Radiation of mammalian cells results in altered cell cycle regulation and altered transcription. The underlying mechanisms that account for these changes are only beginning to be understood. In this application we propose to determine the effect of radiation on histone acetylation and on histone acetyltransferases in order to begin to understand one mechanism through which radiation can alter transcription and chromatin structure. We will focus upon cyclin B1 as a marker for gene expression that is dependent upon histone acetylation and that is inhibited by radiation also noting that the transcription of this gene is crucial for cell cycle progression from G2 into M. In our previous work, we have shown that the radiation induced G2 delay is often accompanied by decreases in cyclin B1 mRNA and protein and that there is a transcriptional component to this regulation. In our recent experiments we have examined the regulation of cyclin B1 transcription through the cell cycle and after radiation. The cell cycle regulation of the cyclin B1 promoter can be conferred by two CCAAT box consensus sequences upstream from the start site. These boxes bind the transcription factor NF-Y and through NF-Y the related histone acetyltransferases GCN5 and P/CAF suggesting that histone acetyl transferase (HAT) activity might play a role in the cell cycle regulation of cyclin B1. The activity of the NF-Y associated HATs varied throughout the cycle with significantly more HAT activity bound to NF-Y in G2 than G1. In addition radiation depressed the levels of NF-Y associated HAT activity. These experiments have suggested that regulation of HAT activity could be a mechanism for regulation of cyclin B1 mRNA both through the cell cycle and after radiation. This leads to the hypothesis that radiation may result in alterations in chromatin structure and transcription mediated by alterations in HAT. In this proposal we describe an approach to studying the effect of radiation on HAT activity and experiments to link the alterations in HAT activity to cyclin B1 transcription.
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