Abstract
Trimethylation of histone H3 lysine 9 (H3K9me3) is crucial for the regulation of gene repression and heterochromatin formation, cell-fate determination and organismal development1. H3K9me3 also provides an essential mechanism for silencing transposable elements1,2,3,4. However, previous studies have shown that canonical H3K9me3 readers (for example, HP1 (refs. 5,6,7,8,9) and MPP8 (refs. 10,11,12)) have limited roles in silencing endogenous retroviruses (ERVs), one of the main transposable element classes in the mammalian genome13. Here we report that trinucleotide-repeat-containing 18 (TNRC18), a poorly understood chromatin regulator, recognizes H3K9me3 to mediate the silencing of ERV class I (ERV1) elements such as LTR12 (ref. 14). Biochemical, biophysical and structural studies identified the carboxy-terminal bromo-adjacent homology (BAH) domain of TNRC18 (TNRC18(BAH)) as an H3K9me3-specific reader. Moreover, the amino-terminal segment of TNRC18 is a platform for the direct recruitment of co-repressors such as HDAC–Sin3–NCoR complexes, thus enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis that disrupts the TNRC18(BAH)-mediated H3K9me3 engagement caused neonatal death in mice and, in multiple mammalian cell models, led to derepressed expression of ERVs, which affected the landscape of cis-regulatory elements and, therefore, gene-expression programmes. Collectively, we describe a new H3K9me3-sensing and regulatory pathway that operates to epigenetically silence evolutionarily young ERVs and exert substantial effects on host genome integrity, transcriptomic regulation, immunity and development.
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Data availability
Next-generation sequencing data have been deposited into the NCBI’s Gene Expression Omnibus database under accession number GSE200839. Coordinates and structural factors for the TNRC18(BAH)–H3K9me3 complex have been deposited into the PDB (code 8DS8). PDB 4DOV was used as a search model for structure determination of the TNRC18(BAH)–H3K9me3 complex. Source data are provided with this paper.
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Acknowledgements
We thank the members of the Wang, Song and Cai laboratories for discussion and technical support; Z. Li and C. Pecot for sharing the reagents used in the work; staff members at the NE-CAT beamlines (GM124165), Advanced Photo Source (DE-AC02-06CH11357) and Argonne National Laboratory for access to the X-ray beamline; staff at institution-affiliated core facilities, including those for imaging, high-throughput deep sequencing, bioinformatics, flow cytometry and sorting, tissue culture and animal-related studies, for their professional assistance of this work. The cores affiliated to UNC Cancer Center are supported in part by the UNC Lineberger Comprehensive Cancer Center Core Support Grant P30-CA016086. This work was supported in part by NIH grants R01CA271603, R01CA268519 and R01CA268384 to G.G.W.; R35GM119721 to J.S.; R24GM137786, P20GM121293 and R01CA236209 to A.J.T., R35GM126900 to B.D.S.; R35GM147286 to J.R.R.; R01CA262903 to L.C.; and U01HL156064 and R35HG011328 to Y.D. J.R.R. is also supported in part by DoD Award W81XWH-19-1-0423. G.G.W. is an American Cancer Society Research Scholar and a Leukemia and Lymphoma Society Scholar.
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S.Z. and J.L. led the biological and structural aspects of this study, respectively, under the supervision of G.G.W. and J.S. S.Z., H.F., Y.G., A.K., L.C. and G.G.W. conducted cell-based characterizations and animal studies. S.Z. and C.X. conducted the protein-complex-related studies. S.D.B., A.J.S., S.G.M. and R.D.E. performed proteomics analysis under the supervision of A.J.T. S.Z. and T.S. conducted CasID under the guidance of G.G.W. and Y.D. S.Z., J.L. and N.T.B. performed biochemical and biophysical assays under the supervision of J.S., B.D.S. and G.G.W. S.Z., B.P., K.L.K., W.G., P.C.K., J.R.R., L.C. and G.G.W. analysed deep-sequencing datasets. G.G.W. and J.S. conceived the idea and designed and supervised the research. S.Z., J.L., J.S. and G.G.W. wrote the manuscript with input from all authors.
