In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation.

The chromatin immunoprecipitation (ChIP) assay (1) is a powerful technique to analyze protein-DNA interactions in a native chromatin context (2–4). ChIP is used to study a broad range of physiological processes including transcriptional regulation, DNA replication, and DNA repair. The assay is useful for a wide range of cells and tissues, including cells derived from yeasts, protozoa, Drosophila, and mammals. The ChIP assay typically comprises four steps: (i) cross-linking proteins to DNA; (ii) chromatin fragmentation; (iii) protein precipitation; and (iv) quantitation (5). The initial crosslinking is used to covalently attach proteins within chromatin, to insure that DNA-protein complexes remain associated through the following steps. The DNA is broken into sizes ranging from approximately 500 bp (achieved by sonication) down to mononucleosomes (achieved by MNase digestion). The DNA is then precipitated, commonly with an antibody to detect the protein of interest, but other means are also used, such as nickel-conjugated beads to precipitate a hexahistidine-tagged protein. The cross-links are then reversed to allow quantitation, using Southern blotting techniques, which directly measure the precipitated DNA, or by PCR, which is either semiquantitative (PCR followed by electrophoresis) or quantitative (PCR in real-time). The most commonly used crosslinking reagent is formaldehyde (HCHO), because it is heat-reversible and is able to cross-link protein-DNA, protein-RNA, and protein-protein. Formaldehyde generates cross-links spanning approximately 2 Å (although commercially available formaldehyde is polymerized, and, thus the actual cross-linking distance is unknown) (1) and is thus most useful to examine proteins directly bound to DNA, such as transcription factors, histones, and their covalent modifications (2–4). However, formaldehyde has a short cross-linking spacer arm and frequently is not effective to examine proteins that are indirectly associated with DNA, such as transcriptional coactivators and corepressors. Thus, it would be generally useful to identify a versatile cross-linking reagent that efficiently reveals proteins more distantly bound to DNA. There have been recent reports of ChIP studies of indirectly bound cofactors using formaldehyde combined with a second cross-linking reagent bearing a longer spacer arm. The binding of the histone deacetylase Rpd3 in the yeast Saccharomyces cerevisiae was mapped using dimethyl adipimidate (DMA) (Figure 1) and formaldehyde (6,7). A second approach using dimethyl 3,3′-dithiobispropionimidate (DTBP) plus formaldehyde In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation