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High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products. Moreover, it provides information on the methylation pattern, e.g., the occurrence of monoallelic methylation. HRM assays have to be calibrated by analyzing DNA methylation standards of known methylation status and mixtures thereof. In general, DNA methylation levels determined by the classical calibration approach, including the whole temperature range in between normalization intervals, are in good agreement with the mean of the DNA methylation status of individual CpGs determined by pyrosequencing (PSQ), the gold standard of targeted DNA methylation analysis. However, the classical calibration approach leads to highly inaccurate results for samples with heterogeneous DNA methylation since they result in more complex melt curves, differing in their shape compared to those of DNA standards and mixtures thereof. Here, we present a novel calibration approach, i.e., temperature-wise calibration. By temperature-wise calibration, methylation profiles over temperature are obtained, which help in finding the optimal calibration range and thus increase the accuracy of HRM data, particularly for heterogeneous DNA methylation. For explaining the principle and demonstrating the potential of the novel calibration approach, we selected the promoter and two enhancers of MGMT, a gene encoding the repair protein MGMT. [ABSTRACT FROM AUTHOR]

Background: High expression of inhibitor of DNA binding 1 (ID1) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation in regulating ID1 transcription is limited. Methods: Immunohistochemistry (IHC), quantitative RT-PCR (RT-qPCR) and Western blotting (WB) were used to determine the expression of ID1. CRISPR-Cas9 was used to generate ID1 or enhancer E1 knockout cell lines. Dual-luciferase reporter assay, chromosome conformation capture assay and ChIP-qPCR were used to determine the active enhancers of ID1. Cell Counting Kit 8, colony-forming, transwell assays and tumorigenicity in nude mice were used to investigate the biological functions of ID1 and enhancer E1. Results: Human CRC tissues and cell lines expressed a higher level of ID1 than normal controls. ID1 promoted CRC cell proliferation and colony formation. Enhancer E1 actively regulated ID1 promoter activity. Signal transducer and activator of transcription 3 (STAT3) bound to ID1 promoter and enhancer E1 to regulate their activity. The inhibitor of STAT3 Stattic attenuated ID1 promoter and enhancer E1 activity and the expression of ID1. Enhancer E1 knockout down-regulated ID1 expression level and cell proliferation in vitro and in vivo. Conclusions: Enhancer E1 is positively regulated by STAT3 and contributes to the regulation of ID1 to promote CRC cell progression and might be a potential target for anti-CRC drug studies. [ABSTRACT FROM AUTHOR]

Understanding the gene regulatory network governing cancer initiation and progression is necessary, although it remains largely unexplored. Enhancer elements represent the center of this regulatory circuit. The study aims to identify the gene expression change driven by copy number variation in enhancer elements of pancreatic adenocarcinoma (PAAD). The pancreatic tissue specific enhancer and target gene data were taken from EnhancerAtlas. The gene expression and copy number data were taken from The Cancer Genome Atlas (TCGA). Differentially expressed genes (DEGs) and copy number variations (CNVs) were identified between matched tumor-normal samples of PAAD. Significant CNVs were matched onto enhancer coordinates by using genomic intersection functionality from BEDTools. By combining the gene expression and CNV data, we identified 169 genes whose expression shows a positive correlation with the CNV of enhancers. We further identified 16 genes which are regulated by a super enhancer and 15 genes which have high prognostic potential (Z-score > 1.96). Cox proportional hazard analysis of these genes indicates that these are better predictors of survival. Taken together, our integrative analytical approach identifies enhancer CNV-driven gene expression change in PAAD, which could lead to better understanding of PAAD pathogenesis and to the design of enhancer-based cancer treatment strategies. [ABSTRACT FROM AUTHOR]

Deciphering the code of cis-regulatory element (CRE) is one of the core issues of current biology. As an important category of CRE, enhancers play crucial roles in gene transcriptional regulations in a distant manner. Further, the disruption of an enhancer can cause abnormal transcription and, thus, trigger human diseases, which means that its accurate identification is currently of broad interest. Here, we introduce an innovative concept, i.e., abelian complexity function (ACF), which is a more complex extension of the classic subword complexity function, for a new coding of DNA sequences. After feature selection by an upper bound estimation and integration with DNA composition features, we developed an enhancer prediction model with hybrid abelian complexity features (HACF). Compared with existing methods, HACF shows consistently superior performance on three sources of enhancer datasets. We tested the generalization ability of HACF by scanning human chromosome 22 to validate previously reported super-enhancers. Meanwhile, we identified novel candidate enhancers which have supports from enhancer-related ENCODE ChIP-seq signals. In summary, HACF improves current enhancer prediction and may be beneficial for further prioritization of functional noncoding variants. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (apoE) is mainly secreted by hepatocytes and incorporated into most plasma lipoproteins. Macrophages, which accumulate cholesterol and are critical for the development of the atherosclerotic plaque, are also an important, albeit smaller, apoE source. Distal regulatory elements control cell-specific activity of the apoE promoter: multienhancers (ME.1/2) in macrophages and hepatic control regions (HCR-1/2) in hepatocytes. A member of AP-1 cell growth regulator, c-Jun regulates the transcription of various apolipoproteins and proinflammatory molecules implicated in atherosclerosis. We aimed to investigate the effect of c-Jun on apoE expression in macrophages versus hepatocytes and to reveal the underlying molecular mechanisms. Herein we show that c-Jun had an opposite, cell-specific effect on apoE expression: downregulation in macrophages but upregulation in hepatocytes. Transient transfections using ME.2 deletion mutants and DNA pull-down (DNAP) assays showed that the inhibitory effect of c-Jun on the apoE promoter in macrophages was mediated by a functional c-Jun binding site located at 301/311 on ME.2. In hepatocytes, c-Jun overexpression strongly increased apoE expression, and this effect was due to c-Jun binding at the canonical site located at −94/−84 on the apoE proximal promoter, identified by transient transfections using apoE deletion mutants, DNAP, and chromatin immunoprecipitation assays. Overall, the dual effect of c-Jun on apoE gene expression led to decreased cholesterol efflux in macrophages resident in the atherosclerotic plaque synergized with an increased level of systemic apoE secreted by the liver to exacerbate atherogenesis. [ABSTRACT FROM AUTHOR]