Introduction

The study of transcriptional processes in higher eukaryotes has been limited by the scarce availability of in vivo assays. This shortage of technical approaches has become more important in light of the emerging notion that the natural chromatin context affects the outcome of transcriptional activation in vivo (1). Chromatin can exert a regulatory effect on transcription by modulating the access of activators to DNA (1). Different posttranslational modifications of specific residues in the N-terminal tails of the nucleosomal histones have been characterized as a signal code that is linked to the active or inactive transcriptional status of promoters (2). These modifications, including acetyla-tion, methylation, phosphorylation, and ubiquitination, are thought to result from the targeted binding to promoters of transcriptional coactivator or core-pressor complexes containing such enzymatic activities. These complexes do not generally possess DNA-binding activity, but are recruited to promoters by their interaction with sequence-specific transcription factors (3). The resulting modifications of the histone tails, in turn, modulate the access of further regulatory complexes to the promoter (4). This subtle regulatory interplay can easily be missed when transcriptional activation or transcription factor binding to target promoters is studied in vitro using naked DNA templates.

In this respect, chromatin immunoprecipitation (ChIP) assays are being quickly established as a powerful method to examine the access of nuclear proteins to their target promoters in the natural chromatin environment, as well as to analyze the posttranslational modifications of the histones that conform the nucleosomes spanning promoters of interest (5-10). This procedure was initially developed for use in Drosophila and yeast, starting with relatively large quantities of cells, by which reason its usefulness in animal tissues from which only limited amounts of cells are available was compromised (5-7). We and others have recently developed small-scale ChIP assays for the study of histone acetylation and transcription factor binding in animal tissues such as mouse pancreatic islets or hepatocytes (11-13).

An outline of the method is provided in Fig. 1. The ChIP procedure rests on the ability of formaldehyde to reversibly crosslink amino and imino groups of both amino acids and DNA that are found within a maximal distance of 2 A from each other (7). This short range of action ensures that the protein-protein or protein-DNA crosslinks generated by these means represent intimate interactions. Furthermore, as its action is immediate, formaldehyde crosslinking provides a snapshot of interactions in the cell at a determined time-point (14). Using specific antibodies to precipitate the protein of interest, either transcription factor or histone, we can pull with it the DNA sequences to which this protein is bound. To this end, DNA should be previously fragmented into small pieces by some methodology (usually sonication) producing random breaks, so that the selected DNA really represents the sequences found in the immediate vicinity of the immunoprecipitated protein. As formaldehyde-generated crosslinks are easily reversible, immunoselected DNA can be separated from the proteins and purified. The immunoprecipitated DNA should be significantly enriched in those sequences associated with the protein of interest. The enrichment of specific DNA sequences in the immunoprecipitate can be easily analyzed by semiquantitative polymerase chain reaction (PCR) using primers recognizing a region of interest.

Some critical points of the ChIP assay are the optimization of the extent of fixation, the sonication of the chromatin sample to obtain adequately sized DNA fragments, and the availability of an antibody that will recognize and immunoprecipitate its epitope when proteins are fixed and partially denatured in formaldehyde. The fixation step needs to be optimized for each new antibody tested by performing a time-course experiment. Overfixation results in the samples becoming refractory to sonication and, consequently, loss of material during centrifugation steps. Excessive fixation could also result in masking of the epitopes and inefficient immunoprecipitation. The optimal size of the sonicated DNA for use in ChIP experiments ranges from 0.4 to 2 kb. Obtaining adequately sized DNA fragments by sonication is critical for the success of the experiment and the interpretation of the results. A ChIP experiment performed with excessively long DNA fragments will result in the selection of DNA sequences that may not be in the immediate vicinity of the protein precipitated. This issue

Islet isolation Formaldehyde fixation

SDS lysis Chromatin sonication

Immunoprecipitation

Reversal of DNA-protein cross-links

Sample analysis (PCR)

Fig. 1. Schematic outline of the ChIP procedure. Islets are fixed right after isolation by incubating in 1% formaldehyde and then lysated in sodium dodecyl sulfate (SDS) buffer. Chromatin is sonicated to obtain small-sized fragments. Sonicated chromatin is immunoprecipitated with an antibody raised against a DNA-binding protein of interest, so that the DNA sequences to which this protein is bound are selected. The presence of a particular sequence in the selected DNA is detected by polymerase chain reaction (PCR).

becomes crucial if high-resolution fine mapping is intended. The availability of a suitable antibody can also represent a crucial factor. When testing a new antibody, pilot immunoprecipitation experiments using unfixed nuclear extracts followed by Western blot analysis can indicate if the antibody is active in the conditions used for immunoprecipitation in the ChIP assay. On the other hand, immunocytochemical experiments can provide indications regarding the ability of an antibody to recognize its epitope in fixed material. Finally, one of the most critical factors for the success of a ChIP experiment is the availability of a robust readout assay to unequivocally determine the existence of enrichment of specific DNA segments in the immunoprecipitate.

The purpose of this chapter is to provide specific guidelines to carry out ChIP experiments with isolated pancreatic islets of Langerhans. The proce-

Fig. 1. Schematic outline of the ChIP procedure. Islets are fixed right after isolation by incubating in 1% formaldehyde and then lysated in sodium dodecyl sulfate (SDS) buffer. Chromatin is sonicated to obtain small-sized fragments. Sonicated chromatin is immunoprecipitated with an antibody raised against a DNA-binding protein of interest, so that the DNA sequences to which this protein is bound are selected. The presence of a particular sequence in the selected DNA is detected by polymerase chain reaction (PCR).

dure is based on several established ChIP protocols (6,7,9,13), but specific variables have been systematically tested to optimize the assay for this particular tissue.

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