In vivo formaldehyde cross-linking: it is time for black box analysis

Formaldehyde cross-linking is an important component of many technologies, including chromatin immunoprecipita- tion and chromosome conformation capture. The procedure remains empirical and poorly characterized, however, despite a long history of its use in research. Little is known about the specificity of in vivo cross-linking, its efficiency and chemical adducts induced by the procedure. It is time to search this black box. We think it is urgent to draw attention to the un- certainty introduced in results obtained by ChIP and other formaldehyde fixation-based approaches by the fact the cross-linking efficiency of various proteins to DNA and to each other is drastically different and, in the case of in vivo cross-linking, may depend on local conditions within different cellular compartments. Current chromatin research is characterized by the fast accumulation of genome-wide data on the distribution of various regulatory proteins along chromosomes. These data are easily accessible through different databases, and much effort has been made to pour more and more data into the pot. Surprisingly, however, not many scientists are concerned about the validity of the chromatin immunoprecipitation (ChIP) approach. The ChIP procedure was developed >15 years ago (1) and, essentially, the original protocol is still used without paying much attention to its inherent problems, even though it has long been felt that 'the devil is in the ChIP details'. The most problematic step is formal- dehyde fixation. It is commonly believed that for- maldehyde can fix any DNA-protein complex. However, this assumption is far from being