Biochemical, biophysical and functional characterization of histone chaperones

Abstract

Nucleosomes, the basic repeating unit of chromatin, are highly dynamic. Nucleosome dynamics allow for various cellular activities such as replication, recombination, transcription and DNA repair, while maintaining a high degree of DNA compaction. Each nucleosome is composed of 147 bp DNA wrapping around a histone octamer. Histone chaperones interact with histones and regulate nucleosome assembly and disassembly in the absence of ATP. To understand how nucleosome dynamics are regulated, it is essential to characterize the functions of histone chaperones. The first project of my doctoral research focused on the comparison of different nucleosome assembly proteins employing various biochemical and molecular approaches. Nucleosome assembly proteins (Nap) are a large family of histone chaperones, including Nap1 and Vps75 in Saccharomyces cerevisiae, and Nap1 (also Nap1L1), Nap1L2-6 (Nap1-like 2-6, with Nap1L4 being Nap2) and Set in metazoans. The functional differences of nucleosome assembly proteins are thus interesting to explore. We show that Nap1, Nap2 and Set bind to histones with similar and high affinities, but Nap2 and Set do not disassemble non-nucleosomal DNA-histone complexes as efficiently as Nap1. Also, nucleosome assembly proteins do not display discrepancies for histone variants or different DNA sequences. In the second project, we identified Spn1 as a novel histone chaperone and look into new functions of Spn1 on the regulation of chromatin structural states. Spn1 was identified as a transcription regulator that regulates post-recruitment of RNA polymerase II in yeast. We demonstrated that Spn1 is a H3/H4 histone chaperone, a novel finding that was not observed previously. Spn1 also interacts with Nap1, and forms ternary complexes with Nap1 and histones. We also show that Spn1 has chromatin assembly activity and N- and C- terminal domains of Spn1 are required for its histone chaperone properties. At the same time, we had an interesting observation that Spn1 potentially has topoisomerase/nuclease activity, which is dependent on magnesium ions. This activity of Spn1 can also help answer questions raised by in vivo assays related to Spn1, including its correlation with telomere length, the heat sensitivity in the reduction of function yeast strains, and the elongated lifespan in the Spn1ΔNΔC strain. Our studies on the functional comparison of nucleosome assembly proteins revealed their distinct roles in the regulation of nucleosome dynamics. Our findings on the histone chaperone functions and nuclease/topoisomerase activities disclosed new roles of Spn1 in chromatin regulation, by regulating histone-DNA interaction and also maintenance of DNA integrity.