| Understanding the complexities of chromosomes beyond the DNA sequence level requires the use of innovative tools and technologies. Epigenetic controls act "on" the genome and are vital to proper cell development. Epigenetic perturbations can lead to diseases such as cancer. This dissertation describes development and use of new technology to better understand epigenetic control, to analyze the dynamics of satellite sequences, to study the effects of aberrant methylation on the organization of these sequences, and to profile DNA methylation states across an entire human chromosome.Existing techniques have provided a myriad of information on chromosome location within the nucleus at a static level. However, in order to grasp the true dynamics of chromosomes as they go in and come out of mitosis, tools are needed to allow for real-time observation. In this dissertation, sequence-specific compounds called polyamides are demonstrated as promising probes for observing the movement of specific genomic locations, specifically satellite repeats concentrated near some human centromeres.The second aspect of this study is DNA methylation. It is well known that gene expression is controlled by various DNA elements such as promoters. However, methylation, a chemical modification of certain DNA sequences, can also have a profound effect on transcriptional activity. In fact, many diseases and developmental abnormalities arise from defects in methylation. This dissertation describes a profiling technique based on microarray technology to measure gross methylation differences between samples across a whole human chromosome. Using this approach, gross regional differences in methylation states are detected in the DNA of cells lacking one or more methylating enzymes and for different human tissues.In a separate study, more traditional cytogenetic techniques were used to analyze the nuclear localization of satellite sequences of patients with a dysfunctional DNA methylating enzyme. These satellite-containing regions did not appear to differ in size in interphase despite obvious cytogenetic abnormalities. However, indirect effects, such as downstream interactions, may still play a role in this phenotype.Epigenetics clearly plays an important, but still poorly understood, role in maintaining genomic stability. Understanding the interplay between DNA sequence and its modifiers leans heavily on novel tools for future discoveries. |
