Rationale:: Misregulation of angiotensin II (Ang II) actions can lead to atherosclerosis and hypertension. Evaluating transcriptomic responses to Ang II in vascular smooth muscle cells (VSMCs) is important to understand the gene networks regulated by Ang II, which might uncover previously unidentified mechanisms and new therapeutic targets. Objective:: To identify all transcripts, including novel protein-coding and long noncoding RNAs, differentially expressed in response to Ang II in rat VSMCs using transcriptome and epigenome profiling. Methods and Results:: De novo assembly of transcripts from RNA-sequencing revealed novel protein-coding and long noncoding RNAs (lncRNAs). The majority of the genomic loci of these novel transcripts are enriched for histone H3 lysine-4-trimethylation and histone H3 lysine-36-trimethylation, 2 chromatin modifications found at actively transcribed regions, providing further evidence that these are bonafide transcripts. Analysis of transcript abundance identified all protein-coding and lncRNAs regulated by Ang II. We further discovered that an Ang II–regulated lncRNA functions as the host transcript for miR-221 and miR-222 , 2 microRNAs implicated in cell proliferation. Additionally, small interfering RNA-mediated knockdown of Lnc-Ang362 reduced proliferation of VSMCs. Conclusions:: These data provide novel insights into the epigenomic and transcriptomic effects of Ang II in VSMCs. They provide the first identification of Ang II–regulated lncRNAs, which suggests functional roles for these lncRNAs in mediating cellular responses to Ang II. Furthermore, we identify an Ang II–regulated lncRNA that is responsible for the production of 2 microRNAs implicated in VSMC proliferation. These newly identified noncoding transcripts could be exploited as novel therapeutic targets for Ang II–associated cardiovascular diseases.

Background: DNA methylation patterns are initiated by de novo DNA methyltransferases DNMT3a/3b adding methyl groups to CG dinucleotides in the hypomethylated genome of early embryos. These patterns are faithfully maintained by DNMT1 during DNA replication to ensure epigenetic inheritance across generations. However, this two-step model is based on limited data. Results: We generated base-resolution DNA methylomes for a series of DNMT knockout embryonic stem cells, with deep coverage at highly repetitive elements. We show that DNMT1 and DNMT3a/3b activities work complementarily and simultaneously to establish symmetric CG methylation and CHH (H = A, T or C) methylation. DNMT3a/3b can add methyl groups to daughter strands after each cycle of DNA replication. We also observe an unexpected division of labor between DNMT1 and DNMT3a/3b in suppressing retrotransposon long terminal repeats and long interspersed elements, respectively. Our data suggest that mammalian cells use a specific CG density threshold to predetermine methylation levels in wild-type cells and the magnitude of methylation reduction in DNMT knockout cells. Only genes with low CG density can be induced or, surprisingly, suppressed in the hypomethylated genome. Lastly, we do not find any association between gene body methylation and transcriptional activity. Conclusions: We show the concerted actions of DNMT enzymes in the establishment and maintenance of methylation patterns. The finding of distinct roles of DNMT1-dependent and -independent methylation patterns in genome stability and regulation of transcription provides new insights for understanding germ cell development, neuronal diversity, and transgenerational epigenetic inheritance and will help to develop next-generation DNMT inhibitors.

Obesity is a highly heritable complex disease that results from the interaction of multiple genetic and environmental factors. Formerly obese individuals are susceptible to metabolic disorders later in life, even after lifestyle changes are made to mitigate the obese state. This is reminiscent of the metabolic memory phenomenon originally observed for persistent complications in diabetic patients, despite subsequent glycemic control. Epigenetic modifications represent a potential mediator of this observed memory. We previously demonstrated that a high fat diet leads to changes in chromatin accessibility in the mouse liver. The regions of greatest chromatin changes in accessibility are largely strain-dependent, indicating a genetic component in diet-induced chromatin alterations. We have now examined the persistence of diet-induced chromatin accessibility changes upon diet reversal in two strains of mice. We find that a substantial fraction of loci that undergo chromatin accessibility changes with a high fat diet remains in the remodeled state after diet reversal in C57BL/6J mice. In contrast, the vast majority of diet-induced chromatin accessibility changes in A/J mice are transient. Our data also indicate that the persistent chromatin accessibility changes observed in C57BL/6J mice are associated with specific transcription factors and histone post-translational modifications. The persistent loci identified here are likely to be contributing to the overall phenotype and are attractive targets for therapeutic intervention.

Metabolic diseases result from multiple genetic and environmental factors. We report here that one manner in which environmental factors can contribute to metabolic disease progression is through modification to chromatin. We demonstrate that high fat diet leads to chromatin remodeling in the livers of C57BL/6J mice, as compared with mice fed a control diet, and that these chromatin changes are associated with changes in gene expression. We further show that the regions of greatest variation in chromatin accessibility are targeted by liver transcription factors, including HNF4α, CCAAT/enhancer-binding protein α (CEBP/α), and FOXA1. Repeating the chromatin and gene expression profiling in another mouse strain, DBA/2J, revealed that the regions of greatest chromatin change are largely strain-specific and that integration of chromatin, gene expression, and genetic data can be used to characterize regulatory regions. Our data indicate dramatic changes in the epigenome due to diet and demonstrate strain-specific dynamics in chromatin remodeling.

PD-L1 has two receptors: PD-1 and CD80. Previous reports assumed that PD-L1 and CD80 interacted in trans, but recent reports showed that only cis PD-L1/CD80 interactions existed, and prevention of cis PD-L1/CD80 interactions on antigen-presenting cells (APCs) reduced antitumor immunity via augmenting PD-L1/PD-1 and CD80/CTLA4 interactions between T and APCs. Here, using tumor-bearing mice capable of cis and trans or trans only PD-L1/CD80 interactions, we show that trans PD-L1/CD80 interactions do exist between tumor and T cells, and the effects of trans PD-L1/CD80 interactions require tumor cell expression of MHC-I and T cell expression of CD28. The blockade of PD-L1/CD80 interactions in mice with both cis and trans interactions or with only trans interactions augments antitumor immunity by expanding IFN-γ–producing CD8 ⁺ T cells and IFN-γ–dependent NOS2-expressing tumor-associated macrophages. Our studies indicate that although cis and trans PD-L1/CD80 interactions may have opposite effects on antitumor immunity, the net effect of blocking PD-L1/CD80 interactions in vivo augments CD8 ⁺ T cell-mediated antitumor immunity.

Significance: Histone modifications associated with gene silencing typically mark large contiguous regions of the genome forming repressive chromatin domain structures. Since the repressive domains exist in close proximity to active regions, maintenance of domain structure is critically important. This study shows that nickel, a nonmutagenic carcinogen, can disrupt histone H3 lysine 9 dimethylation (H3K9me2) domain structures genome-wide, resulting in spreading of H3K9me2 marks into the active regions, which is associated with gene silencing. Our results suggest inhibition of DNA binding of the insulator protein CCCTC-binding factor (CTCF) at the H3K9me2 domain boundaries as a potential reason for H3K9me2 domain disruption. These findings have major implications in understanding chromatin dynamics and the consequences of chromatin domain disruption during pathogenesis.