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Decapping enzyme 1A breaks X-chromosome symmetry by controlling Tsix elongation and RNA turnover

Nature Cell Biology volume 22, pages 1116–1129 (2020)Cite this article

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Abstract

How allelic asymmetry is generated remains a major unsolved problem in epigenetics. Here we model the problem using X-chromosome inactivation by developing “BioRBP”, an enzymatic RNA-proteomic method that enables probing of low-abundance interactions and an allelic RNA-depletion and -tagging system. We identify messenger RNA-decapping enzyme 1A (DCP1A) as a key regulator of Tsix, a noncoding RNA implicated in allelic choice through X-chromosome pairing. DCP1A controls Tsix half-life and transcription elongation. Depleting DCP1A causes accumulation of X–X pairs and perturbs the transition to monoallelic Tsix expression required for Xist upregulation. While ablating DCP1A causes hyperpairing, forcing Tsix degradation resolves pairing and enables Xist upregulation. We link pairing to allelic partitioning of CCCTC-binding factor (CTCF) and show that tethering DCP1A to one Tsix allele is sufficient to drive monoallelic Xist expression. Thus, DCP1A flips a bistable switch for the mutually exclusive determination of active and inactive Xs.

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Fig. 1: BioRBP, a new method to decrypt the RNA proteome of Tsix, identifies DCP1A.
Fig. 2: DCP1A resolves X–X pairs and is required for XCI initiation.
Fig. 3: Allele-specific RNA ablation establishes the essential nature of Tsix RNA.
Fig. 4: Tsix RNA drives formation of X–X pairs and DCP1A resolves the paired state.
Fig. 5: CTCF-degron analysis shows that pairing frequency depends on CTCF levels.
Fig. 6: Tsix RNA sequesters CTCF from chromatin and controls allelic partitioning of CTCF.
Fig. 7: DCP1A regulates Tsix stability and transcription elongation.
Fig. 8: DCP1A breaks X-chromosome symmetry.

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Data availability

Raw and processed sequencing data files have been deposited at the Gene Expression Omnibus under accession number GSE118943. Raw and processed proteomic data files have been deposited at ProteomeXchange under accession number PXD019133. Previously published DRIP-seq data were reanalysed here and the original raw data are available at Gene Expression Omnibus under accession code GSE67581. All other data supporting the findings are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by an Advanced Postdoc Mobility Fellowship from the Swiss National Science Foundation (SNSF) to E.A., an NSF Predoctoral Fellowship and Herschel Smith Fellowship to A.K. and grants from the NIH (R01-GM58839) and the Howard Hughes Medical Institute to J.T.L. We thank B. Nabet and N. S. Gray for advice in generating the CTCF–FKBPdegron line and for the generous gift of dTAG-13; C. Cifuentes-Rojas, M. Blower, A. J. Hernandez, Y. Jeon and A.-S. Reis for technical advice and discussion; D. Trono and I. Barde for lentiviral constructs; E. Heard for sharing the XTetOXTetO/laminB cells; W. Press for mouse management; D. Colognori for oligonucleotides FISH probes; and L. Carrette, Y. Jeon, A. Szanto, J. E. Froberg, R. Aguilar and C. Y. Wang for qPCR primers.

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  1. These authors contributed equally: Hun-Goo Lee, Yong-Woo Lee.

Authors and Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

    Eric Aeby, Hun-Goo Lee, Yong-Woo Lee, Andrea Kriz, Brian C. del Rosario, Hyun Jung Oh & Jeannie T. Lee

  2. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Eric Aeby, Hun-Goo Lee, Yong-Woo Lee, Andrea Kriz, Brian C. del Rosario, Hyun Jung Oh & Jeannie T. Lee

  3. Massachusetts General Hospital Cancer Center, Boston, MA, USA

    Myriam Boukhali & Wilhelm Haas

  4. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Myriam Boukhali & Wilhelm Haas

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Contributions

E.A. and J.T.L. designed the project, analysed data and wrote the paper. E.A. established Tsix allele-specific targeting, Xist and Tsix BioRBP, and performed DCP1A, FISH and ChIP experiments. E.A. performed PRO-seq and RNA-seq experiments. Y.-W.L. performed pairing assays in CTCF–FKBPdegron and XTetOXTetO/laminB cells. H.-G.L. conducted DRIP-seq and PRO-seq bioinformatic analyses with assistance from E.A. A.K. constructed the CTCF–FKBPdegron cell lines. B.C.d.R. performed the apoptosis analysis in mouse embryos with assistance from E.A. H.J.O. performed allelic CTCF ChIP experiments with E.A. Mass spectrometry analysis was conducted by M.B. and W.H.

