the expression of Xist-like, synthetic lncRNAs and by hypomorphic Xist mutants that retain Repeat-A but lack other key silencing domains. Furthermore, we show that recapitulating an interaction between SPEN and Repeat-A on chromatin is insufficient to induce local transcriptional silencing. Thus, in mouse ESCs, Repeat-A is necessary but not sufficient for Xist-induced transcriptional silencing. Instead, Xist-induced transcriptional silencing requires synergy between Repeat-A and other regions of Xist. We hypothesize that the mechanisms that underpin sequestration are relevant to the stabilization of Xist by Repeat-A and its role in recruiting Xist to actively transcribed regions of chromatin. MATERIALS AND METHODS ESC culture E14 and pSM33 ESCs (kind gifts of D. Ciavatta and K. Plath, respectively) were grown on gelatin coated dishes at 37°C in a humidified incubator at 5% CO2. Medium was changed daily and consisted of Dulbecco's modified Eagle's medium high glucose plus sodium pyruvate, 15% ESC qualified fetal bovine serum, 0.1 mM non-essential amino acids, 100 U/ml penicillin-streptomycin, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, and 1:500 LIF conditioned media produced from Lif-1Cα (COS) cells (kind gift of N. Hathaway). ESCs were split at an approximate ratio of 1:6 every 48 h. Rosa26-RMCE cells were grown on gamma-irradiated mouse embryonic fibroblast (MEF) feeder cells plated at ∼1.5 × 106 cells per 10 cm plate. Prior to harvesting of RNA for sequencing, Rosa26-RMCE cells were passaged twice off of MEF feeder cells with a 40-min pre-plate each passage and grown in 70% MEF-conditioned medium supplemented as above. Complete details of protein knockdown and knockout experiments can be found in the Supplemental Methods. TETRIS line generation TETRIS lines were made as described in (30). Briefly, 4 × 105 E14 cells were seeded in a single well of a 6-well plate, and transfected 24 h later with 0.5 μg TETRIS cargo plasmid, 0.5 μg rtTA-cargo plasmid and 1 μg of pUC19-piggyBAC transposase plasmid using Lipofectamine 3000 (Invitrogen) according to manufacturer instructions. Cells were selected for 7–9 days with puromycin (2 μg/ml) and G418 (200 μg/ml) beginning 24 h after transfection. TETRIS assays For each independent TETRIS cell line, six wells of a 24-well plate were seeded at 1 × 105 cells per well. Three of the six wells were induced with 1 μg/ml doxycycline (Sigma) beginning when the cells were plated, and the remaining three wells served as ‘no dox’ controls. After 48 h, the cells were washed with phosphate buffered saline and lysed with 100 μl of passive lysis buffer (Promega) and luciferase activity was measured using Bright-Glo™ Luciferase Assay reagents (Promega) on a PHERAstar FS plate reader (BMG Labtech). Luciferase activity was normalized to total protein concentration in the lysates via Bradford assay (Bio-Rad). Data were plotted in Python (version 3.6.5) using the barplot function of Seaborn (version 0.8.1; https://doi.org/10.5281/zenodo.1313201) with default settings, including generation of 95% confidence intervals by 1000 iterations of bootstrap random sampling with replacement. RNA isolation and RT-qPCR RNA fractionation was performed essentially as in ((32,33); Supplemental Methods). RNA was isolated using Trizol according to manufacturer protocol (Invitrogen). For RT-qPCR assays, equal amounts of RNA (200 ng - 2 μg) were reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with random primers or gene-specific primers (Luc_qPCR4 in Supplementary Table S3). Quantitative polymerase chain reaction (qPCR) was performed using iTaq Universal SYBR Green (Bio-Rad) and custom primers (Supplementary Table S3). Data were plotted in Python (version 3.6.5) using the barplot function of Seaborn (version 0.8.1; https://doi.org/10.5281/zenodo.1313201) with default settings, including generation of 95% confidence intervals by 1000 iterations of bootstrap random sampling with replacement. Stellaris RNA FISH Custom Stellaris® FISH probes were designed against the first 2kb of Xist, firefly luciferase (luc2 in pGL4.10; Promega), and the hygromycin resistance gene (HygroR) using the Stellaris® RNA FISH Probe Designer (Biosearch Technologies, Inc.) and labeled with Quasar® 670 (Xist-2kb) or 570 (luciferase and HygroR) dye. FISH was performed as described in ((34); Supplemental Methods). Recombinase-mediated cassette exchange (RMCE) Complete details can be found in the Supplemental Methods. Briefly, a male F1-hybrid mouse ESC line (derived from a cross between C57BL/6J (B6) and CAST/EiJ (Cast) mice; kind gift of T. Magnuson) was made competent for recombinase-mediated cassette exchange (RMCE) by insertion of a custom homing cassette into the Rosa26 locus via homologous recombination. Xist transgenes were cloned via PCR or recombineering into a custom RMCE-cargo vector and then electroporated along with a plasmid expressing Cre-recombinase into RMCE-competent cells using a Neon® Transfection System (Invitrogen). After selection on hygromycin (150 μg/ml) and ganciclovir (3 μM), individual colonies were picked and genotyped, then selected on G418 (200 μg/ml) after transfection with a pUC19-piggyBAC transposase and a piggyBac-based cargo vector containing an rtTA-expression cassette from (35). RNA sequencing and analysis RNA-seq libraries were prepared using the RNA HyperPrep Kit with RiboErase (Kapa Biosciences) and sequenced on an Illumina NextSeq 500 machine using a 75-cycle high output NextSeq kit (Illumina). Sequencing reads were aligned and processed essentially as in (36,37). Differential expression analysis was performed with DESeq2 (38). To quantify nuclear fraction of transcripts, upper quartile-normalized nuclear and cytosolic counts for each gene were added together and the normalized nuclear count values were divided by the total. All genome-related plots were generated using R (version 3.4.4; (39)). Complete details can be found in the Supplemental Methods. Immunoprecipitation (IP) IP experiments were performed using a protocol from (40). Complete details can be found in the Supplemental Methods. RESULTS Xist-2kb sequesters mRNA on chromatin TETRIS is a transgenic assay that allows the sequence of a lncRNA to be manipulated in a plasmid and then tested for its ability to repress activity of an adjacent reporter gene in a chromatin context. The assay employs the piggyBac transposase to insert a lncRNA expression cassette, a luciferase gene, and a gene conferring resistance to puromycin into the genomes of transfected cells (Figure 1A). Expression of non-repressive lncRNAs in the assay typically causes an ∼2-fold increase in luciferase activity, which we attribute to the proximity of the doxycycline-inducible TRE promoter and the PGK promoter that drives expression of luciferase (30). In contrast, we previously demonstrated that expression of the first 2kb of Xist (Xist-2kb) in TETRIS causes an 80 to 90% reduction of luciferase activity; this silencing depends on Repeat-A and an additional ∼750 nucleotides of sequence located just downstream (region deleted in Δss234 in Supplementary Figure S1A; (30)). Figure 1. View largeDownload slide Xist-2kb represses luciferase at the post-transcriptional level. (A) Schematic of TETRIS Xist-2kb expression construct. ITR, inverted terminal repeat recognized by piggyBac transposase. HS4, chicken βglobin insulator sequence. TRE, tetracycline responsive element (the doxycycline inducible promoter). Xist-2kb, nucleotides 1–2016 of mouse Xist. Luc, firefly luciferase (pGL4.10). PGK and EF1α, constitutive promoters. PuroR, gene encoding resistance to puromycin. Black ovals, location of polyA sites. (B) Representative assay showing Xist-2kb and Hottip effects on luciferase protein activity (see also (30)). Error bars, bootstrap 95% confidence interval of the mean derived from technical duplicate measurements from each of three separate cell platings (biological triplicates). (C) Luciferase mRNA increases upon induction of Xist-2kb and Hottip. Error bars, bootstrap 95% confidence intervals of the mean derived from duplicate qPCR measurements from each of three separate cell platings. (D) Luciferase mRNA increase in Xist-2kb ESCs is specific to the sense strand of the luciferase gene and not due to readthrough transcription from Xist-2kb. Values from sense- and antisense-specific RT reactions are normalized to Gapdh signal from a random primer RT and set relative to no dox values for each strand-specific primer. Error bars, bootstrap 95% confidence intervals around the mean as calculated in (C). Sense (S) and Antisense (AS) relative primer locations are shown in (A). See also Supplementary Figure S1 and Supplementary Table S3. Figure 1. View largeDownload slide Xist-2kb represses luciferase at the post-transcriptional level. (A) Schematic of TETRIS Xist-2kb expression construct. ITR, inverted terminal repeat recognized by piggyBac transposase. HS4, chicken βglobin insulator sequence. TRE, tetracycline responsive element (the doxycycline inducible promoter). Xist-2kb, nucleotides 1–2016 of mouse Xist. Luc, firefly luciferase (pGL4.10). PGK and EF1α, constitutive promoters. PuroR, gene encoding resistance to puromycin. Black ovals, location of polyA sites. (B) Representative assay showing Xist-2kb and Hottip effects on luciferase protein activity (see also (30)). Error bars, bootstrap 95% confidence interval of the mean derived from technical duplicate measurements from each of three separate cell platings (biological triplicates). (C) Luciferase