Formaldehyde cross-linking for studying nucleosomal dynamics

Methods are described for the utilization of formaldehyde as a reversible c ross-linking agent for the characterization of protein-protein and protein- DNA interactions. The methods include a description of procedures to: (1) i solate and characterize transcriptionally active chromatin from cells cross -linked with formaldehyde; (2) study histone mobility during replication an d transcription by the characterization of the formaldehyde-cross-linked hi stone octamer that is isolated from cells labeled with density-labeled amin o acids; and (3) cross-link the in vitro reconstituted histone-DNA complex in order to maintain its structural state during subsequent characterizatio n. Included in these methods are procedures for a second dimensional analys is of protein-protein cross-links in which the monomer components are elect rophoretically resolved in the second dimension. The methods also include p rocedures to selectively reverse protein-DNA crosslinks while maintaining t he protein-protein cross-links. Potential artifacts are also discussed; i.e ., data are presented which indicate that the helical pitch of DNA can be a ltered if the ionic strength is not properly controlled, The stability of t he cross-linked nucleosome in the presence of altered pH or salt/urea conce ntrations is described in order to indicate that there are limitations to p rocedures that can be used for the subsequent characterization of the cross -linked complex, (C) 1999 Academic Press.