Corresponding author

Correspondence to Jeannie T. Lee.

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J.T.L. cofounded Translate Bio and Fulcrum Therapeutics and serves as an advisor to Skyhawk Therapeutics.

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Extended data

Extended Data Fig. 1 Generation of Tsix BioRBP 5’ and 3’ clones.

a, PCR-screening for homozygote integration of 3xPP7 in Tsix. Successful insertion in Clones 1 and 2, evidenced by 97 bp shift of PCR product. Representative result from 3 biological replicates shown. b, Schematic of Southern blot to validate PP7 integration. c, Southern blot analysis of Clones 1 and 2. PP7 probe detects two bands in each clone, reflecting variations in DXPas34 repeat length. ES clones are a Mus/Cas hybrid. Mus repeat is longer. A representative result is shown from 3 biological replicates shown. Unprocessed blots are provided in Source data Extended Data Fig. 1. d, Clone 2 ImmunoFISH shows recruitment of PP7cp-BirA* (cyan) to tagged Tsix RNA (red). Bar: 5 μm. Among the 79 cells examined from one representative replicate, 90% showed colocalized foci. Three independent replicates showed similar results. e, BioRBP application to negative control, Tsix 3’ end. Position of homozygous 3xPP7 insertion is indicated. f, PCR-screening for homozygous integration of 3xPP7 in Tsix 3’ end. Successful insertion in Clone 3, evidenced by 97 bp shift of PCR product. Representative result from 3 biological replicates shown.

Source data

Extended Data Fig. 2 DCP1A interacts with Tsix RNA and resolves X-X pairs.

a, PCR-screening for homozygous integration of AviTag-FLAG at N-terminus of endogenous Dcp1a by CRISPR/Cas9. Successful insertion in Clones F2 and D11, as evidenced by a 99 bp shift of 200 bp WT PCR product. Clones validated by Sanger sequencing and Western blot analysis. Representative result from 3 biological replicates shown. b, Second representative image for immunoFISH experiments shown in Fig. 1i. One of 4 independent replicates shown. c, Western analysis of transient DCP1A knockdown by siRNA in undifferentiated ES cells. RT-qPCR showed 30% residual DCP1A. OCT4 and histone H3 levels are shown. Scr kd: scramble control. Representative result from 3 independent experiments shown. See also Source Data Supplementary Figure 5. d, Fgf5 mRNA levels upon DCP1A knockdown in days 0 and 3 cells. Values normalized to Gapdh RNA at day 0 in Scr kd cells. Mean and s.e. indicated. n=3 biological replicates. e,f, X-X pairing analysis by DNA FISH after DCP1A vs scramble knockdown. Biological replicate #2 for analysis in Fig. 2b,c. A total of three biological replicates was performed with similar results. Whole distributions (e) and cumulative frequencies (f) of inter-Tsix distances shown. Average distances \(\left( {{\bar{\mathrm X}}} \right)\) and p-value (KS-test, two-tailed) are indicated. N, sample size. A representative image with paired and unpaired X-chromosomes is shown. Bar: 5 μm. Statistical source data and unprocessed blots are provided in Source data Extended Data Fig. 2.

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Extended Data Fig. 3 Dcp1a knockout results in persistent X-X pairing and impair Xist upregulation.

a, DCP1A ORF alignment from Sanger sequencing of parental and the two knockout (KO) clones C17 and J15. CRISPR/Cas9 transfection resulted in a short 7 base pairs deletion in both clones and a premature stop codon after DCP1A amino acid 14 (shown in red: stop). b, Western blot analysis confirms complete disappearance of DCP1A protein in both C17 and J15 KO clones. Data shown represent three independent experiments. c–f, X-X pairing analysis by DNA FISH upon DCP1A knockout (DCP1A -/-) vs parental. Biological replicate for the analysis shown in Fig. 2e,f, using DCP1A-KO clone C17. Whole distribution and cumulative frequencies of inter-Tsix distances for differentiation day 2 (c, d) and day 3 (e, f) are shown. Average distances \(\left( {{\bar{\mathrm X}}} \right)\) and p-value (KS-test, two-tailed) are indicated. N=number of analyzed nuclei for each condition in this replicate experiment. In total, the pairing analysis was performed in two biological replicates, two separate clones: KO clones J15 and C17. (g) RNA FISH showing blunted Xist upregulation in day 4 DCP1A-depleted cells. Xist RNA, red. Nuclei, DAPI (blue). Quantification in Fig. 2i. Representative result from 2 independent experiments shown. (h) Impaired Xist expression was not caused by failed ES differentiation. mRNA levels of indicated genes in day 0 and differentiation day 3/4 female ES cells upon DCP1A knockdown. Values normalized to day 0 scramble-treated cells using Gapdh level. Mean and s.e. are indicated, n=3 biological replicates.

