MiCEE is a ncRNA-protein complex that mediates epigenetic silencing and nucleolar organization

Abstract

The majority of the eukaryotic genome is transcribed into noncoding RNAs (ncRNAs), which are important regulators of different nuclear processes by controlling chromatin structure. However, the full extent of ncRNA function has remained elusive. Here we deciphered the function of the microRNA Mirlet7d as a key regulator of bidirectionally transcribed genes. We found that nuclear Mirlet7d binds ncRNAs expressed from these genes. Mirlet7d–ncRNA duplexes are further bound by C1D, which in turn targets the RNA exosome complex and the polycomb repressive complex 2 (PRC2) to the bidirectionally active loci. The exosome degrades the ncRNAs, whereas PRC2 induces heterochromatin and transcriptional silencing through EZH2. Moreover, this multicomponent RNA–protein complex, which we named MiCEE, tethers the regulated genes to the perinucleolar region and thus is required for proper nucleolar organization. Our study demonstrates that the MiCEE complex mediates epigenetic silencing of bidirectionally expressed genes and global genome organization.

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Fig. 1: Mirlet7d binds to ncRNAs in the nucleus and reduces levels of bidirectionally expressed genes.
Fig. 2: Nuclear Mirlet7d reduces ncRNA levels by RNA exosome-mediated degradation.
Fig. 3: C1D binds Mirlet7d ncRNA duplexes.
Fig. 4: Nuclear Mirlet7d reduces cRNA levels by PRC2-mediated transcriptional silencing.
Fig. 5: EXOSC10 mediates interaction between C1D and EZH2.
Fig. 6: MiCEE is required for nucleolar structure and rRNA synthesis.
Fig. 7: MiCEE tethers specific loci to the nucleolus.

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Acknowledgements

We thank R. Bender for technical support; A. Brehm (Philipps-Universität Marburg, Marburg, Germany) and S. Bellusci (ECCPS, Justus Liebig University, Giessen, Germany) for cell lines; N. Oeztuerk, Y. Zhou, X. Yuan, J. Kim, T. Böttger, A. Schneider, M. Looso, K.T. Preissner, R.H. Dammann, and S.A. Shiplu for helpful discussions. GB was funded by the “LOEWE-Initiative der Landesförderung” (Wiesbaden, Germany; III L 4 – 518/15.004 2009) and the “Deutsche Forschungsgemeinschaft” (DFG, Bonn, Germany; BA 4036/1-2). K.R. received a doctoral fellowship from CONACyT-DAAD (PKZ91549687). This work was done in accordance with the competitive growth program at the Kazan Federal University and the Russian Government.

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I.S., A.C., J.C., K.R., S.D., A.M., S.G., S.J., M.K., and G.B. designed and performed the experiments. G.D., W.S., and T.B. were involved in study design. G.B., I.S., J.C., J.G., A.C., K.R., and S.D. designed the study and analyzed the data. G.B., I.S., G.D., T.B., and J.C. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Guillermo Barreto.

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Integrated supplementary information

Supplementary Figure 1 Detection of mature Mirlet7d in the cell nucleus

a, Cell fractionation efficiency shown by WB analysis of the nuclear (Nu) and cytosolic (Cy) fractions of the cell lines used for TaqMan assay based expression analysis of mature Mirlet7d. LMNB1, lamin B1, nuclear marker; ACTB, beta actin, cytosolic marker; TUBA1A, alpha 1a tubulin, cytosolic marker. See Fig. 1a and Supplementary Data Set 2. b, Fluorescence microscopy images of MLE-12 cells after RNA-FISH using probes specific for mature (green) or primary (pri, magenta) Mirlet7d show no co-localization in the nucleus supporting the nuclear localization of mature Mirlet7d. The arrow delineates the position of the staining intensity profiles represented in the plot (bottom, right); blue DAPI staining, nucleus; Scale bar, 20µm. See Fig. 1b. c, Fluorescence microscopy images of MLg cells after Mirlet7d-FISH without (-) or with unlabeled Mirlet7d-probe (+ Competitor) demonstrates the specificity of the Mirlet7d-FISH. Scale bars, 20µm. d, Top, sequence alignment of Mirlet7 family members mature Mirlet7d, Mirlet7a and Mirlet7f. Magenta box indicates non-aligned nucleotides of Mirlet7d with both Mirlet7a and Mirlet7f. Green box indicates aligned nucleotide of nuclear specific miRNA Mir29b with Mirlet7d but not with both Mirlet7a and Mirlet7f. Bottom, TaqMan assay after cellular fractionation of indicated Mirlet7 family members in the nucleus and cytoplasm of MLg and MLE-12 cell lines. Data are shown as means ± s.e.m (n=3 independent experiments; Supplementary Data Set 4). e, Confocal microscopy image of MLg cells after transfection of biotinylated Mirlet7d or Mirlet7f. FITC labeled rabbit anti-Biotin antibody used to visualize the subcellular localization of transfected mature miRNAs. DAPI, nucleus. Scale bars, 20 µm. See Fig. 1b

