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Young LINE-1 transposon 5′ UTRs marked by elongation factor ELL3 function as enhancers to regulate naïve pluripotency in embryonic stem cells

Nature Cell Biology volume 25, pages 1319–1331 (2023)Cite this article

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Abstract

LINE-1s are the major clade of retrotransposons with autonomous retrotransposition activity. Despite the potential genotoxicity, LINE-1s are highly activated in early embryos. Here we show that a subset of young LINE-1s, L1Md_Ts, are marked by the RNA polymerase II elongation factor ELL3, and function as enhancers in mouse embryonic stem cells. ELL3 depletion dislodges the DNA hydroxymethylase TET1 and the co-repressor SIN3A from L1Md_Ts, but increases the enrichment of the Bromodomain protein BRD4, leading to loss of 5hmC, gain of H3K27ac, and upregulation of the L1Md_T nearby genes. Specifically, ELL3 occupies and represses the L1Md_T-based enhancer located within Akt3, which encodes a key regulator of AKT pathway. ELL3 is required for proper ERK activation and efficient shutdown of naïve pluripotency through inhibiting Akt3 during naïve-primed transition. Our study reveals that the enhancer function of a subset of young LINE-1s controlled by ELL3 in transcription regulation and mouse early embryo development.

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Fig. 1: ELL3 represses L1Md_Ts and the nearby genes through directly binding to the L1Md_T 5′ UTRs.
Fig. 2: TET1 and SIN3A are recruited to L1Md_Ts by ELL3.
Fig. 3: The ELL3-regulated L1Md_T 5′ UTRs function as enhancers in naïve mESCs.
Fig. 4: Ell3 KO leads to increased H3K27ac and BRD4 occupancies at the ELL3-bound L1Md_T 5′ UTRs.
Fig. 5: ELL3 depletion impairs ERK activation.
Fig. 6: The ELL3–AKT3 regulatory axis is essential for efficiently silencing naïve pluripotency.
Fig. 7: ELL3 regulates L1Md_T and the AKT3-ERK signalling in murine pre-implantation embryos.
Fig. 8: ELL3 is essential for proper expression of the PrE genes in early embryos.

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Data availability

Deep-sequencing (ChIP–seq, and RNA-seq) data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession numbers GSE208227 and GSE207397. Previously published ELL3, MLL2, SUV39H1, SUV39H2, ZFP281, KAP1, H3K4me1, H3K4me3, H3K9me3, SIN3A, TET1 and SETDB1 ChIP–seq data that were re-analysed here are available under accession codes GSE38148, GSE48172, GSE57092, GSE77115, GSE41903, GSE156261, GSE23943, GSE24841 and GSE126238. Previously published mouse, human and macaque early embryo RNA-seq data and LINE-1 ASO RNA-seq data that were re-analysed here are available under accession codes GSE100939, GSE44183 and GSE86938. 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.

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Acknowledgements

The authors are grateful to the Lin and Luo lab members for helpful discussion of this study. Studies in this manuscript were supported by funds provided by National Key R&D Program of China (2018YFA0800100 and 2018YFA0800101 to C.L.; 2018YFA0800103 to Z.L.), the National Natural Science Foundation of China (32030017 and 31970617 to C.L.; 31970626 to Z.L.; 32100529 to P.X.), Shenzhen Science and Technology Program (JCYJ20210324133602008 and JCYJ20220530160417038 to C.L.; JCYJ20210324133601005 and JCYJ20220530160416037 to Z.L.) and Natural Science Foundation of Yunnan Province (202001BC070001 and 202102AA100053 to K.C.).

Author information

Author notes

  1. These authors contributed equally: Siyan Meng, Xiaoxu Liu, Shiqi Zhu, Peng Xie.

Authors and Affiliations

  1. Key Laboratory of Developmental Genes and Human Disease, School of Life Science and Technology, Southeast University, Nanjing, China

    Siyan Meng, Xiaoxu Liu, Shiqi Zhu, Haitong Fang, Qingyun Pan, Ke Fang, Fanfan Li, Jin Zhang, Zhuanzhuan Che, Guangyao Mao, Fei Sun, Wei Xie, Zhuojuan Luo & Chengqi Lin

