Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo

Nature Genetics volume 49, pages 1502–1510 (2017)Cite this article

Subjects

Abstract

After fertilization, to initiate development, gametes are reprogramed to become totipotent. Approximately half of the mammalian genome consists of repetitive elements, including retrotransposons, some of which are transcribed after fertilization. Retrotransposon activation is generally assumed to be a side effect of the extensive chromatin remodeling underlying the epigenetic reprogramming of gametes. Here, we used a targeted epigenomic approach to address whether specific retrotransposon families play a direct role in chromatin organization and developmental progression. We demonstrate that premature silencing of LINE-1 elements decreases chromatin accessibility, whereas prolonged activation prevents the gradual chromatin compaction that occurs naturally in developmental progression. Preventing LINE-1 activation and interfering with its silencing decreases developmental rates independently of the coding nature of the LINE-1 transcript, thus suggesting that LINE-1 functions primarily at the chromatin level. Our data suggest that activation of LINE-1 regulates global chromatin accessibility at the beginning of development and indicate that retrotransposon activation is integral to the developmental program.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: LINE-1 elements are highly expressed in the early stages of mouse preimplantation development.
Figure 2: Embryos with sustained LINE-1 transcription display developmental arrest.
Figure 3: Targeted repression of LINE-1 after fertilization results in a decreased developmental rate and decreased global transcriptional activity at ZGA.
Figure 4: RNA-seq analysis after targeted repression and activation of LINE-1 shows modest changes in gene expression.
Figure 5: Transcriptional activity of LINE-1 modulates global chromatin accessibility.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Watanabe, T. et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543 (2008).

    CAS  PubMed  Google Scholar 

  2. Tam, O.H. et al. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453, 534–538 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Veselovska, L. et al. Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape. Genome Biol. 16, 209 (2015).

    PubMed  PubMed Central  Google Scholar 

  4. Burton, A. & Torres-Padilla, M.E. Chromatin dynamics in the regulation of cell fate allocation during early embryogenesis. Nat. Rev. Mol. Cell Biol. 15, 723–734 (2014).

    CAS  PubMed  Google Scholar 

  5. Hemberger, M., Dean, W. & Reik, W. Epigenetic dynamics of stem cells and cell lineage commitment: digging Waddington's canal. Nat. Rev. Mol. Cell Biol. 10, 526–537 (2009).

    CAS  PubMed  Google Scholar 

  6. Mayer, W., Niveleau, A., Walter, J., Fundele, R. & Haaf, T. Demethylation of the zygotic paternal genome. Nature 403, 501–502 (2000).

    CAS  PubMed  Google Scholar 

  7. Smith, Z.D. et al. A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 484, 339–344 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Friedli, M. et al. Loss of transcriptional control over endogenous retroelements during reprogramming to pluripotency. Genome Res. 24, 1251–1259 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Akagi, K., Li, J., Stephens, R.M., Volfovsky, N. & Symer, D.E. Extensive variation between inbred mouse strains due to endogenous L1 retrotransposition. Genome Res. 18, 869–880 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Naas, T.P. et al. An actively retrotransposing, novel subfamily of mouse L1 elements. EMBO J. 17, 590–597 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Waterston, R.H. et al. Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002).

    CAS  PubMed  Google Scholar 

  12. Peaston, A.E. et al. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Dev. Cell 7, 597–606 (2004).

    CAS  PubMed  Google Scholar 

  13. Fadloun, A. et al. Chromatin signatures and retrotransposon profiling in mouse embryos reveal regulation of LINE-1 by RNA. Nat. Struct. Mol. Biol. 20, 332–338 (2013).

    CAS  PubMed  Google Scholar 

  14. Ancelin, K. et al. Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation. eLife 5, e08851 (2016).

    PubMed  PubMed Central  Google Scholar 

  15. Sookdeo, A., Hepp, C.M., McClure, M.A. & Boissinot, S. Revisiting the evolution of mouse LINE-1 in the genomic era. Mob. DNA 4, 3 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Moran, J.V. et al. High frequency retrotransposition in cultured mammalian cells. Cell 87, 917–927 (1996).