Supplementary Figure 2 RNA-sequencing after miRNA pulldown

a, Schematic representation of the experimental outline. MLE-12 cells were transfected with biotinylated Mirctrl or Mirlet7d. Nuclear extract from transfected cells was used for the pulldown with streptavidin magnetic beads. Total RNA was extracted from the precipitated material and sequenced. See Fig. 1c-g, S3b-d, 3f, and S6f. b, c, Description of the mir-Pd RNA-seq data sets supports the quality of the experiment. d, Distribution of Mirlet7d-bound ncRNA loci relative to either genome areas (top) or chromosomes (bottom). Base pairs, bp. e, Visualization of representative Mirlet7d targets loci using UCSC genome browser after Mirlet7d-pulldown and RNA-seq (blue). Images of Mirlet7d-pulldown were generated after subtracting the input. Visualization of the same loci after RNA-seq showed reduced levels of both, ncRNA and adjacent cRNA, after Mirlet7d-GOF (magenta) when compared to Mirctrl transfection (green). Images represent mapped sequence tag densities relative to the indicated loci. Black boxes, exons; lines, introns, arrow, transcription direction. See Fig. 1c-g

Supplementary Figure 3 Mirlet7d binds directly to ncRNAs in the cell nucleus

a, qRT-PCR-based analysis of ncRNA and cRNA of the indicated genes in cells after Mirctrl or Mirlet7d transfection. These genes are representative Mirlet7d targets identified in our RNA-seq after miR-Pd. b, Single gene qRT-PCR-based analysis after miRNA-pulldown confirmed RNA-seq results. See Fig. 1c-f. c, Top, agarose gel based analysis showed specific and efficient enzymatic activity of RNA-double-strand specific RNase A, RNA-DNA-hybrid specific RNase H or DNA specific DNase I using plasmid DNA and yeast t-RNA as substrate. Bottom, Fluorescence microscopy images of MLE-12 cells using RNA-DNA hybrid specific antibody with (+) and without (-) RNase H treatment demonstrated RNase H activity. d, Pulldown of ncRNAs by Mirlet7d is RNase A sensitive. qRT-PCR analysis of the indicated ncRNAs after miR-Pd using nuclear extracts from MLE-12 cells transfected with biotinylated Mirctrl or Mirlet7d and treated with RNA-double-strand specific RNase A (RA), RNA-DNA-hybrid specific RNase H (RH) or DNA specific DNase I (D1). e, Endogenous Mirlet7d directly interacts with ncRNAs. qRT-PCR analysis of the indicated ncRNAs after miR-Pd using total RNA from MLE-12 cells. MLE-12 cells were UV crosslinked with 4’-aminomethyltrioxalen, a psoralen-derivative cross-linker prior to the pulldown. Total RNAs were isolated and supplemented with biotinylated Locked Nucleic Acid (LNATM) control (Mirctrl) or antisense Mirlet7d LNA oligonucleotides and streptavidin magnetic beads as indicated. The beads precipitated the LNA-asMirlet7d-bio bound to endogenous Mirlet7d by complementary base pairing; Mirlet7d was UV crosslinked to the ncRNAs supporting the direct interaction between Mirlet7d and ncRNAs. See Fig. 1c-f. In c, representative results from 2 independent experiments (see Supplementary Data Set 2). In all plots, data are shown as means ± s.e.m (n=3 independent experiments); asterisks, P values after one–way ANOVA, ***P˂0.001; **P˂0.01; *P˂0.05; ns, non-significant. The statistical test values of each plot are shown in the Supplementary Data Set 4

