The primary objective of this project this tear has been to determine how a replication-independent histone protein species, H2A.X, results from an apparently replication-dependent gene. The H2A.X gene is the only known situation in which this occurs. The study is of potential importance because understanding the mechanism of this uncoupling is a step towards our goal of being able to manipulate the balance between histone and DNA synthesis for therapeutic benefit. The mRNA is unusual in that it contains stem-loop and U7 snRNP sequence motifs found in mRNAs from replication-dependent histone species, as well as polyA motifs. Two mRNAs exist in proliferating cultures, one terminating at the stem-loop motif and the other at the polyA motifs with a polyA tail. In addition to the altered processing of the H2A.X compared to the H2A.1 transcript, there is also altered regulation of the mRNA stability. The major effort at this time is delineating those sequences and factors which lead to the altered behavior of the H2A.X transcript by analyzing the transcripts of chimeric H2A.X/H2A.1 or otherwise changed H2A.X genes transfected into various cell types. This type of analysis will allow us to progressively localize relevant sequences. One sequence has been found and is being studied. The altered behavior of the H2A.X transcript enables it to escape tight replication-dependence. Elucidating this mechanism may allow the development of techniques disrupting the tight control of histone and DNA synthesis, leading to selective death of tumor cells.
