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.
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 E8.5-9.54,5. 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.
1. Isolation of Embryos
Note: All operations involving mice should be performed in accordance with the appropriate animal care and usage policies and protocols
2. Homogenization of the Isolated Embryo
3. Cross-link Chromatin
4. Sonication
5. Pre-clearing and Immunoprecipitation
6. Washing the Chromatin-protein-bead Complex
Buffer 1:Low salt wash buffer (20 mM Tris-HCl (pH 8.1), 2 mM EDTA, 1% Triton X-100,
150 mM NaCl and 0.1% SDS), 1 ml x 2 washes.
Buffer 2:High salt wash buffer (20 mM Tris-HCl (pH 8.1), 2 mM EDTA, 1% Triton X-100,
500 mM NaCl and 0.1% SDS), 1 ml x 2 washes.
Buffer 3:LiCl salt wash buffer (10 mM Tris-HCl (pH 8.1), 1 mM EDTA, 1% IGEPAL-CA630,
0.25 M LiCl and 1% deoxycholic acid (sodium salt)), 1 ml x 1 wash.
Buffer 4:TE buffer (10 mM Tris-HCl (pH 8.1), 1 mM EDTA), 1 ml x 2 washes.
7. Elution of the Chromatin-antibody Complex
8. Reverse Cross-link and Recover DNA
9. Analysis of Recovered DNA
10. Representative Results
We have used this protocol to perform ChIP from both E8.5 and E9.5 embryos (Figure 2). The results demonstrate that myogenin is present on the myogenin promoter in E8.5 and E9.5 embryos. ChIP purified DNAs were analyzed by conventional PCR (Figure 2A) and by quantitative real-time PCR (Figure 2B). In contrast, there was no indication of myogenin binding to the myogenin promoter in the yolk sac, where myogenin is not expressed. The interferon-γ (IFNγ) promoter, which contains sequences matching the myogenin binding site, was used as a negative sequence control. As expected, myogenin was not bound to the IFNγ promoter in any of the tissue samples tested. In E8.5 and 9.5 embryos, myogenin is specifically expressed in the somites (Figure 3; 6,10), which are the precursors to skeletal muscle. Thus the results indicate that myogenin is bound to the myogenin promoter in the somites at E8.5 and E9.5.
Figure 1. E8.5 embryo dissection. (A) Isolated uterine horn (top panel; higher magnification shown in lower panel). (B) Uterine horn cut with scissors to separate individual implantation sites containing individual embryos. (C) Embryos protruding from uterine tissue during dissection. Arrows mark embryos still covered by extra-embryonic tissue. (D) Two E8.5 embryos from the same litter. The embryo on the left has not begun the process of turning. The embryo on the right is undergoing turning. Far right – representative E9.5 embryo.
Figure 2. ChIP assay demonstrating myogenin binding to the myogenin promoter in E8.5 and E9.5 embryos. (Top) Conventional PCR analysis of 5 ul of DNA purified from ChIP experiments using a myogenin antibody or non-specific IgG was performed with primers that amplify a portion of the myogenin promoter from -79 to +69 relative to the start site of transcription that contains a myogenin binding site located at -12 or primers that amplify a portion of the IFNγ promoter that contains a sequence matching the myogenin binding site located ~ 1075 bp upstream of the transcription start site. The IFNγ primer sequences used were 5′-GCT GAC TCA AGA CCC CGA GGC-3′ and 5′-TGA GGA TGG GGC AGG AGG CC-3′. (Bottom) Quantitative Real-time PCR analysis of the same samples used in (A). The data are plotted as % of input +/- standard deviation.
Figure 3. Myogenin is specifically expressed in the somites of E8.5 and E9.5 embryos. Whole mount in situ hybridization of myogenin shows specific mRNA expression in the somites. Size bar in E8.5 image – 200 μm. Size bar in E9.5 image – 500 μm.