Supplementary Figure 4 Nuclear Mirlet7d interacts with different subunits of the exosome RNA-degrading complex

a, Schematic representation of the experimental outline. Protein analysis after miRNA-pulldown (miR-Pd). The nuclear or cytosolic fractions of MLE-12 cells were supplemented with biotinylated Mirctrl or Mirlet7d. The Ribonucleoprotein complexes (RBP) were precipitated using streptavidin magnetic beads and analyzed either by high-resolution mass spectrometry-based proteomic approach or Western Blot (WB). See Fig. 2a-c. b, Schematic representation of the experimental outline. Chromatin-RNA immunoprecipitation (Ch-RIP). Chromatin from non-transfected or transfected MLE-12 cells was precipitated using specific antibodies. Endogenous RNA was extracted from the precipitates and analyzed by Northern blot assays specific for mature or precursor (pre) Mirlet7d or by TaqMan assays specific for mature or primary (pri) Mirlet7d or by ncRNA-specific qRT-PCR. See Fig. 2d-e, 3e and S6c,d,h. c, Northern blot analysis of mature Mirlet7 after MLE-12 cell fractionation. U6 and tRNAlys was used as marker for nuclear and cytosolic fraction respectively. See Fig. 2d. d, Confocal microscopy of MLg cells after combined Mirlet7d-FISH (green) and EXOSC10-specific immunostaining (magenta) showed nuclear co-localization. DAPI-staining (blue). Scale bar, 20µm. See Fig. 2f. e, Top, WB based assay showed specific and efficient EXOSC10 reduction after transfection of Exosc10-specific shRNA constructs. Bottom, TaqMan based assay showed significant increase of Mirlet7d after transfection of Mirlet7d. f, qRT-PCR-based analysis of indicated ncRNA and cRNA in cells transfected with Mirctrl, Mirlet7d or/and Exosc10-specific shRNA constructs. See Fig. 2g. In c, e, representative results from 3 independent experiments (see Supplementary Data Set 2). In all plots, data are shown as means ± s.e.m (n=3 independent experiments); asterisks, P values after one–way ANOVA, ***P˂0.001; **P˂0.01; *P˂0.05; ns, non-significant. The statistical test values of each plot are shown in the Supplementary Data Set 4

Supplementary Figure 5 Nuclear Mirlet7d reduces ncRNA levels by RNA exosome mediated degradation

a, Visualization of Mafg locus using Exotome browser showed increased level of both ncRNA and cRNA in embryonic stem cells from Exosc10- (Exo10 KO, green) or Exosc3-knockout (Exo3 KO, orange) mice when compared to cells from wild type mice (WT, blue). Exotome browser accessed from (http://rabadan.c2b2.columbia.edu/cgi-bin/hgGateway). Images represent mapped sequence tag densities relative to the indicated loci. Black boxes, exons; lines, introns, arrow, transcription direction. See Fig. 2g-h. b, Small RNA sequencing (small RNA-seq; see Fig. 2h) from the nuclear fraction of MLE-12 cells after Mirctrl (green) or Mirlet7d (magenta) transfection alone or in combination with Exosc10-specific shRNA constructs (purple). UCSC genome browser visualization of RNA degradation products at loci of representative Mirlet7d targets that were identified in our RNA-seq after miR-Pd. Images represent mapped sequence tag densities relative to the indicated loci. Black boxes, exons; lines, introns, arrow, transcription direction. c, Northern blot-based analysis of degradation products of ncMafg (d-ncMafg) and ncChmp2b (d-ncChmp2b) in cells transfected with Mirctrl, Mirlet7d or/and catalytic mutant Exosc10 (Y-A, tyrosine 436 to alanine) construct. See Fig. 2g-h. d, Northern blot-based analysis of degradation product of ncMafg (d-ncMafg) and ncChmp2b (d-ncChmp2b) in cells transfected with Mirctrl, Mirlet7d or/and Exosc10-specific shRNA constructs. See Fig. 2g-h. In c, d, representative results from 2 independent experiments (see Supplementary Data Set 2)

