Stroke triggers dynamic m6A reprogramming of cerebral circular RNAs.

We previously showed that stroke alters circular RNA (circRNA) expression profiles. Many circRNAs undergo epitranscriptomic modifications, particularly methylation of adenosine to form N6-methyladenosine (m6A). This modification significantly influences the circRNA metabolism and functionality. Hence, we currently evaluated if transient focal ischemia in adult C57BL/6J mice alters the m6A methylation of circRNAs. Changes in m6A were profiled in the peri-infarct cortex following immunoprecipitation coupled with microarrays. Correlation and gene ontology analyses were performed to understand the association of m6A changes with circRNA regulation and functional implications after stroke. Many circRNAs showed differential regulation (up or down) after stroke, and this change was highest at 24h of reperfusion. Notably, most circRNAs differentially regulated after stroke also exhibited temporal changes in m6A modification patterns. The majority of circRNAs that showed post-stroke differential m6A modifications were derived from protein-coding genes. Hyper-than hypomethylation of circRNAs was most prevalent after stroke. Gene ontology analysis of the host genes suggested that m6A-modified circRNAs might regulate functions such as synapse-related processes, indicating that m6A epitranscriptomic modification in circRNAs could potentially influence post-stroke synaptic pathophysiology.

ITGB2 is a central hub-gene associated with inflammation and early fibro-atheroma development in a swine model of atherosclerosis.

Background and aim: The complex dynamic interplay between different biological pathways involved in atherosclerosis development has rendered the identification of specific therapeutic targets a challenging quest. We aimed to identify specific genes and mechanistic pathways associated with the early development of fibro-atheromas in a swine model of atherosclerosis. Methods: The Wisconsin Miniature Swine™ model of Familial Hypercholesterolemia (WMS-FH, n = 11) and genetically related WMS controls (WMS-N, n = 11) were used. The infrarenal aorta was harvested from both groups for histopathologic and transcriptomic profiling at 12 months. Bioinformatic analysis was performed to identify hub genes and pathways central to disease pathophysiology. The expression of ITGB2, the top ranked hub gene, was manipulated in cell culture and the expression of interconnected genes was tested. Results: Fibro-atheromatous lesions were documented in all WMS-FH aortic tissues and displayed internal elastic lamina (IEL) disruption, significant reduction of myofibroblast presence and disorganized collagen deposition. No fibro-atheromas were observed in the control group. A total of 266 differentially expressed genes (DEGs) were upregulated in WMS-FH aortic tissues, while 29 genes were downregulated. Top identified hub genes included ITGB2, C1QA, LCP2, SPI1, CSF1R, C5AR1, CTSS, MPEG1, C1QC, and CSF2RB. Overexpression of ITGB2 resulted in elevated expression of other interconnected genes expressed in porcine endothelial cells. Conclusion: In a swine translational model of atherosclerosis, transcriptomic analysis identified ITGB2 as a central hub gene associated inflammation and early fibroatheroma development making it a potential therapeutic target at this stage of disease.

Longitudinal dynamics of circulating miRNAs in a swine model of familial hypercholesterolemia during early atherosclerosis.

Atherosclerosis is a complex progressive disease involving intertwined biological mechanisms. We aimed to identify miRNA expression dynamics at the early stages of atherosclerosis using a large swine model (Wisconsin Miniature Swine, WMS). A total of 18 female pigs; 9 familial hypercholesterolemic (WMS-FH) and 9 normal control swine (WMS-N) were studied. miRNA sequencing was performed on plasma cell-free RNA at 3, 6, and 9 months of age. RT-qPCR validated DE miRNAs in a new cohort of animals (n = 30) with both sexes. Gene ontology and mRNA targets for DE miRNAs were identified. In vivo multimodality imaging and histopathology were performed to document the presence of atherosclerosis at termination. 20, 19, and 9 miRNAs were significantly DE between the groups at months 3, 6, and 9, respectively. Most DE miRNAs and their target genes are involved in human atherosclerosis development. Coronary atherosclerosis was documented in 7/9 WMS-FH pigs. Control animals had no lesions. miR-138, miR-152, miR-190a, and miR-196a showed a significant diagnostic power at month 3, whereas miR-486, miR-126-3p, miR-335, and miR-423-5p were of significant diagnostic power at month 9. In conclusion, specific DE miRNAs with significant discriminatory power may be promising biomarkers for the early detection of coronary atherosclerosis.

The Activation of Protamine 1 Using Epigenome Editing Decreases the Proliferation of Tumorigenic Cells.

