Higher DNA Methylation Levels Research Articles

Studies on the Molecular Basis of Heterosis in Arabidopsis thaliana and Vegetable Crops

Heterosis is an important phenomenon for high-yield crop breeding and is utilized for breeding F1 varieties in horticultural crops. However, its molecular mechanism has not been elucidated, and compared to cereals, heterosis is less explored at the molecular level in horticultural crops. In this review, we compiled the new genetic and epigenetic studies on heterosis in horticultural crops. Because of the difficulty of predicting the level of heterosis from the parental genetic distance, molecular approaches are being used to study its molecular basis in horticultural crops. Transcriptome analyses in vegetables have identified photosynthesis-related genes as important in heterosis. Analysis of noncoding RNAs has suggested their involvement in regulating the heterosis of vegetative and fruit tissues. Quantitative trait locus (QTL) analysis has revealed the association of heterozygosity of a specific locus or multiple loci with heterosis of vegetative and fruit tissues. A higher level of DNA methylation was noted in the heterotic F1 of Brassica rapa leafy vegetables, while the roles of other epigenetic modifications such as histone marks have not been explored.

Transgenerational Herbivory Effects on Performance of Clonal Offspring of the Invasive Plant Alternanthera philoxeroides

Interactions between alien plants and local enemies in introduced ranges may determine plant invasion success. However, little is known about whether herbivory-induced responses are transmitted across vegetative generations of plants and whether epigenetic changes are involved during this process. In a greenhouse experiment, we examined the effects of herbivory by the generalist herbivore Spodoptera litura on the growth, physiology, biomass allocation and DNA methylation level of the invasive plant Alternanthera philoxeroides in the first- (G1), second- (G2) and third-generation (G3). We also tested the effects of root fragments with different branching orders (i.e., the primary- or secondary-root fragments of taproots) of G1 on offspring performance. Our results showed that G1 herbivory promoted the growth of the plants in G2 that sprouted from the secondary-root fragments of G1 but had a neutral or negative effect on the growth of the plants in G2 from the primary-root fragments. The growth of plants in G3 was significantly reduced by G3 herbivory but not affected by G1 herbivory. Plants in G1 exhibited a higher level of DNA methylation when they were damaged by herbivores than when they were not, while neither plants in G2 nor G3 showed herbivory-induced changes in DNA methylation. Overall, the herbivory-induced growth response within one vegetative generation may represent the rapid acclimatization of A. philoxeroides to the unpredictable generalist herbivores in the introduced ranges. Herbivory-induced trans-generational effects may be transient for clonal offspring of A. philoxeroides, which can be influenced by the branching order of taproots, but be less characterized by DNA methylation.

MeCP2 Is an Epigenetic Factor That Links DNA Methylation with Brain Metabolism.

DNA methylation, one of the most well-studied epigenetic modifications, is involved in a wide spectrum of biological processes. Epigenetic mechanisms control cellular morphology and function. Such regulatory mechanisms involve histone modifications, chromatin remodeling, DNA methylation, non-coding regulatory RNA molecules, and RNA modifications. One of the most well-studied epigenetic modifications is DNA methylation that plays key roles in development, health, and disease. Our brain is probably the most complex part of our body, with a high level of DNA methylation. A key protein that binds to different types of methylated DNA in the brain is the methyl-CpG binding protein 2 (MeCP2). MeCP2 acts in a dose-dependent manner and its abnormally high or low expression level, deregulation, and/or genetic mutations lead to neurodevelopmental disorders and aberrant brain function. Recently, some of MeCP2-associated neurodevelopmental disorders have emerged as neurometabolic disorders, suggesting a role for MeCP2 in brain metabolism. Of note, MECP2 loss-of-function mutation in Rett Syndrome is reported to cause impairment of glucose and cholesterol metabolism in human patients and/or mouse models of disease. The purpose of this review is to outline the metabolic abnormalities in MeCP2-associated neurodevelopmental disorders that currently have no available cure. We aim to provide an updated overview into the role of metabolic defects associated with MeCP2-mediated cellular function for consideration of future therapeutic strategies.