  2. Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China

    Siyan Meng, Zhuojuan Luo & Chengqi Lin

  3. School of Biological Science and Medical Engineering, Southeast University, Nanjing, China

    Peng Xie

  4. State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, China

    Quanyong Zhang & Kai Chen

  5. Yunnan Key Laboratory of Primate Biomedical Research, Kunming, China

    Quanyong Zhang & Kai Chen

  6. Department of Prenatal Diagnosis, Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, China

    Yan Wang & Ping Hu

  7. Jiangsu Provincial Key Laboratory of Critical Care Medicine, Southeast University, Nanjing, China

    Zhuojuan Luo

  8. Shenzhen Research Institute, Southeast University, Shenzhen, China

    Zhuojuan Luo & Chengqi Lin

  9. Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing, China

    Chengqi Lin

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Contributions

C.L. and Z.L. initiated the project; S.M. generated the Ell3 KO cell lines, and performed the stem cell phenotypic-, transcriptomic- and epigenomic-related wet experiments; S.Z. performed the murine embryo-related studies; X.L., P.X., Z.L. and C.L. analysed the genome-wide sequencing data; H.F. performed the 5hmC analysis; Q.P. analysed the phenotype of Ell3 KO mice; F.S. performed the enhancer–promoter interaction analysis; K.F. performed the ATAC–seq; F.L., J.Z., G.M., Z.C., Q.Z. and K.F. provided technical assistance; K.C., Y.W., P.H. and W.X. provided the resources; Z.L. and C.L. provided the resources, designed the research, analysed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Zhuojuan Luo or Chengqi Lin.

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Nature Cell Biology thanks Miguel Branco and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Ell3 KO in mESCs.

a, To ablate Ell3 in mESCs, two pairs of sgRNAs were designed to target downstream of the ATG start codon. Introducing the pairs of sgRNAs and Cas9 caused DSBs, which were repaired with indels. The frameshift triggered an immediate premature termination of translation. PTC is an abbreviation for premature termination codon. b, Genomic PCR analyses using the primer sets flanking the cleavage sites verifying the genomic DNA deletion of Ell3. c, Sanger sequencing of the two cut PCR fragments validating the genomic DNA deletion of Ell3. d-g, RT-qPCR (d), RNA-seq (e), western blotting (f) and immunostaining (g) confirming that the two colonies with genomic Ell3 deletion lack ELL3 expression. d, Data are the mean ± SEM from 3 independent experiments. Two-tailed unpaired Student’s t-test was performed. g, Scale bar, 10 µm. h, RNA-seq browser tracks showing RNA levels of Pou5f1, Sox2, Klf4, Nanog, Dppa4 and Dppa2 in WT and Ell3 KO mESCs. i, MA plot showing differentially expressed genes after Ell3 KO in mESCs. Significantly changed genes are shown in dark grey; significantly changed genes bound by ELL3 are shown in orange. j, KEGG pathway analysis of the genes differentially expressed after Ell3 KO. One-sided Fisher’s Exact Test was used for statistical testing with Benjamini-Hochberg multiple testing correction. e, and h,j, Uniquely mapped reads were used for the analysis. Source numerical data and unprocessed scans are available in source data.

Source data

Extended Data Fig. 2 High enrichment of ELL3 at L1Md_Ts.

a, RT-qPCR showing the levels of L1Md A, L1Md T, and L1Md Gf RNAs in WT and Ell3 KO mESCs. b, Pie chart showing the percentage of ELL3 peaks overlapping with a transcription start site (TSS), residing within a gene (inside), or upstream or downstream of the nearest gene. Multi-mapped reads were used for the analysis. c, Heat maps showing ELL3 ChIP-seq profile in mESCs and mappability at the ELL3 bound L1Md_Ts, L1Md_Gfs and non-REs. Shown are ± 5 kb of the center of ELL3 or TET1 peaks. d, Metaplot showing ELL3 ChIP-seq profile along L1Md_T. Mappabilty is also shown. c,d, Multi-mapped and uniquely mapped reads were used for the analysis, as indicated. e, ChIP-qPCR showing loss of the binding of ELL3 to L1Md_T after Ell3 KO. Hemo serves as a negative control for ChIP-qPCR. f, Correlation plot showing the enrichment of ELL3 at L1Md_T in the current study versus in the previous study used a different antibody. g, ChIP-seq browser tracks showing the localization of ELL3 at Akt3_L1. h, Box plots showing the length of all L1Md_Ts (n = 23,233) and the ELL3 bound L1Md_Ts (n = 1,927). Multi-mapped reads were used for the analysis. The box edges in box plots showing 25th and 75th percentiles, the center line showing the 50th percentile, and the bars showing 1.5× the interquartile range (75th percentile -25th percentile). i, MA plot showing differentially expressed genes after Ell3 KO. Significantly up- and down-regulated genes within 50 kb of the ELL3 bound L1Md_Ts are shown in red and blue, respectively. Uniquely mapped reads were used for the analysis. j, RT-qPCR showing the levels of Xlr3a and Xlr4b RNAs in WT and Ell3 KO mESCs. k, ChIP-seq browser tracks showing the localization of ELL3 at the L1Md_T located within the Xlr locus. a,e, and j, Data are the mean ± SEM from 3 independent experiments. Two-tailed unpaired Student’s t-test was performed. f,g, and k, Multi-mapped reads were used for the analysis. Source numerical data are available in source data.