    CAS  PubMed  Google Scholar 

  17. Adey, N.B. et al. Rodent L1 evolution has been driven by a single dominant lineage that has repeatedly acquired new transcriptional regulatory sequences. Mol. Biol. Evol. 11, 778–789 (1994).

    CAS  PubMed  Google Scholar 

  18. Faulkner, G.J. et al. The regulated retrotransposon transcriptome of mammalian cells. Nat. Genet. 41, 563–571 (2009).

    CAS  PubMed  Google Scholar 

  19. Bulut-Karslioglu, A. et al. Suv39h-dependent H3K9me3 marks intact retrotransposons and silences LINE elements in mouse embryonic stem cells. Mol. Cell 55, 277–290 (2014).

    CAS  PubMed  Google Scholar 

  20. Doucet, A.J. et al. Characterization of LINE-1 ribonucleoprotein particles. PLoS Genet. 6, e1001150 (2010).

    PubMed  PubMed Central  Google Scholar 

  21. Miyanari, Y., Ziegler-Birling, C. & Torres-Padilla, M.E. Live visualization of chromatin dynamics with fluorescent TALEs. Nat. Struct. Mol. Biol. 20, 1321–1324 (2013).

    CAS  PubMed  Google Scholar 

  22. Nakamura, T. et al. PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos. Nature 486, 415–419 (2012).

    CAS  PubMed  Google Scholar 

  23. Mathias, S.L., Scott, A.F., Kazazian, H.H. Jr., Boeke, J.D. & Gabriel, A. Reverse transcriptase encoded by a human transposable element. Science 254, 1808–1810 (1991).

    CAS  PubMed  Google Scholar 

  24. Dai, L., Huang, Q. & Boeke, J.D. Effect of reverse transcriptase inhibitors on LINE-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition. BMC Biochem. 12, 18 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Xie, Y., Rosser, J.M., Thompson, T.L., Boeke, J.D. & An, W. Characterization of L1 retrotransposition with high-throughput dual-luciferase assays. Nucleic Acids Res. 39, e16 (2011).

    PubMed  Google Scholar 

  26. Dombroski, B.A., Scott, A.F. & Kazazian, H.H. Jr. Two additional potential retrotransposons isolated from a human L1 subfamily that contains an active retrotransposable element. Proc. Natl. Acad. Sci. USA 90, 6513–6517 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Schultz, R.M. Regulation of zygotic gene activation in the mouse. BioEssays 15, 531–538 (1993).

    CAS  PubMed  Google Scholar 

  29. Schultz, R.M. & Worrad, D.M. Role of chromatin structure in zygotic gene activation in the mammalian embryo. Semin. Cell Biol. 6, 201–208 (1995).

    CAS  PubMed  Google Scholar 

  30. Martens, J.H. et al. The profile of repeat-associated histone lysine methylation states in the mouse epigenome. EMBO J. 24, 800–812 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Burton, A. et al. Single-cell profiling of epigenetic modifiers identifies PRDM14 as an inducer of cell fate in the mammalian embryo. Cell Rep. 5, 687–701 (2013).

    CAS  PubMed  Google Scholar 

  32. Guo, G. et al. Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev. Cell 18, 675–685 (2010).

    CAS  PubMed  Google Scholar 

  33. Jachowicz, J.W., Santenard, A., Bender, A., Muller, J. & Torres-Padilla, M.E. Heterochromatin establishment at pericentromeres depends on nuclear position. Genes Dev. 27, 2427–2432 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Bošković, A. et al. Higher chromatin mobility supports totipotency and precedes pluripotency in vivo. Genes Dev. 28, 1042–1047 (2014).

    PubMed  PubMed Central  Google Scholar 

  35. Aiken, C.E., Swoboda, P.P., Skepper, J.N. & Johnson, M.H. The direct measurement of embryogenic volume and nucleo-cytoplasmic ratio during mouse pre-implantation development. Reproduction 128, 527–535 (2004).

