Quantitative determination of histone modification. H2A acetylation and phosphorylation.

Each variant of histone 2A from mouse L1210 cells separates into at lest three bands on acid-urea gels, an unmodified band b0, and modified bands b1, b2, and, in the case of H2A.Z, b3. By analyzing tryptic peptides from these proteins on 50% acrylamide peptide gels, one can quantitate the fraction of H2A modified in various ways. With this methodology, the b1 form of H2A.1 was found to be a mixture of molecules, two-thirds of which were acetylated at lysine5 and one-third of which were phosphorylated at serine1. The b2 band of H2A.1 contained molecules with phosphorylated serine1, acetylated lysine5, and one or two other sites of modification not found in b1 H2A.1 b2 H2A.1 was not composed solely of molecules each with both a phosphorylated serine1 and an acetylated lysine5. The heteromorphous H2A variant H2A.X can be phosphorylated and acetylated. The phosphorylation site was the same as in H2A.1, but the acetylation site was slightly different. The heteromorphous variant H2A.Z was different from the other H2As in that it was not phosphorylated, and it did not contain the NH2-terminal peptide, which is the phosphorylation site in the .X, .1, and .2 variants. Its acetylation pattern was also different from that of H2A.1 in that two sites, neither of which was found in H2A.1, seem to be involved at the first level of modification.