Lower Methylation Levels Research Articles

P604: CATALASE EXPRESSION IN LEUKEMIA CELLS IS CONTROLLED BY GENETIC AND EPIGENETIC MECHANISMS

Background: Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in Western countries. It is an incurable disease characterized by an extremely variable clinical course and response to treatment. We have recently shown that high catalase (CAT) expression identifies a more aggressive clinical behavior in CLL. However, molecular mechanisms controlling catalase expression in leukemia cells are still poorly characterized. The rs1001179 single nucleotide polymorphism (SNP) as well as DNA methylation in the CAT promoter have been shown to regulate CAT expression in various cell types. Aims: With the aim to characterize regulatory mechanisms underlying differential expression of catalase in CLL, in this study we investigated the role of rs1001179 SNP and CpG Island II methylation encompassing this SNP in the regulation of CAT expression. Methods: Peripheral blood mononuclear cells (PBMC) from 55 CLL patients and 50 healthy donors (HD) were used for the study. CAT mRNA levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). DNA samples were genotyped for the rs1001179 SNP using Restriction Fragment Length Polymorphism (RFLP)-PCR. Bioinformatic analysis was used to predict transcription factor (TF) binding to SNP sequences. Chromatin Immunoprecipitation (ChIP)-qPCR was performed to validate predicted TF binding sites. Methylation levels of CpG sites within the CAT promoter was determined by pyrosequencing of bisulfite-converted DNA. Results: The rs1001179 SNP genotyping showed that CLL cells harboring the T allele exhibited a significantly higher catalase expression compared with cells bearing the CC genotype. Interestingly, bioinformatic analysis predicted that the SNP provide distinct binding sites for various TF, including ETS-1 and GR-β. ChIP assay confirmed that CAT promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-β, but not to the other analyzed TFs. In addition, CLL cells exhibited lower methylation levels within the CAT promoter compared with HD B cells, in line with the higher catalase mRNA and protein levels expressed by CLL versus HD B cells. Moreover, methylation levels negatively correlated with CAT expression in CLL cells. Remarkably, inhibition of methyltransferase activity in leukemic cells induced a significant increase of CAT mRNA and protein levels, thus showing that DNA methylation could control catalase expression in CLL. Moreover, expression of DNA methyl transferase 1 (DNMT1) resulted significantly reduced in CLL cells compared with HD B cells and inversely correlated with CAT expression in CLL, thus suggesting that differences in methylation levels underlying catalase expression could be driven by the DNMT1 enzyme. Finally, modeling analysis showed that the CT/TT genotypes exhibited a lower methylation and a higher CAT expression level, suggesting that the rs1001179 T allele and methylation cooperate in inducing CAT gene expression. Summary/Conclusion: Our data show that SNP as well as methylation of the catalase promoter are involved in controlling catalase expression in CLL. The key advance of this study is to provide new insights into the regulatory mechanisms underlying differential expression of catalase in leukemic cells, which is of clinical relevance in CLL. Moreover, our study may form the basis for future challenges aimed at developing combinatorial therapies targeting catalase regulatory pathways.

Abstract LB118: CEACAM7 as epigenetic prognostic marker in African Americans with lung adenocarcinoma

