wrapped around a histone core octamer (two copies 2 Department of Biochemistry and Molecular Genetics each of histone H3, H4, H2A, and H2B; see Luger et al., University of Virginia Health System 1997 for details). Our understanding of how nucleosomal Charlottesville, Virginia 22908 arrays are packaged into higher-order chromatin fibers, 3 Wellcome Trust Centre for Cell Biology and how distinct functional domains are formed is poor. ICMB An increasing body of evidence suggests that posttrans-University of Edinburgh lational histone modifications (acetylation, phosphoryla-Swann Building tion, methylation, etc.) influence chromatin folding and Mayfield Road have clear functional consequences (reviewed in van Edinburgh EH9 3JR Holde, 1988; Wolffe and Hayes, 1999). In addition, dis-Scotland, United Kingdom tinct patterns of covalently modified histones, by acting 4 Department of Molecular Genetics as a histone code, may serve as signaling platforms to University of Texas M.D. Anderson Cancer Center recruit and bind nuclear factors that mediate down-1515 Holcombe Boulevard. stream functions by unknown mechanisms (reviewed in Houston, Texas 77030 Strahl and Allis, 2000; Turner, 2000; Cheung et al., 2000). 5 Fox Chase Cancer Center Given the drastic changes in the integrity of DNA and 7701 Burholme Avenue the state of chromatin compaction during apoptosis, Philadelphia, Pennsylvania 19111 histone modifications may play a functional role in promoting these changes. To date, histone modifications that are specifically Summary induced during apoptosis have not been clearly defined (reviewed in Cheung et al., 2000; Th'ng, 2001; Jason et DNA in eukaryotic cells is associated with histone proal., 2001). Mitotic chromatin condensation, for example, teins; hence, hallmark properties of apoptosis, such
is associated with histone H3 phosphorylation at serine as chromatin condensation, may be regulated by post-10, but this modification has not been consistently obtranslational histone modifications. Here we report served during apoptotic-induced chromatin condensathat phosphorylation of histone H2B at serine 14 (S14) tion (reviewed in Cheung et al., 2000; Hendzel et al., correlates with cells undergoing programmed cell 1998). Phosphorylation of a relatively minor histone varideath in vertebrates. We identify a 34 kDa apoptosisant, H2A.X, increases during the early stages of DNA induced H2B kinase as caspase-cleaved Mst1 (mamfragmentation in apoptosis (Rogakou et al., 1998, 2000). malian sterile twenty) kinase. Mst1 can phosphorylate However, H2A.X phosphorylation at serine 139 corre-H2B at S14 in vitro and in vivo, and the onset of H2B lates with double-stranded DNA breaks induced by nu-S14 phosphorylation is dependent upon cleavage of merous stimuli, suggesting that this mark acts more as Mst1 by caspase-3. These data reveal a histone modifia DNA-damage sensor rather than an event specifically cation that is uniquely associated with apoptotic chrolinked to the apoptotic process. In mammalian cells, the matin in species ranging from frogs to humans and only core histone modification that has been uniquely provide insights into a previously unrecognized physiassociated with apoptosis is histone H2B phosphorylaological substrate for Mst1 kinase. Our data provide tion. While earlier work showed that H2B phosphorylaevidence for a potential apoptotic "histone code." tion occurs at the N-terminal tail, the specific sites of phosphorylation were not known (Ajiro, 2000; reviewed Introduction in Cheung et al., 2000). Furthermore, H2B phosphorylation and the H2B, but not other histones, amino-terminal Apoptosis is an active process of cell death important for tail is essential for chromatin condensation in Xenopus normal development and homeostasis in multicellular cell-free systems (de la Barre et al., 2001). These data organisms. Formation of pycnotic and condensed chroare consistent with the idea that H2B phosphorylation matin bodies and digestion of DNA into oligonucleosomay be important for apoptotic chromatin condenmal fragments are hallmarks for apoptosis (Wyllie, 1980; sation. Wyllie et al., 1980, 1984). At present, the biochemical Here, using a new phosphospecific antibody (hereafand molecular mechanisms for these nuclear events are ter referred to as β£-Phos (S14) H2B), we demonstrate unclear, whereas upstream cytoplasmic pathways (inthat H2B S14 phosphorylation specifically correlates with the onset of apoptosis in human HL-60 cells. This correlation extends to cells undergoing programmed