    CAS  PubMed  Google Scholar 

  36. Wu, J. et al. The landscape of accessible chromatin in mammalian preimplantation embryos. Nature 534, 652–657 (2016).

    CAS  PubMed  Google Scholar 

  37. Carpenter, A.E., Memedula, S., Plutz, M.J. & Belmont, A.S. Common effects of acidic activators on large-scale chromatin structure and transcription. Mol. Cell. Biol. 25, 958–968 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Kulpa, D.A. & Moran, J.V. Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles. Nat. Struct. Mol. Biol. 13, 655–660 (2006).

    CAS  PubMed  Google Scholar 

  39. Wei, W. et al. Human L1 retrotransposition: cis preference versus trans complementation. Mol. Cell. Biol. 21, 1429–1439 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Ecco, G. et al. Transposable elements and their KRAB-ZFP controllers regulate gene expression in adult tissues. Dev. Cell 36, 611–623 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Svoboda, P. et al. RNAi and expression of retrotransposons MuERV-L and IAP in preimplantation mouse embryos. Dev. Biol. 269, 276–285 (2004).

    CAS  PubMed  Google Scholar 

  42. Ishiuchi, T. et al. Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly. Nat. Struct. Mol. Biol. 22, 662–671 (2015).

    CAS  PubMed  Google Scholar 

  43. Macfarlan, T.S. et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57–63 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Muotri, A.R. et al. Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition. Nature 435, 903–910 (2005).

    CAS  PubMed  Google Scholar 

  45. Chow, J.C. et al. LINE-1 activity in facultative heterochromatin formation during X chromosome inactivation. Cell 141, 956–969 (2010).

    CAS  PubMed  Google Scholar 

  46. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Miyanari, Y. & Torres-Padilla, M.E. Control of ground-state pluripotency by allelic regulation of Nanog. Nature 483, 470–473 (2012).

    CAS  PubMed  Google Scholar 

  48. Malki, S., van der Heijden, G.W., O'Donnell, K.A., Martin, S.L. & Bortvin, A. A role for retrotransposon LINE-1 in fetal oocyte attrition in mice. Dev. Cell 29, 521–533 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Pittoggi, C. et al. Role of endogenous reverse transcriptase in murine early embryo development. Mol. Reprod. Dev. 66, 225–236 (2003).

    CAS  PubMed  Google Scholar 

  50. Nashun, B. et al. Continuous histone replacement by Hira is essential for normal transcriptional regulation and de novo DNA methylation during mouse oogenesis. Mol. Cell 60, 611–625 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Shalek, A.K. et al. Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells. Nature 498, 236–240 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Ramsköld, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777–782 (2012).

    PubMed  PubMed Central  Google Scholar 

  53. Picelli, S. et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat. Methods 10, 1096–1098 (2013).

    CAS  PubMed  Google Scholar 

  54. Trombetta, J.J. et al. Preparation of single-cell RNA-Seq libraries for next generation sequencing. Curr. Protoc. Mol. Biol. 107, 4.22 (2014).

    Google Scholar 

Download references

Acknowledgements

We thank D. O'Carroll (MRC Centre for Regenerative Medicine) for anti-ORF1p; S. Le Gras for initial assembly of the LINE-1 genome and help with the Galaxy platform; G. LeBoulle for customized TaqMan assay design; D. Rodriguez-Terrones for advice on Biomark analysis; A. Fadloun, Y. Miyanari and members of the laboratory of M.-E.T.-P. for helpful discussions; V. Ignatova (Institute of Functional Epigenetics, Helmholtz Centre Munich) for the Gal4-TALE construct; and J. M. Vaquerizas and R. Enriquez-Gasca for advice on RNA-seq. M.-E.T.-P. acknowledges funding from ERC-Stg 280840 'NuclearPotency', the Schlumberger Foundation for Research and Education (2016-Torres-Padilla) and the Helmholtz Gesellschaft. J.W.J was supported as a recipient of a doctoral fellowship from the Ligue Contre le Cancer (2015-Jachowicz); A.B. was supported by a postdoctoral fellowship from H.F.S.P.

