Finding Downstream Target DNAs for Chromatin Proteins by X-CHIP in Drosophila

Excerpt

Formaldehyde cross-linking followed by chromatin immunoprecipitation (X-ChIP) allows mapping of protein--DNA interactions through the ability of formaldehyde to cross-link proteins and nucleic acids in living cells. Formaldehyde is a very reactive dipolar compound that reacts with the amino groups of proteins and amino acids (1, 2). It shows no reactivity, however, toward free double-stranded DNA and thus does not cause the extensive DNA damage seen after prolonged exposure to other cross-linking reagents such as UV. Each formaldehyde molecule has the capacity to interact with two amino groups. Therefore, DNA--protein, protein--protein, and RNA--protein cross-links are rapidly formed after formaldehyde treatment, creating a stable structure that prevents the redistribution of cellular components. Furthermore, a simple heat treatment is sufficient to reverse the reaction equilibrium and to allow isolation of pure DNA for further analysis (3). Formaldehyde cross-linking combined with chromatin immunoprecipitation (IP) is a way of mapping the in vivo distribution of chromatin-associated proteins. As such, this technique is of great value in the analysis of protein--DNA interactions, even more so when studying proteins that do not show specific DNA binding activities in vitro. This has recently been demonstrated for several chromatin-associated proteins from yeast to vertebrates (4–8). Additionally, analysis of the cross-linking pattern not only allows mapping of sites of protein--DNA interaction but can also give an estimation of the relative binding affinity to different sequences across a large genomic region.

In our laboratory, we are applying this technique in a novel approach aimed at the identification of downstream targets for regulatory proteins. Formaldehyde cross-linking coupled to the use of genomic microarrays has already been successfully applied to identify protein targets in the yeast cell nucleus (9). In our approach, we are collaborating with the labs of Florence Maschat, Franck Girard, and Conchita Ferraz at the Institut de Génétique Humaine to directly clone and characterize the immunopreciptiated DNAs to identify targets for several chromatin-associated proteins.