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. Conventional ChIP studies have demonstrated myogenin binding to the myogenin promoter in tissue culture models for myogenesis 21-24. However, there is no evidence that demonstrates that myogenin binds to the myogenin promoter during embryonic skeletal muscle development, though one might predict this to be the case later in embryogenesis based on the down regulation of myogenin expression observed in E15.5 tongue tissue from myogenin deficient mice 25. Thus the data provides evidence that the interaction between myogenin and the myogenin promoter interaction occurs in vivo. An obvious application of this technique is to use it to verify the physiological relevance of prior studies characterizing tissue-specific gene activation tissue performed using culture models. The more interesting application is to use this technique as part of the initial characterization of a new or previously uncharacterized factor to determine the physiological relevance of a specific interaction prior to performing tissue culture studies designed at understanding functional mechanisms.The protocol can also be used to directly compare protein:chromatin interactions occurring at specific developmental stages in mouse models containing an engineered genetic manipulation.
We anticipate that this method will also be useful for time course studies of tissue-specific gene regulation during development. By assessing specific factor:chromatin interactions at different embryonic stages, one could identify the time point at which the factor interaction first occurs, could determine whether the interaction was maintained throughout and beyond the differentiation process or was more transient in nature, and could determine the order of factor binding if multiple factors interact with a specific sequence. Finally, we envision that the protocol could be extended to a re-ChIP procedure 26 whereby the chromatin recovered from one immunoprecipitation is subjected to a second immunoprecipitation with an antibody against a different factor believed to be co-occupying the same piece of chromatin. The re-ChIP protocol provides more conclusive evidence that two distinct factors are present on the same pieces of chromatin; the likely requirement for this modification would be an increase in the amount of starting material.
A remarkable realization that came from our efforts was that the limited amount of starting material was not the impediment to success that one might have predicted, especially given that tissue culture cell based protocols often suggest starting with a homogeneous population of millions or tens of millions of cells 26,27. The success of our efforts speaks to the primary (and often uncontrollable) requirement for antibodies capable of specific immunoprecipitation of the factor under investigation. Once an antibody is identified as capable of immunoprecipitation, the investigator can optimize the ChIP assay by titrating the amount of antibody used. It is important to note that more is not always better; we frequently find that too much antibody, especially with samples of limited quantity, results in higher background binding to irrelevant sequences. In addition, use of immunoglobulin fractions or affinity-purified antibodies often result in lower background than does use of antisera. Background from the control antibody can often be gauged by including a sample containing beads without any antibody.
We conclude that using early stage embryos for ChIP analysis of protein:chromatin interactions that occur during the activation of tissue-specific genes has the potential to provide significant insight into the induction and maintenance of tissue-specific gene expression programs directly within the context of development.
The authors have nothing to disclose.
This work was supported by NIH R01 GM56244 to ANI, which includes funds awarded through the American Recovery and Reinvestment Act of 2009, and by NIH R01 GM87130 to JARP
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
ChIP Assay Kit | Upstate Cell Signaling Solutions, Millipore | 17-295 | ||
Collagenase Type II | Invitrogen | 17101015 | Dilution by 1 x PBS | |
Dulbecco’s modified eagle medium (DMEM) | Gibco Labs, Invitrogen | 12100-061 | High glucose content | |
Dulbecco’s phosphate buffered saline 1X (DPBS) | Gibco Labs, Invitrogen | 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 Labs, Invitrogen | 5000 μg/ml concentration | ||
Protease Inhibitor Cocktail | Sigma-Aldrich | P8340 | ||
Salmon sperm DNA /Protein A agarose | Millipore | 16-157 | ||
myogenin antibody | Santa Cruz Biotechnology, Inc. | sc-576 | ||
Normal rabbit IgG | Millipore | 12-370 | ||
Platinum PCR Supermix | Invitrogen | 11306-016 | ||
GoTaq Q-PCR master mix | Promega | A6001 |