Evolutionary analysis of a complete chicken genome

Microchromosomes are prevalent in nonmammalian vertebrates [P. D. Waters et al., Proc. Natl. Acad. Sci. U.S.A. 118 (2021)], but a few of them are missing in bird genome assemblies. Here, we present a new chicken reference genome containing all autosomes, a Z and a W chromosome, with all gaps closed except for the W. We identified ten small microchromosomes (termed dot chromosomes) with distinct sequence and epigenetic features, among which six were newly assembled. Those dot chromosomes exhibit extremely high GC content and a high level of DNA methylation and are enriched for housekeeping genes. The pericentromeric heterochromatin of dot chromosomes is disproportionately large and continues to expand with the proliferation of satellite DNA and testis-expressed genes. Our analyses revealed that the 41-bp CNM repeat frequently forms higher-order repeats (HORs) at the centromeres of acrocentric chromosomes. The centromere core regions where the kinetochore attaches often encompass telomeric sequence (TTAGGG)n, and in a one of the dot chromosomes, the centromere core recruits an endogenous retrovirus (ERV). We further demonstrate that the W chromosome shares some common features with dot chromosomes, having large arrays of hypermethylated tandem repeats. Finally, using the complete chicken chromosome models, we reconstructed a fine picture of chordate karyotype evolution, revealing frequent chromosomal fusions before and after vertebrate whole-genome duplications. Our sequence and epigenetic characterization of chicken chromosomes shed insights into the understanding of vertebrate genome evolution and chromosome biology.

Analysis of Promoter Methylation of the Bovine FOXO1 Gene and Its Effect on Proliferation and Differentiation of Myoblasts.

This study aimed to explore the regulatory role of FOXO1 promoter methylation on its transcriptional level and unravel the effect of FOXO1 on the proliferation and differentiation of bovine myoblasts. Bisulfite sequencing polymerase chain reaction (BSP) and real-time quantitative PCR were performed to determine the methylation status and transcript levels of the FOXO1 promoter region at different growth stages. BSP results showed that the methylation level in the calf bovine (CB) group was significantly higher than that in the adult bovine (AB) group (p < 0.05). On the other hand, qRT-PCR results indicated that the mRNA expression level in the AB group was significantly higher than that in the CB group (p < 0.05), suggesting a significant decrease in gene expression at high levels of DNA methylation. CCK-8 and flow cytometry were applied to determine the effect of silencing the FOXO1 gene on the proliferation of bovine myoblasts. Furthermore, qRT-PCR and Western blot were conducted to analyze the expression of genes associated with the proliferation and differentiation of bovine myoblasts. Results from CCK-8 revealed that the short hairpin FOXO1 (shFOXO1) group significantly promoted the proliferation of myoblasts compared to the short-hairpin negative control (shNC) group (p < 0.05). Flow cytometry results showed a significant decrease in the number of the G1 phase cells (p < 0.05) and a significant increase in the number of the S phase cells (p < 0.05) in the shFOXO1 group compared to the shNC group. In addition, the expression of key genes for myoblast proliferation (CDK2, PCNA, and CCND1) and differentiation (MYOG, MYOD, and MYHC) was significantly increased at both mRNA and protein levels (p < 0.05). In summary, this study has demonstrated that FOXO1 transcription is regulated by methylation in the promoter region and that silencing FOXO1 promotes the proliferation and differentiation of bovine myoblasts. Overall, our findings lay the foundation for further studies on the regulatory role of epigenetics in the development of bovine myoblasts.

Epigenetic and Genetic Contribution for Expression Bias of Homologous Alleles in Polyploid Sugarcane

DNA methylation regulates gene expression in eukaryotes, but their roles in gene expression changes in polyploids are poorly understood. Here, we comparatively analyzed the DNA methylation profiles and transcriptome maps of four tissues (leaf, rind, pith, and root) in autopolyploid sugarcane (Saccharum spontaneum). The overall DNA methylation levels were relatively equal and were consistent with the similar genome-wide expression levels of homologous alleles among different homologous chromosomes. However, tetrad alleles showed a large proportion of non-balanced alleles (85–89%) compared with balanced alleles (11–15%). For tetrad alleles, highly-expressed alleles showed lower Ka/Ks values than those in lowly-expressed alleles, indicating that dominant (and non-suppressed) alleles were under strong purifying selection pressure compared to non-dominant (and suppressed) alleles. Specifically, higher DNA methylation levels were found in non-balanced alleles compared to balanced alleles, as well as a higher CG to TG substitution rate, suggesting epigenetic and genetic variation associated with transcription divergence. Moreover, among non-balanced alleles, which were associated with DNA methylation changes, many were involved in several processes relevant to agronomic traits, such as responses to stress and carbohydrate transport. Taken together, our results provide unique epigenetic insights into the transcriptional regulation of homologous alleles, which contribute to the key agronomic traits in sugarcane and facilitate epigenetic studies in other polyploid crops.