Supplementary Figure 6 C1D binds Mirlet7d-ncRNA duplexes

a, Electrophoretic mobility shift assays (EMSAs) using biotinylated ncChmp2b-RNA probes and mature Mirlet7d to build RNA-RNA duplexes and increasing amounts of recombinant C1D as indicated. See Fig. 3c-d. b, Nuclear protein extracts of MLE-12 cells, that were treated or untreated with EDTA, were analyzed by immunoprecipitation (IP) using either immunoglobulin G (IgG, as control) or C1D-specific antibodies to precipitate endogenous EXOSC10. Co-IP proteins were analyzed by WB using the indicated antibodies. Input, 5% of material used for the IP. See Fig. 3d. c, qRT-PCR-based analysis of the indicated ncRNAs after C1D- or EXOSC10-specific Ch-RIP from cells transfected with control- (Ctrl), Mirlet7d-antagomir or Mirlet7-sponge. See also Fig. 3e. d, qRT-PCR-based analysis of the indicated ncRNAs after EXOSC5- or EXOSC1-specific Ch-RIP from cells transfected with control- (Ctrl), Mirlet7d-antagomir or Mirlet7-sponge. IgG used as negative control. See Fig. 3e. e, TaqMan assay (left) showed efficient Mirlet7d-reduction after transfection of the Mirlet7d-antagomir or Mirlet7-sponge (spng) when compared to control (Ctrl). qRT-PCR based expression analysis of Hmga2 (right), a well-known mRNA target of Mirlet7d, confirmed the efficient reduction of Mirlet7d after antagomir- or sponge-mediated loss-of-function. See Fig. 3e, 4a-d, 6a-c, 7a, S6c-d, S7, S9d-f, S12c and S14a. f, qRT-PCR-based analysis of the indicated ncRNAs after miR-Pd from cells transfected with Mirctrl-bio, Mirlet7d-bio+shC1d and Mirlet7d-bio+shExosc10. See Fig. 3f, S4e and S6g. g, Top, WB based assay showed specific and efficient C1D reduction after transfection of C1d-specific shRNA constructs. See Fig. 3f. h, qRT-PCR-based analysis of the indicated ncRNAs after FLAG-specific Ch-RIP from cells transfected with control- (Ctrl), FLAG-tagged EXOSC10 wt or deletion mutant 1 (del1). See Fig. 3e-f. In a, b, g, representative results from 2 independent experiments (see Supplementary Data Set 2). In all plots, data are shown as means ± s.e.m (n=3 independent experiments); asterisks, P values after one–way ANOVA, ***P˂0.001; **P˂0.01; *P˂0.05; ns, non-significant. The statistical test values of each plot are shown in the Supplementary Data Set 4

Supplementary Figure 7 PRO-seq-based nascent RNA expression analysis

a-b, Description of the PRO-seq data sets supports the quality of the experiment. c, Visualization of representative Mirlet7d targets loci using UCSC genome browser after PRO-seq in MLE-12 cells showed increased levels of both, ncRNA and adjacent cRNA, after Mirlet7-spng transfection (magenta) when compared to ctrl transfection (green). Drosophila S2 cell line used as spike-in control. Images represent mapped sequence tag densities relative to the indicated loci. Black boxes, exons; lines, introns, arrow, transcription direction. See Fig. 4b-c

