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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 835–841 (2006)Cite this article

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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Figure 1: Quantitation by conventional NChIP of levels of histone modification (bound/unbound ratios) at selected regions of Nanog and the promoter regions of Cdx2 and Gapdh in undifferentiated ES cells (light gray columns), or embryoid bodies formed after 7 d differentiation in culture (black columns).
Figure 2: Outline and initial validation of the CChIP protocol.
Figure 3: Quantitation by CChIP of levels of histone modification (bound/unbound ratio) at selected regions of Nanog and the promoter regions of other genes.
Figure 4: Analysis of histone modifications on selected genes in ICM and trophectoderm by CChIP.

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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

  1. Laura P O'Neill and Matthew D VerMilyea: These authors contributed equally to this work.

Authors and 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

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  1. Laura P O'Neill
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  2. Matthew D VerMilyea
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  3. Bryan M Turner
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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.

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Correspondence to Bryan M Turner.

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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.).

Supplementary information

Supplementary Fig. 1

Levels of H4K16ac and H3K9me2 on silent genes in ES cells and ICM assayed by CChIP. (PDF 12 kb)

Supplementary Table 1

Levels of H4K16 acetylation and H3K9 dimethylation at selected genes and gene regions in ICM and ES cells. (PDF 11 kb)

Supplementary Table 2

Primers used for CChIP and expression analysis. (PDF 11 kb)

Supplementary Note (PDF 16 kb)

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O'Neill, L., VerMilyea, M. & Turner, B. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat Genet 38, 835–841 (2006). https://doi.org/10.1038/ng1820

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