Abstract Background and Objectives of the Study: For the past several decades, lung cancer has had amongst the highest cancer incidence and mortality rates across the world. In the United States, African American (AA) individuals carry a disproportionate share of the lung cancer burden, with higher incidence, later-stage diagnosis, and poorer 5-year overall survival rate when compared with that of Whites. However, there is yet to be a race-specific biomarker for lung cancer. Most genomic data continue to include a small proportion of AA individuals, further contributing to the racial health disparity. Thus, the goal of our study was to conduct a genome-wide analysis to identify lung cancer biomarkers specific for AA individuals, to understand the role that these biomarkers play in overall survival, and to discover how epigenetic modulations may impact outcomes by race. Methods: We utilized clinical and genomic data from The Cancer Genome Atlas (TCGA) for lung adenocarcinoma (LUAD). We conducted a differential expression analysis to identify the differentially expressed genes (DEGs) amongst AA individuals compared with Whites with LUAD. Two-tailed log rank tests with univariate and multivariate Cox proportional hazard models adjusted by age, gender, prior malignancy and stage were used to analyze overall survival for mRNA expression and DNA methylation for each racial subgroup. We investigated the epigenetic modifications that are correlated with the expression of the DEGs that play a significant role in the survival of AA individuals with lung cancer. Results: CEACAM7 was the single gene that was both differentially expressed in AA individuals compared to Whites with LUAD and that had an impact on LUAD survival. CEACAM7 RNA expression showed trend towards significant direct correlation with LUAD stage in AA individuals but not for Whites. High RNA expression of CEACAM7 was associated with shorter median survival (P<0.0001) and increased mortality risk HR: 11.84, 95% CI: 2.69-52.17 for AA individuals with LUAD, but there were no differences in survival time or risk for Whites. We also identified 5 CpG probes inversely correlated with the expression of CEACAM7 (cg22895231, cg25465322, cg01656853, cg17659920, and cg07940585). Each one of them showed that low methylation values had significantly shorter overall median survival. Only cg01656853 and cg07940585 showed association between lower DNA methylation levels and increased mortality risk in multivariate COX regression models. Conclusion: This TCGA study suggests that the expression of CEACAM7 and its associated DNA methylation may play an important role in the survival of AA individuals with LUAD but not for Whites. Further studies are necessary to understand the mechanics of how the epigenetic regulation of CEACAM7 is associated with survival in AA individuals with LUAD and to identify new possible therapeutic targets. Citation Format: Richies Tiv, Ignacio Jusue-Torres, Apurva Mallisetty, Leglys Contreras-Vargas, Ludmila Danilova, Lawrence E. Feldman, Alicia Hulbert. CEACAM7 as epigenetic prognostic marker in African Americans with lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB118.

Bioinformatics analyses proposed xenotropic and polytropic retrovirus receptor 1 as a potential diagnostic and prognostic biomarker and immunotherapeutic target in head and neck squamous cell carcinoma

ObjectiveThe role of Xenotropic and polytropic retrovirus receptor 1 (XPR1), a cell surface receptor for certain types of murine leukemia viruses, in human cancers has been rarely studied. We aimed to evaluate the values of XPR1 as a biomarker and therapeutic target in head and neck squamous cell carcinoma (HNSCC). MethodsBioinformatics tools and online databases, including R packages, ONCOMINE, The Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA), UALCAN, MethSurv, cBioPortal, and TIMER2.0 were applied in this study. ResultsThe mRNA and protein expression of XPR1 is significantly up-regulated in HNSCC tissues compared with normal tissues. The receiver operating characteristic (ROC) curve shows XPR1 has high specificity and accuracy in the diagnosis of HNSCC (AUC = 0.883). Patients with high-level expression of XPR1 have poorer overall survival (OS, P = 0.002), disease-specific survival (DSS, P = 0.014), and progress-free interval (PFI, P = 0.017). UALCAN analysis indicates that the methylation of XPR1 promoter in HNSCC is significantly down-regulated. MethSurve was used to investigate the impact of individual CpG islands on the prognosis of HNSCC patients. Low DNA methylation levels of cg11538848 and cg20948051 and high DNA methylation levels of cg23675362, cg18440470, and cg22026687 are significantly related to poor prognosis. The Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicate that XPR1 is involved in various important biological functions and signaling pathways closely related to cancer. The co-expression analysis of XPR1 and N6-methyladenosine (m6A) RNA methylation regulators shows that XPR1 is significantly related to the expression of main m6A regulators. Immune infiltration analysis shows that the expression of XPR1 is related to certain types of immune infiltrating cells and has a positive correlation with the expression of four immune checkpoint genes, PDCD1LG2, CD274, HAVCR2, and SIGLEC15. ConclusionIn summary, these results indicate that XPR1 is a potential diagnostic and prognostic biomarker and immunotherapy target for HNSCC. This study sheds new light on understanding the formation and development of HNSCC and sets the basis for further studying the role of XPR1 in HNSCC and other types of cancers.

Paired box 8 facilitates the c-MYC related cell cycle progress in TP53-mutation uterine corpus endometrial carcinoma through interaction with DDX5

As a molecular marker of the female reproductive system, Paired Box 8 is widely used in pathological diagnosis of gynecological tumors, but it is not clear whether its expression level is related to the development of uterine corpus endometrial carcinoma and molecular subtype classifications. Here, we show that PAX8 is up-regulated in TP53 mutation category of UCEC, which is result from the low methylation level of PAX8 in UCEC. We have identified that genes connected to ribosome, lysosome, ribosome biogenesis and cell cycle as PAX8 targets and demonstrate that modulation of the PAX8-DDX5 interaction influences c-MYC related cell cycle and cell growth. Our work defines DDX5 as a critical PAX8 co-factor, places the PAX8-DDX5 interaction in biological context, and highlights PAX8 as a key point for development of novel anti-MYC therapies in TP53-mutation UCEC.