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Extended data figures and tables
Extended Data Fig. 1 Unbiased CasID-based proteomic approach identified a previously less-studied nuclear protein TNRC18 as a putative binding protein at ERV regions.
a. Workflow of CasID. A set of sgRNAs targeting representative ERV regions (sgRNA sequence information provided in Supplementary Table 7) were transduced into cells with stable expression of dCas9-BirA*. The sgRNA-based targeting of dCas9-BirA* to ERVs results in the BirA*-mediated biotinylation of proteins in proximity (within approximately 10 nm) in the presence of biotin. Then, biotinylated proteins were enriched by NeutrAvidin affinity purification, followed by mass spectrometry-based protein identification. b. Representative confocal immunofluorescence (IF) microscopy images showing the nuclear localization pattern of TNRC18 in HEK293 cells, probed with either DAPI (left) or anti-TNRC18 antibodies (middle). Scale bars, 10 μm. Data shown represent 3 independent experiments. c. The cDNA of TNRC18 used for mammalian cell expression, which contains an Avi-tag and a GFP tag at the N-terminus and 3×FLAG tag at the C-terminus. d. Representative confocal IF microscopy images for the above tagged TNRC18 in the HEK293 stable expression cells, co-stained with the mouse anti-GFP antibody and the rabbit antibody against endogenous TNRC18 (top panel), or with the rabbit anti-GFP antibody and the mouse anti-FLAG antibody (middle panel), or with the mouse anti-FLAG antibody and the rabbit antibody against endogenous TNRC18 (bottom panel), all of which exhibited a co-localization pattern in the nucleus. Data shown represent 3 independent experiments. e. Immunoprecipitation (IP) of HEK293 cells expressing GFP-3xFLAG-tagged TNRC18, compared to empty vector (EV)-transduced control cells, by using anti-FLAG beads. The IP sample was immunoblotted with anti-FLAG antibody (top panel) or that of endogenous TNRC18 (bottom panel; for TNRC18 protein size, refer to Methods of IP as well). f. RT-qPCR of TNRC18 in the HEK293 cells with knockdown (KD) of endogenous TNRC18 (shTNRC18), compared to scramble controls (shCtrl), and in the cells with endogenous TNRC18 KD followed by the rescued re-expression of shTNRC18-resistant TNRC18 (i.e., shTNRC18_rescue; n = 3 biologically independent experiments). Data were plotted as the mean ± s.d. after normalization to the signals of an internal control (GAPDH) and to those of the control samples. g. Scatter plot showing the indicated transposable element families exhibiting significant expression change, based on RNA-seq profiles of HEK293 cells with shTNRC18, compared to scramble controls (shCtrl) (n = 2 biologically independent experiments). The cut-off of statistical significance is log2 value of fold-change in expression (y-axis) over 0.58 and adjusted P value (x-axis) less than 0.01 for transcripts with basemean read counts over 10. Adjusted P value is calculated by negative binomial model-based methods (DESeq2). h. Classification of endogenous retroviruses in the human cells. Figure adapted from ref. 89, with permission from Elsevier and under a Creative Commons licence CC-BY 4.0, and ref. 14, BioMed Central.
Extended Data Fig. 2 TNRC18 knock-down (KD) or knockout (KO) results in activation of immunity-related genes in HEK293 cells.
a. Sanger sequencing to show frame-shifting mutation and knock-out of TNRC18 in HEK293 cells. b. Western blot to show the knock-out of TNRC18 in HEK293 cells. Vinculin is the sample processing control. c. RNA-seq analysis using unique mapping reads (left) and multi-mapping reads (right) of the indicated HEK293 cells. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. The number of analyzed copies per transposon type: group ‘unique mapping only’ (LTR12 (n = 122 copies), LTR12C (n = 612 copies), LTR12D (n = 54 copies), HERV9-int (n = 58 copies)); group ‘multi-mapping allowed’ (LTR12 (n = 124 copies), LTR12C (n = 678 copies), LTR12D (n = 55 copies), HERV9-int (n = 60 copies)). Sample size of each box plot is also listed in Supplementary Table 8. d. Gene set enrichment analysis (GSEA) revealed enrichment for the indicated pathways in cells with TNRC18 KD (shTNRC18), compared to mock controls (shCtrl) (n = 2 biologically independent experiments). Immunity-related gene sets are labelled in red. The y-axis and x-axis showed the normalized enrichment score (NES) and false discovery rate (FDR) q-values of GSEA, respectively. e. GSEA revealed the positive correlation between activation of genes related to immunity and TNRC18 KD (shTNRC18) in HEK293 cells, compared to those with the scramble controls (shCtrl) or with the rescued re-expression of TNRC18 (shTNRC18_rescue). NES, normalized enrichment score. The P value was calculated by a two-sided empirical phenotype-based permutation test; the false discovery rate q-value is adjusted for gene set size and several hypotheses testing whereas the P value is not. f. GSEA revealing the positive correlation between activation of genes related to immunity and TNRC18 KO in HEK293 cells, compared to WT controls. The P value was calculated by a two-sided empirical phenotype-based permutation test; the false discovery rate q-value is adjusted for gene set size and several hypotheses testing whereas the P value is not.
