The histone proteins are essential components of eukaryotic chromatin. Two molecules each of histones H2A, H2B, H3, and H4 from the core octamer of the nucleosome, and provide the first level of compaction of the DNA in the nucleus. Research over the last five yeast has provided mounting evidence that the histones play essential roles in many aspects of nuclear function, including transcription, replication, recombination, and nuclear division. Thus, determining the functional roles of the histones in vivo is critical for understanding mechanisms of gene expression, genome stability, and chromosome dynamics-processes in which defects are intimately related to causes of cancer, and chromosome abnormalities such as Down Syndrome. This project presents a molecular genetic investigation into the roles of the histone proteins in vivo. It focuses on three major research questions: (1) the role of the histone N-terminal domains in the maintenance of genome integrity; 92) the structure and function of protein interactions within the nucleosomes; and (3) the structure and function of centromeric chromatin and the kinetochore. The N-terminal domain of histone H4 is required to maintain genome integrity. Mutations in this domain cause increased intrinsic DNA damage, activation of DNA damage-inducible gene expression, and DNA damage check- point arrest at mitosis. We will determine how and why this increased DNA damage is incurred by studying mutants in the process. We will ask the N- terminal domains of other histone proteins play a role in this maintenance function. We will test whether the N-terminal domains determine higher- order chromatin compaction and the extent to which this function is related to the maintenance function. We will continue a successful site-directed mutational analysis histone interactions within the nucleosome. Temperature sensitive mutations in H2B-H4 contact sites lead to aberrant gene transcription, a failure to activate transcription of the Gl cyclin genes, and arrest at start. We will determine the genetic basis of this transcription inhibition through further mutational analyses, and the molecular basis through in vitro reconstitution experiments. Genetic evidence from our laboratory showed that proper chromosome segregation requires histone H4 interaction with a novel histone H3 variant related to the mammalian kinetochore antigen CENP-A. We propose that histone H4 and CENP-A form a specialized nucleosome at the centromere and interact with other kinetochore proteins. This model will be tested both genetically and biochemically.
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