Author information

Authors and Affiliations

  1. Institute of Epigenetics and Stem Cells, Helmholtz Zentrum München, München, Germany

    Joanna W Jachowicz, Julien Pontabry & Maria-Elena Torres-Padilla

  2. INSERM U964, U de S, CU de Strasbourg, Illkirch, France

    Joanna W Jachowicz & Maria-Elena Torres-Padilla

  3. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Xinyang Bing, Ana Bošković & Oliver J Rando

Authors
  1. Joanna W Jachowicz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xinyang Bing
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julien Pontabry
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ana Bošković
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Oliver J Rando
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Maria-Elena Torres-Padilla
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.W.J. designed, performed and analyzed most of the experiments in the manuscript. X.B. performed the bioinformatic analysis for the RNA-seq data sets. J.P. generated the pipeline used for 3D image quantification. A.B. generated the libraries and sequencing for the RNA-seq analysis. O.J.R. provided key expertise and supervision on gene expression analysis and interpretation. M.-E.T-P. designed and supervised the study and performed experiments. All authors contributed to the manuscript preparation, read, commented on and approved the manuscript.

Corresponding author

Correspondence to Maria-Elena Torres-Padilla.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Design and characterization of TALEs against LINE-1.

a. TALEs used in this study; mm - mismatches, nt - not tested, * - off-targets predicted within truncated/inactive LTR or non-genic regions. b. Binding affinity of TALE-L1 constructs. TALEs fused or not (control) to VP64 were transfected in 293T cells. Plotted mean of Firefly luciferase activity normalised to Renilla luciferase from independent replicates (transfection experiments); each dot represents 1 transfection experiment. Statistical analysis: Student’s t-test with Welsh correction (two-sided); each p value shown above the compared bars. mCMV: minimal CMV promoter; binding site: TALE binding site. c. Representative images of expression and localisation of Flag-TALE-L1 constructs in mES cells (left) compared to endogenous LINE-1 by DNA-FISH (right). n=2 (independent transfection experiments). Seven different TALE-L1 constructs described in Fig. S1a were detected using an anti-FLAG antibody (green). Maximal projection of Z-confocal sections are shown. Scale bars, 10 μm.

Supplementary Figure 2 Characterization of TALE-L1-VP64 embryos.

a. Immunostaining of TALE-L1-Ctrl and TALE-L1-VP64 constructs using an anti-HA antibody in 4-cell stage embryos after mRNA microinjection at the 2-cell stage. Images are maximal projections of Z-confocal sections of single nuclei. Ni, non-injected embryos; n = number of nuclei analysed. Scale bar,10 μm. b. Summary of the analysis of TALE-Ctrl localisation after microinjection of 2-cell stage blastomeres with mRNA for indicated TALEs (200 ng/μl of each), based on HA-tag immunostaining. nt – not tested, + - TALE strongly detected in the nucleus, +/- - TALE showing weak or undetectable signal. c. Developmental progression of embryos expressing TALE-Ctrls from the indicated TALE cocktails in both blastomeres from the 2-cell (mRNA at 200ng/μl of each). d. ORF1p immunostaining in non-injected 4-cell stage embryos (Ni), or embryos expressing TALE-L1-Ctrl or TALE-L1-VP64. Shown are representative maximal projections of Z-confocal sections. n=number of embryos analysed. Scale bar,10 μm. e. Developmental progression of non-injected embryos (Ni) and embryos expressing TALE2-L1-Ctrl or TALE2-L1-VP64 after mRNAs microinjected (400 ng/μl) in two blastomeres of 2-cell stage embryos. The TALE2-L1 cocktail contains different TALEs than TALE-L1. The % of embryos that reached blastocyst stage (left) and representative images (right) from one of two independent microinjection experiments are shown. Statistical analysis: Z-test; Ni vs L1-VP64: *** p<0.001. f. Immunostaining using an HA antibody in 8-cell stage embryos after microinjections of the indicated TALE constructs from the 2-cell stage. Non-injected embryos (Ni) are shown as a negative control. Images are maximal projections of Z-confocal sections of single nuclei. n = number of nuclei analysed. Scale bar,10 μm. g. Summary of developmental progression of non-injected (Ni) or embryos injected with mRNA coding for the indicated TALEs at the 2-cell stage. Concentrations were corrected for molarity. At least 2 independent microinjection experiments per group. Statistical analysis: Z-test.