Overexpression of ERCC6L correlates with poor prognosis and confers malignant phenotypes of lung adenocarcinoma.

Excision repair cross-complementation group 6 like (ERCC6L) has been reported to be upregulated in a variety of malignant tumors and plays a critical oncogenic role. However, the role and molecular mechanism of ERCC6L in lung adenocarcinoma (LUAD) remain unclear, and were therefore investigated in the present study. Clinical data of patients with LUAD were obtained and bioinformatics analysis was performed to investigate the expression characteristics, prognostic value, and biological function of ERCC6L. In addition, cell function experiments were performed to detect the effect of ERCC6L silencing on the biological behavior of LUAD cells. The results revealed that ERCC6L expression was significantly higher in LUAD tissues vs. normal lung tissues and closely associated with nodal invasion, advanced clinical stage and survival in LUAD. Overexpression of ERCC6L was an independent prognostic biomarker of overall survival, progression-free interval, and disease-specific survival in patients with LUAD. DNA amplification and low methylation levels of ERCC6L suggested regulation at both the genetic and epigenetic levels. The most significant positive genes co-expressed with ERCC6L were mainly enriched in the cell cycle signaling pathway. The major functions of ERCC6L in LUAD cells were positively correlated with the cell cycle, DNA damage, DNA repair, proliferation, invasion and epithelial-mesenchymal transition (EMT). Knockdown of ERCC6L inhibited the proliferative, migratory and invasive abilities of A549 and PC9 cells. It also promoted cell apoptosis, and led to cell cycle arrest in the S phase. ERCC6L may regulate the EMT process through the Wnt/β-catenin and Wnt/Notch 3 signaling pathways, thus regulating the tumorigenesis and progression of LUAD. The overexpression of ERCC6L may be a biological indicator for the diagnosis and prognosis of LUAD. ERCC6L may be a novel molecular target for the treatment of lung cancer.

447-P: C/EBPß-Lin28a Positive Feedback Loop Mediated by C/EBPß Hypomethylation Promotes the Proliferation and Migration of Vascular Smooth Muscle Cells in Restenosis

Background: The main reason for restenosis (RS) after percutaneous transluminal angioplasty is the excessive proliferation and migration of vascular smooth muscle cells (VSMCs) . C/EBPβ plays an important role in the process of vascular remodeling. However, the role of C/EBPβ in RS has not been clarified. Methods: The type 2 diabetes rat models of RS and atherosclerosis (AS) were established to detect the difference in DNA methylation and expression levels of C/EBPβ. Simultaneously, the effect of C/EBPβ on the proliferation and migration of VSMCs was clarified by regulating C/EBPβ and Lin28a in vitro. The effect of decitabine, a demethylating agent, on the expression of C/EBPβ and Lin28a, and the further regulation of VSMCs proliferation and migration was confirmed. Results: C/EBPβ was highly expressed and had a lower methylation level in RS compared with AS. In vitro C/EBPβ overexpression facilitated the proliferation and migration of VSMCs with increased expression of Lin28a. Conversely, C/EBPβ knockdown resulted in opposite effects. Chromatin immunoprecipitation assays further demonstrated that C/EBPβ could directly bind to the promoter of Lin28a. Furthermore, increased expression of C/EBPβ and enhanced proliferation and migration of VSMCs were observed after decitabine treatment. And mechanical stretch promoted the expression of C/EBPβ and Lin28a. Additionally, C/EBPβ expression increased following the overexpression of Lin28a. The reversed result was found in the Lin28a knockdown cells. Conclusion: Our results have demonstrated that C/EBPβ expression is regulated partly by DNA methylation. Meanwhile, as a transcription factor, C/EBPβ promotes the transcription of Lin28a and enhances the proliferation and migration of VSMCs through a positive feedback loop between the two. Our study suggests C/EBPβ-Lin28a axis is a driver of the RS progression, providing a promising therapeutic strategy for RS. Disclosure S.Jiang: n/a. L.Liao: None. J.Dong: None. X.Zhou: n/a. Q.Zhang: None. S.Guo: None. Q.Sheng: None. X.Li: None. R.Zhang: None. C.Xu: None. P.Gong: None. Funding National Natural Science Foundation of China Grants (81800732,81670757,82170824) , Shandong Provincial Medicine and Health Science and Technology Development Program (No.202103060766) and Shandong Provincial Hospital Research incubation Fund (No.2021FY059)