Source data

Extended Data Fig. 4 Allele-specific RNA tagging and ablation establish essential nature of the antisense RNA without interfering with POL-II transcription.

a, Schematic of 3xPP7 (mus allele) and 3xMS2 (cas allele) insertions into Tsix. CRISPR/Cas9 targeted mus-Tsix first. PCR primers for clone screening is indicated (Primers 1 and 2), along with XcmI restriction site. b, PCR-screening showing three positive heterozygous clones for 3xPP7 mus-Tsix insertion. Representative result from 3 independent experiments shown. c, PCR-screening for cas-Tsix insertion of 3xMS2 in C11 clone identified in (b). Positive clones E20 (Clone B) and P20 (Clone A) shown. Representative result from 3 independent experiments shown. d, XcmI digestion confirms the insertion of 3xPP7 (390 bp, mus) and 3xMS2 (digestion pattern: 120+252 bp, cas) into clones E20 and P20. Representative result from 3 independent experiments shown. (e) Allele-specific Tsix RNA levels 6 hours post-LNA nucleofection for independent Clone B. Description as in Fig. 3b. f, Allele-specific Xist RNA level analysis upon Tsix knockdown shown in panel (a) and 24h post nucleofection. Normalized to scramble control. g, Allele-specific Tsix RNA levels 1 and 3 hours post-LNA nucleofection (Clone B). One LNA targeting Tsix mus (PP7) and two LNAs targeting cas (MS2) were used. Normalized to scramble control. h, Allele-specific Xist RNA levels upon Tsix knockdown show a rapid and stable Xist induction at 1 and 3 hours post-nucleofection of Clone B. Xist upregulation is maintained 48-72 hours post-nucleofection. Normalized to scramble control. i, Quantification of Xist RNA clouds upon Tsix knockdown for the analysis in Fig. 3d. Number of nuclei (n) scored across two independent experiments for each clone as shown. In total, 4 biological replicates have been done for this assay with two independent experiments performed for each clone. j, Allele-specific ChIP-qPCR for POL-II-S2- and -S5 phosphorylated isoforms at indicated positions. Negative control IgG pulldown also shown. PCR using primers upstream of LNA-targeted region (Tsix E3). An intergenic region served as negative control (Xneg). Mean and s.e. are indicated, n=3 independent replicates.

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Extended Data Fig. 5 Persistent X-X pairing upon DCP1A depletion and rescue through targeted Tsix degradation.

a, Complete panels for allele-specific Tsix RNA FISH in Clones A and B and parental cell line. See Fig. 4a,b for details. Bar: 5 μm. Representative result from 3 independent experiments for each. b,c, Biological replicate of X-X pairing assayed by RNA FISH upon DCP1A vs scramble (Scr) knockdown. Whole distributions (b) and cumulative frequencies (c) of inter-Tsix distances are shown for Clone A. Average distances \(\left( {{\bar{\mathrm X}}} \right)\) and p-value (KS-test, two-tailed) are indicated. N=sample size. Representative result from 3 independent experiments. d,e, X-X pairing rescue experiment. Pairing assayed by RNA FISH. Biological replicate #2 of experiment described in Fig. 4e–g. See Fig. 4e–g legend for details. Representative result from 3 independent experiments. f,g, X-X pairing rescue experiment, as outlined in Fig. 4e. Pairing assayed by DNA FISH. Biological replicate #1. h,i, Biological replicate #2 for the X-X pairing rescue experiment, as outlined in Fig. 4e–h. X-X pairing analysis by DNA FISH upon DCP1A knockdown and mus Tsix RNA knockdown. Whole distributions (h) and cumulative frequencies (i) of inter-Tsix distances are shown. P-value (KS-test, two-tailed, comparing siDCP1A + ScrLNA and siDCP1A + TsixLNA) is indicated. n=number of nuclei scored for each sample. j, Allele-specific Xist RNA level analysis during concurrent DCP1A and scramble/Tsix knockdown for the experiment in (h, i). Averages from Biological replicate #2 shown. A total of three biological replicates showed similar findings. See Fig. 4h for Biological replicate #1. Statistical source data and unprocessed blots are provided in Source data Extended Data Fig. 5.