Supplementary Figure 3 Characterization of embryos expressing TALE L1-KRAB.

a. Immunostaining with HA antibody in 2-cell stage embryos injected with TALE-L1-KRAB or TALE-L1-Ctrl mRNA at the early zygote stage. Shown are maximal projections of Z-confocal sections of single nuclei. Ni: non-injected; n = number of nuclei analysed. Scale bar, 10 μm. b. ORF1p immunostaining in 2-cell stage non-injected (Ni) or embryos microinjected with TALE-L1-Ctrl or TALE-L1-KRAB mRNA at the zygote stage. Images are maximal projections of single embryos; n = number of embryos analysed. Scale bar, 10 μm. c. Summary of developmental progression of non-injected (Ni) or embryos injected with the indicated mRNA in zygotes. mRNA concentrations were corrected for molarity. Statistical analysis: Z-test. d. Immunostaining with an HA-tag antibody to indicated TALEs at the 2-cell stage, after microinjection in zygotes. Images are maximal projections of Z-confocal sections of single nuclei. Ni: non-injected; n = number of nuclei analysed. Scale bar,10 μm. e. Developmental progression of non-injected embryos (Ni), and embryos microinjected in two blastomeres at the late 2-cell stage with mRNA for TALE-L1-Ctrl or TALE-L1-KRAB. Data are from two independent experiments. Statistical analysis: Z-test.

Supplementary Figure 4 Single-embryo Fluidigm RT–PCR analysis of TALE L1-KRAB-expressing embryos.

a. Relative expression of rDNA genes in non-injected (Ni) and embryos expressing TALE L1-Ctrl or TALE L1-KRAB. Expression was determined in single embryos (n=13 for Ni, n=14 for L1-Ctrl, n=13 for L1-KRAB) and is plotted as mean ± S.D. of absolute Ct values normalised to the mean of GAPDH and Actin-B, averaged from 2 technical replicates. Statistical analysis: one-way ANOVA F(2, 22.9)=0.64, p=0.54 for LSU and F(2, 23.9)=0.33, p=0.72 for SSU. b. Actin-B and GAPDH expression in non-injected or in embryos expressing TALE-L1-Ctrl or TALE-L1-KRAB. The graph shows mean ± S.D. of the Ct values obtained from single embryos (n=13 for Ni, n=14 for L1-Ctrl, n=13 for L1-KRAB) averaged from 2 technical replicates. Statistical analysis: one-way ANOVA F(2, 24)=0.20, p=0.81 for Actin-B and F(2, 23.87)=0.58, p=0.57 for GAPDH. c. Relative expression of indicated repeats in individual 2-cell stage embryos (n= 10 to 14 per group). Each dot represents a single embryo. Statistical analysis: student’s t-test with Welsh correction. d. Expression of indicated repeats and genes in non-injected embryos with and without reverse transcriptase. Shown are Ct values determined in single embryos (n= 10 to 14 embryos per RT+ group and 2 embryos per RT- group), averaged between two technical replicates. e. Relative expression of single copy genes as in c. ZGA genes in LINE-1 proximity are located within 40kb of the predicted TALE-L1 target. Statistical analysis: no significant difference with student’s t-test with Welsh correction.