EPCO-44. HOXD13 GENE PLAYS A ROLE AS A PUTATIVE MASTER EPIGENETIC REGULATOR IN GLIOMA RECURRENCE AND PROGRESSION

Abstract BACKGROUND Adult diffuse glioma is a heterogenous brain tumor group that has malignant potential and recurring properties. Glioma CpG island methylator phenotype (G-CIMP) associated with chromatin remodeling encompasses two different methylation degrees: G-CIMP-high and G-CIMP-low that have high levels of DNA methylation and loss of DNA methylation, respectively. G-CIMP-high confers a more favorable prognosis and better predictive values by altering transcriptional dynamics and the gene expression. METHODS We have performed Chromatin Immunoprecipitation Sequencing (ChIP-Seq) on DNA extracted from fresh frozen primary tumor tissues (G-CIMP-high and G-CIMP-low). We have processed the sequencing data by using FastQC (v.:0.11.5), bwa-mem (v.:0.7.15) and picard tool (v.:2.7.1). The ChIP-seq peaks and the differentially bound peaks were obtained using MACS2 (v.:2.1.1) and DiffBind (v.:3.4.11), respectively. We have also integrated transcriptomic, epigenomic and genomic data. RESULTS Initial results showed that the differentially bound H3K27ac and H3K4me3 peak gains and losses are located mainly in intronic and intergenic regions. The integration between epigenetic and transcriptomic data revealed that the promoter of the HOXD13 gene upregulated in GCIMP-low presents an active regulatory region and is enriched by H3K27ac and H3K4me3 sites. We were able to identify that GCIMP-low, in addition to presenting more downregulated genes than GCIMP-high, also has greater affinity to the H3K27ac peaks. In GCIMP-low, we identified that regions enriched by H3K27ac are highly associated with the presence H3K4me3 marks. CONCLUSION We mapped the enrichment of histone modifications in the TSS of differentially expressed genes in G-CIMP-high and G-CIMP-low identifying both the susceptibility of GIMP-low to interact with H3K27ac and the fact that the progression of GCIMP-high to G-CIMP-low can be explained by the association between H3K27ac gain and gene downregulation. Also, we predicted the HOXD13 gene as a putative master epigenetic regulator of the G-CIMP demethylation process and, consequently, of tumor recurrence and progression, over time.

Characterization of the CpG island methylator phenotype subclass in papillary thyroid carcinoma.

CpG island methylator phenotype (CIMP), characterized by the concurrent and widespread hypermethylation of a cluster of CpGs, has been reported to play an important role in carcinogenesis. Limited studies have explored the role of CIMP in papillary thyroid carcinomas (PTCs). Here, in genome-wide DNA methylation analysis of 350 primary PTCs from the Cancer Genome Atlas database that were assessed using the Illumina HumanMethylation450K platform, our study helps to identify two subtypes displayed markedly distinct DNA methylation levels, termed CIMP (high levels of DNA methylation) and nCIMP subgroup (low levels of DNA methylation). Interestingly, PTCs with CIMP tend to have a higher degree of malignancy, since this subtype was tightly associated with older age, advanced pathological stage, and lymph node metastasis (all P < 0.05). Differential methylation analysis showed a broad methylation gain in CIMP and subsequent generalized gene set testing analysis based on the significantly methylated probes in CIMP showed remarkable enrichment in epithelial mesenchymal transition and angiogenesis hallmark pathways, confirming that the CIMP phenotype may promote the tumor progression from another perspective. Analysis of tumor microenvironment showed that CIMP PTCs are in an immune-depletion status, which may affect the effectiveness of immunotherapy. Genetically, the significantly higher tumor mutation burden and copy number alteration both at the genome and focal level confirmed the genomic heterogeneity and chromosomal instability of CIMP. tumor Corresponding to the above findings, PTC patients with CIMP showed remarkable poor clinical outcome as compared to nCIMP regarding overall survival and progression-free survival. More importantly, CIMP was associated with worse survival independent of known prognostic factors.