mRNA increases upon induction of Xist-2kb and Hottip. Error bars, bootstrap 95% confidence intervals of the mean derived from duplicate qPCR measurements from each of three separate cell platings. (D) Luciferase mRNA increase in Xist-2kb ESCs is specific to the sense strand of the luciferase gene and not due to readthrough transcription from Xist-2kb. Values from sense- and antisense-specific RT reactions are normalized to Gapdh signal from a random primer RT and set relative to no dox values for each strand-specific primer. Error bars, bootstrap 95% confidence intervals around the mean as calculated in (C). Sense (S) and Antisense (AS) relative primer locations are shown in (A). See also Supplementary Figure S1 and Supplementary Table S3. During our initial validation of TETRIS, we performed a control to verify that expression of Xist-2kb caused a level of transcriptional silencing commensurate with the 80% reduction in luciferase protein activity. To our surprise, Xist-2kb expression led to an increase, not decrease, in luciferase mRNA levels, despite the repression of luciferase protein activity (Figure 1B and C). The decrease in luciferase protein was confirmed by western blot (Supplementary Figure S1B), while the increase in luciferase mRNA abundance was characteristic of TETRIS assays in which non-repressive lncRNAs are expressed (see Hottip assays in Figure 1B and C and (30)). The concurrent 80% reduction of luciferase protein activity and elevation of luciferase mRNA levels persisted even after 21 days of Xist-2kb induction (data not shown). Strand-specific RT-qPCR assays confirmed that the increase in luciferase mRNA originated from the PGK promoter driving luciferase and not from a long, read-through transcript originating from the TRE promoter (Figure 1D). We hypothesized that the reduced level of luciferase activity without loss in mRNA abundance was due to physical sequestration of the luciferase mRNA on chromatin. We fractionated cells as in (32,33) and found that upon Xist-2kb induction, the vast majority of total luciferase mRNA co-purified with chromatin, consistent with our hypothesis (Figure 2A). Malat1 RNA and Gapdh mRNA showed no change in distribution after Xist-2kb induction, as expected (Figure 2A). Figure 2. View largeDownload slide Xist-2kb sequesters mRNA on chromatin. (A) Xist-2kb sequesters mRNA from the luciferase (Luc) and puromycin resistance (PuroR) genes on chromatin. Gapdh and Malat1/Xist-2kb are controls to monitor quality of cytoplasmic (Cyt), nucleoplasmic (Nuc) and chromatin (Chr) fractionations. Error bars represent standard deviation propagated across two qPCR technical replicates of duplicate RNA fractionations. (B) Sequestration requires continued expression of Xist-2kb. TETRIS-Xist-2kb cells were grown in the presence of doxycycline for 2 days, prior to splitting at Day 2 and a 2-day release from doxycycline. ESCs were then split into two cultures at Day 4 and doxycycline was re-added to one culture and not the other. Error bars, bootstrap 95% confidence interval of the mean derived from technical duplicate measurements taken from each of three separate platings. (C) Stability of luciferase and PuroR mRNA in TETRIS Xist-2kb and Hottip ESCs with and without dox-induced expression. Cells were either induced or not induced for ∼16 h, then treated with 5 μg/ml actinomycin D. Each point shows the average of technical duplicate qPCR measurements of one of three biological replicate platings normalized to Gapdh and set relative to 0 h on a log2 scale. Linear models were fit to the data, plotted with 95% bootstrap confidence intervals, and used to calculate half-lives (t1/2). To compare regression lines between uninduced (− dox) and induced (+ dox), a single linear regression model was built including an interaction term between time and induction. ***P< 0.001; *P< 0.05, hypothesis test using linear regression model with interaction effect. (D) RT-qPCR analysis of Gapdh RNA and Malat1 lncRNA to assess loading and fractionation quality of RNA prepared from unfractionated parental E14 ESCs (E14 total), unfractionated TETRIS Xist-2kb ESCs (Total) and TETRIS Xist-2kb ESCs fractionated into nuclear (Nuc) and cytoplasmic (Cyt) fractions. TETRIS Xist-2kb ESCs were incubated for 48 h in the absence (− dox) or presence (+ dox) of 1 μg/ml doxycycline prior to harvesting. For each primer pair, RNA abundance values are normalized relative to the average ‘Total −dox’ value, which was arbitrarily set to 1. Each dot represents the RNA abundance from a biological replicate cell plating (n = 1 for E14 total, n = 3 for all others), each calculated as the average of technical triplicate qPCR measurements. Error bars represent standard deviation of biological replicate values. (E andF) Tiled RT-qPCR analysis of luciferase (E) and PuroR (F) mRNAs from the same RNA samples used in (D). Measurements and error bars as in (D). Lack of signal in the ‘E14 total’ sample demonstrates specificity of the primer pairs for luciferase and PuroR mRNA. Primer pair locations are indicated under each plot. Protein-coding sequence of each mRNA is depicted by the thicker lines. (G) Stellaris single-molecule FISH was performed to visualize Xist RNA (red) and luciferase mRNA (green) in Xist-2kb cells with and without 48 h treatment with doxycycline. DAPI-stained nuclei are blue. Scale bar = 10 μm. See also Supplementary Figure S1 and Supplementary Table S3. Figure 2. View largeDownload slide Xist-2kb sequesters mRNA on chromatin. (A) Xist-2kb sequesters mRNA from the luciferase (Luc) and puromycin resistance (PuroR) genes on chromatin. Gapdh and Malat1/Xist-2kb are controls to monitor quality of cytoplasmic (Cyt), nucleoplasmic (Nuc) and chromatin (Chr) fractionations. Error bars represent standard deviation propagated across two qPCR technical replicates of duplicate RNA fractionations. (B) Sequestration requires continued expression of Xist-2kb. TETRIS-Xist-2kb cells were grown in the presence of doxycycline for 2 days, prior to splitting at Day 2 and a 2-day release from doxycycline. ESCs were then split into two cultures at Day 4 and doxycycline was re-added to one culture and not the other. Error bars, bootstrap 95% confidence interval of the mean derived from technical duplicate measurements taken from each of three separate platings. (C) Stability of luciferase and PuroR mRNA in TETRIS Xist-2kb and Hottip ESCs with and without dox-induced expression. Cells were either induced or not induced for ∼16 h, then treated with 5 μg/ml actinomycin D. Each point shows the average of technical duplicate qPCR measurements of one of three biological replicate platings normalized to Gapdh and set relative to 0 h on a log2 scale. Linear models were fit to the data, plotted with 95% bootstrap confidence intervals, and used to calculate half-lives (t1/2). To compare regression lines between uninduced (− dox) and induced (+ dox), a single linear regression model was built including an interaction term between time and induction. ***P< 0.001; *P< 0.05, hypothesis test using linear regression model with interaction effect. (D) RT-qPCR analysis of Gapdh RNA and Malat1 lncRNA to assess loading and fractionation quality of RNA prepared from unfractionated parental E14 ESCs (E14 total), unfractionated TETRIS Xist-2kb ESCs (Total) and TETRIS Xist-2kb ESCs fractionated into nuclear (Nuc) and cytoplasmic (Cyt) fractions. TETRIS Xist-2kb ESCs were incubated for 48 h in the absence (− dox) or presence (+ dox) of 1 μg/ml doxycycline prior to harvesting. For each primer pair, RNA abundance values are normalized relative to the average ‘Total −dox’ value, which was arbitrarily set to 1. Each dot represents the RNA abundance from a biological replicate cell plating (n = 1 for E14 total, n = 3 for all others), each calculated as the average of technical triplicate qPCR measurements. Error bars represent standard deviation of biological replicate values. (E andF) Tiled RT-qPCR analysis of luciferase (E) and PuroR (F) mRNAs from the same RNA samples used in (D). Measurements and error bars as in (D). Lack of signal in the ‘E14 total’ sample demonstrates specificity of the primer pairs for luciferase and PuroR mRNA. Primer pair locations are indicated under each plot. Protein-coding sequence of each mRNA is depicted by the thicker lines. (G) Stellaris single-molecule FISH was performed to visualize Xist RNA (red) and luciferase mRNA (green) in Xist-2kb cells with and without 48 h treatment with doxycycline. DAPI-stained nuclei are blue. Scale bar = 10 μm. See also Supplementary Figure S1 and Supplementary Table S3. The percentage of mRNA that co-purified with chromatin from the puromycin resistance gene (PuroR) also increased upon Xist-2kb induction (Figure 2A), and the functional consequence of PuroR mRNA sequestration was confirmed by observations that Xist-2kb expression inhibited the survival of cells grown in the presence of puromycin (Supplementary Figure S1C). In contrast to the luciferase gene, which is convergently oriented relative to Xist-2kb, the PuroR gene is oriented in tandem. Collectively, these results demonstrate that sequestration induced by Xist-2kb is not exclusive to the luciferase mRNA and show that sequestration can occur regardless of whether target genes are oriented in tandem or convergently to the inducing lncRNA. We also observed a full recovery of luciferase activity after two days growth in the absence of doxycycline, demonstrating that sequestration requires continued