Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Summary

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells ^1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.

We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.

In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.5^4,5. Myogenin is a regulatory factor required for skeletal muscle differentiation ^6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development ^6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

Protocol

1. Isolation of Embryos

Note: All operations involving mice should be performed in accordance with the appropriate animal care and usage policies and protocols

1. Check for the presence of a mating plug in the female mouse the morning after mating and separate the mated females from the stud males by placing them in a different cage. Noon of the day that the mating plug is observed is considered embryonic day 0.5 (E0.5) of development.
2. At E8.5, (or the desired stage, if different), sacrifice the mouse using an approved protocol.
3. Wet the belly of the euthanized animal with 70% ethanol and open t....

Discussion

In the described ChIP protocol, we show that the myogenic regulator myogenin is associated with the myogenin promoter in skeletal muscle precursor tissue present in single E8.5 and E9.5 embryos. Prior studies have extensively characterized myogenin binding to E box containing sequences, beginning with the initial in vitro gel shift experiments utilizing in vitro translated or bacterially produced myogenin and radiolabeled DNA encoding the relevant portion of target gene regulatory sequences ^11-20

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ChIP Assay Kit	Upstate, Millipore	17-295
Collagenase Type II	Invitrogen	17101015	Dilution by 1 x PBS
Dulbecco’s modified eagle medium (DMEM)	GIBCO, by Life Technologies	12100-061	High glucose content
Dulbecco’s phosphate buffered saline 1X (DPBS)	GIBCO, by Life Technologies	14190-144	Calcium chloride free, Magnesium chloride free
Fetal bovine serum (FBS)	Mediatech, Inc.	35-010-CV
Gel extraction kit	QIAquick	28704	50 reaction kit
Penicillin/streptomycin stock solution	GIBCO, by Life Technologies		5000 μg/ml concentration
Protease Inhibitor Cocktail	Sigma-Aldrich	P8340
Salmon sperm DNA /Protein A agarose	EMD Millipore	16-157
myogenin antibody	Santa Cruz Biotechnology, Inc.	sc-576
Normal rabbit IgG	EMD Millipore	12-370
Platinum PCR Supermix	Invitrogen	11306-016
GoTaq Q-PCR master mix	Promega Corp.	A6001