P17.12.B HOXD13 gene plays a role as a putative master epigenetic regulator in glioma recurrence and progression

Abstract Background Adult diffuse glioma is a heterogenous brain tumor group that has malignant potential and recurring properties. Glioma CpG island methylator phenotype (G-CIMP) associated with chromatin remodeling encompasses two different methylation degrees: G-CIMP-high and G-CIMP-low that have high levels of DNA methylation and loss of DNA methylation, respectively. G-CIMP-high confers a more favorable prognosis and better predictive values by altering transcriptional dynamics and inhibiting the gene expression. However, some IDH-mutant G-CIMP-high undergo a demethylation process progressing to G-CIMP-low leading to tumor recurrence and aggressiveness. Material and Methods We have performed Chromatin Immunoprecipitation Sequencing (ChIP-Seq) on DNA extracted from nine (9) fresh frozen primary tumor tissues belonging to G-CIMP-high group and four (4) belonging to G-CIMP-low group. We have processed the sequencing data by using FastQC (v.:0.11.5), bwa-mem (v.:0.7.15) and picard tool (v.:2.7.1). The ChIP-seq peaks and the differentially bound peaks were obtained using MACS2 (v.:2.1.1) and DiffBind (v.:3.4.11), respectively. We have integrated transcriptomic, epigenomic and genomic data to investigate the impact of H3K4me3 and H3K27ac in the epigenome of G-CIMP-low and high tumors. Results Initial results showed that the differentially bound H3K27ac and H3K4me3 peak gains and losses are located mainly in intronic and intergenic regions. The integration between epigenetic and transcriptomic data revealed that the promoter of the HOXD13 gene upregulated in GCIMP-low presents an active regulatory region and is enriched by H3K27ac and H3K4me3 sites. We were able to identify that GCIMP-low, in addition to presenting more downregulated genes than GCIMP-high, also has greater affinity to the H3K27ac peaks. In GCIMP-low, we identified 312 promoter regions significantly enriched by the epigenetic biomarker H3K27ac and 67 promoters with loss of affinity for H3K27ac, with the regions enriched by H3K27ac highly associated with the presence H3K4me3 marks. Functional genomic analysis targeting HOXD13 by using CRISPR in a representative glioma cell line shows that HOXD13 is critical for impairing the proliferative state that drives glioma progression.Conclusion: We mapped the enrichment of histone modifications in the TSS of differentially expressed genes in G-CIMP-high and G-CIMP-low identifying both the susceptibility of GIMP-low to interact with H3K27ac and the fact that the progression of GCIMP-high to G-CIMP-low can be explained by the association between H3K27ac gain and the inhibition of gene expression. Also, we predicted the HOXD13 gene as a putative master epigenetic regulator of the G-CIMP demethylation process and, consequently, of tumor recurrence and progression, over time.

DNA methylation influences the CTCF-modulated transcription of RASSF1A in lung cancer cells.

Ras-association domain family 1A (RASSF1A) is one of the most methylated genes in lung cancer (LC). We investigate whether the high DNA methylation level of RASSF1A can relieve the resistance of RASSF1A to LC by inhibiting RASSF1A's transcription factor binding to RASSF1A. RASSF1A expression in tissues and cells was tested utilizing quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. RASSF1A expression and RASSF1A methylation level in LC cells exposed to 5-Aza-dc were assessed by qRT-PCR and quantitative methylation-specific PCR. The association between CTCF and RASSF1A was assessed using hTFtarget, ChIP, and luciferase reporter gene analysis. The effects of 5-Aza-dc, CTCF, and RASSF1A on cell biological behaviors and epithelial-mesenchymal transition (EMT)-related markers were assessed by cell function experiments and Western blot. Moreover, we constructed the xenograft tumor and pulmonary nodule metastasis models, and assessed tumor volume and weight. RASSF1A expression and pulmonary nodule metastasis were tested utilizing qRT-PCR, Western blot, and H&E staining. RASSF1A was under-expressed in LC tissues and cells. 5-Aza-dc enhanced RASSF1A level and weakened RASSF1A methylation level in LC cells. RASSF1A silencing neutralized 5-Aza-dc-mediated repressing effects on LC cell biological function and EMT. The loss of CTCF binding to RASSF1A in LC cells was associated with DNA methylation. The effect of 5-Aza-dc on RASSF1A level, LC cell malignant behaviors, and EMT-related factors were strengthened by CTCF upregulation. RASSF1A overexpression suppressed LC tumor growth and pulmonary nodule metastasis in vivo. DNAmethylation blocked the modulation of RASSF1A expression by CTCF and relieved the resistance of RASSF1A to LC.