Source data

Extended Data Fig. 3 The enrichment of ELL3 is not affected after Tet1 or Sin3a KD.

a, Correlation plot matrix showing the enrichment of ELL3 and the previously identified LINE-1 regulators at the genome wide scale. Uniquely mapped reads were used for the analysis. b, Pie chart showing the percentage of the TET1 repeat element peaks overlapping with retrotransposons including L1Md_T and L1Md_Gf. c, Heat maps showing CDK9 and AFF4 ChIP-seq profiles in mESCs at the ELL3 bound L1Md_Ts, L1Md_Gfs and non-REs, and the TET1 bound L1Md_As. Shown are ± 5 kb of the center of ELL3 or TET1 peaks. b,c, Multi-mapped reads were used for the analysis. d, ChIP-qPCR showing that the levels of ELL3 remains unchanged at L1Md_T after Sin3a or Tet1 KD. Hemo serves as a negative control for ChIP-qPCR. e, MeDIP-qPCR showing the 5mC levels remain unchanged at the examined regions after Ell3 KO. d,e, Data are the mean ± SEM from 3 independent experiments. Two-tailed unpaired Student’s t-test was performed. Source numerical data are available in source data.

Source data

Extended Data Fig. 4 The ELL3 regulated L1Md_T 5’UTRs are marked by H3K27ac in naïve mESCs.

a, RNA-seq browser tracks showing RNA levels of Rpl7 and Rpl3 after Ell3 KO or LINE-1 ASO treatment in mESCs. Uniquely mapped reads were used for the analysis. b, RT-qPCR showing that L1Md_A and L1Md_Gf remain unchanged after Akt3_L1 CRISPRa. c, Heat maps showing H3K27ac profiles at the ELL3 bound L1Md_T, L1Md_Gf, and non-RE regions, and the ELL3 free but TET1 bound L1Md_A regions in naïve or primed mouse ESCs, and different stages of embryos and indicated tissues. d, ChIP-seq browser tracks showing increased H3K27ac levels, and hMeDIP-seq browser tracks showing reduced 5hmC levels after Ell3 KO at the L1Md_T near to Lipo3, or within Sema3c. Multi-mapped reads were used for the analyses. Virtual 4 C analyses showing that the promoters of Lipo3and Aldh1a2 are able to physically interact with their nearby L1Md_Ts, respectively. e, Browser tracks showing that the L1Md_Ts within Drp2 and Mast2 are not bound by ELL3, and that physical interaction between the L1Md_Ts within these two genes and their promoters are not observed. d,e, Blue bars indicating the view points, and orange bars indicating the putative enhancer regions. Multi-mapped reads were used for the analysis unless otherwise indicated. f, Diagram illustrating the positions of the primers used to amplify the regions in the vicinity of the Akt3 promoter. g, qPCR showing that neither downstream nor upstream region of the Akt3 promoter is enriched in the HA ChIP in the Akt3_L1 CRISPRa cells. b, and g, Data are the mean ± SEM from 3 independent experiments. Two-tailed unpaired Student’s t-test was performed. Source numerical data are available in source data.

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Extended Data Fig. 5 BRD4 and YY1 are involved in the regulation of the ELL3 bound L1Md_Ts.

a, MA plot showing differentially expressed genes after JQ1 treatment in mESCs. Genes significantly up-regulated after Ell3 KO are shown in red. Uniquely mapped reads were used for the analysis. b, RT-qPCR showing RNA levels of L1Md_T, L1Md_Gf, Akt3, Xlr3a and Aldh1a2 after JQ1 treatment in WT and Ell3 KO mESCs. Data are the mean ± SEM from 3 independent experiments. Two-tailed unpaired Student’s t-test was performed. c,d, ChIP-qPCR showing YY1 occupancies at the examined regions after Ell3 KO (c) or JQ1 treatment (d). e, RT-qPCR showing RNA levels of L1Md_T, L1Md_Gf, Akt3, Xlr3a and Aldh1a2 after Yy1 KD. b-e, Data are the mean ± SEM from 3 independent experiments. Two-tailed unpaired Student’s t-test was performed. Source numerical data are available in source data.

