The primary objective of this project is to determine how a replication-- independent histone protein species, H2A.X, results from an apparently replication-dependent gene. This paradoxical situation may allow us a greater understanding of the regulation of histone synthesis and replication and may help us to manipulate the balance between histone and DNA synthesis for therapeutic benefit. We have described three aspects by which the H2A.X gene is unusual. The first is that it yields two mRNA's, one terminating at the conserved stem-loop sequence found in all replication-linked histone mRNA's, and a second terminating in a polyA motif about 1 kb further downstream. The proportion of each mRNA form varies greatly among cell lines; IMR90 cells contain 80% polyA form while Jurkat cells contain 75% stem-loop form. The two mRNA's do not explain the paradox because in some cell lines the stemloop mRNA predominates. The paradox was further resolved by the findings that there is also altered regulation of the H2A.X stem-loop mRNA with respect to DNA and protein synthesis. During this year, our studies of histone gene specific transcription and mRNA stability have shown that the explanation for the altered regulation of the H2A.X stemloop mRNA is due to differences in the regulation of H2A.X gene transcription. H2A.X gene transcription is only slightly affected by changes in DNA and protein synthesis while the rate of transcription of replication-linked genes decreases 5-10 fold in the former case and increases about two-fold in the latter. These findings show the importance of gene transcription relative to mRNA stability in the linkage of histone and DNA synthesis. A manuscript describing these findings has been submitted.
