Epigenomic Profiling Advises Therapeutic Potential of Leukotriene Receptor Inhibitors for a Subset of Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype, with a poor survival rate compared to others subtypes. For a long time, chemotherapy was the only systemic treatment for TNBC, and the identification of actionable molecular targets might ultimately improve the prognosis for TNBC patients. We performed a genome-wide analysis of DNA methylation at CpG islands on a collection of one hundred ten breast carcinoma samples and six normal breast tissue samples using reduced representation bisulfite sequencing with the XmaI restriction enzyme (XmaI-RRBS) and identified a subset of TNBC samples with significant hypomethylation at the LTB4R/LTB4R2 genes' CpG islands, including CpG dinucleotides covered with cg12853742 and cg21886367 HumanMethylation 450K microarray probes. Abnormal DNA hypomethylation of this region in TNBC compared to normal samples was confirmed by bisulfite Sanger sequencing. Gene expression generally anticorrelates with promoter methylation, and thus, the promoter hypomethylation detected and confirmed in our study might be revealed as an indirect marker of high LTB4R/LTB4R2 expression using a simple methylation-sensitive PCR test. Analysis of RNA-seq expression and DNA methylation data from the TCGA dataset demonstrates that the expression of the LTB4R and LTB4R2 genes significantly negatively correlates with DNA methylation at both CpG sites cg12853742 (R = -0.4, p = 2.6 × 10-6; R = -0.21, p = 0.015) and cg21886367 (R = -0.45, p = 7.3 × 10-8; R = -0.24, p = 0.005), suggesting the upregulation of these genes in tumors with abnormal hypomethylation of their CpG island. Kaplan-Meier analysis using the TCGA-BRCA gene expression and clinical data revealed poorer overall survival for TNBC patients with an upregulated LTB4R. To this day, only the leukotriene inhibitor LY255283 has been tested on an MCF-7/DOX cell line, which is a luminal A breast cancer molecular subtype. Other studies compare the effects of Montelukast and Zafirlukast (inhibitors of the cysteinyl leukotriene receptor, which is different from LTB4R/LTB4R2) on the MDA-MB-231 (TNBC) cell line, with high methylation and low expression levels of LTB4R. In our study, we assess the therapeutic effects of various drugs (including leukotriene receptor inhibitors) with the DepMap gene effect and drug sensitivity data for TNBC cell lines with hypomethylated and upregulated LTB4R/LTB4R2 genes. LY255283, Minocycline, Silibinin, Piceatannol, Mitiglinide, 1-Azakenpaullone, Carbetocin, and Pim-1-inhibitor-2 can be considered as candidates for the additional treatment of TNBC patients with tumors demonstrating LTB4R/LTB4R2 hypomethylation/upregulation. Finally, our results suggest that the epigenetic status of leukotriene B4 receptors is a novel, potential, predictive, and prognostic biomarker for TNBC. These findings might improve individualized therapy for TNBC patients by introducing new therapeutic adjuncts as anticancer agents.

Epigenetic disruptions in the offspring hypothalamus in response to maternal infection.

DNA methylation is an epigenetic modification that can alter gene expression, and the incidence can vary across developmental stages, inflammatory conditions, and sexes. The effects of viral maternal viral infection and sex on the DNA methylation patterns were studied in the hypothalamus of a pig model of immune activation during development. DNA methylation at single-base resolution in regions of high CpG density was measured on 24 individual hypothalamus samples using reduced representation bisulfite sequencing. Differential over- and under-methylated sites were identified and annotated to proximal genes and corresponding biological processes. A total of 120 sites were differentially methylated (FDR-adjusted p-value < 0.05) between maternal infection or sex groups. Among the 66 sites differentially methylated between groups exposed to inflammatory signals and control, most sites were over-methylated in the challenged group and included sites in the promoter regions of genes SIRT3 and NRBP1. Among the 54 differentially methylated sites between females and males, most sites were over-methylated in females and included sites in the promoter region of genes TNC and EIF4G1. The analysis of the genes proximal to the differentially methylated sites suggested that biological processes potentially impacted include immune response, neuron migration and ensheathment, peptide signaling, adaptive thermogenesis, and tissue development. These results suggest that translational studies should consider that the prolonged effect of maternal infection during gestation may be enacted through epigenetic regulatory mechanisms that may differ between sexes.

DNA methylation of microRNA-365-1 induces apoptosis of hair follicle stem cells by targeting DAP3.

