Analysis of Histone and Histone Chaperone Nuclear Import

The nuclear import of proteins is a fundamental process required for the proper formation and function of chromatin. Analysis of the nuclear import pathways of the histones, the major protein component of chromatin, and the histone chaperones that mediate their assembly into nucleosomes is important for understanding how chromatin assembly is regulated by the cell. Histones H3 and H4 are acetylated in the cytoplasm prior to their nuclear import, but detailed study of the impact of these modifications has not been completed. Mutational analysis of lysines within each NLS suggested that acetylation is not necessary, and in fact unfavorable, for nuclear transport. Here it is shown that the H3 NLS interacts with a single karyopherin while the H4 NLS interacts with multiple karyopherins. In addition to the karyopherins, it was determined that cytosolic H3/H4 associates with the HAT-B complex and the histone chaperone Asf1. Evidence is presented that Asf1 may serve as a transport cofactor for H3/H4 nuclear import. Co-import of Asf1 with H3/H4 could represent a link between H3/H4 import and chromatin assembly. Asf1 also contains a classical NLS that appears to provide nuclear import independent of co-import with histones. The nuclear import pathways of histone variants have not been determined. Here, the nuclear import pathways of H2A variant Htz1 were analyzed. Cytosolic Htz1 was found to be associated with Kap114, Kap123 and histone chaperone Nap1. Nap1 was previously shown to be a transport cofactor for H2A/H2B import, thus Nap1 may also play a role as a transport cofactor for iii Htz1 import. Interestingly, the Htz1 chaperone Chz1 does not seem to function in Htz1 import and is in fact imported by a separate pathway. NAP1 and CHZ1 were found to have different genetic interactions with HTZ1, pointing to non-redundant functions of these chaperones in the cell. Taken together, the work put forth in this dissertation examines how the nuclear import and cellular functions of histone chaperones and histone acetyltransferases intersect with the nuclear import of histones H3/H4 and Htz1. iv DEDICATION I dedicate this work to Kellie. Your love, support, and affection are what made this possible.

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