An ambitious EC-funded research initiative on epigenetics advancing towards systems biology 142

Chromatin-Immunoprecipitation (ChIP)

Antonio Porro and Giovanni Perini

Introduction

Cross-linking Chromatin Immunoprecipitation (ChIP) has become a popular method to detect the in vivo binding of proteins to DNA. In general the protein/DNA complex is fixed with formaldehyde, a cross-linking agent that, because of its short spacer arm (about 2 A), generates reversible covalent links, mainly between proteins and DNA. Subsequently, chromatin is fragmented by sonication and DNA protein complexes are selectively immunoprecipitated with antibodies raised against proteins of interest. Covalent links are reversed and proteins are removed from DNA through phenol/chloroform extraction. Following DNA purification, specific DNA regions supposed to be engaged with proteins of interest are analyzed by semi-quantitative PCR or real time PCR. Although very powerful, this method may present some limits when applied to nuclear proteins that, although being tightly associated with chromatin DNA, are not directly bound to it. For example, recent studies have shown that c-Myc can repress transcription of the p21CIP/WAF gene without binding DNA directly but through interaction with different transcription factors such as Sp1 and Miz-1 that recognize specific DNA sequences in the p21CIP/WAF promoter (Gartel et al., 2001 and Wu e al., 2003).  […]

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Antonio Porro and Giovanni Perini

University of Bologna – Department of Biology – University of Bologna – Via Francesco Selmi 3 – 40126 Bologna, Italy

Haico van Attikum & Jennifer Cobb

Introduction

ChIP (chromatin immunoprecipitation) is a powerful tool that allows one to determine whether and where a protein or protein modification is associated with chromatin in vivo. The technique starts with a formaldehyde treatment of cells to crosslink protein-protein and protein-DNA complexes. After cross-linking, the cells are lysed and crude extracts are sonicated to shear the DNA. Proteins together with crosslinked DNA are immunoprecipitated. Protein-DNA crosslinks in the immunoprecipitate and input (non-immunoprecipitated whole cell extract) are then reversed and the DNA fragments are purified. Real-time quantitative PCR can then be used to amplify the region where either a protein or protein modification is present. DNA fragments of this genomic locus should be enriched in the immunoprecipitate compared to that in the input (which represents all portions of the genome). This protocol has been successfully used in the Gasser lab to study proteins at replication forks and chromosomal DNA double-strand breaks (DSBs) in budding yeast (Saccharomyces cerevisiae) (Cobb et al., 2003; van Attikum et al., 2004).

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Haico van Attikum & Jennifer Cobb

Friedrich Miescher Institute for Biomedical Research – Maulbeerstrasse 66 – 4058 Basel, Switzerland

Laura O’Neill & Bryan Turner

Introduction

Chromatin immunoprecipitation (ChIP) arguably represents the most powerful application of antibody technology to epigenetic research. It allows analysis of patterns of histone modification and non-histone protein distribution across genomic regions and underpins large scale epigenetic mapping projects. However, conventional ChIP generally requires at least 107 cells, which limits its applicability. To address this, we have developed a new protocol, CChIP, based on the use of carrier chromatin, that allows detailed and reproducible epigenetic analysis of as few as 100 cells. The procedure has been validated with primary mouse embryo material, but should be applicable to cells from various sources, including tissue biopsies and FACS-sorted cell populations. The protocol given here is for analysis of histone modifications in native, unfixed chromatin prepared by micrococcal nuclease digestion. CChIP will undoubtedly be applied to formaldehyde cross-linked chromatin, and thereby used to locate non-histone chromatin proteins, but the generally lower efficiency of precipitation with cross-linked chromatin is likely to increase the numbers of cell required.

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Laura O’Neill & Bryan Turner

University of Birmingham – Institute of Biomedical Research – The Medical School – Birmingham – B15 2TT, UK

David Umlauf, Yuji Goto, Katia Delaval, Alexandre Wagschal, Philippe Arnaud & Robert Feil

Introduction

In cells and tissues, the histone proteins that constitute the nucleosomes can present multiple post-translational modifications (Luger & Richmond, 1998), such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, and lysine ubiquitination. On their own, or in combination, these covalent modifications on the core histones are thought to play essential roles in chromatin organisation and gene expression in eukaryotes (Hebbes et al., 1994; O'Neill & Turner, 1995; Grunstein, 1998; Turner, 2000; Jenuwein & Allis, 2001). Importantly, patterns of histone modifications may be somatically conserved and can, thereby, maintain locus-specific repression/activity in defined lineages, or throughout development. Indirect immuno-fluorescence studies on cultured cells have been pivotal in unravelling the roles of histone modifications. These studies have been highly informative on the functions of specific histone modifications in, for instance, pericentric chromatin condensation (Peters, et al., 2001; Maison, et al., 2002) and X-chromosome inactivation (Heard et al., 2001, Boggs et al., 2002, Peters et al., 2002) in mammals (H3 and H4 deacetylation, and H3-K9 methylation). However, particularly in mammalian model systems, it remains poorly understood how histone modifications are organised at specific chromosomal regions and genes. To address in detail what happens at specific sites in vivo, chromatin immuno-precipitation (ChIP) is the method of choice. Here, we describe how ChIP can be performed on native chromatin extracted from cells, or tissues, to analyse histone methylation and acetylation at specific chromosomal sites. In addition, we present different PCR-based methods that allow the analysis of a locus of interest in chromatin precipitated with antibodies to specific histone marks. Should you require a literature reference, please, quote an earlier paper by our group, where these methodologies were originally described (Umlauf et al., 2003).

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David Umlauf, Yuji Goto, Katia Delaval, Alexandre Wagschal, Philippe Arnaud & Robert Feil

Institute of Molecular Genetics – CNRS UMR-5535 – University of Montpellier II – 1919, route de Mende – 34293 Montpellier cedex 5, France

Werner Aufsatz

Introduction

Eukaryotic chromatin is a complex of DNA and associated histone proteins which are involved in the higher order packaging of DNA into chromosomes. The chromatin state of a given DNA sequence influences transcriptional activity and replication timing and is regulated by potentially reversible covalent modifications of DNA and histones. Histone modifications at conserved lysine and arginine residues within the flexible N-terminal tails, such as phosphorylation, acetylation and methylation, specify a code which serves as an interaction platform with specific domains of chromatin-associated proteins. The immunoprecipitation (IP) of crosslinked chromatin with antibodies specific for certain histone modifications (chromatin immunoprecipitation; ChIP), followed by PCR to detect a potential enrichment or depletion of a DNA sequence of interest within IP fractions, constitutes an elegant and direct method to query specific chromatin states of individual genes and is already routinely used in many labs. In contrast to animal cells, however, plant cells have a rigid cell wall which poses limitations to the simple utilization of protocols established for animals. In this protocol, I describe the method used in our laboratory to study histone modifications in the plant model organism Arabidopsis thaliana. This protocol is an adapted version of the original procedure published by Lawrence and co-workers (Lawrence et al., 2004).

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Werner Aufsatz

Gregor Mendel Institute of Molecular Plant Biology – Austrian Academy of Sciences – Dr. Bohrgasse 3 – A-1030 Vienna, Austria

Email feedback to: Werner.Aufsatz@gmi.oeaw.ac.at