A PHOSPHATASE INHIBITOR ENHANCES THE DNASE-I SENSITIVITY OF ACTIVE CHROMATIN

Although it is well-known that active domains of chromatin have elevated DNase I sensitivity, it can be difficult to observe preferential sensitivity in many cell types. We show that the DNase I sensitivity of active chromatin is enhanced some 10-fold by treating nuclei with the phosphatase inhibitor p-(chloromercuri)benzenesulfonic acid (CMBS) whereas DNase I sensitivity in inactive domains is only 3-fold higher. We further show that CMBS-enhanced DNase I sensitivity is associated with at least two histone modifications. First, the negatively charged CMBS molecule becomes covalently attached to the thiol groups on histone H-3. Second, histone H2A phosphorylation is significantly elevated in treated nuclei. The phosphorylation data along with other results point to the possibility that H2A phosphorylaation plays a role in enhancing preferential DNase I sensitivity. Whatever the mechanism, CMBS treatment of nuclei followed by DNase I digestion provides a novel and reproducible assay for probing the chromatin structure of active domains.