expression of Xist-2kb (Figure 2B). The sequestration of luciferase and PuroR mRNAs was accompanied by changes in mRNA stability. Upon expression of Xist-2kb, the half-life of luciferase mRNA dropped ∼75%, from 14.9 to 3.9 h, and the half-life of PuroR mRNA dropped ∼50%, from 17 to 8.2 h (Figure 2C). Expression of Hottip had no effect on the stability of luciferase or PuroR mRNA (Figure 2C). Translation is thought to be a major mechanism through which mRNAs are stabilized in cells (41), so the reduced half-lives are consistent with a shift of luciferase and PuroR mRNA to the chromatin fraction upon expression of Xist-2kb. It was unclear whether partial or full-length luciferase and PuroR mRNAs were sequestered in the nucleus upon Xist-2kb expression. Using RT-qPCR with primer pairs targeting multiple regions of each mRNA, we found that Xist-2kb expression caused changes in signal along the length of luciferase and PuroR mRNAs that were similar between primer pairs (Figure 2D–F). Upon induction of Xist-2kb, cytoplasmic levels of luciferase mRNA decreased 3- to 4-fold, coincident with the observed 4- to 7-fold increases in nuclear signals (Figure 2E). Relatedly, cytoplasmic levels of PuroR mRNA decreased 6- to 8-fold upon Xist-2kb expression, while the nuclear levels of PuroR remained unchanged (Figure 2F). The exception to these patterns was a 12- and 4-fold increase in nuclear signal at the 5′ ends of the luciferase and PuroR transcripts, respectively, which may be consistent with the accumulation of short 5′ mRNA products via an increase in promoter-proximal pausing upon addition of doxycycline (Figure 2E and F; (42)). We also note that the apparent increase in overall levels of luciferase mRNA were only detected when the ‘Luc 4’ primer pair was used (Figure 2E; Figure 1C luciferase data were also obtained using the ‘Luc 4’ primer pair). Taken together, these data suggest that although full-length luciferase and PuroR mRNAs are still produced in the presence of Xist-2kb, their export to the cytoplasm is greatly hindered. We hypothesized that luciferase mRNA was specifically sequestered near the site of Xist-2kb transcription. To test this hypothesis, we used a single-molecule sensitivity FISH assay from Stellaris (34). In Xist-2kb ESCs untreated with doxycycline, luciferase mRNA was broadly dispersed throughout the cytoplasm, consistent with its ongoing export from the nucleus and active translation (Figure 2G). In contrast, upon Xist-2kb induction, the cytoplasmic signal was lost, and we observed the appearance of foci of luciferase mRNA that co-localized with Xist-2kb in the nucleus. These results suggest that sequestration occurs in cis, in regions that accumulate Xist-2kb upon addition of doxycycline (Figure 2G). Sequestration depends on GC-rich sequence elements in Repeat-A and can be induced by Xist-like, synthetic lncRNAs In previous work, we demonstrated that repression of luciferase by Xist-2kb in TETRIS depended specifically on the GC-rich individual repeats in Repeat-A, and not its U-rich spacers, as well as three stably structured elements located just downstream of Repeat-A and their intervening sequence ((30); Figure 3A). Consistent with these data, we found that deletion of the GC-rich portions of Repeat-A, the stably structured region, or all of Repeat-A and the downstream structures each abrogated sequestration of luciferase and PuroR mRNA (ΔrA (no GC), Δss234, and ΔrA234, respectively; Figure 3A and B). Relative to Xist-2kb, these deletions led to an apparent destabilization of the mutant Xist transcripts coincident with a minor reduction in their association with chromatin (Figure 3C and D). Collectively, these data show that the same genetic elements in Repeat-A that are required for repression by full-length Xist (15) are required for sequestration of luciferase mRNA. Figure 3. View largeDownload slide Sequestration depends on GC-rich sequence elements in Repeat-A and can be induced by Xist-like, synthetic lncRNAs. (A) Schematic of Xist-2kb showing the location of Repeat-A, Repeat-F, stably structured elements (SS; (6)) and mutants that reduce repressive activity in TETRIS (30). (B) mRNA sequestration requires the GC-rich portions of Repeat-A and three stably structured elements and their intervening sequence located downstream. Measurements and error bars as in Figure 2A. (C) Xist RNA is induced to a lesser extent in deletion mutants. Error bars, bootstrap 95% confidence intervals of the Gapdh-normalized mean derived from duplicate qPCR measurements from each of four separate cell platings. (D) Xist RNA is less chromatin-associated in Xist mutant TETRIS lines. Measurements and error bars as in Figure 2A, but showing only results from doxycycline-induced (+) cells. Wild-type Xist-2kb localization data from Figure 2A are included again for comparison. (E) Representative luciferase assay data from TETRIS-Empty, -Xist-2kb, and the six synthetic lncRNAs from (30). Measurements and error bars as in Figure 1B. Each synthetic lncRNA is 1650 nucleotides long. Similarity of each synthetic lncRNA to Xist-2kb measured by Pearson similarity at kmer length k = 6 as in (30) is listed in parenthesis. (F) Subcellular localization of Luciferase mRNA, Malat1 lncRNA and Gapdh mRNA upon expression of TETRIS-Empty, -Xist-2kb and the six synthetic lncRNAs from (30). Error bars represent standard deviation propagated across two technical replicates of qPCR from a single fractionation experiment. Data shown are representative of three independent fractionation experiments. See also Supplementary Table S3. Figure 3. View largeDownload slide Sequestration depends on GC-rich sequence elements in Repeat-A and can be induced by Xist-like, synthetic lncRNAs. (A) Schematic of Xist-2kb showing the location of Repeat-A, Repeat-F, stably structured elements (SS; (6)) and mutants that reduce repressive activity in TETRIS (30). (B) mRNA sequestration requires the GC-rich portions of Repeat-A and three stably structured elements and their intervening sequence located downstream. Measurements and error bars as in Figure 2A. (C) Xist RNA is induced to a lesser extent in deletion mutants. Error bars, bootstrap 95% confidence intervals of the Gapdh-normalized mean derived from duplicate qPCR measurements from each of four separate cell platings. (D) Xist RNA is less chromatin-associated in Xist mutant TETRIS lines. Measurements and error bars as in Figure 2A, but showing only results from doxycycline-induced (+) cells. Wild-type Xist-2kb localization data from Figure 2A are included again for comparison. (E) Representative luciferase assay data from TETRIS-Empty, -Xist-2kb, and the six synthetic lncRNAs from (30). Measurements and error bars as in Figure 1B. Each synthetic lncRNA is 1650 nucleotides long. Similarity of each synthetic lncRNA to Xist-2kb measured by Pearson similarity at kmer length k = 6 as in (30) is listed in parenthesis. (F) Subcellular localization of Luciferase mRNA, Malat1 lncRNA and Gapdh mRNA upon expression of TETRIS-Empty, -Xist-2kb and the six synthetic lncRNAs from (30). Error bars represent standard deviation propagated across two technical replicates of qPCR from a single fractionation experiment. Data shown are representative of three independent fractionation experiments. See also Supplementary Table S3. We recently recognized that different lncRNAs can encode similar function through different spatial arrangements of related sequence motifs (30). As a part of that study, we designed a series of synthetic lncRNAs with varying levels of non-linear sequence similarity to Xist-2kb, and we demonstrated using the TETRIS assay that the synthetic lncRNAs repressed luciferase activity in a manner that was directly proportional to their non-linear similarity to Xist-2kb. The synthetic lncRNAs had no linear homology to Xist, each other, or any region in the mouse or human genome (30). Here, we examined whether these synthetic lncRNAs, like Xist-2kb, repressed luciferase activity in TETRIS by sequestering mRNA on chromatin. Indeed, we observed a direct correlation between the repressive activity of synthetic lncRNAs in TETRIS (Figure 3E) and their ability to sequester luciferase mRNA on chromatin (Figure 3F). Thus, in addition to Xist, other lncRNAs are also capable of sequestering nearby mRNA on chromatin. Further, their ability to sequester nearby mRNA is correlated to their non-linear sequence similarity to Xist-2kb. Xist hypomorphs sequester nearby mRNA on chromatin as single-copy insertions Under the conditions we use to make standard TETRIS ESC lines, approximately five copies of the Xist-2kb/luciferase cargo DNA are randomly inserted into the genome of each cell that survives the selection process (30). We sought to determine if Xist-2kb could sequester the mRNA of nearby genes when inserted as a single copy into a defined chromosomal locus. Insertion into a defined locus would also allow us to determine the extent to which Xist-induced sequestration can spread along a single chromosome. In parallel, such a system would allow us to directly compare the extent to which Xist-2kb silenced gene expression relative to full-length Xist as well as to hypomorphic Xist mutants that lacked different subsets of key functional domains. To these ends, we established an RMCE system in the Rosa26 locus (43). We created a Rosa26 targeting vector that contained a lox66 site, a puromycin-Herpes-Simplex-Virus-thymidine-kinase fusion protein and a lox2272 site followed by a polyadenylation