Unexpected Low DNA Methylation in Transposable Elements at the 5′-CCGG Sites in Three Fruit Tree Cultivars

DNA methylation of three cultivars, each of the fruit tree species pear, plum and apple, was analyzed by the methylation-sensitive amplified polymorphism (MSAP) marker. All three fruit tree cultivars were found to contain apparently lower levels of methylation at the 5′-CCGG sites than all other plant species, such as rice and wheat, studied by the same method. Sequencing of the representative loci isolated from the MSAP profiles indicated that both protein-coding genes and transposable elements (TEs) were involved in low methylation. Gel blotting using isolated MSAP fragments and fragment mixtures representing two major types of TEs (copia- and gypsy-like) as hybridization probes confirmed the unexpected low DNA methylation levels at the 5′-CCGG sites in these three fruit tree genomes. Our results suggest that the three asexually propagated perennial fruit trees may indeed contain unusual lower levels of DNA methylation, especially in TEs at the 5′-CCGG sites. Additionally, our results may also suggest that the often used MSAP marker, which targets only one kind of specific methylation-sensitive sites recognized by a pair of isoschizomers (e.g., 5′-CCGG by HpaII/MspI), is not always representative of other cytosine sites (e.g., CHH) or CG sites other than those of 5′-CCGGs in some plant species.

Recurrence- and Malignant Progression-Associated Biomarkers in Low-Grade Gliomas and Their Roles in Immunotherapy.

Despite a generally better prognosis than high-grade glioma (HGG), recurrence and malignant progression are the main causes for the poor prognosis and difficulties in the treatment of low-grade glioma (LGG). It is of great importance to learn about the risk factors and underlying mechanisms of LGG recurrence and progression. In this study, the transcriptome characteristics of four groups, namely, normal brain tissue and recurrent LGG (rLGG), normal brain tissue and secondary glioblastoma (sGBM), primary LGG (pLGG) and rLGG, and pLGG and sGBM, were compared using Chinese Glioma Genome Atlas (CGGA) and Genotype-Tissue Expression Project (GTEx) databases. In this study, 296 downregulated and 396 upregulated differentially expressed genes (DEGs) with high consensus were screened out. Univariate Cox regression analysis of data from The Cancer Genome Atlas (TCGA) yielded 86 prognostically relevant DEGs; a prognostic prediction model based on five key genes (HOXA1, KIF18A, FAM133A, HGF, and MN1) was established using the least absolute shrinkage and selection operator (LASSO) regression dimensionality reduction and multivariate Cox regression analysis. LGG was divided into high- and low-risk groups using this prediction model. Gene Set Enrichment Analysis (GSEA) revealed that signaling pathway differences in the high- and low-risk groups were mainly seen in tumor immune regulation and DNA damage-related cell cycle checkpoints. Furthermore, the infiltration of immune cells in the high- and low-risk groups was analyzed, which indicated a stronger infiltration of immune cells in the high-risk group than that in the low-risk group, suggesting that an immune microenvironment more conducive to tumor growth emerged due to the interaction between tumor and immune cells. The tumor mutational burden and tumor methylation burden in the high- and low-risk groups were also analyzed, which indicated higher gene mutation burden and lower DNA methylation level in the high-risk group, suggesting that with the accumulation of genomic mutations and epigenetic changes, tumor cells continued to evolve and led to the progression of LGG to HGG. Finally, the value of potential therapeutic targets for the five key genes was analyzed, and findings demonstrated that KIF18A was the gene most likely to be a potential therapeutic target. In conclusion, the prediction model based on these five key genes can better identify the high- and low-risk groups of LGG and lay a solid foundation for evaluating the risk of LGG recurrence and malignant progression.

Single-Molecule Sequencing Enables Long Cell-Free DNA Detection and Direct Methylation Analysis for Cancer Patients.

