采用适用于小细胞群体的染色质免疫沉淀方案对早期胚胎进行表观遗传学特征分析。

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

作者信息

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

机构信息

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

出版信息

Nat Genet. 2006 Jul;38(7):835-41. doi: 10.1038/ng1820. Epub 2006 Jun 11.

DOI:10.1038/ng1820

PMID:16767102

Abstract

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.

摘要

染色质免疫沉淀（ChIP）可确定蛋白质及其修饰在基因组中的分布，但受所需细胞数量的限制（理想情况下>10^7）。在此，我们描述了一种使用载体染色质和PCR的方法，即“载体”ChIP（CChIP），可用于分析少至100个细胞。我们通过CChIP在小鼠胚胎干细胞、培养囊胚的内细胞团（ICM）和滋养外胚层中检测了关键调控基因（如Nanog、Pou5f1（也称为Oct4）和Cdx2）上的组蛋白修饰。如预期的那样，激活和沉默修饰（H4乙酰化和H3K9甲基化）分别标记了活跃和沉默的启动子，并且我们发现源自CChIP（1000个胚胎干细胞）和传统ChIP（5×10^7个胚胎干细胞）的值之间具有密切相关性。对在ICM和胚胎干细胞中均沉默的基因（Cdx2、Cfc1、Hhex和Nkx2 - 2，也称为Nkx）的研究表明，沉默标记的强度在胚胎干细胞中相对减弱，这表明在适应培养过程中，某些沉默成分可能有所松弛。