CHROMATIN AND DNA FUNCTION: RECURRING QUESTIONS AND EVOLVING ANSWERS
Open Access
- Author:
- Wang, Xi
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 03, 2003
- Committee Members:
- Robert T Simpson, Committee Chair/Co-Chair
Jerry L Workman, Committee Member
Song Tan, Committee Member
Andrew Thomas Henderson, Committee Member
Hong Ma, Committee Member - Keywords:
- chromatin
DNA
transcription
chromatin mapping techniques - Abstract:
- In this thesis, in vivo analyses are presented to better understand the specific parameters by which gene transcription is regulated in the context of chromatin. A novel DNase I probing assay is established and employed to detect both histone-DNA and non-histone-DNA interactions in living cells. By introducing a bovine pancreatic DNase I gene into yeast under control of a galactose responsive promoter, we mapped chromatin structure at nucleotide resolution in whole cells without isolation of nuclei. The validity and efficacy of the strategy are demonstrated by footprinting a labile repressor bound to its operator. Investigation of the inter-nucleosome linker regions in several types of repressed domains has revealed different degrees of protection in cells, relative to isolated nuclei. These different structural signatures likely reflect the in vivo chromatin architectures that result in different biological behaviors of these domains. Moreover, this strategy has been applied to map active promoters and suggested that TBP, and possible other transcription factors, are persisting at some, if not most, active promoters through multiple transcription cycles in vivo. This conclusion was supported by chromatin immunoprecipitation (ChIP) assays. Unique chromatin structure characterizes cell type gene regions, including the a cell-specific gene domains in yeast. In this study, the componential and structural information of chromatin along the MFA1 gene, one of the a cell-specific genes, was investigated comprehensively by employing multiple approaches. Employing minichromosome affinity purification (MAP) and electron microscopy (EM) techniques, we observed this domain as a highly compact higher order chromatin structure. By doing Western blot, ChIP, and knock-out assays, we detected the presence of Tup1p and Hho1p in this domain, and their possible roles have also been discussed.