Analysis of circulating tumor DNA has become increasingly important as a tool for cancer care. However, the focus of previous studies has been on short fragments of DNA. Also, bisulfite sequencing, a conventional approach for methylation analysis, causes DNA degradation, which is not ideal for the assessment of long DNA properties and methylation patterns. This study attempted to overcome such obstacles by single-molecule sequencing. Single-molecule real-time (SMRT) sequencing was used to sequence plasma DNA. We performed fragment size and direct methylation analysis for each molecule. A methylation score concerning single-molecule methylation patterns was used for cancer detection. A substantial proportion of plasma DNA was longer than 1 kb with a median of 16% in hepatocellular carcinoma (HCC) patients, hepatitis B virus carriers, and healthy individuals. The longest plasma DNA molecule in the HCC patients was 39.8 kb. Tumoral cell-free DNA (cfDNA) was generally shorter than nontumoral cfDNA. The longest tumoral cfDNA was 13.6 kb. Tumoral cfDNA had lower methylation levels compared with nontumoral cfDNA (median: 59.3% vs 76.9%). We developed and analyzed a metric reflecting single-molecule methylation patterns associated with cancer, named the HCC methylation score. HCC patients displayed significantly higher HCC methylation scores than those without HCC. Interestingly, compared to using short cfDNA (area under the receiver operating characteristic [ROC] curve, AUC: 0.75), the use of long cfDNA molecules greatly enhanced the discriminatory power (AUC: 0.91). A previously unidentified long cfDNA population was revealed in cancer patients. The presence and direct methylation analysis of these molecules open new possibilities for cancer liquid biopsy.

Changes in global DNA methylation under climatic stress in two related grasses suggest a possible role of epigenetics in the ecological success of polyploids

Polyploidization drives the evolution of grasses and can result in epigenetic changes, which may have a role in the creation of new evolutionary lineages and ecological speciation. As such changes may be inherited, they can also influence adaptation to the environment. Populations from different regions and climates may also differ epigenetically; however, this phenomenon is poorly understood. The present study analyzes the effect of climatic stress on global DNA methylation based on a garden collection of two related mountain grasses (the narrow endemic diploid Festuca tatrae and the more widely distributed mixed-ploidy F. amethystina) with different geographic ranges and ecological niches. A lower level of DNA methylation was observed for F. tatrae, while a higher mean level was obtained for the diploid and tetraploid of F. amethystina; with the tetraploids having a higher level of global methylated DNA than the diploids. The weather conditions (especially insolation) measured 24 h prior to sampling appeared to have a closer relationship with global DNA methylation level than those observed seven days before sampling. Our findings suggest that the level of methylation during stress conditions (drought, high temperature and high insolation) may be significantly influenced by the ploidy level and bioclimatic provenance of specimens; however an important role may also be played by the intensity of stress conditions in a given year.

Analysis of DNA Repair-Related Prognostic Function and Mechanism in Gastric Cancer.

DNA repair mechanisms have been proven to be essential for cells, and abnormalities in DNA repair could cause various diseases, such as cancer. However, the diversity and complexity of DNA repair mechanisms obscure the functions of DNA repair in cancers. In addition, the relationships between DNA repair, the tumor mutational burden (TMB), and immune infiltration are still ambiguous. In the present study, we evaluated the prognostic values of various types of DNA repair mechanisms and found that double-strand break repair through single-strand annealing (SSA) and nonhomologous end-joining (NHEJ) was the most prognostic DNA repair processes in gastric cancer (GC) patients. Based on the activity of these two approaches and expression profiles, we constructed a HR-LR model, which could accurately divide patients into high-risk and low-risk groups with different probabilities of survival and recurrence. Similarly, we also constructed a cancer-normal model to estimate whether an individual had GC or normal health status. The prognostic value of the HR-LR model and the accuracy of the cancer-normal model were validated in several independent datasets. Notably, low-risk samples, which had higher SSA and NHEJ activities, had more somatic mutations and less immune infiltration. Furthermore, the analysis found that low-risk samples had higher and lower methylation levels in CpG islands (CGIs) and open sea regions respectively, and had higher expression levels of programmed death-ligand 1 (PD-L1) and lower methylation levels in the promoter of the gene encoding PD-L1. Moreover, low-risk samples were characterized primarily by higher levels of CD4+ memory T cells, CD8+ naive T cells, and CD8+ TEM cells than those in high-risk samples. Finally, we proposed a decision tree and nomogram to help predict the clinical outcome of an individual. These results provide an improved understanding of the complexity of DNA repair, the TMB, and immune infiltration in GC, and present an accurate prognostic model for use in GC patients.

