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Genomic instability during reprogramming by nuclear transfer is DNA replication dependent

Abstract

Somatic cells can be reprogrammed to a pluripotent state by nuclear transfer into oocytes, yet developmental arrest often occurs. While incomplete transcriptional reprogramming is known to cause developmental failure, reprogramming also involves concurrent changes in cell cycle progression and nuclear structure. Here we study cellular reprogramming events in human and mouse nuclear transfer embryos prior to embryonic genome activation. We show that genetic instability marked by frequent chromosome segregation errors and DNA damage arise prior to, and independent of, transcriptional activity. These errors occur following transition through DNA replication and are repaired by BRCA1. In the absence of mitotic nuclear remodelling, DNA replication is delayed and errors are exacerbated in subsequent mitosis. These results demonstrate that independent of gene expression, cell-type-specific features of cell cycle progression constitute a barrier sufficient to prevent the transition from one cell type to another during reprogramming.

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Figure 1: Genomic instability after human somatic NT.
Figure 2: Segregation errors are pre-mitotic in origin.
Figure 3: Mitotic segregation defects due to DNA damage arising from progression through DNA replication.
Figure 4: Nuclear remodelling is necessary for normal mitotic segregation.
Figure 5: DNA damage following progression through DNA replication in the second cell cycle.
Figure 6: DNA damage is repaired by BRCA1.

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Acknowledgements

This research was supported by the New York Stem Cell Foundation (NYSCF) and a New York State Stem Cell Science (NYSTEM) IIRP Award no. C026184 and the John M. Driscoll, Jr, M. D. Children’s Fund. D.E. is a NYSCF-Robertson Investigator. We thank W. Kearns (Shady Grove Center for Preimplantation Genetic Diagnosis) for initial help with karyotyping. G.C. is supported by the Agency for Science, Technology and Research (A STAR) International Fellowship. We thank A. Ciccia and W.-W. Tee for critical comments on the manuscript.

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Authors and Affiliations

Authors

Contributions

G.C. and D.E. designed and performed the experiments, analysed the data and wrote the manuscript with input from all authors. D.E. conceived and supervised the studies and performed NT. J.A. and B.D.B. provided T cells. N.T. performed karyotype analysis of blastomeres. D.B. and R.B. provided Brca1 mutant mice and helped with data interpretation and manuscript writing. M.V.S. was involved in all aspects of oocyte donation, including consent and oocyte retrieval.

Corresponding author

Correspondence to Dieter Egli.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Genetic instability in human nuclear transfer embryos.

(a) Normal chromosome segregation at the second meiotic division upon transfer of a somatic cell genome. (bd) Anaphase at the first mitosis. b) The arrow points to a chromosome fragment. (c) The arrows point the direction of chromosome movement. Note the splitup of chromosomes into different groups, and a chromosome fragment (arrowhead). n = 142 (IVF), 138 (NT), 111 (NT, caffeine pulse) embryos. P = 0.0000 (Student’s t-test). (d) lagging chromosomes occurring in pairs (arrows). (e) A single confocal section of the cell shown in (d). Note the indentation (arrowhead) in the anaphase chromosomes, likely as a result of strain from a resolving chromosome bridge (arrow). (f) Formation of nuclei at telophase of the first mitosis. Note the formation of multiple nuclei. (g) 6-cell NT embryo with four blastomeres (one not visible) in mitosis on day 4 after transfer. Arrows point to the metaphase plate, arrowheads to chromosomes that fail to integrate. (h) Blastomere arrested in mitosis. Abnormal chromosome condensation prevents the formation of a metaphase plate. DNA damage is evident from phosphorylated γH2AX staining. The three channels were shifted relative to each other for visibility. (i,j) Examples of multinucleation in blastomeres. pH3 indicates phosphorylation on serine 10 of histone H3. (k) Another example of an embryo arrested with DNA damage, at entry into mitosis. (l,m) RPA foci in an interphase nucleus, and centromere staining. (np) Examples of RPA and phosphorylated γH2AX in NT blastomeres. Scale bar: 10 μm.

Supplementary Figure 2

SNP array analysis of copy number and heterozygosity in NT blastomeres. Each of the four clusters is derived from a different embryo. Only cells with successful biopsy and array analysis are shown.

Supplementary Figure 3 Different types of mitotic chromosomal defects in nuclear transfer embryos.

(a) Immunostaining of NT embryos at the first mitosis reconstituted using different donor cell types (ES cells, T-cells and cumulus cells). (b) Percentage of embryos (parthenotes and NT embryos), activated with either latrunculin A or cytochalasin B, with chromosomal segregation defects at the first mitosis. n = 64 (Parthenotes, Latrunculin A), 202 (Parthenotes, Cytochalasin B), 170 (NT, Latrunculin A) and 129 (NT, Cytochalasin B) embryos. Scale bar: 10 μm.

Supplementary Figure 4 Cell cycle of somatic donor cells.

Cell cycle profile by flow cytometry of naïve T-cells used for transfer. Note that most cells are in G1 of the cell cycle.

Supplementary Figure 5 DNA damage is reduced in G2, following exit from S-phase, and is independent of transcription.

NT embryos and parthenotes were incubated in EdU for 1 h at the following time points (17 h, 22 h and 28h postactivation). Bars represent the mean percentage of NT embryos that are undergoing DNA replication (EdU positive) at the respective time points. n = 12 (P, 17h), 13 (NT, 17h), 12 (P, 22h), 12 (NT, 22h), 5 (P, 28h) and 12 (NT, 28h) embryos.] b) DNA damage (no. of γH2AX) in NT embryos and parthenotes at the respective time points in a. Line represents mean number of foci in n = 12 (P, 17h), 13 (NT, 17h), 12 (P, 22h), 12 (NT, 22 h), 5 (P, 28h) and 12 (NT, 28h) number of embryos. P < 0.01; ns = not significant (Student’s t-test). (c) Quantification of γH2AX, RPA and Rad51 foci at 30h versus 42h after nuclear transfer and activation. Lines represent the median no. of foci in n = 23 (untreated, γH2AX) and 25 (α-amanitin, γH2AX),n = 56 (untreated, RPA), 69 (α-amanitin, RPA), 34 (untreated, Rad51) and 42 ((α-amanitin, Rad51) number of embryos. Arrowhead indicates outlier with significant DNA damage. (d) DNA replication (EdU incorporation, top panel) and nascent RNA transcription (5-ethynyluridine, EU incorporation, bottom panel) in untreated and α-amanitin treated NT embryos. Arrowheads point to EU labelled foci indicating high transcriptional activity. (e) RPA, Rad51 and γH2AX staining in untreated and α-amanitin treated NT embryos. (fh) Quantification of γH2AX, RPA and Rad51 foci in untreated and α-amanitin treated embryos. Line represents median number of foci. n = 23 (γH2AX, untreated), 25 (γH2AX, α-amanitin treated), 56 (RPA32, untreated), 69 (RPA32, α-amanitin treated), 34 (Rad51, untreated) and 42 (Rad5, α-amanitin treated) embryos. Scale bar: 5 μm and 10 μm (inserts).

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Chia, G., Agudo, J., Treff, N. et al. Genomic instability during reprogramming by nuclear transfer is DNA replication dependent. Nat Cell Biol 19, 282–291 (2017). https://doi.org/10.1038/ncb3485

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