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Abstract

Ten-Eleven-Translocation 5-methylcytosine dioxygenases 1-3 (TET1-3) convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC), using O2 as a co-substrate. Contrary to expectations, hypoxia induces 5-hmC gains in MYCN-amplified neuroblastoma (NB) cells through TET1 up-regulation. There were several unanswered questions from this work including why this phenotype was unique to MYCN-amplified NB and how the transcription factor HIF-1 interacts with TET1 gene and TET1 protein. Additionally, it was unclear if 5-hmC enrichment in hypoxic response genes and subsequent augmentation of gene expression mediated aggressive hypoxic phenotypes characteristic of NB cell lines. In my doctoral work, I show that MYCN directly controls TET1 expression in normoxia via binding of two genomic sites within the first and second introns of TET1. Further investigation revealed that MYCN specifically drives expression of the full-length TET1 transcript. In hypoxia, HIF-1 augments TET1 expression through the same binding sites identified as MYCN binding sites in the previous chapter. Unexpectedly, in gene edited cells that lack the binding site, TET1 protein still increased over respective normoxic protein. Subsequently, hypoxic 5-hmC levels were also induced compared to normoxic levels. To determine the rate of TET1 turnover, I performed TET1 degradation assays to determine TET1 protein half-life. In normoxia, TET1 had a half-life of 20 hours while hypoxic TET1 had a half-life of 40 hours. I found that a binding interaction between HIF-1 and TET1 was the source of increased TET1 stability in hypoxia. To determine how the 5-hmC landscape changes between 0 and 48 hours I performed hMe-SEAL at 0, 6, 12, 24, 48, and 72 hours. Bioinformatic analyses of 5-hmC distribution and RNA-sequencing data from hypoxic cells implicated genes important for cell migration as hypoxia targets, including CXCR4. Following this I performed migration assays and mouse xenograft experiments to determine if 5-hmC gains impacted NB pathogenesis. Treatment of MYCN-amplified NB cells with a CXCR4 antagonist resulted in slower migration in hypoxia, suggesting that inclusion of a CXCR4 antagonist into NB treatment regimens could be beneficial for children with MYCN-amplified NBs. When parental and S1/2 SK-N-BE(2) cells were injected into athymic mice, S1/2 tumors grew slower compared to controls, indicating 5-hmC may mediate tumor growth as well. Overall, I show how MYCN and HIF-1 regulate the TET1 gene and TET1 protein, and subsequently, how the 5-hmC epigenetic landscape is regulated in NB.

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