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KCNQ1OT1 promotes genome-wide transposon repression by guiding RNA–DNA triplexes and HP1 binding

Nature Cell Biology volume 24, pages 1617–1629 (2022)Cite this article

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Abstract

Transposon (de)repression and heterochromatin reorganization are dynamically regulated during cell fate determination and are hallmarks of cellular senescence. However, whether they are sequence specifically regulated remains unknown. Here we uncover that the KCNQ1OT1 lncRNA, by sequence-specific Hoogsteen base pairing with double-stranded genomic DNA via its repeat-rich region and binding to the heterochromatin protein HP1α, guides, induces and maintains epigenetic silencing at specific repetitive DNA elements. Repressing KCNQ1OT1 or deleting its repeat-rich region reduces DNA methylation and H3K9me3 on KCNQ1OT1-targeted transposons. Engineering a fusion KCNQ1OT1 with an ectopically targeting guiding triplex sequence induces de novo DNA methylation at the target site. Phenotypically, repressing KCNQ1OT1 induces senescence-associated heterochromatin foci, transposon activation and retrotransposition as well as cellular senescence, demonstrating an essential role of KCNQ1OT1 to safeguard against genome instability and senescence.

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Fig. 1: The lncRNA KCNQ1OT1 binds HP1 and suppresses H3K9me3 foci.
Fig. 2: Genomic targets of KCNQ1OT1 lncRNA and overlap with heterochromatin marks in HEK293T cells.
Fig. 3: Repeat-rich and non-repeat-rich regions of KCNQ1OT1 have different protein interactomes.
Fig. 4: Decreased DNA methylation and increased L1 transposition in K rep KO HEK293T cells.
Fig. 5: KCNQ1OT1 target genomic sequences are guided by RNA–dsDNA triplex.
Fig. 6: The RNA–dsDNA triplex contributes to KCNQ1OT1 binding to genomic DNA.
Fig. 7: KCNQ1OT1 repression induces cellular senescence.

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Data availability

Sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE208017. The protein mass spectrometry data have been deposited to ProteomeXchange under the accession code PXD035996. Previously published data that were reanalysed here are available: KCNQ1OT1 expression in the human brain (National Omics Data Encyclopedia, OEP001041) and blood (GEO, GSE106670), H3K9me2 ChIP–seq data in HEK293T cells (GEO, GSE67317), H3K27me3 ChIP–seq data in HEK293T cells (GEO, GSE97326), and RNA-seq data in proliferating, early senescent and deep senescent human lung fibroblast cells (GEO, GSE109700). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

The WinMine package was used for Bayesian network inference. Triplex Domain Finder characterizes the triplex-forming potential between RNA and DNA regions47. For high-confidence protein–lncRNA interactions analyses, MiST (https://github.com/kroganlab/mist), SAINTexpress (v3.6.3), BioGRID database (release 4.1.190) and the CORUM database (accessed 24 December 2020) were used40,41,47,58,59.

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Acknowledgements

We thank J. Wang (Sun Yat-Sen University) for providing L1 retrotansposation vector and B. Zhu (Institute of Biophysics) for critical reading of the manuscript and invaluable suggestions, the technology platform of the National Center for Protein Sciences, Peking University for mass spectrometry and flow cytometry analysis, and the Center for Quantitative Biology, Peking University for microscopy imaging. This work was supported by grants from the National Natural Science Foundation of China (grant nos 92049302 and 32088101), China Ministry of Science and Technology (grant no. 2020YFA0804000) and Shanghai Municipal Science and Technology Major Project (grant no. 2017SHZDZX01) to J.-D.J.H.