cassette (Supplementary Figure S2A). The targeting construct was electroporated into F1-hybrid, male ESCs that were derived from a cross between C57BL/6J (B6) and CAST/EiJ (Cast) mice, and Southern blot was used to confirm insertion of the construct into the correct locus on the B6 allele of selected clones (Supplementary Figure S2B). In parallel, we created a cargo vector that contained a lox71 site, a lncRNA-expression cassette driven by a doxycycline-inducible promoter, a constitutively expressed hygromycin B resistance gene lacking a polyadenylation signal and a lox2272 site (Supplementary Figure S2C). Electroporation of the cargo vector along with Cre recombinase into our F1-hybrid RMCE cells, followed by positive selection on hygromycin B and negative selection on ganciclovir, generates a small number of surviving clones that harbor cargo vectors inserted in the desired orientation in Rosa26 (Supplementary Figure S2D; not shown). We employed this RMCE system to create four separate ESC lines that expressed different versions of inducible Xist transgenes from Rosa26 (Figure 4A): one line expressed the Xist-2kb transgene, another line expressed full-length Xist from its endogenous DNA sequence (not a spliced transgene), another line expressed the first 5.5kb of Xist (‘Xist-5.5kb’), which includes the Repeat-B and Repeat-C domains of Xist known to recruit PRC1 (8), and a final line expressed Xist-2kb fused to the final 2 exons of Xist (‘Xist-2kb+6,7’). The final two exons of Xist include Repeat-E and are essential for proper Xist localization and PRC2 recruitment to the inactive X (9,44,45). As a negative control, we created a control ESC line that underwent recombination but lacked any Xist insertion (‘empty’). We then used piggyBac-mediated transgenesis to insert the reverse-tetracycline transactivator (rtTA; (35)) into select clones of each genotype, to allow doxycycline-inducible expression of each cargo RNA. RNA FISH and RT-qPCR verified the doxycycline-inducible expression of each transgene in each clone (Figure 4B and not shown). We note that despite several attempts, we were unable to clone an Xist-5.5kb construct that contained all ∼36 repeats in Repeat-B; the construct used for this study contained ∼14 repeats (Figure 4A and Supplementary Figure S3A). Figure 4. View largeDownload slide Xist hypomorphs sequester nearby mRNA on chromatin as single-copy insertions. (A) Schematic of endogenous Xist locus with Xist insertions. Repeats A-F and exon locations are shown. (B) Stellaris single-molecule FISH for Xist RNA (red) in cells expressing Xist-2kb, Xist-5.5kb, Xist-2kb+6,7 or full-length Xist inserted at Rosa26 with and without doxycycline. DAPI-stained nuclei are blue. Scale bar = 10 μm. (C) Xist expression in each ESC line. Reads per kilobase per million (RPKM) values were calculated using reads aligned to the endogenous Xist locus divided by length of inserted transcript in kb divided by total aligned reads in millions for each dataset. (D) Venn diagram showing overlap between significantly repressed genes in Xist-5.5kb, Xist-2kb+6,7 and full-length Xist cells. (E) Nuclear ratio of reads mapping to HygroR. ***P< 0.0001, Tukey HSD post-hoc analysis of significant differences by ANOVA. (F) Nuclear ratio of allele-specific reads mapping to Setd5. Note the difference in y-axis compared to (E). ***P< 0.0001; *P< 0.01, Tukey HSD post-hoc analysis of significant differences by ANOVA. (G) Stellaris single-molecule FISH was performed to visualize Xist-2kb RNA (red) and HygroR mRNA (green) in cells with Xist-2kb inserted at Rosa26 with and without 48 h treatment with doxycycline. DAPI-stained nuclei are blue. Scale bar = 10 μm. (H) Normalized nuclear counts relative to empty-cargo ESCs for hygromycin resistance (HygroR) mRNA. (I) Allele-specific normalized nuclear counts relative to empty-cargo ESCs for Setd5 mRNA on the B6 allele. (J) Hygromycin resistance (HygroR) mRNA levels measured by RT-qPCR from total cellular RNA and normalized to Gapdh. Cells expressing empty-cargo or Xist transgenes were induced (1 μg/ml doxycycline) for 2 days prior to RNA extraction. Expression in each line is set relative to that in empty-cargo ESCs. Error bars, bootstrap 95% confidence intervals of the mean derived from duplicate qPCR measurements from each of two (Xist-full length), three (empty-cargo and Xist-2kb) or four (Xist-5.5kb and Xist-2kb+6,7) separate cell platings. (K) Similar to (J) but for Setd5 mRNA. See also Supplementary Figures S2–5 and Supplementary Tables S1 and S2. Figure 4. View largeDownload slide Xist hypomorphs sequester nearby mRNA on chromatin as single-copy insertions. (A) Schematic of endogenous Xist locus with Xist insertions. Repeats A-F and exon locations are shown. (B) Stellaris single-molecule FISH for Xist RNA (red) in cells expressing Xist-2kb, Xist-5.5kb, Xist-2kb+6,7 or full-length Xist inserted at Rosa26 with and without doxycycline. DAPI-stained nuclei are blue. Scale bar = 10 μm. (C) Xist expression in each ESC line. Reads per kilobase per million (RPKM) values were calculated using reads aligned to the endogenous Xist locus divided by length of inserted transcript in kb divided by total aligned reads in millions for each dataset. (D) Venn diagram showing overlap between significantly repressed genes in Xist-5.5kb, Xist-2kb+6,7 and full-length Xist cells. (E) Nuclear ratio of reads mapping to HygroR. ***P< 0.0001, Tukey HSD post-hoc analysis of significant differences by ANOVA. (F) Nuclear ratio of allele-specific reads mapping to Setd5. Note the difference in y-axis compared to (E). ***P< 0.0001; *P< 0.01, Tukey HSD post-hoc analysis of significant differences by ANOVA. (G) Stellaris single-molecule FISH was performed to visualize Xist-2kb RNA (red) and HygroR mRNA (green) in cells with Xist-2kb inserted at Rosa26 with and without 48 h treatment with doxycycline. DAPI-stained nuclei are blue. Scale bar = 10 μm. (H) Normalized nuclear counts relative to empty-cargo ESCs for hygromycin resistance (HygroR) mRNA. (I) Allele-specific normalized nuclear counts relative to empty-cargo ESCs for Setd5 mRNA on the B6 allele. (J) Hygromycin resistance (HygroR) mRNA levels measured by RT-qPCR from total cellular RNA and normalized to Gapdh. Cells expressing empty-cargo or Xist transgenes were induced (1 μg/ml doxycycline) for 2 days prior to RNA extraction. Expression in each line is set relative to that in empty-cargo ESCs. Error bars, bootstrap 95% confidence intervals of the mean derived from duplicate qPCR measurements from each of two (Xist-full length), three (empty-cargo and Xist-2kb) or four (Xist-5.5kb and Xist-2kb+6,7) separate cell platings. (K) Similar to (J) but for Setd5 mRNA. See also Supplementary Figures S2–5 and Supplementary Tables S1 and S2. To determine if any of the Xist sequences sequester nearby mRNAs when expressed from the Rosa26 locus, and to determine how far along the chromosome the sequestration spread, we induced Xist transgene expression for 3 days and sequenced RNA purified from cytoplasmic and nuclear fractions. In parallel, we treated empty-cargo ESCs with doxycycline for 3 days and sequenced RNA purified from cytoplasmic and nuclear fractions. Expression of the Xist transgenes was verified by examining reads mapping to the endogenous Xist locus (which is not expressed in these cells; Supplementary Figure S3B), and total counts were used to calculate RPKM values for each Xist transgene. All transgene RNA localized in the nucleus (Supplementary Figure S3C), and expression levels were only slightly lower for the Xist hypomorphs than for full-length Xist (Figure 4C). Next, we examined the extent to which cytoplasmic gene expression was silenced by the different Xist transgenes (Supplementary Figure S4A–H). In cells expressing full-length Xist, we detected 2404 genes that were differentially expressed between empty-cargo ESCs after 3 days of doxycycline treatment. On the B6 allele of chr6—the chromosome that harbors the Xist transgene at the Rosa26 locus—214 genes were differentially expressed (Supplementary Figure S4A). Of these, 205 genes shifted in the downward direction and 9 shifted in the upward direction, consistent with repression by full-length Xist (Supplementary Figure S4A). On the Cast allele of chr6, only 34 genes changed, with similar numbers going up (21 genes) and down (13 genes; Supplementary Figure S4B). The 2175 remaining differentially expressed genes throughout the genome also consisted of similar numbers of genes that were up- and downregulated (1012 and 1163, respectively). Thus, as expected, insertion of full-length Xist into Rosa26 caused chromosome-level repression of genes in cis, and also caused gene expression changes genome-wide due to secondary effects of Xist expression. Expression of Xist-5.5kb and Xist-2kb+6,7 also led to silencing of genes along the B6 allele of chr6, but at a reduced level relative to full-length Xist. Xist-5.5kb expression led to differential expression of 98 genes on the B6 allele of chr6, 76 of which were shifted down (Supplementary Figure S4C; Cast allele Supplementary Figure S4D). Xist-2kb+6,7 expression led to differential expression of 88 genes on the B6 allele of chr6, 65 of which were shifted down (Supplementary Figure S4E; Cast allele Supplementary Figure S4F). The majority of genes repressed by the two hypomorphs were also repressed by full-length Xist (69 of 76 for Xist-5.5kb and 64 of 65 for Xist-2kb+6,7). Forty-nine genes were silenced by all three transgenes (Figure 4D). The