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Extended Data Fig. 6 AKT3 is a downstream effector of ELL3.

a, Functional annotation of the genes up- or down-regulated after Akt3 KD (one-sided Fisher’s Exact test with Benjamini-Hochberg multiple testing correction). b, Volcano plot showing differentially expressed retrotransposons after Akt3 KD. Red and blue dots highlighting significance with FC ≥ 1.5 and P-value < 0.05. Multi-mapped reads were used for the analysis (two-sided Wald test with Benjamini-Hochberg multiple testing correction). c, RNA-seq browser tracks showing that T, Hoxa1, Zscan4d and Zscan4f RNA levels are oppositely regulated by ELL3 and AKT3. Uniquely mapped reads were used for the analysis. d, RT-qPCR showing RNA levels of MERVL, Zscan4a, Zscan4b and Dux after AKT3 OE. Data are the mean ± SEM from 3 independent experiments. Two-tailed unpaired Student’s t-test was performed. e, Heat map showing the expression of the genes highlighted in Fig. 5a after Akt3 KD or Ell3 KO. The genes directly bound by ELL3 are highlighted in blue. f-h, Western blot analysis showing the levels of AKT1, AKT2, AKT3, p-AKT1/2/3, p-RAF S259, ERK, p-ERK1/2 after AKT3 OE (f), Akt3_L1 CRISPRa (g) after 2i withdrawal, or Ell3 KO (h) after FA treatment. TUBULIN was used as a loading control. The experiment was repeated 3 times with similar results. Source numerical data and unprocessed scans are available in source data.

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Extended Data Fig. 7 ELL3 regulates mESC naïve-primed transition.

a, Bright field microscopic image of control and Akt3_L1 CRISPRa mESCs cultured in 2i/LIF or FA medium for indicated time. The experiment was repeated over 3 times with similar results. Scale bar, 30 µm. b, MA plot showing differentially expressed genes after Ell3 KO in FA treated mESCs. Significantly up- and down-regulated genes are shown in red and blue, respectively. Uniquely mapped reads were used for the analysis. c-d, RT-qPCR showing the levels of RNA from the examined genes in AKT3 OE (c) or Akt3_L1 CRISPRa (d) mESCs before and after FA treatment. Data are the mean ± SEM from 3 independent experiments. Two-tailed unpaired Student’s t-test was performed. e, Representative immunostaining images showing the levels of NANOG, ESRRB and PITX2 after AKT3 OE in control and AKT3 OE mESCs before and after FA treatment (left panel). Scale bar, 10 µm. Statistical plots showing log2 of fluorescence intensity (right panel). Each dot representing a single cell. Cells were collected from 3 independent experiments. Two-tailed unpaired Student’s t-test was performed. Source numerical data are available in source data.

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Extended Data Fig. 8 ELL3 regulates L1Md_T in pre-implantation embryos.

a, Line graphs showing ELL and ELL2 expression in human (left panel), rhesus macaque (middle panel) and mouse (right panel) oocyte, pronucleus, 2 cell embryo, 4 cell embryo, 8 cell embryo and morula. Uniquely mapped reads were used for the analysis. Error bands indicate 95% confidence intervals. b, Heat map showing the expression of the ELL3 regulated retrotransposons in different stages of mouse pre-implantation embryos. Multi-mapped reads were used for the analysis. c, Representative immunostaining images showing the levels of AKT3 and p-ERK1/2 in control and Ell3 KD blastocysts, and that MK2206 treatment can restore the level of p-ERK1/2 in Ell3 KD blastocysts. The experiment was repeated over 3 times with similar results. Scale bar, 20 μm.

Extended Data Fig. 9 ELL3 is required for ICM lineage choice.

a, Representative images showing TUNEL intensity in control and Ell3 KD 2 C, 4 C embryos and morulas. Scale bar, 20 μm (upper panel). Box plots showing the normalized TUNEL intensity in control and Ell3 KD 2 C, 4 C embryos and morulas (lower panel). b, Representative immunostaining images showing Ell3 KD efficiency in blastocysts. Scale bar, 20 μm. c, Box plots showing the RNA levels of Nanog and Dppa2 in control and Ell3 KD blastocysts. d-e, Representative immunostaining images showing the levels of OCT4 (d) and SOX2 (e) in control and Ell3 KD blastocysts. Scale bar, 20 μm. a-e, Each dot representing an embryo. Embryos were collected from 3 independent experiments. Box centres indicate median, boundaries represent 25th and 75th percentiles, and error bars represent maximum and minimum values. Two-tailed unpaired Student’s t-test was performed. Source numerical data are available in source data.

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Extended Data Fig. 10 ELL3 plays a role in mouse embryo development.

a, Diagram illustrating the Ell3 KO strategy in mice, and the primers designed for genotyping. b, Genomic PCR analyses using the primer sets to genotype the embryos from intercrossing between genotypes of Ell3fl/fl and Ell3-/+; CAG-iCre mice. Source unprocessed scans are available in source data.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. Number of REs regulated by ELL3. Supplementary Table 2. Primers/oligos used in the current study.

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Meng, S., Liu, X., Zhu, S. et al. Young LINE-1 transposon 5′ UTRs marked by elongation factor ELL3 function as enhancers to regulate naïve pluripotency in embryonic stem cells. Nat Cell Biol 25, 1319–1331 (2023). https://doi.org/10.1038/s41556-023-01211-y

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