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

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.

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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.

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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.

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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.

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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)

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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

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