Genome-wide analysis of epigenetics and alternative promoters in cancer cells

Genome-wide approaches, such as ChIP-chip, have been widely applied to explore the patterns of epigenetic markers and the interactions between DNA and proteins. Compared to candidate gene studies, the application of epigenomic and genomic tools in these fields provides more comprehensive understanding of normal and abnormal events in cells, such as those biological changes promoting cancer development.; In the first part, I studied the relations between two well-known epigenetic markers, DNA methylation and histone modifications. Previous studies of individual genes have shown that in a self-enforcing way, dimethylation at histone 3 lysine 9 (dimethyl-H3K9) and DNA methylation cooperate to maintain a repressive mode of inactive genes. Less clear is whether this cooperation is generalized in mammalian genomes, such as the mouse genome. Here I use epigenomic tools to simultaneously interrogate chromatin modifications and DNA methylation in a mouse leukemia cell line, L1210. Histone modifications on H3K9 and DNA methylation in L1210 were profiled by both global CpG island array and custom mouse promoter array analysis. I used chromatin immunoprecipitation microarray (ChIP-chip) to examine acetyl-H3K9 and dimethyl-H3K9. I found that the relative level of acetyl-H3K9 at different chromatin positions has a wider range of distribution than that of dimethyl-H3K9. I then used differential methylation hybridization (DMH) and restriction landmark genome scanning (RLGS) to analyze the DNA methylation status of the same targets investigated by ChIP-chip. The results of epigenomic profiling, which have been independently confirmed for individual loci, show an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing. In contrast to the previous notion, dimethyl-H3K9 seems to be less distinct in specifying silencing for the genes tested. This study demonstrates in L1210 leukemia cells a diverse relationship exists between histone modifications and DNA methylation in the maintenance of gene silencing. Acetyl-H3K9 shows an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing as expected. However, dimethyl-H3K9 seems to be less distinct in relation to promoter methylation. Meanwhile, a combination of epigenomic tools is of help in understanding the heterogeneity of epigenetic regulation, which may further our vision accumulated from single-gene studies.; In the second part, I profiled the multiple promoter usage in breast cancer cells. Various independent lines of evidence have suggested that a large fraction of human genes have multiple independently regulated promoters with distinct transcription start sites. Understanding which promoter is employed in which cellular condition is key to unraveling gene regulatory networks within the cell. To this end, we have designed a custom microarray platform that tiles roughly 35,000 alternative putative promoters from nearly 7,000 genes in the human genome. To demonstrate the utility of this platform, I have analyzed the pattern of promoter usage in E2-treated and untreated MCF7 cells and show widespread usage of alternative promoters. Most intriguingly, I show that the downstream promoter in E2-sensitive multiple promoter genes tends to be very close to the 3'-terminus of the gene sequence, suggesting exotic mechanisms of expression regulation in these genes.; Taken together, I showed genome-wide diverse relations between DNA methylation and histone modifications in leukemia cell line L1210. I also characterized the alternative promoter usage in breast cell line MCF-7.