Histone H3K36me2 and H3K36me3 form a chromatin platform essential for DNMT3A-dependent DNA methylation in mouse oocytes

Establishment of the DNA methylation landscape of mammalian oocytes, mediated by the DNMT3A-DNMT3L complex, is crucial for reproduction and development. In mouse oocytes, high levels of DNA methylation occur exclusively in the transcriptionally active regions, with moderate to low levels of methylation in other regions. Histone H3K36me3 mediates the high levels of methylation in the transcribed regions; however, it is unknown which histone mark guides the methylation in the other regions. Here, we show that, in mouse oocytes, H3K36me2 is highly enriched in the X chromosome and is broadly distributed across all autosomes. Upon H3K36me2 depletion, DNA methylation in moderately methylated regions is selectively affected, and a methylation pattern unique to the X chromosome is switched to an autosome-like pattern. Furthermore, we find that simultaneous depletion of H3K36me2 and H3K36me3 results in global hypomethylation, comparable to that of DNMT3A depletion. Therefore, the two histone marks jointly provide the chromatin platform essential for guiding DNMT3A-dependent DNA methylation in mouse oocytes.

SPATA16 promoter hypermethylation and downregulation in male infertility.

Spermatogenesis associated 16 (SPATA16) gene plays an important role in acrosome formation. In this study, we analysed SPATA16 promoter methylation in 29 oligozoospermic infertile and 16 normozoospermic fertile sperm samples and in testicular biopsy from 16 non-obstructive azoospermic and 2 obstructive azoospermic individuals. Next, we analysed SPATA16 level in sperm from 8 oligozoospermic infertile, 6 normozoospermic fertile, 9 IVF failed normozoospermic and 10 IVF successful normozoospermic samples by immunoblotting. This was followed by the analysis of SPATA16 expression in testicular biopsy from azoospermic individuals (n=8) in comparison to normozoospermic individuals (n=2). Oligozoospermic infertile sperm samples showed significantly higher methylation in the SPATA16 promoter region. Similarly, testicular biopsy from azoospermic men also showed significantly higher level of DNA methylation. Sub-group analysis of infertile sperm and testicular biopsy samples showed a direct correlation between DNA methylation and the degree of spermatogenic impairment. Oligozoospermic infertile samples and IVF failed samples showed reduced SPATA16 expression in comparison to normozoospermic fertile and IVF successful samples, respectively. Human biopsy analysis showed a significant decrease in SPATA16 expression in hypospermatogenesis, maturation arrest and Sertoli cell only syndrome. In conclusion, hypermethylation in SPATA16 promoter shows a highly significant correlation with infertility, which is consistent with its down-regulation in infertility.

5-Aza-dC promotes T-cell acute lymphoblastic leukemia cell invasion via downregulation of DNMT1 and upregulation of MMP-2 and MMP-9

5-Aza-2'-deoxycytidine (5-Aza-dC) is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells, and can restore the expression of their target genes in Jurkat cells. The goal of this study was to discern the potential effect of 5-Aza-dC on the invasion of T-ALL cells in acute lymphoblastic leukemia (ALL). The role of matrix metallopeptidase (MMP)-2, MMP-9, and DNMT1 in cell invasion was determined using loss- and gain-of-function investigations in Jurkat- and Sup-T1-R cells. A nude mouse model of ALL was established for further exploration of their roles in vivo. MMP-2 and MMP-9 exhibited high expression and low DNA methylation levels in 5-Aza-dC-resistant T-ALL cells. DNMT1 was poorly expressed in 5-Aza-dC-resistant T-ALL cells and exhibited decreased enrichment in the promoter region of MMP-2 and MMP-9. Silencing of MMP-2 and MMP-9 or DNMT1 overexpression reduced T-ALL cell invasion. After treatment of Sup-T1 cells with 5-Aza-dC, MMP-2 and MMP-9 presented with reduced DNA methylation levels but increased expression, and DNMT1 expression was identified to be suppressed. Further, in vivo assays revealed that DNMT1 alleviated T-ALL by reducing the expression of MMP-2 and MMP-9 in vivo. All in all, 5-Aza-dC activates MMP-2 and MMP-9 expression by reducing DNMT1-dependent DNA methylation levels and, hence, promotes the invasion of T-ALL cells.

DNA Demethylation Switches Oncogenic ΔNp63 to Tumor Suppressive TAp63 in Squamous Cell Carcinoma.