Author information

Author notes

  1. These authors contributed equally: Xiaoli Zhang, Quanlong Jiang, Jiyang Li, Shiqiang Zhang.

Authors and Affiliations

  1. Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Center for Quantitative Biology (CQB), Peking University, Beijing, China

    Xiaoli Zhang, Shiqiang Zhang, Jiaqi Tan & Jing-Dong J. Han

  2. CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Quanlong Jiang, Jiyang Li, Shiqiang Zhang, Yaqiang Cao & Donghong Cai

  3. University of Chinese Academy of Sciences, Beijing, China

    Quanlong Jiang, Jiyang Li, Shiqiang Zhang, Yaqiang Cao & Donghong Cai

  4. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Jiyang Li

  5. Department of Pharmacology, Nanjing University of Chinese Medicine, Nanjing, China

    Xian Xia

  6. CAS Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

    Jiekai Chen

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Contributions

X.Z. and J.-D.J.H. designed experiments. J.-D.J.H., X.Z. and J.C. designed analyses. X.Z. and J.T. performed wet experiments. Q.J., J.L., S.Z., Y.C., X.X. and D.C. performed computational analyses, and X.Z., J.-D.J.H., Q.J., J.L. and S.Z. wrote the manuscript.

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Correspondence to Jing-Dong J. Han.

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Nature Cell Biology thanks Edward Chuong, John Sedivy and Rory Johnson for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 LncRNA KCNQ1OT1 binds HP1 and suppresses H3K9me3 foci in mouse NIH3T3 cells.

a, Expression rank of XIST, KCNQ1OT1, AIRN, and HOTTIP among all lncRNAs derived from HEK293T RNA-seq data. b, RIP performed using antibodies against control immunoglobulin G (IgG) or H3K9me3 in mouse NIH3T3 cells. c, In vitro RNA pull down with biotinylated DNA probes complementary to KCNQ1OT1 (K probe) or control probes (lacZ probe) followed by western blotting. This experiment was repeated three times with similar results. d, Expression of KCNQ1OT1 in control (transfected with empty vector, EV) and K KD cells (left, HEK293T; right, NIH3T3). e, Left, increase of H3K9me3 foci in KCNQ1OT1 KD 3T3 cells shown by immunofluoresence against H3K9me3. Nuclei were counterstained with DAPI. Scale bar, 5 μm. Right, quantification of heterochromatin foci. n = 94 (EV) and 110 (KD) cells pooled across three independent experiments. The bound of a box shows 25% (lower quartile) and 75% (upper quartile) of all values in the group. The centre of the box shows the median value. Values outside middle 50% value (Interquartile Range, or IQR) but within 1.5xIQR are shown as whiskers, otherwise as black dots. All values are shown as grey spots. Data in a and d are shown as mean ± s.e.m. (n = 3 independent experiments), and as fold enrichment relative to input. ACTB, U6 snRNA, and NEAT1 are used as negative controls for RIP-qPCR. Statistical significance was determined by two-tailed Student’s t-test, *p < 0.05, ** p < 0.01, *** p < 0.001. Source numerical data and unprocessed blots are available in Source Data.

Source data

Extended Data Fig. 2 KCNQ1OT1 lncRNA binding regions overlap heterochromatin marks in HEK293T cells.

a, Length distribution of DNA sequences bound by KCNQ1OT1. b, Number of KCNQ1OT1 ChIRP–seq peaks ≥ 1 bp overlapped with at least one repeat or no repeats on each chromosome. c, Compared to 1000 times random sampling of the same number of genomic fragments of the exact same length distribution as KCNQ1OT1 ChIRP–seq peaks (panel 2a), the total 90.8% overlap of KCNQ1OT1 ChIRP–seq peak length to repeat elements is significantly larger than those generated by random sampling with binomial distribution test p < 2.2e-16, empirical p < 0.001. d, Validation of 3 randomly selected KCNQ1OT1 targeted L1 repeats by ChIRP–qPCR. e, ChIP-qPCR of H3K9me3 in HEK293T cells at the same 3 L1 repeats shown in d and at one negative control region. f, Flow-chart of sequential ChIRP–ChIP. g, Sequential ChIRP–ChIP-qPCR showing H3K9me3 at 3 randomly selected KCNQ1OT1 lncRNA associated L1 repeats regions and one negative control (NC) region. h, Genome-browser tracks of H3K9me3 ChIP–seq, MeDIP-seq, and ChIRP–seq alignments at two representative repeats regions, including L1 and Alu. i, Expression of KCNQ1OT1 in control (transfected with empty vector, EV) and KCNQ1OT1 activation (transfected with gRNA expression vector) cells. j, DNA methylation status on a representative KCNQ1OT1 binding L1 element (qPCR region: chr1:65,797,529-65,797,674) in EV, EV-ASO, and K activation-ASO cells detected by MeDIP-qPCR. Data in d, e, g, i, and j are mean ± s.e.m. (n = 3 independent experiments). Statistical significance was determined by two-tailed Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001. Source numerical data are available in Source Data.