Supplementary Figure 8 Nuclear Mirlet7d reduces cRNA levels by PRC2 mediated transcriptional silencing

a, Top, WB based assay showed specific and efficient EZH2 reduction after transfection of Ezh2-specific shRNA constructs. Bottom, TaqMan based assay showed significant increase of Mirlet7d after transfection of Mirlet7d. b, Top, WB based assay showed specific and efficient EED reduction after transfection of Eed2-specific shRNA constructs. Bottom, TaqMan based assay showed significant increase of Mirlet7d after transfection of Mirlet7d. c, Top, WB based assay showed specific and efficient SUZ12 reduction after transfection of Suz12-specific shRNA constructs. Bottom, TaqMan based assay showed significant increase of Mirlet7d after transfection of Mirlet7d. d, qRT-PCR-based analysis of indicated ncRNA and cRNA in cells transfected with Mirctrl, Mirlet7d or/and Ezh2-specific shRNA constructs. See Fig. 4f-g. e, qRT-PCR-based analysis of indicated ncRNA and cRNA in cells transfected with Mirctrl, Mirlet7d or/and Eed2-specific shRNA constructs. f, qRT-PCR-based analysis of indicated ncRNA and cRNA in cells transfected with Mirctrl, Mirlet7d or/and Suz12-specific shRNA constructs. g, Top, WB based assay showed specific and efficient EZH2 enzymatic activity inhibition after treatment of EZH2-specific inhibitor (EZH2inh, UNC1999). Bottom, TaqMan based assay showed significant increase of Mirlet7d after transfection of Mirlet7d. h, qRT-PCR-based analysis of indicated ncRNA and cRNA in cells transfected with Mirctrl or Mirlet7d or/and treatment with EZH2inh. See also Fig. 4f-g. In a-c, g, representative results from 2 independent experiments (see Supplementary Data Sets 2 and 3). In all plots, data are shown as means ± s.e.m (n=3 independent experiments); asterisks, P values after one–way ANOVA, ***P˂0.001; **P˂0.01; *P˂0.05; ns, non-significant. The statistical test values of each plot are shown in the Supplementary Data Set 4

Supplementary Figure 9 EXOSC10 mediates interaction between C1D and EZH2

a, Nuclear protein extracts of MLE-12 cells were analyzed by immunoprecipitation (IP) using either immunoglobulin G (IgG, as control) or EXOSC10-specific antibodies to precipitate endogenous EXOSC10. Co-purifying proteins were analyzed by WB using the indicated antibodies. Input, 5% of material used for the IP prior to LC-MS/MS analysis. See Fig. 5a-b. b, Sequential ChIP (ChIP-reChIP) analysis of the Mafg- and Chmp2b-promoters using chromatin isolated from MLE-12 cells after transfection of control (Ctrl) plasmid or expression constructs for EXOSC10 fl, del1 or del4. See Fig. 5g. c, qRT-PCR-based analysis of ncRNA and cRNA of the indicated genes in cells after transfection of control (Ctrl) plasmid or expression constructs for EXOSC10 fl, del1 or del4. See Fig. 5g. d,e, Confocal microscopy after proximity ligation assay using EXOSC10- and EZH2-specific antibodies in MLg cells that were Ctrl or Mirlet7-sponge transfected. Squares show details at higher magnification. Scale bars, 20 µm. See Figure S6e. f, Nuclear protein extracts of MLE-12 cells that were transfected with control- (Ctrl), Mirlet7d-antagomir (anti), Mirlet7-sponge (spng) or C1d-specific shRNA construct were analyzed by immunoprecipitation (IP) using either immunoglobulin G (IgG, as control) or EXOSC10-specific antibodies to precipitate endogenous EXOSC10. Co-IP proteins were analyzed by WB using the indicated antibodies. Input, 5% of material used for the IP. See also Figures S6e,g. g, Nuclear protein extracts of MLE-12 cells, that were treated or untreated with RNaseA or DNase1, were analyzed by immunoprecipitation (IP) using either immunoglobulin G (IgG, as control) or EXOSC10-specific antibodies to precipitate endogenous EXOSC10. Co-IP proteins were analyzed by WB using the indicated antibodies. Input, 5% of material used for the IP. See Fig. 6a-b. f, qRT-PCR-based analysis of the indicated ncRNAs after miR-Pd from cells transfected with biotinylated Mirctrl or biotinylated Mirlet7d. Nuclear protein lysate of biotinylated Mirlet7d transfected cells were treated with Proteinase K before miR-Pd as indicated. See Fig. 6a-b. h, ChIP analysis of Mafg- and Chmp2b-promoters using the indicated antibodies in MLE-12 cells transfected with Mirctrl, Mirlet7d or/and C1d-specific shRNA constructs. See Fig. 6c. In a, d-g, representative results from 2 independent experiments (see Supplementary Data Set 3). In all plots, data are shown as means ± s.e.m (n=3 independent experiments); asterisks, P values after one–way ANOVA, ***P˂0.001; **P˂0.01; *P˂0.05; ns, non-significant. The statistical test values of each plot are shown in the Supplementary Data Set 4

