One of the fundamental challenges in cancer is to detect the regulators of gene expression changes during cancer progression. Through transcriptional silencing of critical cancer-related genes, epigenetic change such as DNA methylation plays a crucial role in cancer. In addition, miRNA, another major component of epigenome, is also a regulator at the post-transcriptional levels that modulate transcriptome changes. However, a mechanistic role of synergistic interactions between DNA methylation and miRNA as epigenetic regulators on transcriptomic changes and its association with clinical outcomes such as survival have remained largely unexplored in cancer. In this study, we propose an integrative framework to identify epigenetic interactions between methylation and miRNA associated with transcriptomic changes. To test the utility of the proposed framework, the bladder cancer data set, including DNA methylation, miRNA expression, and gene expression data, from The Cancer Genome Atlas (TCGA) was analyzed for this study. First, we found 120 genes associated with interactions between the two epigenomic components. Then, 11 significant epigenetic interactions between miRNA and methylation, which target E2F3, CCND1, UTP6, CDADC1, SLC35E3, METRNL, TPCN2, NACC2, VGLL4, and PTEN, were found to be associated with survival. To this end, exploration of TCGA bladder cancer data identified epigenetic interactions that are associated with survival as potential prognostic markers in bladder cancer. Given the importance and prevalence of these interactions of epigenetic events in bladder cancer it is timely to understand further how different epigenetic components interact and influence each other.

Regulation of gene expression by DNA methylation in gene promoter regions is well studied; however, the effects of methylation in the gene body (exons and introns) on gene expression are comparatively understudied. Recently, hypermethylation has been implicated in the inclusion of alternatively spliced exons; moreover, exon recognition can be enhanced by recruiting the methyl-CpG-binding protein (MeCP2) to hypermethylated sites. This study examines whether the methylation status of an intron is correlated with how frequently the intron is retained during splicing using DNA methylation and RNA sequencing data from breast cancer tissue specimens in The Cancer Genome Atlas. Interestingly, hypomethylation of introns is correlated with higher levels of intron expression in mRNA and the methylation level of an intron is inversely correlated with its retention in mRNA from the gene in which it is located. Furthermore, significant population differences were observed in the methylation level of retained introns. In African-American donors, retained introns were not only less methylated compared to European-American donors, but also were more highly expressed. This underscores the need for understanding epigenetic differences in populations and their correlation with breast cancer is an important step toward achieving personalized cancer care. Implications: This research contributes to the understanding of how epigenetic markers in the gene body communicate with the transcriptional machinery to control transcript diversity and differential biological response to changes in methylation status could underlie some of the known, yet unexplained, disparities in certain breast cancer patient populations. Mol Cancer Res; 16(3); 461–9. ©2018 AACR.

At least 90% of human genes are alternatively spliced. Alternative splicing has an important function regulating gene expression and miss-splicing can contribute to risk for human diseases, including Alzheimer’s disease (AD). We developed a splicing decision model as a molecular mechanism to identify functional exon skipping events and genetic variation affecting alternative splicing on a genome-wide scale by integrating genomics, transcriptomics, and neuroimaging data in a systems biology approach. In this study, we analyzed RNA-Seq data of hippocampus brain tissue from Alzheimer’s disease (AD; n = 24) and cognitively normal elderly controls (CN; n = 50) and identified three exon skipping events in two genes (RELN and NOS1) as significantly associated with AD (corrected p-value < 0.05 and fold change > 1.5). Next, we identified single-nucleotide polymorphisms (SNPs) affecting exon skipping events using the splicing decision model and then performed an association analysis of SNPs potentially affecting three exon skipping events with a global cortical measure of amyloid-β deposition measured by [18F] Florbetapir position emission tomography (PET) scan as an AD-related quantitative phenotype. A whole-brain voxel-based analysis was also performed. Two exons in RELN and one exon in NOS1 showed significantly lower expression levels in the AD participants compared to CN participants, suggesting that the exons tend to be skipped more in AD. We also showed the loss of the core protein structure due to the skipped exons using the protein 3D structure analysis. The targeted SNP-based association analysis identified one intronic SNP (rs362771) adjacent to the skipped exon 24 in RELN as significantly associated with cortical amyloid-β levels (corrected p-value < 0.05). This SNP is within the splicing regulatory element, i.e., intronic splicing enhancer. The minor allele of rs362771 conferred decreases in cortical amyloid-β levels in the right temporal and bilateral parietal lobes. Our results suggest that exon skipping events and splicing-affecting SNPs in the human hippocampus may contribute to AD pathogenesis. Integration of multiple omics and neuroimaging data provides insights into possible mechanisms underlying AD pathophysiology through exon skipping and may help identify novel therapeutic targets.

