Aberrant Polycomb Activity In Melanocyte Development And Disease

Abstract

Alterations to the epigenome are known to be drivers of tumorigenesis. Polycomb repressive complex 2 (PRC2) catalyzes methylation at Histone 3 Lysine 27 (H3K27), a modification that results in chromatin compaction and repression of target gene expression. In human melanoma, components of PRC2 are subject to both gain and loss of function alterations. To investigate the role of PRC2 in melanoma, we utilized a zebrafish model in which melanocytes harbor a loss of function mutation in tp53 and express oncogenic BRAF-V600E. We find that overexpression of activating PRC2 alleles have no effect on the rate of melanoma formation, but that overexpression of a PRC2 loss of function allele (H3.3K27M) accelerates melanoma onset. We have characterized gene expression and chromatin structure changes in H3.3K27M melanomas by RNA-seq and histone mass spectrometry. These studies suggest that PRC2 inhibition may not be therapeutically beneficial for some patients with melanoma. To identify PRC2 targets in melanocytes, we utilized NIH Epigenomics Roadmap Data to identify genes marked by H3K27me3 in melanocytes but not fibroblasts. We nominated FOXD1 as a critical PRC2 target which is extensively marked by H3K27me3 in melanocytes. We show that foxd1 is expressed in neural crest cells, silenced in melanocytes and aberrantly expressed in a subset of human melanomas. We characterized the results of foxd1 deregulation during embryogenesis and have mapped the local and distal cis-regulatory elements which are essential for foxd1 expression. To perform loss of function studies, we have examined the requirements to achieve an optimal rate of CRISPR/Cas9 genome editing. We have found that synthetic chemically modified guide RNAs (cmgRNAs) are highly active in vivo, leading to increased editing efficiency compared to in vitro synthesized gRNAs. We use cmgRNAs to generate loss of function alleles, delete regions of DNA greater than 100KB and to perform homology-directed repair. Collectively, these studies contribute to our understanding of epigenetic targets in melanoma, gene expression regulation, and optimal methods of genome editing.