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Extended Data Fig. 6 Evidence against the Stochastic Model for allelic choice.

a, Time-course analysis of XCI establishment during peri-implantation stages of mouse embryonic development demonstrates XCI taking place during 24h time-window (From E5.5 to E6.5). H3K27me3 (Xi marker) and DAPI staining shown. Epi: epiblast. Bar: 50 μm. Representative results shown from 4 independent female embryos of each stage showed similar results. b, XCI establishment results in random activation of one of two X-chromosomes. Schematic of possible outcomes for the mouse cross between a male carrying a Tsix deletion and a wildtype female, resulting empirically in skewed inactivation of Tsix-deleted paternal allele. Two possible mechanisms by which the non-random pattern could arise are shown. Model 1 (Deterministic): Tsix controls X-chromosome choice for inactivation. Model 2: Tsix does not directly control choice but solely prevents inactivation of the second X-chromosome. M, maternal X; P, paternal X.

Extended Data Fig. 7 XTetOXTetO /TetR-EGFP-LaminB1 “tethering” system is not suitable to test pairing’s effect on XCI.

a, Nuclear periphery localization of stably expressed TetR-EGFP-LaminB1 fusion proteins. Bar: 5 μm. One replicate of two shown. b, Whole distributions of inter-Xic allelic differences in untethered (+Dox) versus tethered (-Dox) conditions during cell differentiation using adherent growth conditions. Χ, Average distances. P-values (KS test, two-tailed) determined significance of the difference between day 0 versus days 1-4 timepoints. Sample size (n) = number of cells counted for each timepoint in one biological replicate. Two biological replicates for adherent growth showed similar results. Two additional biological replicates using the suspension growth method also showed similar results (panels g-i below). c, Cumulative frequencies from 0.0 to 0.2 uM distance for the experiment shown in (b). P, two-tailed Fisher’s exact test comparing differences between day 0 versus days 1-4, for the distribution between 0-2 uM. d, Cell clumping due to adherent growth conditions for the experiments shown in (b, c). Two replicates showed similar clumping. Xist RNA (red). Nuclei, DAPI (blue). Bar: 5 μm. e, Xist RNA FISH on different days of differentiation, with or without Dox treatment. 100 cells counted for each sample. f, Alternative growth conditions under suspension culture as embryoid bodies. Cells were dispersed prior to FISH analysis. Xist RNA (red). Nuclei, DAPI (blue). Bar: 5 μm. Sample size (n) = number of cells counted for each timepoint in one biological replicate. Two biological replicates for suspension growth showed similar results. Two additional biological replicates using the adherent growth method also showed similar results (panels b-e above). g, Whole distributions of inter-Xic allelic differences in untethered (+Dox) versus tethered (-Dox) conditions during cell differentiation for the experiment shown in f, Χ, average distance. P-values (KS test, two-tailed) determined significance of difference between day 0 versus day 1-4 timepoints. h, Cumulative frequencies from 0.0 to 0.2 uM distance. P, two-tailed Fisher’s exact test comparing differences between day 0 versus days 1-4, for the distribution between 0-2 uM. (i) Xist RNA FISH on different days of differentiation, +/-Dox treatment. 100 nuclei counted for each sample. Statistical source data are provided in Source data Extended Data Fig. 7.

Source data

Extended Data Fig. 8 DCP1A limits transcription pause-release of Tsix.