Source data

Extended Data Fig. 3 Binding proteins and repetitive elements expression changes in K non-rep KO and K rep KO versus WT HEK293T cells.

a, Diagram showing regions of KCNQ1OT1 deleted in K non-rep KO and K rep KO cells. b, Strand-specific RNA-seq tracks show the expression of KCNQ1 and KCNQ1OT1 in WT, K non-rep KO, K rep KO, EV, and K KD HEK293T cells. c, Correlation analysis of the ChOP-MS dataset shows good correlation between biological replicates. Spearman’s rank correlation coefficient (rho) and p-value between two replicate samples are shown. KCNQ1OT1 denotes KCNQ1OT1 probe, LacZ denotes LacZ probe. d, Expression of randomly selected AluY elements that are targeted by KCNQ1OT1 assessed by RT-qPCR. e, Expression of randomly selected L1 elements that are targeted by KCNQ1OT1 assessed by RT-qPCR. f, Expression of L1HS in two different K rep KO clonal cell lines detected by RT-qPCR. g, DNA methylation status on a representative KCNQ1OT1 binding L1 element in WT, K rep KO clone-1, and K rep KO clone-2 cells detected by MeDIP-qPCR. The qPCR primers amplify unique sequence within the repetitive element (chr1:65,797,529-65,797,674). h, Significance of DNMT1, DNMT3A and HP1α in WT, K non-rep KO and K rep KO cells (use NSAF for one-tailed paired Student’s t-test). Data in d, e, f, and g are mean ± s.e.m. (n = 3 independent experiments). Statistical significance was determined by two-tailed Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001. Source numerical data are available in Source Data.

Source data

Extended Data Fig. 4 KCNQ1OT1 binding at L1 and Alu elements that contain triplex-forming sequences.

a, In vivo triplex pulldown by transfecting biotinylated normal L1 (containing poly(A)) or mutant L1 RNA (containing poly(G)) targeting the site indicated in Fig. 5d (left panel) into HEK293T cells. RNA-associated DNA was analysed by qPCR and normalized to input DNA. The qPCR primers amplify unique sequences within the repetitive elements (L1, chr11:93,426,919-93,427,079; Alu, chr7:105,405,644-105,405,823). b and c, Probability density of number of triplexes on KCNQ1OT1 with each L1 (b) and Alu (c) subfamilies in each 100 bp interval along the KCNQ1OT1 genes. d, Average input and H3K9me3 ChIP–seq signal at the triplexes predicted on L1 and Alu elements. e, Saturation analysis for ChIRP–seq data. Peak numbers detected at the indicated percentage of reads randomly sampled. Data in a are shown as mean ± s.e.m. (n = 3 independent experiments). Statistical significance was determined by two-tailed Student’s t-test, ** p < 0.01, *** p < 0.001. Source numerical data are available in Source Data.

Source data

Supplementary information

Reporting Summary

Peer Review File

Supplementary Table 1

Sequences of primers and probes, Alu subfamilies and proteins bound by KCNQ1OT1.

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Zhang, X., Jiang, Q., Li, J. et al. KCNQ1OT1 promotes genome-wide transposon repression by guiding RNA–DNA triplexes and HP1 binding. Nat Cell Biol 24, 1617–1629 (2022). https://doi.org/10.1038/s41556-022-01008-5

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