Maternal iron status in early pregnancy and DNA methylation in offspring: an epigenome-wide meta-analysis

BackgroundUnbalanced iron homeostasis in pregnancy is associated with an increased risk of adverse birth and childhood health outcomes. DNA methylation has been suggested as a potential underlying mechanism linking environmental exposures such as micronutrient status during pregnancy with offspring health. We performed a meta-analysis on the association of maternal early-pregnancy serum ferritin concentrations, as a marker of body iron stores, and cord blood DNA methylation. We included 1286 mother–newborn pairs from two population-based prospective cohorts. Serum ferritin concentrations were measured in early pregnancy. DNA methylation was measured with the Infinium HumanMethylation450 BeadChip (Illumina). We examined epigenome-wide associations of maternal early-pregnancy serum ferritin and cord blood DNA methylation using robust linear regression analyses, with adjustment for confounders and performed fixed-effects meta-analyses. We additionally examined whether associations of any CpGs identified in cord blood persisted in the peripheral blood of older children and explored associations with other markers of maternal iron status. We also examined whether similar findings were present in the association of cord blood serum ferritin concentrations with cord blood DNA methylation.ResultsMaternal early-pregnancy serum ferritin concentrations were inversely associated with DNA methylation at two CpGs (cg02806645 and cg06322988) in PRR23A and one CpG (cg04468817) in PRSS22. Associations at two of these CpG sites persisted at each of the follow-up time points in childhood. Cord blood serum ferritin concentrations were not associated with cord blood DNA methylation levels at the three identified CpGs.ConclusionMaternal early-pregnancy serum ferritin concentrations were associated with lower cord blood DNA methylation levels at three CpGs and these associations partly persisted in older children. Further studies are needed to uncover the role of these CpGs in the underlying mechanisms of the associations of maternal iron status and offspring health outcomes.

Preliminary study on the role of the C5orf46 gene in renal cancer

BackgroundC5orf46 has been found to have antibacterial and anti-inflammatory effects via sequencing and microarray technologies, but its effects on cancer are unclear. MethodsC5orf46 expression in renal cancer patients and cell lines was measured by quantitative polymerase chain reaction (qPCR). RNA sequencing data and clinicopathological information from renal cancer patients extracted from The Tumor Genome Atlas (TCGA) were analyzed to evaluate the prognostic value of C5orf46. The role of C5orf46 in vitro was verified by migration, proliferation and apoptosis experiments in renal cancer cell lines. Furthermore, the transcriptome of renal cancer cell lines with C5orf46 knocked down was sequenced to analyze potential signaling network pathways. Finally, the possible mechanisms of C5orf46 involvement in renal cancer development were analyzed by evaluating the immune microenvironment, mutation status and methylation levels. ResultsC5orf46 was highly expressed in renal cancer and was an independent prognostic factor. In vitro cell experiments showed that inhibition of C5orf46 expression could reduce renal cancer cell proliferation and migration and increase apoptosis. Transcriptomic sequencing after knockdown of C5orf46 in renal cancer cells revealed that it is involved in the malignant phenotype and immune microenvironment regulation of renal cancer. Finally, public databases suggest that C5orf46-related immune cell infiltration, mutational potential, and low methylation levels may contribute to poor prognosis in renal cancer. ConclusionThese findings suggest that C5orf46 is associated with renal cancer progression and could be a potential target for improving renal cancer prognosis.

Vitamin B12 Transport Genes and Nonsyndromic Cleft Lip With or Without Cleft Palate in Chile.

The aims of this study were to assess the association between polymorphisms within genes involved in vitamin B12 transport and nonsyndromic cleft lip with or without cleft palate (NSCL/P) and global DNA methylation in Chile. From 247 cases and 453 controls, we obtained variant genotypes for CBLIF, CUBN, AMN, ABCC1, CD320, and TCN2 from a single nucleotide polymorphisms array. Global DNA methylation in 95 controls was obtained through LINE-1 methylation. After multiple comparison corrections, only rs780807 in CUBN remains associated with NSCL/P at dominant model (OR 0.564, p-value = 0.0006, q-value = 0.0450). Carriers of protective allele showed lower levels of DNA methylation than non-carriers (p = 0.0259). Further studies are necessary in order to explain relations with the phenotype and DNA methylation due to the absence of functional evidence for rs780807 in CUBN.

Placental Abnormalities are Associated With Specific Windows of Embryo Culture in a Mouse Model.

