Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Marisa S. Bartolomei

Abstract

Genomic imprinting is an epigenetic phenomenon in which genes are monoallelicaly expressed according to their parent-of-origin. Imprinted expression entails marking parental chromosomes so that a specific parental allele is stably repressed or expressed. Differential DNA methylation is essential for marking and regulating imprinted genes and is often found at imprinting control regions (ICRs). These DNA methylation imprints must be maintained throughout early development despite genome-wide epigenetic reprogramming to allow for stable allelic expression in differentiated tissues. Moreover, marking of the alleles must be erased in the germline so that establishment of sex-specific marks can occur during gametogenesis. These processes are critical for normal imprinting, however, the precise mechanisms and factors involved remain largely unknown. Of particular concern, environmental perturbations occurring during times of epigenetic reprogramming have been reported to disrupt imprinting. In this dissertation I investigate both cis and trans mechanisms by which DNA methylation confers imprints and how environmental stresses can disrupt imprinted regulation. I show that decreased CpG content at the endogenous paternal H19 ICR in mouse renders the ICR unable to silence paternal H19, indicating a cis-regulatory role for CpG density in imprinted regulation of H19. I also investigate the role that methyl-CpG-binding domain (MBD) proteins, involved in DNA methylation dependent repression, have in genomic imprinting. Through analysis of Mbd1 and Mbd2 mutant mice, I find that individual MBD proteins are dispensable for normal imprinting. In a collaborative effort to identify factors necessary for resetting of imprints in germ cells, we examine the cooperative function of Ten-eleven-translocation (TET)1 and TET2 in the erasure of imprints, and show that both TET1 and TET2 are required for demethylation at imprinted loci in the germline. Furthermore, as a collaborative effort, we investigate possible deregulation of imprints upon environmental stress through analysis of spermatogonial stem cells (SSCs) undergoing aging and cryopreservation. We find that stressed SSCs stably maintain methylation imprints and can produce sperm to be used in intracytoplasmic sperm injection that result in normal offspring. These results provide novel insights into mechanisms involved in normal imprint establishment and maintenance, as well as the stability of these marks despite environmental perturbations.

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