This proposal involves the characterization of a transcriptional activator located in the protein coding regions of two highly expressed histone genes The replication-dependent histone genes belong to a highly conserved gene family which code for the basic proteins which organize newly synthesized DNA into nucleosomes. These genes are turned """"""""up"""""""" coordinately by a cellular signal(s) at the onset of DNA synthesis in the eukaryotic cell and are among the most highly expressed mammalian genes. Stable transfections of in-frame mouse histone gene constructs in hamster cells have shown that a coding region activating sequence (CRAS) identified in both the H2a.2 and H3.2 genes functions in an orientation-dependent, but position-independent manner. That is, the H3.2 CRAS element functions when moved 700 nts 5' of its original location in the H3.2 gene (in the distal promoter), and also functions when moved into the H2a.2 gene which has had its CRAS deleted. Since identical mRNA molecules are produced from the H3.2 deletion gene and the deletion gene with the CRAS moved into the distal promoter, effects of the CRAS deletion upon mRNA stability or other post-transcriptional events are completely ruled out. Gel mobility shift analysis shows that the H3 an H2a CRAS sequences compete with each other for binding of nuclear proteins consistent with the interpretation that the same proteins interact with bot sequences. This data combined with that showing the restoration of normal expression to the H2a CRAS deletion gene by the H3 CRAS supports the hypothesis that the same proteins are involved in regulation of these two (and perhaps all) replication-dependent histone genes.
The specific aims o this proposal include 1. identification of the exact nucleotides in the H3 CRAS which are required for high-level expression by oligonucleotide- directed mutagenesis: 2. purification of the protein(s) shown to bind specifically to the CRAS and cloning of the gene(s) which code for these proteins; 3. examination of the role that the CRAS plays in activation of histone gene transcription at the onset of DNA synthesis; and 4. construction of deletion clones using a mouse H2b gene from chromosome 13 t confirm the hypothesis that the CRAS is a common transcriptional control element in the control of all replication-dependent histone genes; Control of histone gene expression is a critical component of control of cell growt and likely is sensitive to cellular signals involved in the onset of cancer For example, chemotherapeutic drugs such as 5-fluorodeoxyuridine (which inhibits rapidly growing cells by inhibiting DNA synthesis) have immediate effects on histone gene expression. Understanding how the histone gene CRA activates transcription of this gene family will add to the understanding o the complex system of cellular signals which control cell growth and proliferation.
