Elucidating the role of 5-hydroxymethylcitosine for the function of Polycomb Repressive Complex 2
Mentor Name: Mark Applebaum
Currently, only 60% of children with high-risk neuroblastoma are cured, emphasizing the desperate need for more effective therapies. MYCN-amplified neuroblastoma is selectively dependent on Polycomb Repressive Complex 2 (PRC2), which catalyzes the tri-methylation of lysine 27 of histone 3 (H3K27me3). There is also 5-hydroxymethylcytosine (5-hmC), a stable intermediate generated in the process of removing methyl groups from DNA cytosines, a process catalyzed by the TET1/2/3 family of proteins. In addition to showing that MYCN regulates TET1 expression, we unexpectedly found that genes targeted by PRC2 that should be transcriptionally repressed had higher 5-hmC deposition in high-risk compared to low-risk tumors and in cfDNA from patients with metastatic and relapsed disease. Thus, we hypothesized a cooperative function between 5-hmC and PRC2 at transcriptionally repressed genes that mediates a de-differentiated state in MYCN-amplified neuroblastoma. This project focuses on elucidating the cooperative mechanisms between 5-hmC and PRC2 that enables transcriptional repression to identify targetable pathways that promotes high-risk neuroblastoma. To test this, we will establish the role of the TET enzymes in facilitating the deposition of H3K27me3 at genes necessary for cellular differentiation. We will generate a CRISPR knockout of all three TET enzymes individually and in combination to remove 5-hmC from neuroblastoma cells. We will evaluate H3K27 and DNA methylation, 5-hmC, and transcriptional changes to determine the role of 5-hmC in the aberrant recruitment and function of the PRC2 in aggressive neuroblastoma.