Extended Data Fig. 3 TNRC18 KO using four different human cancer cell lines of epithelial origins.
a. Bulk tissue gene expression for TNRC18 in the GTEx Analysis Release V890. Expression values are shown in TPM (Transcripts Per Million), calculated from a gene model with isoforms collapsed to a single gene. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. b-c. Sanger sequencing (b) and Western blot (c) to show frame-shifting mutation and KO of TNRC18 in the indicated edited cells, in comparison to WT. Vinculin is the sample processing control. For TNRC18 protein size, see Methods as well. d. GSEA revealed enrichment for the indicated pathways in SNU-1 cells (left) and NCI-H23 cells (right) with TNRC18 KO, compared to WT controls. Immunity-related gene sets are labelled in red. NES, normalized enrichment score. The P value was calculated by a two-sided empirical phenotype-based permutation test; the false discovery rate q-value is adjusted for gene set size and several hypotheses testing whereas the P value is not.
Extended Data Fig. 4 TNRC18 and H3K9me3 co-localize at ERV regions.
a. Scatter plot showing correlation between signals of CUT&RUN for endogenous TNRC18 in parental HEK293 cells (x-axis; using anti-TNRC18 antibody) and those for GFP-TNRC18 following its stable expression into the HEK293 cells (y-axis; using anti-GFP antibody). Pearson correlation coefficient is shown. b. Pie chart showing distribution of the indicated TE annotation using the TNRC18 CUT&RUN peaks annotated as TE by ChIPpeakAnno. c. Box plots showing the log2 values of fold-changes in signals of CUT&RUN for the indicated protein, relative to IgG controls, at different TE classes in HEK293 cells. CUT&RUN for TNRC18 was conducted with antibody of endogenous TNRC18 in parental HEK293 cells, or antibody of GFP in cells stably expressed with GFP-TNRC18. CUT&RUN for H3K9me3 was performed by using two independent antibodies, from either Abcam or Active Motif Inc. (AM). The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. d. Heatmap of anti-GFP CUT&RUN signals in HEK293 cells stably expressed with GFP-TNRC18 (1st column), or those for TNRC18, probed by antibody of endogenous TNRC18, in either parental HEK293 (2nd column), HeLa (3rd column) or K562 cells (4th column), across ±5 kb from the most confident TNRC18 peaks defined in HEK293 cells (n = 7545; peaks common to GFP-TNRC18 [1st column) and TNRC18 [2nd column] in HEK293 cells were used). e. IGV tracks showing the CUT&RUN signal for TNRC18 in the indicated cells at the representative ERV (left), promoter-TSS (middle) or intergenic target site (right). TSS, transcription start site. f,g. Heatmap for the CUT&RUN signals of TNRC18 and H3K9me3 (probed with two independent antibodies), across ±5 kb from those TNRC18 peaks that were annotated as TEs (f) or LTRs (g). h. Motif search analysis revealing the most enriched motifs at the TNRC18 (top panel) and GFP-TNRC18 peaks (bottom panel) in HEK293 cells.
Extended Data Fig. 5 TNRC18BAH is a conserved domain binding specifically to H3K9me3.
a. Sequence alignment of TNRC18BAH among different species. The secondary structures of human TNRC18BAH are indicated on top. The H3K9me3-binding pocket residues are indicated by red asterisks, and the rest of H3-binding sites are indicated by filled black circles at the bottom. b. SDS-PAGE image of the purified recombinant protein of TNRC18BAH used for biochemical and structural studies. c. ITC fitting parameters of TNRC18BAH binding to various histone peptides. NDB, no detectable binding. d. ITC fitting curves of TNRC18BAH against the indicated histone peptides trimethylated at different lysine sites. e. Original ITC binding curves of the recombinant TNRC18BAH protein against histone peptides with the indicated modification.