a, RNAseq analysis of Tsix population in wildtype (Parental) vs Dcp1a-/- clone J15 day 0 female ES cells. Blue track, difference in FPM between mutant and wildtype. FPM scales shown in brackets. b, Differential expression analysis by RNAseq comparing Dcp1a-/- clone C17 to Parental line. c, Differential expression analysis by RNAseq comparing Dcp1a-/- clone J15 to Parental line. No significant changes can be detected between clone C17 and J15. d, Shown at FPM scale of 0-30, PRO-seq analysis of Tsix transcription in Parental vs Dcp1a-/- clone C17 day 0 cells. Green track, difference in FPM between mutant and wildtype. Purple arrow, main POLII pause site. e, Shown at FPM scale of 0-330, PRO-seq analysis of Tsix transcription in Parental vs Dcp1a-/- clone C17 day 0 cells. Green track, difference in FPM between mutant and wildtype. Purple arrow, main POLII pause site. f, Genome-wide metagene analysis and heat map of PROseq: Dcp1a-/- versus wildtype parental cells. Signal ratio is shown with strand specificity. Transcription start site (TSS) and transcription termination site (TTS) +/- 1 kb were used with gene body size normalized to 5 kb. Two biological replicates showed similar results. g, Increase in genome-wide pausing is reproducible in two Dcp1a-/- clones. Box plots showing pausing index ratios between wildtype and Clone J15 are drawn with n=500 genes for each group. Top 500 genes showing increased pausing in Clone C17 are compared to two random sets of 500 genes. Upper whiskers are defined at 75 percentile (Q3) + 1.5 * Interquartile Range (IQR) and lower whiskers are defined at the minima or 25 percentile (Q1) +1.5 * IQR whichever is the larger, where IQR is defined as Q3-Q1. Values outside of the upper and lower whiskers are indicated as open circles. Statistical source data are provided in Source data Extended Data Fig. 8.

Source data

Extended Data Fig. 9 PROseq analysis show appropriate changes in pluripotency and early cell differentiation markers.

PROseq analysis of Eef1a1 a, Rex1 b, Nanog c, and Oct6 d, transcription in differentiating Tsix +/- female hybrid ES cells3. Undifferentiated ES cells (day 0) and differentiating cells (day 3 and day 7) are shown. Strand-specific analysis: Watson strand (+, red) and Crick strand (-, blue). Two biological replicates (Repli1, 2) are shown. Scale is indicated at top (same for all lanes of one gene).

Extended Data Fig. 10 DCP1 inhibits full-length elongation of Tsix transcript.

a, PROseq analysis of Tsix and Xist transcription in differentiating Tsix+/- female hybrid ES cells3. Undifferentiated ES cells (day 0) and differentiating cells (day 3 and day 7) are shown. Strand-specific analysis: Watson strand (+, red) and Crick strand (-, blue). Dashed line indicates region magnified in Extended Data Fig. 8d. Biological replicate 1. b, Shown at an FPM scale of 0-1250, PROseq analysis of Tsix and Xist transcription in differentiating Tsix+/- female hybrid ES cells. Undifferentiated ES cells (day 0) and differentiating cells (day 3 and day 7) are shown. Strand-specific analysis: Watson strand (+, red) and Crick strand (-, blue). Two biological replicates are shown. c, Shown at an FPM scale of 0-200, PROseq analysis of Tsix and Xist transcription in differentiating Tsix+/- female hybrid ES cells. Undifferentiated ES cells (day 0) and differentiating cells (day 3 and day 7) are shown. Strand-specific analysis: Watson strand (+, red) and Crick strand (-, blue). Two biological replicates are shown. d, PROseq analysis of Tsix and Xist transcription in differentiating day 7 Tsix+/- female hybrid ES cells. Allele-specific analysis: Xa (cas allele, light blue), Xi (mus allele, pink). Strand-specific analysis: Watson strand (+, Tsix) and Crick strand (-, Xist). Biological replicate 1. e, PROseq analysis of Tsix and Xist transcription in differentiating Tsix+/- female hybrid ES cells. Differentiating cells (day 3 and day 7) are shown. Allele-specific analysis: Xa (cas allele, light blue), Xi (mus allele, pink). Strand-specific analysis: Watson strand (+, Tsix) and Crick strand (-, Xist). Biological replicate 2.

Supplementary information

Reporting Summary

Supplementary Tables

Table 1: list of selected candidates identified to interact with Tsix RNA, applying BioRBP. Table 2: proteomics dataset identified to interact with Tsix RNA, applying BioRBP. Table 3: proteomics of negative control for Tsix RNA. Table 4: proteomics dataset with relative enrichment between Tsix BioRBP and negative control. Table 5: list and sequence of oligonucleotides used in the study.

Source data

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Aeby, E., Lee, HG., Lee, YW. et al. Decapping enzyme 1A breaks X-chromosome symmetry by controlling Tsix elongation and RNA turnover. Nat Cell Biol 22, 1116–1129 (2020). https://doi.org/10.1038/s41556-020-0558-0

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