Supplementary Figure 10 C1D-EXOSC10 interaction anchors the exosome to the nucleolus

a, MLg cells were transfected with a control (Ctrl) or C1d-specific shRNA construct. Transfected cell were analyzed by confocal microscopy after double immunostaining using EXOSC10- and FBL-specific antibodies. DAPI, nuclear staining. Scale bar, 10 µm. The arrow delineates the position of the staining intensity profiles represented in the plots (right). See also Figures S6g. b, MLg cells were transfected with EXOSC10 full length (fl) or deletion mutant 1 (del1). Transfected cells were analyzed as in (a). Scale bar, 20 µm. c, ChIP analysis of Mafg- and Chmp2b-promoters using the indicated antibodies in MLE-12 cells transfected with Mirctrl, Mirlet7d or/and C1d-specific shRNA constructs. See Figure S6g. In a-b, representative results from 3 independent experiments. In all plots, data are shown as means ± s.e.m (n=3 independent experiments); asterisks, P values after one–way ANOVA, ***P˂0.001; **P˂0.01; *P˂0.05; ns, non-significant. The statistical test values of each plot are shown in the Supplementary Data Set 4

Supplementary Figure 11 MiCEE is required for nucleolar structure

a, MLg cells were transfected with a control (Ctrl) or C1d-specific shRNA constructs. Transfected cells were analyzed by confocal microscopy after double immunostaining using FBL- and H3K27me3-specific antibodies. Arrows, perinucleolar region. DAPI, nuclear staining. Scale bars, 10µm. See Fig. 6a and S6g. b, MLg cells were treated with a control (Ctrl, DMSO) or EZH2 inhibitor (EZH2inh, UNC1999). Treated cell were analyzed by confocal microscopy after double immunostaining using FBL- and H3K27me3-specific antibodies. Arrows, perinucleolar region. DAPI, nuclear staining. Scale bars, 10µm. See Fig. 6a and S8g. c, Schematic representation of the Mirlet7d locus (left) explaining the strategy for depletion of the Mirlet7d using the CRISPR/Cas9 technology. Arrows show the direction of expression of Mirlet7. Arrowheads represent guide RNAs for Cas9 binding (magenta) and primers for screening of Mirlet7d -/- cells (blue). Representative results of genotyping of wildtype (+/+) and Mirlet7d depleted (-/-) MLg cells (middle). TaqMan assay based expression analysis of the indicated Mirlet7 family members in the wildtype (+/+) and Mirlet7d depleted (-/-) MLg cells (right). In a-c, representative results from 3 independent experiments (see Supplementary Data Sets 3 and 4)

Supplementary Figure 12 MiCEE is required for rRNA synthesis

a, q-RT-PCR-based analysis of pre-rRNA in MLE-12 cells either treated with EZH2 inhibitor (EZH2inh, UNC1999) or transfected with a control (Ctrl) shRNA constructs or specific for Eed (shEed) or Suz12 (shSuz12). See Fig. 6b-c and S8b,c,g. b, q-RT-PCR-based analysis of pre-rRNA in MLE-12 cells transfected with a Mirctrl or Mirlet7d. Pre-rRNA, precursor 47/45S rRNA. qRT-PCR primer pair against pre-rRNA were made within the external transcribed spacer (5’-ETS). c, Nuclear run-on q-RT-PCR-based nascent RNA expression analysis of pre-rRNA in MLE-12 cells transfected with a control (Ctrl), Mirlet7d or Mirlet7-sponge (spng) construct. See Fig. 6b-c and S6e. d, Schematic representation of the experimental outline. Chromatin isolation by microRNA purification (miR-ChIRP). The chromatin was isolated, crosslinked, sonicated and supplemented with biotinylated Locked Nucleic Acid (LNATM) antisense control (asMirctrl) or antisense Mirlet7d LNA oligonucleotides and streptavidin magnetic beads as indicated. The beads precipitated the LNA-asMirlet7d-bio bound Mirlet7d by complementary base pairing and thereby Mirlet7d associated chromatin. See Fig. 6d-e and S13. e, f, qPCR-based analysis of single gene promoter after Mir-ChIRP using biotinylated Locked Nucleic Acid (LNATM) antisense control (asMirctrl) or antisense Mirlet7d LNA oligonucleotides (e) and transfecting biotinylated Mirctrl or Mirlet7d (f) confirmed ChIRP-seq results. See Fig. 6d-e and S13. In all plots, data are shown as means ± s.e.m (n=3 independent experiments); asterisks, P values after one–way ANOVA, ***P˂0.001; **P˂0.01; *P˂0.05; ns, non-significant. The statistical test values of each plot are shown in the Supplementary Data Set 4