loss of nuclear as well as cytoplasmic RNA-seq signal at these genes indicated that, along with full-length Xist, the hypomorphic Xist transgenes induced transcriptional silencing (Supplementary Figure S5A–C). In contrast, when comparing cytoplasmic expression between Xist-2kb and empty-cargo ESCs, we only detected three differentially expressed genes genome-wide (including Xist), all on the B6 allele of chr6 (Supplementary Figure S4G; Cast allele Supplementary Figure S4H). These data indicate that expression of Xist-2kb does not cause chromosome-level changes in gene expression. However, when we examined cytoplasmic and nuclear levels of the two genes nearest to the transgene insertion site—the gene conferring hygromycin resistance (∼1kb downstream; HygroR), and Setd5 (∼5kb downstream)—we found strong evidence of Xist-induced sequestration, specifically in cells expressing Xist hypomorphic transgenes. The nuclear fraction of reads deriving from HygroR was significantly increased by expression of Xist-2kb, Xist-5.5kb, and Xist-2kb+6,7, but not by full-length Xist (Figure 4E), consistent with sequestration of HygroR mRNA. Similarly, the nuclear fraction of reads mapping to the B6 allele of the Setd5 gene was significantly increased by expression of Xist-2kb and Xist-5.5kb, but not by Xist-2kb+6,7 or full-length Xist (Figure 4F). Single-molecule FISH confirmed that HygroR mRNA co-localized in the nucleus with Xist-2kb, providing additional support that sequestration occurs in cis, near the site of Xist transcription (Figure 4G). Examination of nuclear RNA levels relative to empty-cargo control indicated that in Xist-2kb and Xist-5.5kb cells, sequestration of HygroR and Setd5 was not accompanied by transcriptional silencing (Figure 4H and I). In contrast, the same analysis showed that in Xist-2kb+6,7 cells, sequestration of HygroR occurred jointly with transcriptional silencing (Figure 4H). Sequestration remained limited to the genes adjacent to the Xist-insertion locus; we found no evidence for sequestration of genes along the rest of chr6 (Supplementary Figure S5D and not shown). Next, we used RT-qPCR to determine if sequestration was accompanied by a change in total levels of mRNA produced from sequestered genes. Indeed, relative to empty-cargo control, total mRNA levels of HygroR were decreased by more than 70% by all Xist hypomorphs that induced HygroR sequestration (Xist-2kb, Xist-5.5kb, and Xist-2kb+6,7; Figure 4J). Total mRNA levels of Setd5 were also modestly decreased by one of two Xist hypomorphs that induced Setd5 sequestration (decreased by Xist-2kb but not by Xist-5.5kb; Figure 4K). Consistent with these results, expression of Xist-2kb in TETRIS led to an overall reduction in PuroR mRNA levels (Figure 2F) and significantly reduced luciferase and PuroR mRNA stability (Figure 2C). Together, these results indicate that sequestration can decrease total levels of mRNA without causing transcriptional silencing, presumably because mRNA is degraded at a higher rate in the nucleus compared to the cytoplasm. We conclude that hypomorphic versions of Xist can sequester nearby mRNAs when expressed from a single chromosomal locus, that full-length Xist does not sequester mRNAs to a similar extent, and that sequestration remains local and can reduce mRNA levels of target genes. Lack of evidence for stable mRNA sequestration at the onset of X-inactivation It remained possible that sequestration of target mRNAs by full-length Xist occurred transiently, at the earliest stages of Xist-induced gene silencing, and was not detectable after three days of Xist induction. To test this idea, we sequenced cytoplasmic and nuclear RNA 3, 5 and 24 h after induction of Xist in pSM33 cells, a male ESC line in which the endogenous promoter of Xist has been replaced with a doxycycline-inducible one (27). We found that 291 out of 421 expressed genes on the X were significantly silenced following 24 h of Xist expression in pSM33 cells (Supplementary Figure S6A). However, we were unable to detect evidence for chromosome-level sequestration at any time-point post-Xist induction (Supplementary Figure S6B and C). Thus, at the time-points profiled, gene silencing by full-length Xist does not appear to be accompanied by the stable sequestration of mRNA on chromatin. Xist-2kb associates with SPEN and RBM15, but neither are required for sequestration Having established that expression of Xist-2kb in mouse ESCs does not induce transcriptional silencing, as would have been expected based on current models for Repeat-A function, we next sought to determine if Xist-2kb still bound SPEN, a key cofactor required for Repeat-A-induced transcriptional silencing (7,18–20). We also examined whether Xist-2kb bound RBM15, another Repeat-A binding protein shown to be important for Xist-induced silencing (19,46). We used a formaldehyde-based immunoprecipitation (IP) approach to determine whether SPEN and RBM15 associated with the Xist-2kb RNA (40). Indeed, IP of both SPEN and RBM15 from crosslinked and sonicated cell extracts robustly retrieved RNA corresponding to Repeat-A, but did not retrieve RNA upstream or downstream of Repeat-A within Xist-2kb. The enrichment of Repeat-A was lost in IP from ΔrA234 cells (Figures 3A and 5A and B). Thus, two Repeat-A binding proteins required for Xist-induced transcriptional silencing, SPEN and RBM15, also associate with Xist-2kb in a Repeat-A-dependent manner. Figure 5. View largeDownload slide SPEN and RBM15 associate with Xist-2kb but are not required for repression in TETRIS. (A) Xist-2kb diagram showing the location of Repeat-A and three stably structured elements (ss2–4) as well as the qPCR primers used in RNA IP. (B) Immunoprecipitation verifies Repeat-A-dependent association of SPEN and RBM15 in TETRIS-Xist2kb ESCs. IPs were performed in the parent cell line, E14, which does not express Xist at appreciable levels but does express the antisense lncRNA Tsix, in TETRIS-Xist-2kb ESCs with and without a 48 h treatment of doxycycline and in Xist-ΔrA234 ESCs after a 48 h treatment with doxycycline. RT-qPCR signal from RNA prepared from each IP was plotted relative to input RNA from Xist-2kb +dox cells. Error bars, bootstrap 95% confidence interval of the mean derived from triplicate qPCR measurements from a representative IP experiment. In all, the SPEN IP was conducted four times and the RBM15 IP was conducted twice. (C) Spen mRNA is absent in TETRIS Xist-2kb lines made in two independent lines of homozygous SPEN deletion cells. Error bars, bootstrap 95% confidence intervals of the mean derived from duplicate qPCR measurements from each of two separate cell platings. (D) Luciferase assay showing that SPEN deletion has no effect on repression of luciferase protein activity by expression of Xist-2kb in TETRIS. Error bars, bootstrap 95% confidence interval of the mean derived from technical duplicate measurements from each of four separate cell platings. (E) Western blots confirm knockdown of RBM15, RBM15B and EZH2 in TETRIS Xist-2kb lines with and without doxycycline treatment. ‘rtTA’: non-targeting gRNA sequence (negative control); ‘Both’: simultaneous targeting of both RBM15 and RBM15B. (F) Luciferase assay showing that none of the knockdowns influence repression of luciferase protein activity by expression of Xist-2kb in TETRIS. Error bars, bootstrap 95% confidence interval of the mean derived from technical duplicate measurements from each of three separate cell platings. See also Supplementary Figure S7 and Supplementary Table S3. Figure 5. View largeDownload slide SPEN and RBM15 associate with Xist-2kb but are not required for repression in TETRIS. (A) Xist-2kb diagram showing the location of Repeat-A and three stably structured elements (ss2–4) as well as the qPCR primers used in RNA IP. (B) Immunoprecipitation verifies Repeat-A-dependent association of SPEN and RBM15 in TETRIS-Xist2kb ESCs. IPs were performed in the parent cell line, E14, which does not express Xist at appreciable levels but does express the antisense lncRNA Tsix, in TETRIS-Xist-2kb ESCs with and without a 48 h treatment of doxycycline and in Xist-ΔrA234 ESCs after a 48 h treatment with doxycycline. RT-qPCR signal from RNA prepared from each IP was plotted relative to input RNA from Xist-2kb +dox cells. Error bars, bootstrap 95% confidence interval of the mean derived from triplicate qPCR measurements from a representative IP experiment. In all, the SPEN IP was conducted four times and the RBM15 IP was conducted twice. (C) Spen mRNA is absent in TETRIS Xist-2kb lines made in two independent lines of homozygous SPEN deletion cells. Error bars, bootstrap 95% confidence intervals of the mean derived from duplicate qPCR measurements from each of two separate cell platings. (D) Luciferase assay showing that SPEN deletion has no effect on repression of luciferase protein activity by expression of Xist-2kb in TETRIS. Error bars, bootstrap 95% confidence interval of the mean derived from technical duplicate measurements from each of four separate cell platings. (E) Western blots confirm knockdown of RBM15, RBM15B and EZH2 in TETRIS Xist-2kb lines with and without doxycycline treatment. ‘rtTA’: non-targeting gRNA sequence (negative control); ‘Both’: simultaneous targeting of both RBM15 and RBM15B. (F) Luciferase assay showing that none of the knockdowns influence repression of luciferase protein activity by expression of Xist-2kb in TETRIS. Error bars, bootstrap 95% confidence interval of the mean derived from technical