Assisted Reproductive Technologies (ART) employ gamete/embryo handling and culture in vitro to produce offspring. ART pregnancies have an increased risk of low birth weight, abnormal placentation, pregnancy complications, and imprinting disorders. Embryo culture induces low birth weight, abnormal placental morphology, and lower levels of DNA methylation in placentas in a mouse model of ART. Whether preimplantation embryos at specific stages of development are more susceptible to these perturbations remains unresolved. Accordingly, we performed embryo culture for several discrete periods of preimplantation development and following embryo transfer, assessed fetal and placental outcomes at term. We observed a reduction in fetal:placental ratio associated with two distinct windows of preimplantation embryo development, one prior to the morula stage and the other from the morula to blastocyst stage, whereas placental morphological abnormalities and reduced imprinting control region methylation were only associated with culture prior to the morula stage. Extended culture to the blastocyst stage also induces additional placental DNA methylation changes compared to embryos transferred at the morula stage, and female concepti exhibited a higher loss of DNA methylation than males. By identifying specific developmental windows of susceptibility, this study provides a framework to optimize further culture conditions to minimize risks associated with ART pregnancies.

PMEPA1 Serves as a Prognostic Biomarker and Correlates with Immune Infiltrates in Cervical Cancer.

Emerging studies have demonstrated that Prostate transmembrane protein androgen induced 1 (PMEPA1) plays crucial roles in the carcinogenesis of many developing human tumors. However, the clinical significance of PMEPA1 expression in cervical cancer (CC) and its contribution to cancer immunity have not been investigated. In this study, we identified PMEPA1 as a survival-related gene in CC based on TCGA datasets. Univariate and multivariate analysis showed that PMEPA1 expression was an independent predictor for overall survival in CC patients. We could observe a strong negative correlation between PMEPA1 expression and PMEPA1 methylation. Two CpG sites of PMEPA1 were associated with overall survival, and one CpG site of PMEPA1 was associated with progression-free survival. The low level of PMEPA1 methylation was associated with advanced clinical stage of CC patients. KEGG assays revealed the genes associated with PMEPA1 expression were mainly enriched in several tumor-related pathways. Increased PMEPA1 expressions were observed to be positively related to high immune infiltration levels in several immune cells. Finally, the pan-cancer assays revealed that PMEPA1 expression was associated with the overall survival of UVM, PAAD, LUSC, BLCA, CESC, and LUAD. Taken together, PMEPA1 is a prognosis-related biomarker for multiple cancer types, especially CC. PMEPA1 is involved in tumor immunity, suggesting PMEPA1 may be a potential immunotherapeutic target in CC.

Validation of reliable safe harbor locus for efficient porcine transgenesis.

Transgenic technology is now widely used in biomedical and agricultural fields. Transgenesis is commonly achieved through random integration which might cause some uncertain consequences. The site-specific integration could avoid this disadvantage. This study aimed to screen and validate the best safe harbor (SH) locus for efficient porcine transgenesis. First, the cells carrying the EGFP reporter construct at four different SH loci (ROSA26, AAVS1, H11 and COL1A1) were achieved through CRSIPR/Cas9-mediated HDR. At the COL1A1 and ROSA26 loci, a higher mRNA and protein expression of EGFP was detected, and it was correlated with a lower level of DNA methylation of the EGFP promoter, hEF1α. A decreased H3K27me3 modification of the hEF1α promoter at the COL1A1 locus was also detected. For the safety of transgenesis at different SH locus, we found that transgenesis could relatively alter the expression of the adjacent endogenous genes, but the influence was limited. We also did not observe any off-target cleavage for the selected sgRNAs of the COL1A1 and ROSA26 loci. In conclusion, the COL1A1 and ROSA26 were confirmed to be the best two SH loci with the COL1A1 being more competitive for porcine transgenesis. This work would greatly facilitate porcine genome engineering and transgenic pig production.

Pollution induces epigenetic effects that are stably transmitted across multiple generations.

