Analyses of histone phosphorylation during apoptosis and DNA damage.

摘要

Apoptosis is an active process of cell death that is important for normal development and homeostasis in multicellular organisms. One of the hallmarks for apoptosis is the change in the chromatin structure such as the formation of condensed chromatin bodies and DNA fragmentation into pieces of oligonucleosomal length. However, the biochemical and molecular mechanisms for these events are still unclear. One mechanism that can affect chromatin structure is the covalent modification of histones, which normally bind to DNA and form the nucleosome, the fundamental unit of chromatin. Recently, this mode of altering chromatin has been linked to many DNA templated processes such as mitosis, transcription, replication and silencing. Using phospho-specific antibodies, I found that histone H2B serine 14 (S14) phosphorylation correlates with the onset of apoptotic chromatin condensation and DNA fragmentation. This correlation was extended to other cell lines tested, as well as cells undergoing programmed cell death during Xenopus tail resorption. I identified the 34 kDa-induced H2B kinase that fractionates with H2B (S14) kinase activity 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 novel histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans. Moreover, my data provide new insights into a previously unrecognized physiological substrate for Mst1 kinase, and provide evidence for a potential apoptotic 'histone code.'; In addition, I found that histone H4 at serine 1 (S1) phosphorylation is associated with DNA damage response in both yeast and human cells. This phosphorylation event does not seem to be essential for cells to survive through DNA damage; however, it might be important for other response such as shutting off transcription. To further understand this phosphorylation, I have detected an MMS-induced 40 kDa H4 kinase. The identity of this kinase will help identify the roles that H4 S1 phosphorylation might play in DNA damage repair. Hence, my work has identified another novel chromatin modification linked to the DNA damage response pathway.