Sarah A. Tincher Thesis Defense: Project Description
Title: DNA Methylation Patterns of Specific L1 Loci on the Short Arm of Chromosome 21
While 10-15% of the human genome is composed of heterochromatic DNA, these regions are not included in the completed genome sequence. We are using the short arm of chromosome 21 (HC21p) as a model for understanding the structure and function of heterochromatin. While LINE-1 (L1) retrotransposons are underrepresented in heterochromatin, including HC21p, there is a disproportionate number of full length L1s on HC21p. L1 elements are also overrepresented on the X chromosome, and it has been proposed that these L1s are involved in the assembly of heterochromatic nuclear compartments during X chromosome inactivation. DNA methylation at the L1 promoter is a host defense mechanism to suppress L1 activity, but L1 promoters on the inactive X are hypomethylated.
I hypothesized that the L1s on HC21p may be similarly involved in organizing the heterochromatin in that genomic region, possibly through changes in DNA methylation. L1 structure and function, including promoter methylation, have not previously been studied in heterochromatic regions of the genome like HC21p. Using bisulfite sequencing PCR on both chromosome-specific hybrid cell lines and leukocytes, I examined the promoter methylation status of four specific L1s on HC21p. I also included in my analysis two control L1s from the euchromatic HC21q, as well as LRE3, a highly active L1 on HC2. I found that the L1 promoters on HC21p are substantially less methylated than those in the control loci. Four CpG sites in the L1 promoter have been specifically implicated in the suppression of L1 expression by DNA methylation. In most of the HC21p L1 promoters these sites are either absent or hypomethylated compared to the same sites in the control loci. These observations all suggest that L1s may be influencing heterochromatin formation on HC21p.
Additionally, due to evidence that some L1s become hypomethylated in cancer cells, the same HC21p L1 loci were analyzed in a small number of prostate cancer cell lines to determine if changes in methylation at those loci could be relevant to the development of a cancer biomarker. The results show that the L1s on HC21p are overall hypomethylated compared to the HC21q control loci, but the extent varies between loci.
I would first like to express my gratitude to my advisor, Dr. Jeffrey Doering, for his unwavering support throughout my years at Loyola University Chicago. Thank you to my fellow students in the Doering lab, as well as the staff and faculty in the Department of Biology, particularly my committee members, Dr. Howard Laten and Dr. Stefan Kanzok, for their input and moral support throughout this entire process. Finally, thank you to my family and close friends for their continuous encouragement throughout the thesis writing process.
Sarah Tincher was born and raised in the Northwest suburbs of Chicago, Illinois. She received her Bachelor of Science degree in Biology in 2012 at Loyola University Chicago and continued her studies there to pursue a Master of Science in Biology. Currently, Sarah is in her second year of a PhD program at University of Illinois at Chicago, where she investigates the role of nitric oxide in the regulation of epigenetic mechanisms in cancer.
Dr. Jeffrey Doering
Dr. Stefan Kanzok
Dr. Howard Laten