The Study of Non-Nucleosomal Chromatin Particles and the Development of a Simple and Versatile System for the ATP-Dependent Assembly of Chromatin
Chromatin is a nucleoprotein complex that is responsible for both the organization of the genome as well as the regulation of DNA-utilizing processes. The basic repeating unit of chromatin is the nucleosome, which consists of approximately 147 bp of DNA wrapped around a core histone octamer. Non-nucleosomal chromatin particles also exist and are of interest due to their localization at regions of active chromatin. They are thought to prime chromatin for the initiation of DNA-utilizing processes, such as replication, repair, and transcription. For this dissertation, I developed methods to investigate two non-nucleosomal chromatin particles, the H3C110 particle and the prenucleosome, in cells using thiol-specific reagents and antibodies targeting acetylated H3K56, respectively. In addition, the formation of the prenucleosome was further investigated using a previously published in vitro chromatin assembly system with histone chaperone NLP in lieu of NAP1. The investigation into the H3C110 particles and prenucleosomes in vivo were ultimately discontinued due to a lack of enrichment of active chromatin. However, further in vitro work with the prenucleosome revealed that prenucleosomes can be formed with either NAP1 or NLP. This finding ultimately led to the development of a simplified and versatile in vitro chromatin assembly system. In this system, NLP has been substituted for NAP-1 and ATPase ISWI for ACF. Both proteins are synthesized in bacteria and easily purified making the system simpler to set up. In addition, this system can assemble chromatin with a variety of histones and DNA and can also incorporate histone H1 and nonhistone chromosomal protein HMGN2 into the chromatin. The simplicity and versatility of this system will allow a broader range of scientists to assemble customized chromatin for structural and functional analysis.