Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations.
Chromatin immunoprecipitation (ChIP) defines the genomic distribution of proteins and their modifications but is limited by the cell numbers required (ideally >10(7)). 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 x 10(7) 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.
O'Neill
Laura P
LP
Chromatin and Gene Expression Group, Institute of Biomedical Research, University of Birmingham Medical School, Birmingham B15 2TT, UK.
VerMilyea
Matthew D
MD
Turner
Bryan M
BM
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D002452:Cell Count; D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D044127:Epigenesis, Genetic; D020868:Gene Silencing; D005809:Genes, Regulator; D006657:Histones / Q000378:metabolism; D000090322:Homeobox Protein Nkx-2.2; D051379:Mice; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism
D006657:Histones; D000090322:Homeobox Protein Nkx-2.2; C000715627:Nkx2-2 protein, mouse