RECOVERY OF TRANSCRIPTIONALLY ACTIVE CHROMATIN RESTRICTION FRAGMENTS BY BINDING TO ORGANOMERCURIAL-AGAROSE MAGNETIC BEADS - A RAPID AND SENSITIVE METHOD FOR MONITORING CHANGES IN HIGHER-ORDER CHROMATIN STRUCTURE DURING GENE ACTIVATION AND REPRESSION

The unfolding of nucleosomes along transcriptionally active DNA sequen ces uncovers previously shielded cysteinyl-thiol groups of histone H3 molecules located at the center of the nucleosome core. This change in conformation and SH reactivity of nucleosomes along transcribed DNA s equences makes it possible to separate active from inactive nucleosome s by mercury affinity chromatography. The binding of thiol-reactive nu cleosomes to an organomercurial-agarose column has been shown previous ly to reflect, with accuracy, both the timing and extent of transcript ion of the associated DNA sequences (Chen, T. A., and Allfrey, V. G. ( 1987) Proc. Natl. Acad. Sci. U. S. A. 84,5252-5256). Here, we extend t his experimental approach to the analysis of higher order chromatin st ructures. Large chromatin fragments released by treating isolated nucl ei with restriction endonucleases are fractionated on mercurated agaro se magnetic beads that capture nucleosomes with accessible histone H3 thiols, but do not react with the hidden H3 thiols of the compactly be aded nucleosomes of inactive genes. The SH-reactive domains of c-myc a nd other genes are rapidly separated from the non-SH-reactive restrict ion fragments by the magnetic bead technique. The new method also over comes a major limitation of mercurated agarose column chromatography, which is not suitable for studies of higher order chromatin structure because large chromatin fragments occlude the mercury column; occlusio n is not a problem in magnetic separations using suspended mercurated agarose beads. Here, we describe the synthesis of mercurated agarose m agnetic beads with high capacity for SH groups and test their applicat ion to the recovery of chromatin restriction fragments of c-myc and th e growth arrest gene gas1.