The TP63 gene encodes two major protein variants; TAp63 contains a p53-like transcription domain and consequently has tumor suppressor activities whereas ΔNp63 lacks this domain and acts as an oncogene. The two variants show distinct expression patterns in normal tissues and tumors, with lymphocytes and lymphomas/leukemias expressing TAp63, and basal epithelial cells and some carcinomas expressing high levels of ΔNp63, most notably squamous cell carcinomas (SCC). Whilst the transcriptional functions of TAp63 and ΔNp63 isoforms are known, the mechanisms involved in their regulation are poorly understood. Using squamous epithelial cells that contain high levels of ΔNp63 and low/undetectable TAp63, the DNA demethylating agent decitabine (5-aza-2’-deoxycytidine, 5-dAza) caused a dose-dependent increase in TAp63, with a simultaneous reduction in ΔNp63, indicating DNA methylation-dependent regulation at the isoform-specific promoters. The basal cytokeratin KRT5, a direct ΔNp63 transcriptional target, was also reduced, confirming functional alteration of p63 activity after DNA demethylation. We also showed high level methylation of three CpG sites in the TAP63 promoter in these cells, which was reduced by decitabine. DNMT1 depletion using inducible shRNAs partially replicated these effects, including an increase in the ratio of TAP63:ΔNP63 mRNAs, a reduction in ΔNp63 protein and reduced KRT5 mRNA levels. Finally, high DNA methylation levels were found at the TAP63 promoter in clinical SCC samples and matched normal tissues. We conclude that DNA methylation at the TAP63 promoter normally silences transcription in squamous epithelial cells, indicating DNA methylation as a therapeutic approach to induce this tumor suppressor in cancer. That decitabine simultaneously reduced the oncogenic activity of ΔNp63 provides a “double whammy” for SCC and other p63-positive carcinomas. Whilst a variety of mechanisms may be involved in producing the opposite effects of DNA demethylation on TAp63 and ΔNp63, we propose an “either or” mechanism in which TAP63 transcription physically interferes with the ability to initiate transcription from the downstream ΔNP63 promoter on the same DNA strand. This mechanism can explain the observed inverse expression of p63 isoforms in normal cells and cancer.

Abstract LB035: Understanding the mechanism of neuropilin2 upregulation in advanced prostate cancer

Abstract Background: Prostate cancer management involves administering Androgen Deprivation Therapy (ADT). Patients with advanced form of the disease become resistant to ADT and progress to a lethal phenotype called Castration Resistant Prostate Cancer (CRPC). Neuropilin-2 is upregulated in advanced prostate cancer. It has been implicated in decreasing the overall survival of such patients by inducing therapy-resistance. Significance: Understanding how NRP2 is differentially expressed will uncover molecular pathways that can be targeted to develop a specific treatment strategy for better management of patients. Experimental Design: We checked NRP2 expression in different prostate cancer cell lines {LNCaP, C4-2 and C4-2 P53/RB1 null cells (DKD cells)} and the level of methylation of the NRP2 promoter in these cells by pyrosequencing. We demethylated the NRP2 promoter by treatment with 5’Azacytidine and checked NRP2 mRNA expression. We analyzed the NRP2 promoter activity under demethylated conditions by transfecting cells with a demethylated NRP2 promoter construct using a GLuc dual-luciferase assay. Using Biobase we found DNA sequences for binding of transcription factors within the NRP2 promoter. RNA-Seq, RT-PCR was performed to identify potential transcription factors of NRP2(highly expressed in DKD cells). To validate SOX2 as a transcription factor for NRP2, we tested its binding to NRP2 promoter by ChIP-q-PCR. Requirement of SOX2 in transcribing NRP2 mRNA in C4-2 and DKD cells was tested by both knocking down or ectopically expressing SOX2. To determine whether demethylation of NRP2 promoter is a prerequisite for SOX2 binding, SOX2 was overexpressed in LNCaP cells following 5’Azacytidine and monitored the change in the NRP2 expression. Results: NRP2 expression was low in LNCaP cells. It steadily increased in C4-2 cells with a steep rise in the DKD cells. Pyrosequencing showed a high level of DNA methylation in LNCaP cells compared to C4-2 cells. On treatment with 5’Azacytidine, NRP2 mRNA expression was upregulated in the LNCaP cells and it significantly rose in the C4-2 cells. Our dual-luciferase assay showed us that in C4-2 cells, the promoter activity of NRP2 upon demethylation went up causing a significant rise in Luciferase expression. RNA-seq and RT-PCR analysis showed high SOX2 levels in DKD cells indicating that SOX2 might play a role in elevating NRP2 expression in these cells. ChIP q-PCR indicated enriched binding of SOX2 in the NRP2 promoter in DKD cells. On silencing SOX2 in DKD cells, the expression of NRP2 was decreased. Analogous to this finding, overexpressing SOX2 in C4-2 cells caused significant rise in NRP2 expression, whereas in LNCaP cells, there was no change. Demethylation of NRP2 promoter and simultaneous overexpression of SOX2 in LNCaP lead to a significant increase in NRP2 mRNA suggesting the requirement of demethylated NRP2 promoter for SOX2 function. Conclusion: The differential expression of NRP2 in advanced prostate cancer is a two-step process of initial demethylation of the DNA in the NRP2 promoter region, followed by binding of SOX2 Citation Format: Sanika Bodas, Ridwan Islam, Sreyashi Bhattacharya, Juhi Mishra, Dipanwita Das, Michael Muders, Samikshan Dutta, Benjamin A. Teply, Kaustubh Datta. Understanding the mechanism of neuropilin2 upregulation in advanced prostate cancer [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 LB035.