Background: DNA methylation is a crucial epigenetic alteration involved in diverse biological processes and diseases. Nevertheless, the precise role of DNA methylation in chemotherapeutic drug-induced alopecia remains unclear. This study examined the role and novel processes of DNA methylation in regulating of chemotherapeutic drug-induced alopecia. Methods: A mouse model of cyclophosphamide (CTX)-induced alopecia was established. Hematoxylin-eosin staining and immunohistochemical staining for the Ki67 proportion and a mitochondrial membrane potential assay (JC-1) were performed to assess the structural integrity and proliferative efficiency of the hair follicle stem cells (HFSCs). Immunofluorescence staining and real-time fluorescence quantitative PCR (RT-qPCR) were performed to determine the expression levels of key HFSC markers, namely Lgr5, CD49f, Sox9, CD200, and FZD10. Differential DNA methylation levels between the normal and CTX-induced model groups were determined through simple methylation sequencing and analyzed using bioinformatics tools. The expression levels of miR-365-1, apoptosis markers, and DAP3 were detected through RT-qPCR and western blotting. In parallel, primary mouse HFSCs were extracted and used as a cell model, which was constructed using 4-hydroperoxycyclophosphamide. The luciferase reporter gene assay was conducted to confirm miR-365-1 binding to DAP3. To measure the expression of relevant indicators, superoxide dismutase (SOD) and malondialdehyde (MDA) kits were used. Methylation-specific PCR (MS-PCR) was performed to determine DNA methylation levels. The regulatory relationship within HFSCs was confirmed through plasmid overexpression of miR-365-1 and DAP3. Result: In the alopecia areata model, a substantial number of apoptotic cells were observed within the hair follicles on the mouse backs. Immunofluorescence staining revealed that the expression of HFSC markers significantly reduced in the CTX group. Both RT-qPCR and western blotting demonstrated a noteworthy difference in DNA methyltransferase expression. Simple methylation sequencing unveiled that DNA methylation substantially increased within the dorsal skin of the CTX group. Subsequent screening identified miR-365-1 as the most differentially expressed miRNA. miR-365-1 was predicted and confirmed to bind to the target gene DAP3. In the CTX group, SOD and ATP expression markedly reduced, whereas MDA levels were significantly elevated. Cellular investigations revealed 4-HC-induced cell cycle arrest and decreased expression of HFSC markers. MS-PCR indicated hypermethylation modification of miR-365-1 in the 4-HC-induced HFSCs. The luciferase reporter gene experiment confirmed the binding of miR-365-1 to the DAP3 promoter region. miR-365-1 overexpression dramatically reduced apoptotic protein expression in the HFSCs. However, this effect was slightly reversed after DAP3 overexpression in lentivirus. Conclusion: This study explored the occurrence of miR-365-1 DNA methylation in chemotherapeutic drug-induced alopecia. The results unveiled that miR-365-1 reduces cell apoptosis by targeting DAP3 in HFSCs, thereby revealing the role of DNA methylation of the miR-365-1 promoter in chemotherapeutic drug-induced alopecia.

Wemics: A Single-Base Resolution Methylation Quantification Method for Enhanced Prediction of Epigenetic Regulation.

DNA methylation, an epigenetic mechanism that alters gene expression without changing DNA sequence, is essential for organism development and key biological processes like genomic imprinting and X-chromosome inactivation. Despite tremendous efforts in DNA methylation research, accurate quantification of cytosine methylation remains a challenge. Here, a single-base methylation quantification approach is introduced by weighting methylation of consecutive CpG sites (Wemics) in genomic regions. Wemics quantification of DNA methylation better predicts its regulatory impact on gene transcription and identifies differentially methylated regions (DMRs) with more biological relevance. Most Wemics-quantified DMRs in lung cancer are epigenetically conserved and recurrently occurred in other primary cancers from The Cancer Genome Atlas (TCGA), and their aberrant alterations can serve as promising pan-cancer diagnostic markers. It is further revealed that these detected DMRs are enriched in transcription factor (TF) binding motifs, and methylation of these TF binding motifs and TF expression synergistically regulate target gene expression. Using Wemics on epigenomic-transcriptomic data from the large lung cancer cohort, a dozen novel genes with oncogenic potential are discovered that are upregulated by hypomethylation but overlooked by other quantification methods. These findings increase the understanding of the epigenetic mechanism by which DNA methylation regulates gene expression.