Supplementary Figure 13 Distribution of PNH associated domains (PNHADs)

a, Distribution of normalized mapped reads from Mirlet7d-ChIRP-seq respective to the gene body and a ± 2 kb region to transcription start (TSS) and termination (TTS) sites. Norm tags, tag density normalized to input and represented as log2 values. Genes used for the analysis are the Mirletd7 targets identified in our RNA-seq after Mirlet7d-Pd shown in Fig. 1f. b, Aggregate plot for Mirctrl-, Mirlet7d-ChIRP-seq, H3K27me3-, UBF-ChIP-seq and input peaks in a ± 2 kb region respective to Mirlet7d peaks. Norm tags, mapped reads normalized to 10 million using annotatePeaks.pl from Homer. c, UCSC genome browser visualization of chromosomes (left) containing specific loci (right and bottom) enriched after Mirlet7d-ChIRP, UBF- and H3K27me3-ChIP. We hypothesized that the Mirlet7d-UBF-H3K27me3-common loci were PNH associated domains (PNHADs). Images represent indicated loci; black boxes, exons; lines, introns; arrow, transcription direction. Sites enriched with Mirlet7d, UBF and H3K27me3 (common sites) are represented as black boxes at the bottom of each loci. See Fig. 7d-e and S11d-f

Supplementary Figure 14 MiCEE tethers specific loci to the nucleolus

a, Sequential ChIP (ChIP-reChIP) analysis of the Mafg- and Nolc1-promoters using FBL- and H3K27me3-specific antibodies. FBL-specific antibody used for nucleolus enrichment. IgG used as a negative control. The analyzed chromatin was isolated from MLE-12 cells after transfection of control (Ctrl) or Mirlet7-sponge (spng) plasmid (left); from MLE-12 cells after transfection of control (Ctrl) plasmid or expression constructs for EXOSC10 fl or del1 (middle); and from wildtype (+/+) and ncMafg-depleted (-/-) MLE-12 cells (right). See Fig. 7a-e. In all bar plots, data are shown as means ± s.e.m (n=3 independent experiments); asterisks, P values after one–way ANOVA, ***P˂0.001; **P˂0.01; *P˂0.05; ns, non-significant. b, Quantification of nucleolus associated Mafg and Chmp2b loci. Confocal microscopy images after fluorescent in situ hybridization using a DNA probe (DNA-FISH) specific against the genomic region containing the Mafg loci or Chmp2b loci in wildtype (+/+) and ncMafg-depleted (-/-) MLE-12 cells. Experiment was performed 5 times and in each experiment 250 nuclei were counted. See Fig. 7f. Boxes, interquartile ranges; whiskers, maximum and minimum values; horizontal lines, medians; asterisks, P values after Welch Two sample t-test, ***P˂0.001; **P˂0.01; *P˂0.05; ns, non-significant. The statistical test values of each plot are shown in the Supplementary Data Set 4

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Supplementary Note 1 and Supplementary Tables 1 and 2

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Singh, I., Contreras, A., Cordero, J. et al. MiCEE is a ncRNA-protein complex that mediates epigenetic silencing and nucleolar organization. Nat Genet 50, 990–1001 (2018). https://doi.org/10.1038/s41588-018-0139-3

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