The overall objective of this project is to elucidate flow replication-dependent and replication-independent histone synthesis are interrelated during the chromosome replication cycle of proliferating cells and in nonproliferating cells. Such knowledge may be useful in developing procedures with selective toxicity for tumor cells by, as one example, disrupting the balance between histone synthesis and DNA synthesis. (1) Last year we described studies into the response of the 20 or so members of a histone gene family to DNA replication. The evidence indicated that there is a unified histone gene response to replication that is independent of cell type. Using these results, we have initiated studies on the use of non-radioactive nucleic acid probes to determine histone mRNA concentrations in situ in cell cultures, in tissues and tumors. One goal of this study is to determine whether probes to his tone mRNA species can be used as a reliable assay for the proliferation index of tumors. (2) The studies on the regulation of H2A.X and H2A.Z gene transcription are completed (Two manuscripts submitted). These two genes both have CCAAT boxes which bind common factors, while the replication- linked H2A.1a gene promoter contains similar CCAAT boxes which appear to bind different factors. Thus there appears to be common regulatory elements in the two basal promoters even though the H2A.Z gene contains introns and has polyadenylated transcripts while the H2A.X gene lacks introns and has transcripts that terminate in the conserved histone stemloop. (3) The human H2A.Z gene has been localized to chromosome 4q23 with a pseudogene on chromosome 21. The human H2A.X gene has been localized to chromosome 11q23, a region often translocated in leukemias. This H2A.X gene is very close to the gene for hydroxymethylbilane synthase, an enzyme involved in porphyrin synthesis; the 3' end of the mature mRNA species of the two genes are within 330 bp of each other. (4) Screening of cell lines in the DTP Cell Screen has uncovered one cell line that is a natural overproducer of H2A.X. We are studying the regulation of H2A.X synthesis in this cell line, which may account for about 50% of the total H2A synthesis.