Extended Data Fig. 6 Structural basis of TNRC18BAH binding to H3K9me3 peptide and biochemical analysis of TNRC18BAH binding to H3Kc9me3-modified nucleosome.
a. Crystal structures of the two color-coded human TNRC18BAH molecules in one asymmetric unit, with the chain identifiers labeled (chain A and B). Each TNRC18BAH molecule is complexed with one H3K9me3 peptide (chain C or D). The TNRC18BAH-H3K9me3 complex with the best model-to-map fit (chain B and D) was selected for structural analysis. b. Electrostatic surface view of human TNRC18BAH bound to the H3K9me3 (yellow sticks). (left) The Fo-Fc omit map of the H3K9me3 peptide, contoured at 1.5σ level, is shown as magenta mesh. (right) The surface patch enriched with basic residues is indicated as the potential binding site to nucleosome/DNA. c. ITC binding curves of TNRC18BAH against histone peptides with the indicated modification. d. ITC binding curves of the indicated TNRC18BAH mutants against the H3K9me3 peptide. e. Structural comparison of ORC1BAH-H4K20me2 (electrostatic surface and stick representation; PDB 4DOW), DNMT1BAH1-H4K20me3 (electrostatic surface and stick representation; PDB 7LMK) and BAHCC1BAH-H3K27me3 (ribbon and stick representation; PDB 6VIL). f. Structural superposition of TNRC18BAH with the nucleosome core particle (NCP)-bound yeast Sir3 BAH domain (Sir3BAH; PDB 4KUD) reveals that the basic patch of TNRC18BAH (Extended Data Fig. 6b, right panel) corresponds to a similar region of Sir3BAH involved in binding to the acidic patch of NCP. g. Assessment of purified H3Kc9me3-modified NCP on a native 5% TBE gel. The positions of reconstituted NCP and biotinylated 601 DNA are labeled on the right. h. The BLI kinetic curves for the TNRC18BAH-NCP binding. The concentrations of TNRC18BAH used for each kinetic measurement are indicated. The Kd value and s.d. were derived from two independent measurements.
Extended Data Fig. 7 A H3K9me3-binding-defective mutation of TNRC18BAH leads to the activated expression of the LTR12 family TEs, TINATs and interferon-stimulated genes.
a. Sanger sequencing verified homozygous knock-in (KI) mutation of TNRC18W2858A, introduced by the CRISPR-Cas9-based gene editing, in HEK293 cells. b. RT-qPCR and Western blot of TNRC18 in HEK293 cells, either wild type (WT) or with homozygous mutation of TNRC18W2858A. Data were plotted as the mean ±s.d. after normalization to the signals of an internal control, GAPDH, and to those of WT cells (n = 3 independent experiments). Vinculin is the sample processing control. c. GSEA revealed the positive correlation between activation of the indicated immunity-related gene sets and the H3K9me3-binding-defective mutation of TNRC18 (W2858A), relative to WT, in HEK293 cells (n = 2 independent experiments). The P value was calculated by a two sided empirical phenotype-based permutation test; the false discovery rate q-value is adjusted for gene set size and several hypotheses testing whereas the P value is not. d-e. Overall expression levels of genes associated with all TNRC18 peaks (d, left), those with TNRC18-bound promoter/TSS regions (d, right), or genes close to (within 50 kb) the TNRC18-bound LTRs (e), based on RNA-seq profiling of HEK293 cells, either WT (left) or carrying the TNRC18W2858A homozygous mutation (right). VST, variance-stabilizing transformation. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. The P value was calculated by two-sided Wilcoxon test. f, Averaged distance to the nearest TNRC18/H3K9me3-bound LTRs from genes exhibiting either down-regulation (left), no expression change (stable; middle) or up-regulation (right) in HEK293 cells carrying the TNRC18W2858A homozygous mutation, relative to WT, based on RNA-seq. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. P value was calculated by two-sided Wilcoxon test. Sample size of each box plot is listed in Supplementary Table 8. g, RT-qPCR of representative treatment-induced non-annotated TSSs (TINATs40) in HEK293 cells carrying the TNRC18W2858A homozygous mutation, relative to WT (n = 3 independent experiments; *P < 0.05; **P < 0.01, two-sided t-test). Data were plotted as the mean ±s.d. after normalization to signals of GAPDH and then to those of WT. The exact P value is shown in Supplementary Table 8. h. Scatter plot showing Pearson correlation of the CAGE-seq profiles of HEK293 cells, WT or with the TNRC18W2858A homozygous mutation (n = 2 independent experiments). Rep-1/2, biological replicate 1 or 2. i-j. IGV view of CAGE-seq profiles at the indicated gene (i) or long non-coding RNA (lncRNA; j) containing a nearby LTR12 in HEK293 cells, WT or with the TNRC18W2858A homozygous mutation.
