Pan-Cancer DNA Hypermethylation Leads to Transcriptional Upregulation of Homeobox Oncogene

Cancers have long been recognized to be not only genetically but also epigenetically distinct from their tissues of origin. Although genetic alterations underlying oncogene upregulation have been well studied, to what extent epigenetic mechanisms, such as DNA methylation, can also induce oncogene expression remains unknown. Here, through pan-cancer analysis of 4,182 genome-wide profiles, including whole-genome bisulfite sequencing (WGBS) data from 30 normal tissues and 35 solid tumors, we discovered a strong correlation between gene-body hypermethylation of DNA methylation canyons (broad under-methylated regions) and overexpression of ~43% homeobox genes, many of which are also oncogenes. To gain insights into the cause-and-effect relationship, we used a newly developed dCas9-SunTag-DNMT3A system to methylate genomic sites of interest. The locus-specific hypermethylation of gene-body canyon, but not promoter, of homeobox oncogene DLX1 and POU3F3, can direct increase its gene expression. Together, our pan-cancer analysis followed by functional validation reveals DNA hypermethylation as a novel epigenetic mechanism for homeobox oncogene upregulation. Locus-specific DNA methylation of DLX1 and POU3F3 gene-bodies was conducted using our dCas9-SunTag-DNMT3A system. In brief, doxycycline-inducible lentiviral particles of dCas9-SunTag-p2A-BFP and scFv-sfGFP-DNMT3A were transduced in human embryonic kidney cell line (HEK293T). The Single clones of idCas9SunTag, idCas9SunTag+iscFvDNMT3A and idCas9SunTag+iscFvDNMT3AE756A was purified. Lentiviral particles of sgDLX1+sgPOU3F3-puromycin and sgPOU3F3-puromycin were also generated and transduced in previously generated inducible dCas9-SunTag-DNMT3A cells. Transduced cells were treated with 2μg/ml puromycin for seven consecutive days and cultured in 2μg/ml doxycycline for another thirty days. SgRNA primers were listed as following: DLX1-F 5’-CACCGGGCGGACTCGGAGAAGAGCA-3’, DLX1-R 5’-AAACTGCTCTTCTC CGAGTCCGCCC-3’, POU3F3-F: 5’-CACCGCGGCGGCGGGGGCGGCGCAG-3’, POU3F3-R: 5’ -AAACCTGCGCCGCCCCCGCCGCCGC-3’. Bisulfite sequencing to identify the loci DNA methylation changes in homeobox gene DLX1 and POU3F3 promoter and gene body using Illumina HiSeq 2000