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Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations

Nature Genetics volume 38, pages 835841 (2006) | Download Citation

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Abstract

Chromatin immunoprecipitation (ChIP) defines the genomic distribution of proteins and their modifications but is limited by the cell numbers required (ideally >107). Here we describe a protocol that uses carrier chromatin and PCR, 'carrier' ChIP (CChIP), to permit analysis of as few as 100 cells. We assayed histone modifications at key regulator genes (such as Nanog, Pou5f1 (also known as Oct4) and Cdx2) by CChIP in mouse embryonic stem (ES) cells and in inner cell mass (ICM) and trophectoderm of cultured blastocysts. Activating and silencing modifications (H4 acetylation and H3K9 methylation) mark active and silent promoters as predicted, and we find close correlation between values derived from CChIP (1,000 ES cells) and conventional ChIP (5 × 107 ES cells). Studies on genes silenced in both ICM and ES cells (Cdx2, Cfc1, Hhex and Nkx2-2, also known as Nkx) show that the intensity of silencing marks is relatively diminished in ES cells, indicating a possible relaxation of some components of silencing on adaptation to culture.

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Acknowledgements

We are grateful to S. Parnell for help with preparation of mRNA from small numbers of cells, to V. Gupta for advice on statistical analyses and to J. Frampton and W. Reik for comments on the manuscript. L.P.O. is a Royal Society Research Fellow and M.D.V. is supported by a PhD studentship from the University of Birmingham Medical School. This work was funded by Cancer Research UK and the Biotechnology and Biological Sciences Research Council (BBSRC).

Author information

Author notes

    • Laura P O'Neill
    •  & Matthew D VerMilyea

    These authors contributed equally to this work.

Affiliations

  1. Chromatin and Gene Expression Group, Institute of Biomedical Research, University of Birmingham Medical School, Birmingham B15 2TT, UK.

    • Laura P O'Neill
    • , Matthew D VerMilyea
    •  & Bryan M Turner

Authors

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Contributions

L.P.O.: validation and development of CChIP technique, study design, planning and carrying out experiments, data analysis, writing paper. M.D.V.: growth and dissection of embryos, carrying out experiments, data analysis, assistance in writing paper. B.M.T.: concept, study design, planning experiments, data analysis, writing paper.

Competing interests

The CChIP procedure described in this paper is the subject of British patent application number 0601538.2 (B.M.T., L.P.O.).

Corresponding author

Correspondence to Bryan M Turner.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Levels of H4K16ac and H3K9me2 on silent genes in ES cells and ICM assayed by CChIP.

  2. 2.

    Supplementary Table 1

    Levels of H4K16 acetylation and H3K9 dimethylation at selected genes and gene regions in ICM and ES cells.

  3. 3.

    Supplementary Table 2

    Primers used for CChIP and expression analysis.

  4. 4.

    Supplementary Note

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DOI

https://doi.org/10.1038/ng1820

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