Supplementary Figure 5 RNA-seq expression analysis in TALE L1-KRAB- and TALE-L1-VP64-expressing embryos.

a. Euclidean distance between samples using a composite of all genes with at least 10 TPM across all samples analysed by RNAseq, plotted as a cluster heatmap. 8C – 8-cell, 2C – 2-cell; NI – non-injected, Ctrl – TALE L1-Ctrl, VP64 – TALE L1-VP64, KRAB - TALE L1-KRAB. b. Heatmap of all analysed replicates of TALE L1-Ctrl (Ctrl) and TALE L1-KRAB (Krab) expressing embryos with genes expressed at 2-cell, 4-cell, 8-cell, and inner cell mass (ICM). Gene lists were extracted from Wu et al. (ref. 36) and Deng et al (DOI: 10.1126/science.1245316) Log2 scale is across rows to indicate overall variability for each gene across all samples. n=7 groups for L1-Ctrl and n=6 groups for L1-KRAB. Note that the transcriptomes of Krab group is similar to Ctrl embryos (closer to the Log2Fold change 0, on the colour scale). c. Heatmap as in b, for all analysed replicates of TALE-L1-Ctrl (Ctrl) and L1-VP64 (VP) expressing embryos and genes expressed at 2-cell, 4-cell, 8-cell, and inner cell mass (ICM).

Supplementary Figure 6 Description of DNase I in situ assay and characterization of TALE L1-DEL-expressing embryos.

a. Experimental conditions for DNase I treatment followed by TUNEL detection. Fluorescence intensity of TUNEL signal normalised to nuclear volume was quantified in 2-cell stage embryos after treatment with increasing DNase I concentrations (n<10 embryos per condition). The horizontal line indicates the threshold below which DNase I treatment has no effect on TUNEL intensity. Each dot is a single nucleus, the mean is indicated. b. Immunostaining of TALE L1-DEL with an HA antibody in 8-cell stage embryos upon mRNA microinjection at the 2-cell stage. Images shown are maximal projections of Z-confocal sections of single nuclei. Ni: non-injected; n=number of nuclei analysed. c. RNA-FISH of 8-cell stage nuclei from non-injected (Ni) embryos and embryos expressing TALE L1-Ctrl or TALE-L1-DEL. Images are maximal projections of Z-confocal sections of single nuclei. n=number of nuclei analysed. d. ORF1p immunostaining in 8-cell stage non-injected (Ni) or embryos expressing TALE L1-DEL. Images are maximal Z-confocal sections projections. n: number of embryos analysed. e. Representative images of maximal Z-confocal sections projections of single nuclei after DNase I treatment and TUNEL detection. n=number of nuclei analysed. Scale bar, 10 μm. f. Levels of γH2A.X in 8-cell stage embryos expressing TALE L1-Ctrl, TALE L1-DEL, TALE L1-VP64 or non-injected (Ni). Maximal projections of Z-confocal sections of single nuclei are shown. n=number of nuclei analysed. Quantification of fluorescence γH2A.X intensity normalised to nuclear volume is shown on the right. Statistical analysis: one-way ANOVA with Tukey post-hoc test; * p<0.05. Scale bar, 10 μm. g. Quantification of nuclear volume of 2-cell stage embryos microinjected with the indicated mRNAs at the zygote stage. The median value is indicated and each dot represents a single nucleus. Statistical analysis: one-way ANOVA, ns=not significant. h. Bright-field images of representative embryos as described in Figure 5f. Scale bar, 10 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6. (PDF 1563 kb)

Life Sciences Reporting Summary (PDF 130 kb)

Supplementary Table 1

TALE binding sites. (XLSX 654 kb)

Supplementary Table 2

List of Taqman assays used in the study. (XLSX 10 kb)

Supplementary Table 3

Fluidigm raw data. (XLSX 163 kb)

Supplementary Table 4

Differentially expressed genes at 2-cell and 8-cell stage embryos. (XLSX 2955 kb)

Supplementary Table 5

Sequencing samples and reads. (XLSX 11 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jachowicz, J., Bing, X., Pontabry, J. et al. LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo. Nat Genet 49, 1502–1510 (2017). https://doi.org/10.1038/ng.3945

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.3945

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research