Eukaryotic organisms have developed a variety of mechanisms to regulate translation post-transcriptionally, including but not limited to the use of miRNA silencing in many species. One method of post-transcriptional regulation is through miRNAs that bind to the 3′ UTRs to regulate mRNA abundance and influence protein expression. Therefore, the diversity of mRNA 3′ UTRs mediating miRNA binding sites influence miRNA-mediated regulation. Alternative polyadenylation, by shortening mRNA isoforms, increases the diversity of 3′ UTRs; moreover, short mRNA isoforms elude miRNA-medicated repression. Because no current prediction methods for putative miRNA target sites consider whether or not 1) splicing-informed miRNA binding sites and/or 2) the use of 3′ UTRs provide higher resolution or functionality, we sought to identify not only the genome-wide impact of using exons in mRNA 3′ UTRs but also their functional connection to miRNA regulation and clinical outcomes in cancer. With a genome-wide expression of mRNA and miRNA quantified by 395 bladder cancer cases from The Cancer Genome Atlas (TCGA), we 1) demonstrate the diversity of 3′ UTRs affecting miRNA efficiency and 2) identify a set of genes clinically associated with mRNA expression in bladder cancer. Knowledge of 3′ UTR diversity will not only be a useful addition to current miRNA target prediction algorithms but also enhance the clinical utility of mRNA isoforms in the expression of mRNA in cancer. Thus, variability among cancer patient’s variability in molecular signatures based on these exon usage events in 3′ UTR along with miRNAs in bladder cancer may lead to better prognostic/treatment strategies for improved precision medicine.

TREM2 is among the most well-known Alzheimer’s disease (AD) risk genes; however, the functional roles of its AD-associated variants remain to be elucidated, and most known risk alleles are low-frequency variants whose investigation is challenging. Here, we utilized a splicing-guided aggregation method in which multiple low-frequency TREM2 variants were bundled together to investigate the functional impact of those variants on alternative splicing in AD. We analyzed whole genome sequencing (WGS) and RNA-seq data generated from cognitively normal elderly controls (CN) and AD patients in two independent cohorts, representing three regions in the frontal lobe of the human brain: the dorsolateral prefrontal cortex (CN = 213 and AD = 376), frontal pole (CN = 72 and AD = 175), and inferior frontal (CN = 63 and AD = 157). We observed an exon skipping event in the second exon of TREM2, with that exon tending to be more frequently skipped (p = 0.0012) in individuals having at least one low-frequency variant that caused loss-of-function for a splicing regulatory element. In addition, genes differentially expressed between AD patients with high vs. low skipping of the second exon (i.e., loss of a TREM2 functional domain) were significantly enriched in immune-related pathways. Our splicing-guided aggregation method thus provides new insight into the regulation of alternative splicing of the second exon of TREM2 by low-frequency variants and could be a useful tool for further exploring the potential molecular mechanisms of multiple, disease-associated, low-frequency variants.

Clusterin (CLU) is one of the risk genes most associated with late onset Alzheimer’s disease (AD), and several genetic variants in CLU are associated with AD risk. However, the functional role of known AD risk genetic variants in CLU has been little explored. We investigated the effect of an AD risk variant (rs7982) in the 5th exon of CLU on alternative splicing by using an integrative approach of brain-tissue-based RNA-Seq and whole genome sequencing data from Accelerating Medicines Partnership—Alzheimer’s Disease (AMP-AD). RNA-Seq data were generated from three regions in the temporal lobe of the brain—the temporal cortex, superior temporal gyrus, and parahippocampal gyrus. The rs7982 was significantly associated with intron retention (IR) of the 5th exon of CLU; as the number of alternative alleles (G) increased, the IR rates decreased more significantly in females than in males. Our results suggest a sex-dependent role of rs7982 in AD pathogenesis via splicing regulation.