Chromatin-Based Regulation and Maintenance of the Human Genome

• Other Authors: Sexton, Brittany Sloane (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Biology; Molecular biology; Cytology
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-9449

Nucleosome distributions are critically important in regulating access to the eukaryotic genome. Cells with different physiologies have strikingly similar nucleosome distributions. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. Factors regulating the maintenance of the basal state as well as changes in nucleosome distribution following a response must be investigated. In our first set of experiments we used the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) as a model system for stimulus-induced nucleosome distribution changes. We measured nucleosome distribution at high temporal resolution in human cells at the 2 kb flanking the transcription start sites (TSSs) of hundreds immunity-related loci, using microarray technology, during the reactivation of KSHV. We show that nucleosome redistribution peaks at 24 hours post KSHV reactivation and that the nucleosomal redistributions are widespread and transient. To clarify the role of DNA sequence in these nucleosomal redistributions, we compared the genes with altered nucleosome distribution to a sequence-based computer model and in vitro assembled nucleosomes. We demonstrate that both the predicted model and the assembled nucleosome distributions are concordant with the majority of nucleosome redistributions at 24 hours post KSHV reactivation. We suggest a model in which loci are held in an unfavorable chromatin architecture and "spring" to a transient intermediate state directed by DNA sequence information. We propose that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. The surprising findings that nucleosome redistributions are widespread, transient, and DNA-directed shift the current perspective regarding regulation of nucleosome distribution in humans. We next wanted to affirm and extend our previous observations regarding the widespread and transient nature of nucleosome redistributions during viral reactivation. We tested if this widespread nucleosome remodeling was a genome wide event or limited solely to the hundreds of immunity-related loci measured by microarray. We measured nucleosome distributions at high temporal resolution following KSHV reactivation using our newly developed mTSS-seq technology, which maps nucleosome distribution at the TSS of all human genes. Nucleosomes underwent widespread changes in organization 24 hours after KSHV reactivation and returned to their basal nucleosomal architecture 48 hours after KSHV reactivation. 72% of the loci with translationally remodeled nucleosomes have nucleosomes that moved to positions encoded by the sophisticated underlying DNA sequence. We demonstrated that these widespread alterations in nucleosomal architecture potentiated regulatory factor binding. These descriptions of nucleosomal architecture changes have allowed us to propose a new hierarchical model for chromatin-based regulation of genome response. Given that we discovered that nucleosome distributions are widespread and transient, it was important for us to understand the forces maintaining the basal state. We It would be interesting to understand the forces and factors that maintain nucleosome architecture in a basal state and regenerate it following a response, such as KSHV reactivation. An appealing group of candidates that might maintain and regenerate the nucleosome architecture in its basal state is the transcriptional machinery. We identified RNA polymerase II (RNA Pol II) as a likely candidate as it is found throughout the genome and not always associated with transcription. We next were interested in the role RNA Pol II plays in the maintenance of chromatin structure. We measured nucleosome distributions in response to RNA Pol II inhibition by δ-amanitin treatment. Nucleosome distribution changes, following RNA Pol II inhibition, were widespread and the TSSs with nucleosome distribution changes were enriched for RNA Pol II independent of it's role it plays in active transcription. This work gives new insight into understanding the role of chromatin structure regulates and maintains the human genome. === A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Spring Semester, 2015. === April 9, 2015. === Chromatin, KSHV, microarray, next generation sequencing, nucleosome, RNA Pol II === Includes bibliographical references. === Jonathan H. Dennis, Professor Directing Dissertation; Michael G. Roper, University Representative; Hank W. Bass, Committee Member; P. Bryant Chase, Committee Member; Debra A. Fadool, Committee Member.