Extended Data Fig. 8 Compared to WT mice, those harboring the homozygous H3K9me3-binding-defective mutation of TNRC18BAH exhibited severe phenotypes of neonatal lethality and dwarfism in adult surviving animals, as well as ERV de-repression in their organs and MEF cells.
a. Sanger sequencing results verified the genotype of mice, either WT (w/w) or with the Tnrc18W2745A/Y2747A heterozygous (w/m) or homozygous (m/m) mutation. b. Measurement of body size and weight at the indicated time point post-birth showing that the Tnrc18W2745A/Y2747A homozygous mice (female, n = 3; male, n = 3) were smaller, compared to their WT (female, n = 5; male, n = 4) and heterozygous (female, n = 10; male, n = 10) littermates, both in the cohort of females (left) and males (right). Data are presented as mean value ± SEM. c. Scatter plot showing the indicated TE families that exhibit significant expression change, based on RNA-seq profiles of different primary tissue samples isolated from mice with the Tnrc18W2745A/Y2747A homozygous mutation (n = 6, two replicated experiments for three mice), compared to those of WT littermates (n = 4, two replicated experiments for two mice). Adjusted P value is calculated by negative binomial model-based methods (DESeq2). d. GSEA revealed the positive correlation between activation of the indicated immunity-related gene sets in the lung and the Tnrc18W2745A/Y2747A homozygous mutation, relative to WT controls. The P value was calculated by a two-sided empirical phenotype-based permutation test; the false discovery rate q-value is adjusted for gene set size and several hypotheses testing whereas the P value is not. e. GSEA revealed enrichment for the indicated pathways in the liver of mice with the Tnrc18W2745A/Y2747A homozygous mutation, compared to WT controls. Immunity-related gene sets are labelled in red. The P value was calculated by a two-sided empirical phenotype-based permutation test; the false discovery rate q-value is adjusted for gene set size and several hypotheses testing whereas the P value is not. f. Western blot of TNRC18 in WT MEF cells and those with the Tnrc18W2745A/Y2747A homozygous mutation. Vinculin is the sample processing control. g. Heatmap showing TE families with significant expression change, based on RNA-seq profiles of MEFs harboring the Tnrc18W2745A/Y2747A homozygous mutation versus WT (n = 2 independent experiments). The cut-off of statistical significance is log2 value of fold-change in expression over 0.58 and adjusted P value less than 0.01 for transcripts with basemean counts over 10. h. GSEA revealed the positive correlation between activation of the indicated interferon response-related gene sets and homozygous Tnrc18W2745A/Y2747A mutation in MEF cells, relative to WT (n = 2 independent experiments). Immunity-related gene sets are labelled in red. The P value was calculated by a two sided empirical phenotype-based permutation test; the false discovery rate q-value is adjusted for gene set size and several hypotheses testing whereas the P value is not.
Extended Data Fig. 9 TNRC18 binds corepressors, mediating transcriptional repression.
a. Scatter plots of the indicated TNRC18-interacting proteins identified by BioID in HEK293 (x-axis) and HeLa cells (y-axis). The mass spectrometry data following BioID in HeLa cells is shown in Supplementary Table 5. b. Pearson correlation plot using the IF signals of the indicated protein in HEK293 cells. c. Structural model of the Sin3A’s PAH domain (green) in complex with the TNRC18 peptide (cyan). The structure was predicted via PHYRE2 (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index). The model of the complex was generated by the Coot program, using the structure of mouse Sin3A PAH1-SAP25 SID complex (PDB 2RMS) as template. d. Sanger sequencing (top) and Western blot (bottom) for the TNRC18 L760A KI mutation introduced into HEK293 cells. Vinculin is the sample processing control. e. Scatter plot showing the indicated TE families that exhibit significant expression change, based on RNA-seq profiles of HEK293 cells with the TNRC18 L760A mutation versus WT controls. Adjusted P value is calculated by negative binomial model-based methods (DESeq2). f. RT-qPCR of the indicated TEs in HEK293 cells with TNRC18 L760A mutation versus WT controls (n = 3 independent experiments; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided t-test). Data were plotted as the mean ±s.d. after normalization to signals of GAPDH and then to those of WT. The exact P value is shown in Supplementary Table 8. g. GSEA revealed enrichment for the indicated pathways in HEK293 cells with the TNRC18 L760A mutation versus WT controls. Immunity-related gene sets are labelled in red. The P value was calculated by a two sided empirical phenotype-based permutation test; the false discovery rate q-value is adjusted for geneset size and several hypotheses testing whereas the P value is not. h. RT-qPCR of the indicated immunity-related genes in WT and TNRC18-L760A-mutated HEK293 cells (n = 3 independent experiments; *P < 0.05; ***P < 0.001; ****P < 0.0001, two-sided t-test). Data were plotted as the mean ±s.d. after normalization to signals of GAPDH and then to those of WT. The exact P value is shown in Supplementary Table 8. i. Heatmap of CUT&RUN signals for TNRC18, HDAC2, TRIM28 and SETDB1 in HEK293 cells, across ±5 kb from the TNRC18 peaks (n = 7545; defined as the common peaks of TNRC18 and GFP-TNRC18, based on CUT&RUN in HEK293 cells). j. IGV tracks showing the CUT&RUN signals of the indicated protein at the reported TRIM28 target gene in HEK293 cells. k. The motif search analysis revealed the binding motifs of KRAB-ZnF proteins to be enriched at the TNRC18 peaks.