DNA methyltransferases (DNMT) and histone deacetylases (HDAC) inhibitors are used as cancer epigenome drugs. However, these epigenetic drugs lack targeting specificity and could risk inducing genome instability and the expression of oncogenes. Therefore, there is a need to develop new therapeutic strategies where specific cancer genes can be targeted for silencing or activation. The CRISPR/dCas9 system represents a promising, powerful therapeutic tool because of its simplicity and specificity. Protamine 1 (PRM1) is exclusively expressed in sperm and has a vital role in the tight packaging of DNA, thus inducing transcriptional silencing in sperm cells. We hypothesized that the activation of the PRM1 gene in tumorigenic cells would lead to DNA condensation and reduce the proliferation of these cells. To test our hypothesis, we transfected human embryonic kidney cells 293T with a dCas9-P300 plasmid that adds acetyl groups to the promoter region of PRM1 via specific gRNAs plasmids. RNA-Seq analysis of transfected cells revealed high specificity of targeted gene activation. PRM1 expression resulted in a significant decrease in cell proliferation as measured by the BrdU ELISA assay. To confirm that the activation of PRM1 was due to acetyl groups deposited to H3K27, a ChIP-qPCR was performed. The acetylation of the PRM1 promoter region targeted by dCas9-p300 in transfected cells was higher than that of the control cells. Interestingly, the targeted promoter region for acetylation showed reduced DNA methylation. These findings demonstrate the efficacy of epigenome editing in activating PRM1 in non-expressing tumorigenic cells, which could be used as a promising therapeutic strategy in cancer treatment.

Paternal diet induces transgenerational epigenetic inheritance of DNA methylation signatures and phenotypes in sheep model.

Transgenerational epigenetic inheritance (TEI) requires transmission of environmentally induced epigenetic changes and associated phenotypes to subsequent generations without continued exposure to the environmental factor that originated the change. TEI is well-established in plants and Caenorhabditis elegans; however, occurrence in mammals is debated and poorly understood. Here, we examined whether paternal diet from weaning to puberty-induced changes in sperm DNA methylation that were transmitted to subsequent generations. Over 100 methylated cytosines, environmentally altered in the F0 generation, were inherited by the F1 and F2 generations. Furthermore, the F0 paternal diet was associated with growth and male fertility phenotypes in subsequent generations. Differentially methylated cytosines were correlated with gene expression. Our results demonstrate that some sperm methylation sites may escape DNA methylation erasure and are transmitted to subsequent generations despite the 2 waves of epigenetic programming: in primordial germ cells and in embryos after fertilization. These results advance our understanding of the complex relationships between nature and nurture.

Integrative analysis of methylomic and transcriptomic data in fetal sheep muscle tissues in response to maternal diet during pregnancy.

BACKGROUND: Numerous studies have established a link between maternal diet and the physiological and metabolic phenotypes of their offspring. In previous studies in sheep, we demonstrated that different maternal diets altered the transcriptome of fetal tissues. However, the mechanisms underlying transcriptomic changes are poorly understood. DNA methylation is an epigenetic mark regulating transcription and is largely influenced by dietary components of the one-carbon cycle that generate the methyl group donor, SAM. Therefore, in the present study, we tested whether different maternal diets during pregnancy would alter the DNA methylation and gene expression patterns in fetal tissues. RESULTS: Pregnant ewes were randomly divided into two groups which received either hay or corn diet from mid-gestation (day 67 ± 5) until day 131 ± 1 when fetuses were collected by necropsy. A total of 1516 fetal longissimus dorsi (LD) tissues were used for DNA methylation analysis and gene expression profiling. Whole genome DNA methylation using methyl-binding domain enrichment analysis revealed 60 differentially methylated regions (DMRs) between hay and corn fetuses with 39 DMRs more highly methylated in the hay fetuses vs. 21 DMRs more highly methylated in the corn fetuses. Three DMRs (LPAR3, PLIN5-PLIN4, and the differential methylation of a novel lincRNA) were validated using bisulfite sequencing. These DMRs were associated with differential gene expression. Additionally, significant DNA methylation differences were found at the single CpG level. Integrative methylome and transcriptome analysis revealed an association between gene expression and inter-/intragenic methylated regions. Furthermore, intragenic DMRs were found to be associated with expression of neighboring genes. CONCLUSIONS: The findings of this study imply that maternal diet from mid- to late-gestation can shape the epigenome and the transcriptome of fetal tissues, and putatively affect phenotypes of the lambs.

Male fertility status is associated with DNA methylation signatures in sperm and transcriptomic profiles of bovine preimplantation embryos.

BACKGROUND: Infertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing. RESULTS: Embryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate < 1%. A total of 65 genes were upregulated in high fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions. CONCLUSIONS: Despite similar morphology and development to the blastocyst stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.