CelFiE-ISH: a probabilistic model for multi-cell type deconvolution from single-molecule DNA methylation haplotypes.

Deconvolution methods infer quantitative cell type estimates from bulk measurement of mixed samples including blood and tissue. DNA methylation sequencing measures multiple CpGs per read, but few existing deconvolution methods leverage this within-read information. We develop CelFiE-ISH, which extends an existing method (CelFiE) to use within-read haplotype information. CelFiE-ISH outperforms CelFiE and other existing methods, achieving 30% better accuracy and more sensitive detection of rare cell types. We also demonstrate the importance of marker selection and of tailoring markers for haplotype-aware methods. While here we use gold-standard short-read sequencing data, haplotype-aware methods will be well-suited for long-read sequencing.

Development of Restricted and Repetitive Behaviors from 2-19: Stability and Change in Repetitive Sensorimotor, Insistence on Sameness, and Verbal Behaviors in a Longitudinal Study of Autism.

This study examined trajectories of repetitive sensorimotor (RSM), insistence on sameness (IS), and verbal RRBs from ages 2-19 in a well-characterized longitudinal cohort. We also tested the factor structure of the ADI-R restricted and repetitive behavior (RRB) domain at age 19 and the inclusion of a verbal RRBs factor, in addition to previously identified RSM and IS factors, at ages 9 and 19. The ADI-R was administered to 193 participants at five timepoints from 2 to 19. Confirmatory factor analysis (CFA) was used to examine the factor structure of ADI-R RRB data. Change in RRB subtypes was examined using group-based trajectory modeling and multilevel modeling. RSM and IS behaviors generally decreased from 2 to 19, with some participants experiencing increases from ages 2-9. 46.46% of participants experienced increasing verbal RRBs from 2 to 9, followed by a plateau from 9 to 19. The remaining participants had few to no verbal RRBs throughout development. Multilevel modeling indicated ADOS CSS scores were associated with change in RSM trajectories only. Child IQ was not associated with change in any RRB trajectories. CFA indicated previously-derived RSM and IS factors fit age 19 ADI-R data well. The inclusion of a third factor, verbal RRBs, worsened goodness-of-fit statistics and was ultimately omitted. Our findings suggest the RRB factor structure of the ADI remains consistent into early adulthood and illustrate developmental continuity and change in RRBs. For autistic individuals with fluent speech, RRB presentation may be influenced by verbal ability in ways that are highly variable, but clinically meaningful.

Methylation changes of liver DNA during the formation of gallstones.

Aim: To explore the overall methylation changes in liver tissues during the formation of gallstones, as well as the key pathways and genes involved in the process. Methods: Reduced-representation bisulfite sequencing and RNA sequencing were conducted on the liver tissues of mice with gallstones and control normal mice. Results: A total of 8705 differentially methylated regions in CpG and 1410 differentially expressed genes were identified. The joint analysis indicated that aberrant DNA methylation may be associated with dysregulated gene expression in key pathways such as cholesterol metabolism and bile secretion. Conclusion: We propose for the first time that methylation changes in some key pathway genes in liver tissue may be involved in the formation of gallstones.

Molecular adaptations in response to exercise training are associated with tissue-specific transcriptomic and epigenomic signatures.

Regular exercise has many physical and brain health benefits, yet the molecular mechanisms mediating exercise effects across tissues remain poorly understood. Here we analyzed 400 high-quality DNA methylation, ATAC-seq, and RNA-seq datasets from eight tissues from control and endurance exercise-trained (EET) rats. Integration of baseline datasets mapped the gene location dependence of epigenetic control features and identified differing regulatory landscapes in each tissue. The transcriptional responses to 8 weeks of EET showed little overlap across tissues and predominantly comprised tissue-type enriched genes. We identified sex differences in the transcriptomic and epigenomic changes induced by EET. However, the sex-biased gene responses were linked to shared signaling pathways. We found that many G protein-coupled receptor-encoding genes are regulated by EET, suggesting a role for these receptors in mediating the molecular adaptations to training across tissues. Our findings provide new insights into the mechanisms underlying EET-induced health benefits across organs.

DNA Methylation Profiling in Genetically Selected Clarias magur (Hamilton, 1822) Provides Insights into the Epigenetic Regulation of Growth and Development.