Extended Data Fig. 10 The TNRC18BAH-H3K9me3 engagement mediates optimal recruitment of TNRC18 and associated co-repressors, maintaining a silenced chromatin state at targets.
a. Sanger sequencing to verify the CRISPR-Cas9-induced frameshift and KO of SETDB1 in HEK293 cells. b. Left: Western blot of SETDB1, TNRC18 and H3K9me3 in WT and SETDB1-KO HEK293 cells. Right: Western blot of SETDB1 in HEK293 cells with WT TNRC18, TNRC18 KO and TNRC18 W2858A mutation. Vinculin and Tubulin are the sample processing control. The representative results from 3 independent experiments are shown. c. Heatmap of TNRC18 CUT&RUN signal across ±5 kb at the TNRC18 peaks region in WT and SETDB1-KO HEK293 cells. d. Immunoblotting for H3K9me3, histone H3 acetylation (H3ac) and total H3 in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A. Data shown are the representative results from 3 independent experiments. e. The heatmap of CUT&RUN signals of H3ac (left) and H3K9me3 (right) in HEK293 cells, either WT or with the homozygous mutation of TNRC18W2858A, across ±5 kb from the called peaks. f. Box plot showing the log2 values of normalized CUT&RUN sequencing counts in HEK293 cells with homozygous TNRC18W2858A mutation, compared to WT, at the indicated different TE classes. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. g. Global methylation levels of WT and TNRC18W2858A-mutated HEK293 cells. h. DNA methylation levels of WT and TNRC18W2858A-mutated HEK293 cells at SINE, LTR7Y, LTR12C and CpG_island. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers indicate 1.5× the interquartile range. Sample size of each box plot is listed in Supplementary Table 8. i. The dual-luciferase reporter-based system for assaying the activity of full-length TNRC18 fused to the GAL4’s DNA binding domain (GAL4-DBD), relative to GAL4-DBD alone (n = 6 biologically independent samples; data presented as the mean ±s.d.; ****P < 0.0001, two-sided t-test). The luciferase signals of cells with GAL4-DBD-TNRC18 fusion were first normalized to those of internal control (Renilla luciferase) and then normalized to those of cells with GAL4-DBD alone. The exact P value is shown in Supplementary Table 8.
Supplementary information
Supplementary Information
Supplementary Note 1 and additional references, and Supplementary Fig. 1 (uncropped immunoblots from Extended Data Figs.).
Supplementary Table 1
Positive hits identified by CasID.
Supplementary Table 2
TE families showing significant changes in RNA-seq, related to Fig. 1.
Supplementary Table 3
Differentially expressed genes showing significant changes in RNA-seq, related to Fig. 1.
Supplementary Table 4
TE families and differentially expressed genes showing significant changes in RNA-seq, related to Fig. 4 and Extended Data Fig. 8.
Supplementary Table 5
BioID identified proteins that interact with TNRC18 in HEK293 and HeLa stable expression cells.
Supplementary Table 6
TE families and differentially expressed genes showing significant changes in RNA-seq, related to Extended Data Fig. 9.
Supplementary Table 7
Information on the reagents used in this study.
Supplementary Table 8
P values and sample sizes for the main figures and Extended Data.
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Zhao, S., Lu, J., Pan, B. et al. TNRC18 engages H3K9me3 to mediate silencing of endogenous retrotransposons. Nature 623, 633–642 (2023). https://doi.org/10.1038/s41586-023-06688-z
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DOI: https://doi.org/10.1038/s41586-023-06688-z
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