Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Marisa S. Bartolomei

Second Advisor

Gerd Blobel

Abstract

Genomic imprinting is a mammalian-specific phenomenon where gene expression is regulated differently between maternally- or paternally-inherited alleles. As most imprinted genes are important growth regulators, proper expression of these genes is crucial for normal development in humans and mice. A well-known H19/IGF2 imprinted cluster harbors two growth regulators with opposite functions, H19 and IGF2. The maternally expressed H19 gene is a long noncoding RNA implicated in growth suppression, while the paternally expressed IGF2 gene is a major growth factor in multiple developmental pathways. Dysregulation of the imprinted H19/IGF2 cluster is associated with Beckwith-Wiedemann Syndrome (BWS) and Silver-Russel Syndrome (SRS). Although both are primarily represented by abnormal growth, BWS and SRS show substantial variability in the severity of symptoms among patients. The mechanism underlying how the dysregulated H19/IGF2 expression leads to a specific pathological defect is still unknown. BWS and SRS phenotypes have been largely attributed to aberrant IGF2 expression because the exact role of H19 has been difficult to discern due to its coupled regulation with IGF2. Additionally, because of the mosaic nature of the epimutations in human patients, it has been challenging to understand the contribution of abnormally expressed H19 and IGF2 to the tissue-specific BWS/SRS pathologies. To dissect the effect of H19/IGF2 dysregulation in various lineages, we utilized two model systems. In chapter 2, we present the human induced pluripotent stem cells (hiPSCs) that are derived from BWS patient fibroblasts with paternal uniparental disomy of chromosome 11 (pUPD11). Our iPSC clones maintained proper imprinting in H19/IGF2 locus with an expected epigenetic profile of pUPD11. Differentiation of hiPSCs into a hepatic lineage enabled us to examine the effect of pUPD11 in a tissue type that is prone to BWS-related tumors. Through the transcriptomic profiling of pUPD11 and non-pUPD11 hepatocytes, we could identify the target pathways that are affected by pUPD11 and likely contributing to the increased occurrence of hepatoblastoma and growth anomalies in BWS. In chapter 3, we utilized mouse models with subtle perturbations of H19 and Igf2 expression to address the independent roles of H19 and Igf2 in SRS-like pathologies. We started with a previously described humanized mouse model that substituted the human H19 imprinting control region for the corresponding mouse region (H19+/hIC1), which exhibited H19 overexpression and Igf2 depletion. These mice had severe developmental defects in the heart and placenta, which may significantly contribute to the previously described perinatal lethality and severe growth restriction. While normalizing H19 expression was not sufficient for the full rescue of the growth restriction and lethality, altering both H19 and Igf2 expression restored viability, although certain cardiac defects were still retained. Our results demonstrate that physiological levels of H19 and Igf2 are crucial for normal cardiac and placental development. Overall, this work emphasizes the importance of precise regulation of H19 and IGF2 expression in embryonic growth and tissue formation. These results enhance our understanding of how molecular defects in the H19/IGF2 cluster lead to the characteristic BWS and SRS phenotypes, further helping to establish molecular subtype-specific therapeutic strategies.

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