Abstract 3744: Differential Orthopedia Homeobox (OTP) expression in pulmonary carcinoids is associated with changes in DNA methylation

Abstract Introduction: Limited number of tumor types have been examined for Orthopedia Homeobox (OTP) expression. In pulmonary carcinoids, loss of expression is a strong indicator of poor prognosis. Here, we investigated OTP expression in 37 different tumor types, and the association between OTP expression and DNA methylation levels in lung neuroendocrine neoplasms. Methods: We analyzed publicly available multi-omics data (whole-exome-, whole-genome-, RNA sequencing, Epic-850K-methylation array) of 58 typical-, 27 atypical carcinoids, 69 large cell neuroendocrine carcinoma and 51 small cell lung cancer patients and TCGA (The Cancer Genome Atlas) data of 33 tumor types. 850K-methylation analysis was cross validated using targeted pyrosequencing on 35 carcinoids. Results: Results showed bimodality of OTP expression in carcinoids (OTPhigh versus OTPlow group, likelihood-ratio test p=1.5x10-2), with the OTPhigh group specific to pulmonary carcinoids while absent from all other cohorts analyzed. Significantly different DNA methylation levels were observed between OTPhigh and OTPlow carcinoids in 12/34 OTP infinium probes (fdr&amp;lt;0.05 &amp; β-value effect size&amp;gt;0.2). Overall, OTPlow carcinoids harbor a high DNA methylation level as compared to OTPhigh carcinoids. OTPlow carcinoids showed a significantly worse overall survival (logrank test p=0.0052). Gene set enrichment analysis for somatically mutated genes associated with hallmarks of cancer showed robust enrichment of three hallmarks in the OTPlow group, i.e., sustaining proliferative signaling, evading growth suppressor, and genome instability and mutation. Conclusion: High OTP expression is a unique feature of pulmonary carcinoids with a favorable prognosis. In poor prognostic patients, OTP expression is lost, most likely due to changes in DNA methylation levels. Citation Format: Laura Moonen, Lise Mangiante, Daphne J. Leunissen, Lisa M. Lap, Aurelie Gabriel, Lisa M. Hillen, Guido M. Roemen, Alexander Koch, Manon van Engeland, Anne-Marie C. Dingemans, Matthieu Foll, Nicolas Alcala, Lynnette Fernandez-Cuesta, Jules L. Derks, Ernst-Jan M. Speel. Differential Orthopedia Homeobox (OTP) expression in pulmonary carcinoids is associated with changes in DNA methylation [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 3744.

Epigenome-Wide Association Study Identified VTI1A DNA Methylation Associated With Accelerometer-Assessed Physical Activity.

Health benefits of physical activity (PA) may be mediated by DNA methylation alterations. The purpose of the current study was to comprehensively identify CpG sites whose methylation levels were associated with accelerometer-assessed total PA in a general Japanese population. The study participants were from the baseline survey of Saga Japan Multi-institutional Collaborative Cohort. PA was objectively measured by a single-axis accelerometer for 7 d. We used a two-stage strategy. In the discovery stage, we performed a meta-analysis of two epigenome-wide association studies of total PA in 898 individuals (a combination of random sample ( n = 507) and case-control study sample ( n = 391)). Peripheral blood DNA methylation levels were measured using Infinium EPIC or HM450 arrays. In the replication stage, we subsequently examined whether CpG sites significantly associated ( P < 1 × 10 -5 ) with total PA were replicated in another sample ( n = 1711), in which methylation levels were measured by pyrosequencing. A multiple linear regression was performed to determine the cross-sectional association between total PA and methylation levels with adjustment for potential confounders, including body mass index. A fixed-effects model was used in the meta-analysis. Correlations between total PA-associated DNA methylation and several inflammatory markers, such as high-sensitivity C-reactive protein, were also conducted. In the meta-analysis, nine CpG sites were significantly associated with total PA ( P < 1 × 10 -5 ). Among the nine sites, one site cg07030336 (annotated to VTI1A/ZDHHC6 gene) was successfully replicated ( P = 0.009). The current study showed that greater accelerometer-assessed total PA was associated with higher DNA methylation levels at cg07030336 ( VTI1A/ZDHHC6 ) in the general population. In addition, we found a divergent relationship between the methylation levels at cg07030336 and several inflammatory biomarkers.

