Letter | Published:

Co-repressor CBFA2T2 regulates pluripotency and germline development

Nature volume 534, pages 387390 (16 June 2016) | Download Citation

Abstract

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs)1,2, precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own ‘latent’ form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2, 3, 4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell- and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2/ mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional ‘passenger’ role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency.

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Accessions

Primary accessions

Gene Expression Omnibus

Data deposits

Sequencing data have been deposited in the Gene Expression Omnibus under accession number GSE71676.

References

  1. 1.

    , & A molecular programme for the specification of germ cell fate in mice. Nature 418, 293–300 (2002)

  2. 2.

    , & Genetic and epigenetic regulators of pluripotency. Cell 128, 747–762 (2007)

  3. 3.

    et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996)

  4. 4.

    et al. Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nature Genet. 40, 1016–1022 (2008)

  5. 5.

    et al. A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature 468, 316–320 (2010)

  6. 6.

    , , , & Sequence-specific regulator Prdm14 safeguards mouse ESCs from entering extraembryonic endoderm fates. Nature Struct. Mol. Biol . 18, 120–127 (2011)

  7. 7.

    et al. PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells. Cell Stem Cell 12, 368–382 (2013)

  8. 8.

    et al. A PRC2-dependent repressive role of PRDM14 in human embryonic stem cells and induced pluripotent stem cell reprogramming. Stem Cells 31, 682–692 (2013)

  9. 9.

    et al. Tsix RNA and the germline factor, PRDM14, link X reactivation and stem cell reprogramming. Mol. Cell 52, 805–818 (2013)

  10. 10.

    et al. RUNX1 translocations and fusion genes in malignant hemopathies. Future Oncol. 7, 77–91 (2011)

  11. 11.

    et al. CBFA2T2 and C20orf112: two novel fusion partners of RUNX1 in acute myeloid leukemia. Leukemia 24, 1516–1519 (2010)

  12. 12.

    et al. The t(8;21) translocation in acute myeloid leukemia results in production of an AML1-MTG8 fusion transcript. EMBO J. 12, 2715–2721 (1993)

  13. 13.

    & CBFA2T1, a gene rearranged in human leukemia, is a member of a multigene family. Genomics 52, 332–341 (1998)

  14. 14.

    et al. The tetramer structure of the Nervy homology two domain, NHR2, is critical for AML1/ETO’s activity. Cancer Cell 9, 249–260 (2006)

  15. 15.

    & ETO interacting proteins. Oncogene 23, 4270–4274 (2004)

  16. 16.

    et al. A stable transcription factor complex nucleated by oligomeric AML1-ETO controls leukaemogenesis. Nature 500, 93–97 (2013)

  17. 17.

    et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 49–55 (2008)

  18. 18.

    et al. Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice. Genes Dev. 22, 1617–1635 (2008)

  19. 19.

    , , , & Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44, 23–28 (2006)

  20. 20.

    et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 2281–2308 (2013)

  21. 21.

    et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008)

  22. 22.

    , & Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nature Protocols 9, 1956–1968 (2014)

  23. 23.

    et al. Mtgr1 is a transcriptional corepressor that is required for maintenance of the secretory cell lineage in the small intestine. Mol. Cell. Biol. 25, 9576–9585 (2005)

  24. 24.

    et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013)

  25. 25.

    et al. Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. Genes Dev. 19, 815–826 (2005)

  26. 26.

    et al. Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice. Dev. Biol. 278, 440–458 (2005)

  27. 27.

    et al. Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature 452, 877–881 (2008)

  28. 28.

    et al. ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation. Elife 4, e10150 (2015)

  29. 29.

    , , , & Transient and stable transgene expression in human embryonic stem cells. Stem Cells 25, 1521–1528 (2007)

  30. 30.

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

  31. 31.

    , , & Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3. Mol. Cell 14, 183–193 (2004)

  32. 32.

    , & Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983)

  33. 33.

    , & Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression. Genes Dev . 14, 804–816 (2000)

  34. 34.

    et al. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation. Science 347, 1017–1021 (2015)

  35. 35.

    , & Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols 4, 44–57 (2009)

  36. 36.

    , , , & DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26, 136–138 (2010)

  37. 37.

    et al. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells 27, 543–549 (2009)

  38. 38.

    , , , & Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer. Proc. Natl Acad. Sci. USA 103, 16406–16411 (2006)

  39. 39.

    et al. Chromatin-modifying enzymes as modulators of reprogramming. Nature 483, 598–602 (2012)

  40. 40.

    & Examining histone posttranslational modification patterns by high-resolution mass spectrometry. Methods Enzymol. 512, 3–28 (2012)

  41. 41.

    et al. EpiProfile quantifies histone peptides with modifications by extracting retention time and intensity in high-resolution mass spectra. Mol. Cell. Proteomics 14, 1696–1707 (2015)

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Acknowledgements

We thank P. Andrews and X.-J. Sun for providing plasmids. We are grateful to L. Vales, M. E. Torres-Padilla and L. Bu for critical comments, H. Zheng for MS analysis, and D. Hernandez, C. Leek, M. Yamaji, A. Paradkar and M. Alu for excellent technical assistance. The work was supported by the Howard Hughes Medical Institute (HHMI) and National Institutes of Health (NIH; RO1GM064844-12) (D.R.). B.A.G. acknowledges funding from NIH grant R01GM110174. T.T. was supported by the HHMI, NIH, Starr Foundation, and Tri-Institutional Stem Cell Initiative. M.Y. was a recipient of a Japan Society for the Promotion of Science (JSPS) Research Fellowship.

Author information

Affiliations

  1. Howard Hughes Medical Institute, New York University School of Medicine, New York, New York 10016, USA

    • Shengjiang Tu
    • , Varun Narendra
    • , Luis Alejandro Rojas
    •  & Danny Reinberg
  2. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York 10016, USA

    • Shengjiang Tu
    • , Varun Narendra
    • , Luis Alejandro Rojas
    •  & Danny Reinberg
  3. Howard Hughes Medical Institute, Laboratory for RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA

    • Masashi Yamaji
    •  & Thomas Tuschl
  4. Skirball Institute of Biomolecular Medicine, Department of Cell Biology and Helen L. and Martin S. Kimmel Center for Biology and Medicine, New York University School of Medicine, New York, New York 10016, USA

    • Simon E. Vidal
    •  & Matthias Stadtfeld
  5. Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Xiaoshi Wang
    •  & Benjamin A. Garcia
  6. Rodent Genetic Engineering Core, NYU School of Medicine, New York, New York 10016, USA

    • Sang Yong Kim

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Contributions

S.T. designed and performed majority of experiments; V.N. analysed ChIP-seq and RNA-seq data; S.E.V., S.T. and M.S. performed iPSC reprogramming experiments; X.W. and B.A.G. quantified histone modifications. S.Y.K. did CRISPR zygotic injection. S.T., S.Y.K., M.Y. and L.A.R. characterized mice. M.Y., S.T. and T.T. designed and performed PGC experiments. S.T., V.N., M.Y. and D.R. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Danny Reinberg.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    This table contains PRDM14 and CBFA2T2 target genes identified by ChIP-seq in NCCIT cell.

  2. 2.

    Supplementary Table 2

    This table contains differentially expressed genes in Prdm14 and Cbfa2t2 Knockout lines.

  3. 3.

    Supplementary Table 3

    This table contains qPCR and PCR primers used in ChIP-qPCR and off-target verification.

  4. 4.

    Supplementary Table 4

    This table contains PRDM14 and CBFA2T2 target genes identified by ChIP-seq in mouse ESC.

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figure 1, the uncropped western blot images.

About this article

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DOI

https://doi.org/10.1038/nature18004

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