Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy

We have described procedures for collecting, processing, and analyzing kinetic data obtained by photobleaching microscopy of GFP-tagged chromatin proteins in nuclei of cultured living cells. These procedures are useful for characterizing the in vivo binding of chromatin proteins to their natural template--unperturbed, native chromatin in an intact cell nucleus. These techniques have revealed several generalizations that significantly change our view of the nucleus. At the qualitative level, it has become clear that almost all chromatin proteins bind only transiently to their targets. More importantly, the combined use of in vivo microscopy and kinetic, computational analysis allows analysis of the kinetics of protein binding in vivo. These methods should prove useful in the further in vivo investigation of the molecular mechanisms involved in genome organization and expression.