DNA in eukaryotic cells is associated with histone proteins; hence, hallmark properties of apoptosis, such as chromatin condensation, may be regulated by posttranslational histone modifications. Here we report that phosphorylation of histone H2B at serine 14 (S14) correlates with cells undergoing programmed cell death in vertebrates. We identify a 34 kDa apoptosis-induced H2B kinase as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans and provide insights into a previously unrecognized physiological substrate for Mst1 kinase. Our data provide evidence for a potential apoptotic "histone code."

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The histone 1 (H1) fraction of HeLa S-3 cells contains two principal subtypes, H1A (Mr approximately 21 000) and H1B (Mr approximately 22 000). In G1 cells, the H1 molecules are distributed among several phosphorylation states, most H1A molecules containing 0 or 1 phosphate groups and most H1B molecules containing 0, 1, 2, or 3 phosphate groups. Both subtypes undergo a general increase in phosphorylation levels of approximately 1 P/mol during the S phase and a further increase or 3--4 P/mol during mitosis. These two increases affect most of the H1 molecules and thus reflect phosphorylations occurring widely throughout the chromatin, presumably in association with replication and mitotic chromosome condensation. During all these periods, multiple phosphorylation levels of H1 molecules persist, as does the phosphorylation differential between H1A and H1B. Thus, there appear to be phosphorylation states that only some of the H1 molecules occupy, a fact that may be related to the conformational diversity in interphase and mitotic chromatin. The existence of differences between H1A and H1B phosphorylation states throughout the cell cycle, and within a single cell type, is in accord with the hypothesis that the H1 subtypes are functionally distinct, such that subtype-specific phosphorylations contribute to the control of chromatin organization.

Histone phosphorylation was investigated in several mammalian cells undergoing apoptosis (human HL-60 and HeLa, mouse FM3A and N18 cells, and rat thymocytes). Among the four nucleosomal core histones (H2A, H2B, H3, and H4), H2B, which is not usually phosphorylated in quiescent or growing cells, was found to be phosphorylated after treatment with various apoptotic inducers. The H2B was phosphorylated around the time when nucleosomal DNA fragmentation was initiated and, like this fragmentation, was completely blocked with Z-Asp-CH 2 -DCB, an inhibitor of ICE or ICE-like caspase. The involved single phosphopeptide of H2B proved to be phosphorylatable in vitro with a protein kinase C, and the site Ser-32 was tentatively identified. Despite typical apoptotic chromatin condensation, the H3 phosphorylation was at a low level, and the sites where phosphorylation did occur did not include any mitosis-specific phosphopeptides. Phosphorylation of H4 was increased, but the other two histone proteins (H1 and H2A) were not appreciably changed. These observations imply that 1) H2B phosphorylation occurs universally in apoptotic cells and is associated with apoptosis-specific nucleosomal DNA fragmentation, 2) chromatin condensation in apoptosis occurs by a different biochemical mechanism from those operating during mitosis or premature chromosome condensation, and 3) this unique phosphorylation of H2B is a useful biochemical hallmark of apoptotic cells.

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