It has been hypothesized that the effects of pollutants on phenotypes can be passed to subsequent generations through epigenetic inheritance, affecting populations long after the removal of a pollutant. But there is still little evidence that pollutants can induce persistent epigenetic effects in animals. Here, we show that low doses of commonly used pollutants induce genome‐wide differences in cytosine methylation in the freshwater crustacean Daphnia pulex. Uniclonal populations were either continually exposed to pollutants or switched to clean water, and methylation was compared to control populations that did not experience pollutant exposure. Although some direct changes to methylation were only present in the continually exposed populations, others were present in both the continually exposed and switched to clean water treatments, suggesting that these modifications had persisted for 7 months (>15 generations). We also identified modifications that were only present in the populations that had switched to clean water, indicating a long‐term legacy of pollutant exposure distinct from the persistent effects. Pollutant‐induced differential methylation tended to occur at sites that were highly methylated in controls. Modifications that were observed in both continually and switched treatments were highly methylated in controls and showed reduced methylation in the treatments. On the other hand, modifications found just in the switched treatment tended to have lower levels of methylation in the controls and showed increase methylation in the switched treatment. In a second experiment, we confirmed that sublethal doses of the same pollutants generate effects on life histories for at least three generations following the removal of the pollutant. Our results demonstrate that even low doses of pollutants can induce transgenerational epigenetic effects that are stably transmitted over many generations. Persistent effects are likely to influence phenotypic development, which could contribute to the rapid adaptation, or extinction, of populations confronted by anthropogenic stressors.

Epigenetic states of genes controlling immune responsiveness in bovine chronic mastitis

Abstract Mastitis is a common disease in dairy cows, causing substantial economic losses. The leading cause of elevated milk somatic cell count (SC), which is the best indicator for detecting mastitis, is the invasion of pathogens. A major pathogen responsible for bovine mastitis is Staphylococcus aureus, a member of the coagulase-positive staphylococci. Some strains of coagulase-negative staphylococci can also be a cause of clinical or subclinical mastitis. Our study used bisulfite sequencing PCR (BSP) to detect the methylation status of nine candidate genes (CCL2, HCK, F11R, CD8A, PDIA3, LGMN, HSPA1A, IL18 and NFKBIA). We investigated the mechanisms associated with overexpression of these genes, in the mammary gland secretory tissue of cows diagnosed with mastitis and infected with coagulase-positive or coagulase-negative staphylococci. The results showed no changes at the DNA methylation level between the mastitis (CoPS and CoNS) and control groups (H), except for in the HCK region, where the observed differences between the CoPS and H groups were statistically significant. The low methylation level of the CpG sequence seems not to correspond to the previously observed increased activity of these genes, suggesting that mechanisms other than DNA methylation may control mRNA expression at the analyzed loci.

Multigenerational Exposure to Heat Stress Induces Phenotypic Resilience, and Genetic and Epigenetic Variations in Arabidopsis thaliana Offspring.

Plants are sedentary organisms that constantly sense changes in their environment and react to various environmental cues. On a short-time scale, plants respond through alterations in their physiology, and on a long-time scale, plants alter their development and pass on the memory of stress to the progeny. The latter is controlled genetically and epigenetically and allows the progeny to be primed for future stress encounters, thus increasing the likelihood of survival. The current study intended to explore the effects of multigenerational heat stress in Arabidopsis thaliana. Twenty-five generations of Arabidopsis thaliana were propagated in the presence of heat stress. The multigenerational stressed lineage F25H exhibited a higher tolerance to heat stress and elevated frequency of homologous recombination, as compared to the parallel control progeny F25C. A comparison of genomic sequences revealed that the F25H lineage had a three-fold higher number of mutations [single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs)] as compared control lineages, suggesting that heat stress induced genetic variations in the heat-stressed progeny. The F25H stressed progeny showed a 7-fold higher number of non-synonymous mutations than the F25C line. Methylome analysis revealed that the F25H stressed progeny showed a lower global methylation level in the CHH context than the control progeny. The F25H and F25C lineages were different from the parental control lineage F2C by 66,491 and 80,464 differentially methylated positions (DMPs), respectively. F25H stressed progeny displayed higher frequency of methylation changes in the gene body and lower in the body of transposable elements (TEs). Gene Ontology analysis revealed that CG-DMRs were enriched in processes such as response to abiotic and biotic stimulus, cell organizations and biogenesis, and DNA or RNA metabolism. Hierarchical clustering of these epimutations separated the heat stressed and control parental progenies into distinct groups which revealed the non-random nature of epimutations. We observed an overall higher number of epigenetic variations than genetic variations in all comparison groups, indicating that epigenetic variations are more prevalent than genetic variations. The largest difference in epigenetic and genetic variations was observed between control plants comparison (F25C vs. F2C), which clearly indicated that the spontaneous nature of epigenetic variations and heat-inducible nature of genetic variations. Overall, our study showed that progenies derived from multigenerational heat stress displayed a notable adaption in context of phenotypic, genotypic and epigenotypic resilience.