DNA methylation is an epigenetic alteration that impacts gene expression without changing the DNA sequence affecting an organism's phenotype. This study utilized a reduced representation bisulfite sequencing (RRBS) approach to investigate the patterns of DNA methylation in genetically selected Clarias magur stocks. RRBS generated 249.22 million reads, with an average of 490,120 methylation sites detected in various parts of genes, including exons, introns, and intergenic regions. A total of 896 differentially methylated regions (DMRs) were identified; 356 and 540 were detected as hyper-methylated and hypo-methylated regions, respectively. The DMRs and their association with overlapping genes were explored using whole genome data of magur, which revealed 205 genes in exonic, 210 in intronic, and 480 in intergenic regions. The analysis identified the maximum number of genes enriched in biological processes such as RNA biosynthetic process, response to growth factors, nervous system development, neurogenesis, and anatomical structure morphogenesis. Differentially methylated genes (DMGs) such as myrip, mylk3, mafb, egr3, ndnf, meis2a, foxn3, bmp1a, plxna3, fgf6, sipa1l1, mcu, cnot8, trim55b, and myof were associated with growth and development. The selected DMGs were analyzed using real-time PCR, which showed altered mRNA expression levels. This work offers insights into the epigenetic mechanisms governing growth performance regulation in magur stocks. This work provides a valuable resource of epigenetic data that could be integrated into breeding programs to select high-performing individuals.

Barriers to an Autistic Identity: How RRBs may Contribute to the Underdiagnosis of Females.

Background: Autistic females are frequently underdiagnosed, misdiagnosed, and/or diagnosed later in life. Restricted and Repetitive Behaviors (RRBs) are increasingly critical for diagnosis and yet are commonly rated lower in females. Whether this reflects genuinely lower levels of these traits, or if female-typical RRBs have a different phenotypic presentation that may not register on current quantitative measurement tools is unclear. Methods: Twenty-one autistic females and 21 autistic males matched on chronological age and FSIQ completed the AQ, ADOS-2, and ADI-R. Items from the ADOS-2 and ADI-R were selected that were relevant to the four areas of restricted and repetitive behavior in the DSM-5 diagnostic criteria of autism. Using a mixed-methods analytical approach, scores and comments on these measures were compared between sexes to better characterize RRBs in autistic females. Results: There were no sex differences on the AQ, which broadly assesses autistic traits. When analyzed by the four DSM-5 RRB criteria, there were no sex differences on the ADI-R when using traditional algorithm scoring that narrows questions down to those that are more sensitive and specific in capturing autism in research samples with a high proportion of males. When incorporating additional items relevant to the DSM-5 to identify sex changes in a broader pool of items, females scored higher on stereotyped movements and speech. Females also engaged in more sensory behaviors during the ADOS-2. Qualitative analyses indicated that females were more likely to engage in stereotyped body rocking and spinning, stereotyped behaviors when anxious, to show major reactions to changes, repetitive language including listing and counting, and sensory behaviors, especially in the tactile domain. Conclusion: Exploratory findings highlight sex differences in RRBs that may help enhance diagnostic clarity for females. Higher tactile sensory behaviors in females suggests there may be increased diagnostic sensitivity for females with the updated DSM-5 that now includes sensory components as part of the diagnostic criteria.

Integration of RRBS and RNA-seq unravels the regulatory role of DNMT3A in porcine Sertoli cell proliferation.

DNMT3A participates in de novo methylation, yet its impact on the proliferation of testicular Sertoli cells remains unclear. Development-specific methylation has been proven to be associated with cellular development. Therefore, in this study, we simulated DNMT3A expression pattern during testicular development by DNMT3A interference. Then, RRBS and RNA-seq were used to decipher DNMT3A regulatory mechanisms on Sertoli cell proliferation. Immunofluorescence staining revealed the expression of DNMT3A in the Sertoli cells of the prepubertal testis. DNMT3A was demonstrated to inhibit the cell cycle and proliferation of Sertoli cells, while promoting cell apoptosis. After transfected with DNMT3A interference, a total of 560 DEGs and 2,091 DMGs produced by DNMT3A interference were identified between two treated groups, respectively. Integrating the results from RRBS and RNA-seq, the overlapping genes between DMGs and DEGs were found to be enriched in the Gene Ontology (GO) terms related to cellular development and the Apelin signaling pathway. The present study demonstrated the impact of DNMT3A on the proliferation of porcine testicular Sertoli cells, suggesting that DNMT3A primarily acts through the Apelin signaling pathway. These findings provide valuable insights into how DNMT3A influences testicular development and health, offering new perspectives.