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 H3. Second, histone H2A phosphorylation is significantly elevated in treated nuclei. The phosphorylation data along with other results point to the possibility that H2A phosphorylation 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.
Feng
J L
JL
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-2007.
Irving
J
J
Villeponteau
B
B
D002731:4-Chloromercuribenzenesulfonate / Q000737:chemistry; D000199:Actins / Q000737:chemistry; D001345:Autoradiography; D015139:Blotting, Southern; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D003850:Deoxyribonuclease I / Q000378:metabolism*; D006657:Histones / Q000737:chemistry; D006868:Hydrolysis; D009693:Nucleic Acid Hybridization; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D013438:Sulfhydryl Compounds
D000199:Actins; D002843:Chromatin; D006657:Histones; D013438:Sulfhydryl Compounds; D002731:4-Chloromercuribenzenesulfonate; D004247:DNA; D003850:Deoxyribonuclease I; D010744:Phosphoric Monoester Hydrolases