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一个标志，可能是由于表观遗传改变所致。 5-羟甲基胞嘧啶 (5-hmC) 是一种表观遗传修饰，尚未在胰腺癌中进行研究。在低传代胰腺癌细胞系、原发性患者来源的异种移植物和胰腺对照队列中，使用 hmC-seal 对富含 5-hmC 的位点进行全基因组分析，结果揭示了肿瘤组织中显着改变的模式。差异羟甲基化区域优先影响基因组的已知调控区域，特别是与已知的 H3K4me1 增强子重叠。此外，利用氧化亚硫酸氢盐测序对胞嘧啶甲基化和羟甲基化进行碱基对解析分析，并通过 ATAC-seq 与胰腺癌和对照样本中的染色质可及性以及 RNA-seq 的基因表达相关联。 5-hmC 在染色质的开放区域特异性富集，并且 5-hmC 的增加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 。具体来说，在大多数肿瘤样本中， BRD4过度表达并在增强子区域获得 5-hmC。从功能上讲， BRD4启动子处 5-hmC 的获得与报告基因检测和初级样品中转录本表达的增加相关。此外，阻断 BRD4 可抑制体内胰腺癌的生长。总之，5-hmC 的重新分配和致癌增强子的优先富集是人类胰腺癌的一种新型调节机制。