duplicate measurements from each of three separate cell platings. See also Supplementary Figure S7 and Supplementary Table S3. To determine whether Xist-induced sequestration requires SPEN or RBM15, TETRIS assays using Xist-2kb were performed in ESCs following CRISPR-mediated deletion (SPEN) or depletion (RBM15) of the proteins. For SPEN, ∼40kb of the gene, including its major RNA-binding domains (21), was targeted for deletion by CRISPR (Supplementary Figure S7A). This deletion is known to cause complete failure of XCI and is expected to comprise a null mutant (20). Deletion was confirmed in select clones by PCR of genomic DNA and RT-qPCR (Supplementary Figure S7B and Figure 5C), which showed the expected loss of Spen mRNA expression in the deleted region. TETRIS assays performed in two independent SPEN deletion lines showed that SPEN deletion had no effect on Xist-induced sequestration (Figure 5D). For RBM15, polyclonal cell populations were generated that carried sgRNAs targeting a doxycycline-inducible Cas9 to numerous locations in gene exons of Rbm15, leading to significant depletion of the protein product (Figure 5E and Supplementary Figure S7C). RBM15B, a paralog of RBM15 which can compensate for its role in Xist-mediated repression (46), was also depleted individually and in combination with RBM15 (Figure 5E and Supplementary Figure S7D). EZH2, which we did not expect to be involved in Xist-induced sequestration, was also depleted as a negative control (Figure 5E and Supplementary Figure S7E). As a second control, we performed TETRIS assays in cells which express Cas9, rtTA, and a sgRNA that lacked a genic targeting sequence (rtTA, Figure 5E). Relative to control cells that expressed the non-targeting sgRNA, TETRIS assays in knockdown cell lines showed no reduction of luciferase protein repression (Figure 5F). Thus, individual knockdown of several proteins required for transcriptional silencing by Xist – SPEN, RBM15, RBM15B and EZH2 – does not affect silencing of luciferase in TETRIS. DISCUSSION Reductionist systems are ubiquitously employed in biology to simplify complex systems into constituent parts. Such systems have fundamentally advanced our understanding of many biological processes, including transcription, splicing and epigenetic phenomena such as position effect variegation and XCI. Using two reductionist assays similar in nature to those that have been used in many prior studies in mouse ESCs to identify seminal aspects of Xist biology (7,8,15–20,27), we found, quite unexpectedly, that expression of the first 2kb of Xist was insufficient to induce transcriptional silencing of nearby genes. Instead, Xist-2kb silenced adjacent genes at the post-transcriptional level, by sequestering their RNA on chromatin. Sequestration was rapidly reversible upon loss of Xist-2kb expression, and, critically, depended on the same sequences within Repeat-A that are needed to induce transcriptional silencing by full-length Xist: its conserved, GC-rich segments, but not its intervening U-rich spacers (Figures 1 and 2; (15)). Sequestration of RNAs produced from genes located at variable distances and in different orientations relative to Xist-2kb, as well as the lack of sequestration in cells expressing Hottip or empty-cargo control RNAs, indicates that sequestration is not an effect of convergent transcription. Our ability to observe sequestration of multiple target RNAs by Xist-2kb, as well as sequestration of a single target RNA by multiple synthetic lncRNAs, indicates that sequestration is unlikely to require extensive base pairing between lncRNA and target and supports its dependence on a protein intermediary. In the context of the Rosa26 locus, there was not a clear relationship between sequestration and expression levels of Xist transgenes. Knockdown of our top candidates, SPEN and RBM15, had no effect on sequestration, suggesting the process may be mediated by other proteins. We found no evidence of ongoing sequestration at time points both early and late after induction of full-length Xist, but we did observe local sequestration by two Xist hypomorphs that were capable of chromosome-scale transcriptional silencing. These data suggest that stable sequestration of target genes is not an obligate intermediate during Xist-induced gene silencing. However, along with our experiments done with synthetic lncRNAs, they indicate that sequestration is not an action limited to Xist-2kb. Our study provides another example of a connection between lncRNAs and pathways that mediate the export of RNA from the nucleus. For reasons that are unclear, many of the proteins that Xist requires for its function have roles in RNA export (19). Moreover, the lncRNAs Malat1 and Neat1 have been shown to sequester RNAs in specific regions of the nucleus, and, intriguingly, at the level of k-mers, Malat1 and Neat1 are more similar to Xist than they are similar to most other lncRNAs (30,47–49). A recent study found that release of a lncRNA from chromatin coincided with increased transcription from a nearby protein-coding gene (50). Thus, in the mammalian nucleus, multiple connections exist between lncRNAs, RNA export and transcription. These connections extend even to the fission yeast Schizosaccharomyces pombe, where a block in RNA export during meiosis induces locus-specific formation of heterochromatin (51). Thus, in ways that are not yet understood, it is conceivable that Xist functions in part by interfering with RNA export. It is possible that sequestration of RNA occurs during the earliest phases of Xist-induced gene silencing, but that sequestration is undetectable under steady-state conditions because it destabilizes RNA and is followed immediately by transcriptional silencing. Consistent with this notion, sequestration was only apparent upon expression of Xist mutants with attenuated silencing capabilities, and sequestration reduced overall levels of target transcripts. In TETRIS, Xist-2kb reduced the total levels of PuroR mRNA by 4-fold without changing its nuclear levels and Xist-2kb also reduced the stability of luciferase and PuroR mRNA (Figure 2F and C). Similarly, in the Rosa26 locus, Xist-2kb and Xist-5.5kb reduced total levels of HygroR mRNA by 5-fold without reducing nuclear levels (Figure 4J and H). A separate study, performed in human cells, found that as few as two individual monomers from Repeat-A were sufficient to reduce the total mRNA levels of a nearby GFP reporter by half (52). While this reduction in GFP mRNA was interpreted as transcriptional silencing, the decrease could have been due to a Repeat-A induced block in nuclear export leading to destabilization of GFP mRNA. Together, these examples are not inconsistent with the possibility that a block in nuclear export of target genes (i.e. sequestration) occurs transiently during XCI, coincident with the onset of Xist-induced transcriptional silencing. Sequestration would reduce overall levels of mRNA from sequestered genes, and would be difficult to distinguish from transcriptional silencing unless RNA localization or half-life was analyzed at the appropriate time-point. However, because expression of Xist-2kb was not associated with transcriptional silencing, nor did sequestration require the critical silencing factor SPEN, nor did sequestration spread beyond genes that were directly adjacent to hypomorphic Xist insertion sites, we favor the hypothesis that the mechanisms that underlie sequestration are more directly related to the splicing, post-transcriptional stabilization and/or localization of Xist to actively transcribed regions of chromatin. All of these events, as well as sequestration, depend on a functional Repeat-A element (25–28). It is possible that the same proteins that cause Repeat-A to sequester nearby RNA on chromatin help tether Xist to actively transcribed loci through interactions with RNAs produced from soon-to-be-repressed genes. Simultaneously or independently, the proteins required for sequestration may protect the unusually large exons in Xist (>7.5kb) from unintended splicing or degradation. Studies to address these hypotheses are ongoing in our laboratory. At last, in the simplest model for XCI, Xist recruits SPEN via Repeat-A, which, in turn, recruits HDAC3 to silence transcription over the X chromosome (18). Our data definitively demonstrate that in mouse ESCs, recapitulating a SPEN/Repeat-A interaction is insufficient to induce transcriptional silencing by Xist, highlighting a critical gap in our understanding of the mechanism through which Repeat-A functions to silence gene expression. Even though Xist-2kb is retained on chromatin and binds SPEN in Repeat-A-dependent fashion, it fails to induce transcriptional silencing, even of nearby genes. However, fusion of Xist-2kb to the remaining first 5.5kb of Xist, which in our construct included Repeat-C and a portion of the essential PRC1 recruitment domain Repeat-B (Figure 4A and Supplementary Figure S3A; (8)), or the fusion of Xist-2kb with the final two exons of Xist, which lacks a PRC1 recruitment domain but contains Repeat-E and additional downstream sequence elements (6,9,44,45), both conferred near-equal transcriptional silencing capability in an isogenic context (Figure 4 and Supplementary Figures S3 and S4). Thus, in mouse ESCs, Repeat-A is necessary for Xist-induced transcriptional silencing, but it is not sufficient. Silencing requires synergy between Repeat-A and additional downstream sequence elements within Xist. It will be important to