Transcriptional deregulation of oncogenic pathways is a hallmark of cancer and can be due to epigenetic alterations. 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification that has not been studied in pancreatic cancer. Genome-wide analysis of 5-hmC-enriched loci with hmC-seal was conducted in a cohort of low-passage pancreatic cancer cell lines, primary patient-derived xenografts, and pancreatic controls and revealed strikingly altered patterns in neoplastic tissues. Differentially hydroxymethylated regions preferentially affected known regulatory regions of the genome, specifically overlapping with known H3K4me1 enhancers. Furthermore, base pair resolution analysis of cytosine methylation and hydroxymethylation with oxidative bisulfite sequencing was conducted and correlated with chromatin accessibility by ATAC-seq and gene expression by RNA-seq in pancreatic cancer and control samples. 5-hmC was specifically enriched at open regions of chromatin, and gain of 5-hmC was correlated with up-regulation of the cognate transcripts, including many oncogenic pathways implicated in pancreatic neoplasia, such as MYC, KRAS, VEGFA, and BRD4. Specifically, BRD4 was overexpressed and acquired 5-hmC at enhancer regions in the majority of neoplastic samples. Functionally, acquisition of 5-hmC at BRD4 promoter was associated with increase in transcript expression in reporter assays and primary samples. Furthermore, blockade of BRD4 inhibited pancreatic cancer growth in vivo. In summary, redistribution of 5-hmC and preferential enrichment at oncogenic enhancers is a novel regulatory mechanism in human pancreatic cancer.

致癌途径的转录失调是癌症的标志，可能是由于表观遗传学改变。5-羟甲基胞嘧啶（5-hmC）是一种在胰腺癌中尚未研究的表观遗传修饰。在一组低通行胰腺癌细胞系，原发患者来源的异种移植物和胰腺对照中，对含5-hmC的5-hmC丰富的基因座进行了全基因组分析。结果显示，肿瘤组织中的模式发生了显着变化。不同的羟甲基化区域优先影响基因组的已知调节区域，特别是与已知的H3K4me1增强子重叠。此外，进行了氧化亚硫酸氢盐测序的胞嘧啶甲基化和羟甲基化碱基对分辨率分析，并将其与胰腺癌和对照样品中ATAC-seq的染色质可及性以及RNA-seq的基因表达相关。5-hmC在染色质的开放区域特异富集，并且5-hmC的获得与同源转录本的上调相关，包括与胰腺肿瘤相关的许多致癌途径，例如MYC，KRAS，VEGFA和BRD4。具体而言，在大多数肿瘤样品中，BRD4过表达并在增强子区域获得了5-hmC的表达。在功能上，在BRD4启动子处获得5-hmC与报告基因测定和主要样品中转录表达的增加有关。此外，BRD4的阻滞在体内抑制胰腺癌的生长。总之，在致癌增强子上5-hmC的重新分布和优先富集是人类胰腺癌的新型调节机制。