Formaldehyde Cross-Linking for Studying Nucleosomal Dynamics

Abstract

Methods are described for the utilization of formaldehyde as a reversible cross-linking agent for the characterization of protein–protein and protein–DNA interactions. The methods include a description of procedures to: (1) isolate and characterize transcriptionally active chromatin from cells cross-linked with formaldehyde; (2) study histone mobility during replication and transcription by the characterization of the formaldehyde-cross-linked histone octamer that is isolated from cells labeled with density-labeled amino acids; and (3) cross-link thein vitroreconstituted histone–DNA complex in order to maintain its structural state during subsequent characterization. Included in these methods are procedures for a second dimensional analysis of protein–protein cross-links in which the monomer components are electrophoretically resolved in the second dimension. The methods also include procedures to selectively reverse protein–DNA cross-links while maintaining the protein–protein cross-links. Potential artifacts are also discussed; i.e., data are presented which indicate that the helical pitch of DNA can be altered if the ionic strength is not properly controlled. The stability of the cross-linked nucleosome in the presence of altered pH or salt/urea concentrations is described in order to indicate that there are limitations to procedures that can be used for the subsequent characterization of the cross-linked complex.