define the molecular mechanisms that underlie this synergy in future works. More broadly, the potential for similar forms of synergy between protein interaction modules in RNA is widespread in the mammalian transcriptome. For example, many of the proteins that Xist binds in cells are considered to be ‘splicing factors’ that have well-documented roles in RNA processing and export (7,8,18,26,53). The Xist cofactor HNRNPK binds thousands of different positions within thousands of RNA transcripts yet only a subset of the binding events are directly associated with changes in splicing (54,55). Still, we presume that HNRNPK binding in its own capacity is not sufficient to cause transcripts to induce Xist-like gene silencing. Similarly, SPEN family proteins are known to be involved in the nuclear export and 3′-end processing of mRNAs (56–59), and SPEN itself appears to associate with thousands of different transcripts in ESCs and in mouse trophoblast stem cells ((21,60); Supplementary Table S4; our unpublished data). Presumably, the majority of these associations do not cause Xist-like transcriptional silencing events. Understanding the properties that confer regulatory function to RNAs will require the study of their protein binding modules in isolation and in combination. DATA AVAILABILITY All genomic data, including raw sequencing files and processed data files, are available at GEO accession number GSE120197. Genomic data are also available in wiggle tracks at UCSC genome browser at the following links: Rosa26-RMCE fractionation data: http://genome.ucsc.edu/s/davidlee/rosa26_xist_fractions_lee_2019 sm33 fractionation data: http://genome.ucsc.edu/s/davidlee/sm33_fractions_lee_2019 SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank UNC colleagues for discussion, and D. Ciavatta, N. Hathaway, M. Magnuson, T. Magnuson and K. Plath for reagents. Author Contributions: D.M.L, J.B.T, and J.M.C. conceived the study, D.O.C., S.R.B, and J.M.C. designed the RMCE strategy, D.M.L, J.B.T, R.E.C., K.I., M.D.S., S.R.B, and J.M.C. designed and performed experiments, and D.M.L, J.B.T., and J.M.C. wrote the paper. FUNDING National Institutes of Health (NIH) [GM121806; T32 GM007092 to D.M.L., R.E.C., in part]; March of Dimes Foundation, Basil O’Connor Award [#5100683]; National Cancer Institute [T32 CA217824 to J.B.T.; P30 CA016086, in part]; Lineberger Comprehensive Cancer Center and UNC Department of Pharmacology (to J.M.C.). Funding for open access charge: NIH [GM121806]. Conflict of interest statement. D.O.C. is employed by, has equity ownership in and serves on the board of directors of TransViragen, the company contracted by UNC-Chapel Hill to manage its Animal Models Core Facility. The authors declare no other competing interests. Notes Present address: Kaoru Inoue, National Institute for Environmental Health Sciences, Research Triangle Park, NC 27709, USA. REFERENCES 1. Kopp F. , Mendell J.T. Functional classification and experimental dissection of long noncoding RNAs . Cell . 2018 ; 172 : 393 – 407 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Tsai M.C. , Manor O. , Wan Y. , Mosammaparast N. , Wang J.K. , Lan F. , Shi Y. , Segal E. , Chang H.Y. Long noncoding RNA as modular scaffold of histone modification complexes . Science . 2010 ; 329 : 689 – 693 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Lu Z.P. , Zhang Q.C. , Lee B. , Flynn R.A. , Smith M.A. , Robinson J.T. , Davidovich C. , Gooding A.R. , Goodrich K.J. , Mattick J.S. et al. . RNA duplex map in living cells reveals higher-order transcriptome structure . Cell . 2016 ; 165 : 1267 – 1279 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Liu F. , Somarowthu S. , Pyle A.M. Visualizing the secondary and tertiary architectural domains of lncRNA RepA . Nat. Chem. Biol. 2017 ; 13 : 282 – 289 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Somarowthu S. , Legiewicz M. , Chillon I. , Marcia M. , Liu F. , Pyle A.M. HOTAIR forms an intricate and modular secondary structure . Mol. Cell . 2015 ; 58 : 353 – 361 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Smola M.J. , Christy T.W. , Inoue K. , Nicholson C.O. , Friedersdorf M. , Keene J.D. , Lee D.M. , Calabrese J.M. , Weeks K.M. SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells . Proc. Natl. Acad. Sci. U.S.A. 2016 ; 113 : 10322 – 10327 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Chu C. , Zhang Q.C. , da Rocha S.T. , Flynn R.A. , Bharadwaj M. , Calabrese J.M. , Magnuson T. , Heard E. , Chang H.Y. Systematic discovery of Xist RNA binding proteins . Cell . 2015 ; 161 : 404 – 416 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Pintacuda G. , Wei G. , Roustan C. , Kirmizitas B.A. , Solcan N. , Cerase A. , Castello A. , Mohammed S. , Moindrot B. , Nesterova T.B. et al. . hnRNPK recruits PCGF3/5-PRC1 to the Xist RNA B-repeat to establish polycomb-mediated chromosomal silencing . Mol. Cell . 2017 ; 68 : 955 – 969 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Ridings-Figueroa R. , Stewart E.R. , Nesterova T.B. , Coker H. , Pintacuda G. , Godwin J. , Wilson R. , Haslam A. , Lilley F. , Ruigrok R. et al. . The nuclear matrix protein CIZ1 facilitates localization of Xist RNA to the inactive X-chromosome territory . Genes Dev. 2017 ; 31 : 876 – 888 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Sunwoo H. , Wu J.Y. , Lee J.T. The Xist RNA-PRC2 complex at 20-nm resolution reveals a low Xist stoichiometry and suggests a hit-and-run mechanism in mouse cells . Proc. Natl. Acad. Sci. U.S.A. 2015 ; 112 : E4216 – E4225 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Brockdorff N. , Ashworth A. , Kay G.F. , McCabe V.M. , Norris D.P. , Cooper P.J. , Swift S. , Rastan S. The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus . Cell . 1992 ; 71 : 515 – 526 . Google Scholar Crossref Search ADS PubMed WorldCat 12. Brown C.J. , Hendrich B.D. , Rupert J.L. , Lafreniere R.G. , Xing Y. , Lawrence J. , Willard H.F. The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus . Cell . 1992 ; 71 : 527 – 542 . Google Scholar Crossref Search ADS PubMed WorldCat 13. Nesterova T.B. , Slobodyanyuk S.Y. , Elisaphenko E.A. , Shevchenko A.I. , Johnston C. , Pavlova M.E. , Rogozin I.B. , Kolesnikov N.N. , Brockdorff N. , Zakian S.M. Characterization of the genomic Xist locus in rodents reveals conservation of overall gene structure and tandem repeats but rapid evolution of unique sequence . Genome Res. 2001 ; 11 : 833 – 849 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Yen Z.C. , Meyer I.M. , Karalic S. , Brown C.J. A cross-species comparison of X-chromosome inactivation in Eutheria . Genomics . 2007 ; 90 : 453 – 463 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Wutz A. , Rasmussen T.P. , Jaenisch R. Chromosomal silencing and localization are mediated by different domains of Xist RNA . Nat. Genet. 2002 ; 30 : 167 – 174 . Google Scholar Crossref Search ADS PubMed WorldCat 16. Kohlmaier A. , Savarese F. , Lachner M. , Martens J. , Jenuwein T. , Wutz A. A chromosomal memory triggered by Xist regulates histone methylation in X inactivation . PLoS Biol. 2004 ; 2 : e171 . Google Scholar Crossref Search ADS PubMed WorldCat 17. Chaumeil J. , Le Baccon P. , Wutz A. , Heard E. A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced . Genes Dev. 2006 ; 20 : 2223 – 2237 . Google Scholar Crossref Search ADS PubMed WorldCat 18. McHugh C.A. , Chen C.K. , Chow A. , Surka C.F. , Tran C. , McDonel P. , Pandya-Jones A. , Blanco M. , Burghard C. , Moradian A. et al. . The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3 . Nature . 2015 ; 521 : 232 – 236 . Google Scholar Crossref Search ADS PubMed WorldCat 19. Moindrot B. , Cerase A. , Coker H. , Masui O. , Grijzenhout A. , Pintacuda G. , Schermelleh L. , Nesterova T.B. , Brockdorff N. A Pooled shRNA Screen Identifies Rbm15, Spen, and Wtap as Factors Required for Xist RNA-Mediated Silencing . Cell Rep. 2015 ; 12 : 562 – 572 . Google Scholar Crossref Search ADS PubMed WorldCat 20. Monfort A. , Di Minin G. , Postlmayr A. , Freimann R. , Arieti F. , Thore S. , Wutz A. Identification of spen as a crucial factor for Xist function through forward genetic screening in haploid embryonic stem cells . Cell Rep. 2015 ; 12 : 554 – 561 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Cirillo D. , Blanco M. , Armaos A. , Buness A. , Avner P. , Guttman M. , Cerase A. , Tartaglia G.G. Quantitative predictions of protein interactions with long noncoding RNAs . Nat. Methods . 2016 ; 14 : 5 – 6 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Ariyoshi M. , Schwabe J.W. A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling . Genes Dev. 2003 ; 17 : 1909 – 1920 . Google Scholar Crossref Search ADS PubMed WorldCat 23. Oswald F. , Kostezka U. , Astrahantseff K. , Bourteele S. , Dillinger K. , Zechner U. , Ludwig L. , Wilda M. , Hameister H. , Knochel W. et al. . SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway . EMBO J. 2002 ; 21 : 5417 – 5426 . Google Scholar Crossref Search ADS PubMed WorldCat 24. Shi Y. , Downes M. , Xie W. , Kao H.Y. , Ordentlich P. , Tsai C.C. , Hon M. , Evans R.M. Sharp, an inducible cofactor that integrates nuclear receptor repression and activation . Genes Dev. 2001 ; 15 : 1140 – 1151 . Google Scholar Crossref Search ADS PubMed WorldCat 25. Hoki Y. , Kimura N. , Kanbayashi M. , Amakawa Y. , Ohhata T. , Sasaki H. , Sado T. A proximal conserved repeat in the Xist gene is essential as a genomic element for X-inactivation in mouse . Development . 2009 ; 136 : 139 – 146 . Google Scholar Crossref Search ADS PubMed WorldCat 26. Royce-Tolland M.E. , Andersen A.A. , Koyfman H.R. , Talbot D.J. , Wutz A. , Tonks I.D. , Kay G.F. , Panning B. The A-repeat links ASF/SF2-dependent Xist RNA processing with random choice during X inactivation . Nat. Struct. Mol. Biol. 2010 ; 17 : 948 – 954 . Google Scholar Crossref Search ADS PubMed WorldCat 27. Engreitz J.M. , Pandya-Jones A. , McDonel P. , Shishkin A. , Sirokman K. , Surka C. , Kadri S. , Xing J. , Goren A. , Lander E.S. et al. . The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome . Science . 2013 ; 341 : 1237973 . Google Scholar Crossref Search ADS PubMed WorldCat 28. Chigi Y. , Sasaki H. , Sado T. The 5′ region of Xist RNA has the potential to associate with chromatin through the A-repeat . RNA . 2017 ; 23 : 1894 – 1901 . Google Scholar Crossref Search ADS PubMed WorldCat 29. Ha N. , Lai L.T. , Chelliah R. , Zhen Y. , Yi Vanessa S.P. , Lai S.K. , Li H.Y. , Ludwig A. , Sandin S. , Chen L. et al. . Live-cell imaging and functional dissection of xist RNA reveal mechanisms of X chromosome inactivation and reactivation . iScience . 2018 ; 8 : 1 – 14 . Google Scholar Crossref Search ADS PubMed WorldCat 30. Kirk J.M. , Kim S.O. , Inoue K. , Smola M.J. , Lee D.M. , Schertzer M.D. , Wooten J.S. , Baker A.R. , Sprague D. , Collins D.W. et al. . Functional classification of long non-coding RNAs by k-mer content . Nat. Genet. 2018 ; 50 : 1474 – 1482 . Google Scholar Crossref Search ADS PubMed WorldCat 31. Hoki Y. , Ikeda R. , Mise N. , Sakata Y. , Ohhata T. , Sasaki H. , Abe K. , Sado T. Incomplete X-inactivation initiated by a hypomorphic Xist allele in the mouse . Development . 2011 ; 138 : 2649 – 2659 . Google Scholar Crossref Search ADS PubMed WorldCat 32. Bhatt D.M. , Pandya-Jones A. , Tong A.J. , Barozzi I. , Lissner M.M. , Natoli G. , Black D.L. , Smale S.T. Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions . Cell . 2012 ; 150 : 279 – 290 . Google Scholar Crossref Search ADS PubMed WorldCat 33. Wysocka J. , Reilly P.T. , Herr W. Loss of HCF-1-chromatin association precedes temperature-induced growth arrest of tsBN67 cells . Mol. Cell Biol. 2001 ; 21 : 3820 – 3829 . Google Scholar Crossref Search ADS PubMed WorldCat 34. Dunagin M. , Cabili M.N. , Rinn J. , Raj A. Visualization of lncRNA by single-molecule fluorescence in situ hybridization . Methods Mol. Biol. 2015 ; 1262 : 3 – 19 . Google Scholar Crossref Search ADS PubMed WorldCat 35. Gossen M. , Freundlieb S. , Bender G. , Muller G. , Hillen W. , Bujard H. Transcriptional activation by tetracyclines in mammalian cells . Science . 1995 ; 268 : 1766 – 1769 . Google Scholar Crossref Search ADS PubMed WorldCat 36. Calabrese J.M. , Starmer J. , Schertzer M.D. , Yee D. , Magnuson T. A survey of imprinted gene expression in mouse trophoblast stem cells . G3 (Bethesda) . 2015 ; 5 : 751 – 759 . Google Scholar Crossref Search ADS PubMed WorldCat 37. Calabrese J.M. , Sun W. , Song L. , Mugford J.W. , Williams L. , Yee D. , Starmer J. , Mieczkowski P. , Crawford G.E. , Magnuson T. Site-specific silencing of regulatory elements as a mechanism of X inactivation . Cell . 2012 ; 151 : 951 – 963 . Google Scholar Crossref Search ADS PubMed WorldCat 38. Love M.I. , Huber W. , Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biol. 2014 ; 15 : 550 . Google Scholar Crossref Search ADS PubMed WorldCat 39. Team R.C. R: A Language and Environment for Statistical Computing . 2018 ; Vienna R Foundation for Statistical Computing . Google Preview WorldCat COPAC 40. Raab J.R. , Smith K.N. , Spear C.C. , Manner C.J. , Calabrese J.M. , Magnuson T. SWI/SNF remains localized to chromatin in the presence of SCHLAP1 . Nat. Genet. 2019 ; 51 : 26 – 29 . Google Scholar Crossref Search ADS PubMed WorldCat 41. Presnyak V. , Alhusaini N. , Chen Y.H. , Martin S. , Morris N. , Kline N. , Olson S. , Weinberg D. , Baker K.E. , Graveley B.R. et al. . Codon optimality is a major determinant of mRNA stability . Cell . 2015 ; 160 : 1111 – 1124 . Google Scholar Crossref Search ADS PubMed WorldCat 42. Adelman K. , Lis J.T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans . Nat. Rev. Genet. 2012 ; 13 : 720 – 731 . Google Scholar Crossref Search ADS PubMed WorldCat 43. Chen S.X. , Osipovich A.B. , Ustione A. , Potter L.A. , Hipkens S. , Gangula R. , Yuan W.P. , Piston D.W. , Magnuson M.A. Quantification of factors influencing fluorescent protein expression using RMCE to generate an allelic series in the ROSA26 locus in mice . Dis. Model Mech. 2011 ; 4 : 537 – 547 . Google Scholar Crossref Search ADS PubMed WorldCat 44. Sunwoo H. , Colognori D. , Froberg J.E. , Jeon Y. , Lee J.T. Repeat E anchors Xist RNA to the inactive X chromosomal compartment through CDKN1A-interacting protein (CIZ1) . Proc. Natl. Acad. Sci. U.S.A. 2017 ; 114 : 10654 – 10659 . Google Scholar Crossref Search ADS PubMed WorldCat 45. Yamada N. , Hasegawa Y. , Yue M. , Hamada T. , Nakagawa S. , Ogawa Y. Xist exon 7 contributes to the stable localization of Xist RNA on the Inactive X-Chromosome . PLos Genet. 2015 ; 11 : e1005430 . Google Scholar Crossref Search ADS PubMed WorldCat 46. Patil D.P. , Chen C.K. , Pickering B.F. , Chow A. , Jackson C. , Guttman M. , Jaffrey S.R. m(6)A RNA methylation promotes XIST-mediated transcriptional repression . Nature . 2016 ; 537 : 369 – 373 . Google Scholar Crossref Search ADS PubMed WorldCat 47. Chen L.L. , Carmichael G.G. Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem cells: functional role of a nuclear noncoding RNA . Mol. Cell . 2009 ; 35 : 467 – 478 . Google Scholar Crossref Search ADS PubMed WorldCat 48. Prasanth K.V. , Prasanth S.G. , Xuan Z. , Hearn S. , Freier S.M. , Bennett C.F. , Zhang M.Q. , Spector D.L. Regulating gene expression through RNA nuclear retention . Cell . 2005 ; 123 : 249 – 263 . Google Scholar Crossref Search ADS PubMed WorldCat 49. Tripathi V. , Ellis J.D. , Shen Z. , Song D.Y. , Pan Q. , Watt A.T. , Freier S.M. , Bennett C.F. , Sharma A. , Bubulya P.A. et al. . The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation . Mol. Cell . 2010 ; 39 : 925 – 938 . Google Scholar Crossref Search ADS PubMed WorldCat 50. Ntini E. , Louloupi A. , Liz J. , Muino J.M. , Marsico A. , Orom U.A.V. Long ncRNA A-ROD activates its target gene DKK1 at its release from chromatin . Nat. Commun. 2018 ; 9 : 1636 . Google Scholar Crossref Search ADS PubMed WorldCat 51. Reyes-Turcu F.E. , Grewal S.I. Different means, same end-heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission yeast . Curr. Opin. Genet. Dev. 2012 ; 22 : 156 – 163 . Google Scholar Crossref Search ADS PubMed WorldCat 52. Minks J. , Baldry S.E. , Yang C. , Cotton A.M. , Brown C.J. XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells . Epigenet.Chromatin . 2013 ; 6 : 23 . Google Scholar Crossref Search ADS WorldCat 53. Minajigi A. , Froberg J.E. , Wei C. , Sunwoo H. , Kesner B. , Colognori D. , Lessing D. , Payer B. , Boukhali M. , Haas W. et al. . A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation . Science . 2015 ; 349 : 282 . Google Scholar Crossref Search ADS WorldCat 54. Huelga S.C. , Vu A.Q. , Arnold J.D. , Liang T.Y. , Liu P.P. , Yan B.Y. , Donohue J.P. , Shiue L. , Hoon S. , Brenner S. et al. . Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins . Cell Rep. 2012 ; 1 : 167 – 178 . Google Scholar Crossref Search ADS PubMed WorldCat 55. Van Nostrand E.L. , Pratt G.A. , Shishkin A.A. , Gelboin-Burkhart C. , Fang M.Y. , Sundararaman B. , Blue S.M. , Nguyen T.B. , Surka C. , Elkins K. et al. . Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP) . Nat. Methods . 2016 ; 13 : 508 – 514 . Google Scholar Crossref Search ADS PubMed WorldCat 56. Hornyik C. , Terzi L.C. , Simpson G.G. The spen family protein FPA controls alternative cleavage and polyadenylation of RNA . Dev. Cell . 2010 ; 18 : 203 – 213 . Google Scholar Crossref Search ADS PubMed WorldCat 57. Lindtner S. , Zolotukhin A.S. , Uranishi H. , Bear J. , Kulkarni V. , Smulevitch S. , Samiotaki M. , Panayotou G. , Felber B.K. , Pavlakis G.N. RNA-binding motif protein 15 binds to the RNA transport element RTE and provides a direct link to the NXF1 export pathway . J. Biol. Chem. 2006 ; 281 : 36915 – 36928 . Google Scholar Crossref Search ADS PubMed WorldCat 58. Majerciak V. , Uranishi H. , Kruhlak M. , Pilkington G.R. , Massimelli M.J. , Bear J. , Pavlakis G.N. , Felber B.K. , Zheng Z.M. Kaposi's sarcoma-associated herpesvirus ORF57 interacts with cellular RNA export cofactors RBM15 and OTT3 to promote expression of viral ORF59 . J. Virol. 2011 ; 85 : 1528 – 1540 . Google Scholar Crossref Search ADS PubMed WorldCat 59. Uranishi H. , Zolotukhin A.S. , Lindtner S. , Warming S. , Zhang G.M. , Bear J. , Copeland N.G. , Jenkins N.A. , Pavlakis G.N. , Felber B.K. The RNA-binding motif protein 15B (RBM15B/OTT3) acts as cofactor of the nuclear export receptor NXF1 . J. Biol. Chem. 2009 ; 284 : 26106 – 26116 . Google Scholar Crossref Search ADS PubMed WorldCat 60. Chen C.K. , Blanco M. , Jackson C. , Aznauryan E. , Ollikainen N. , Surka C. , Chow A. , Cerase A. , McDonel P. , Guttman M. Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing . Science . 2016 ; 354 : 468 – 472 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
TI - A 5′ fragment of Xist can sequester RNA produced from adjacent genes on chromatin
JF - Nucleic Acids Research
DO - 10.1093/nar/gkz432
DA - 2019-07-26
UR - https://www.deepdyve.com/lp/oxford-university-press/a-5-fragment-of-xist-can-sequester-rna-produced-from-adjacent-genes-on-DX0awm1Qcp
SP - 7049
VL - 47
IS - 13
DP - DeepDyve
ER -