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.

PCDH17 is regulated by methylation of DNMT3B and affects the malignant biological behavior of HCC through EMT

Hepatocellular carcinoma (HCC) is one of the malignant tumors most frequently encountered in the clinic. Studies have shown that the abnormal expression of various genes leads to the malignant progression of tumors, and the modification in DNA methylation can cause a change in gene expression. Increasing evidence has shown that abnormal expression of PCDH17 is found in many human cancers. However, its functional role in HCC remains unexplored. Herein, we found that PCDH17 was expressed at low levels in HCC tissues and cell lines. There is a significant correlation between low expression of PCDH17 and poor prognosis of HCC patients. Increased expression of PCDH17 significantly suppressed cell proliferation, migration and invasion in HCC. The low expression of PCDH17 was due to its high DNA methylation level, and changing the expression of DNMT3B significantly affect the DNA methylation level of PCDH17 and increase its protein expression. Furthermore, methylation of PCDH17 regulated by DNMT3B affects the malignant biological behavior of HCC through EMT. In conclusion, PCDH17 participates in malignant biological behavior of HCC and that DNMT3B plays an important role in the regulation of PCDH17 methylation, which affects the malignant progression of HCC.

Pan-Cancer Integrated Analysis Identification of SASH3, a Potential Biomarker That Inhibits Lung Adenocarcinoma Progression.

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3) is an adaptor protein expressed mainly in lymphocytes, and plays significant roles in T-cell proliferation and cell survival. However, its expression level, clinical significance, and correlation with tumor-infiltrating immune cells across cancers remain unclear. In this study, we comprehensively examined the expression, dysregulation, and prognostic significance of SASH3, and the correlation with clinicopathological parameters and immune infiltration in pan-cancer. The mRNA and protein expression status of SASH3 were determined by TCGA, GTEx, and UALCAN. Kaplan–Meier analysis utilized the prognostic values of SASH3 in diverse cancers. The association between SASH3 expression and gene mutation, DNA methylation, immune cells infiltration, immune checkpoints, tumor mutation burden (TMB), and microsatellite instability (MSI) were analyzed using data from the TCGA database. High expression of SASH3 was not only linked to poor OS in ESCC, LAML, LGG, and UVM, but also associated with better OS in CESC, HNSC, LUAD, SARC, SKCM, THYM, and UCEC. As for DSS, a high level of SASH3 correlated with adverse DSS in ESCC, LGG, and UVM, and lowly expressed SASH3 was associated with shorter OS in CESC, HNSC, LUAD, SARC, SKCM, and UCEC. The results of Cox regression and nomogram analyses confirmed that SASH3 was an independent factor for LUAD prognosis. Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) results showed that SASH3 was involved in natural killer cell-mediated cytotoxicity, Th17 cell differentiation, PD-L1 expression and PD-1 checkpoint pathway in cancer, NF-kappa B signaling pathway, B-cell receptor signaling pathway, and Toll-like receptor signaling pathway. SASH3 expression was correlated with TMB in 28 cancer types and associated with MSI in 22 cancer types, while there was a negative correlation between SASH3 expression and DNA methylation in diverse human cancer. The high DNA methylation level of SASH3 was correlated with better OS in KIRC and UVM, and associated with poor OS in SKCM. Moreover, we uncover that SASH3 expression was positively associated with the stroma score in 27 cancer types, the microenvironment score, and immune score in 32 cancer types, 38 types of immune cells in 32 cancer types, the 45 immune stimulators, 24 immune inhibitors, 41 chemokines, 18 receptors, and 21 major histocompatibility complex (MHC) molecules in 33 cancer types. Finally, forced SASH3 expression inhibited lung adenocarcinoma (LUAD) cell proliferation and cell migration. Our findings confirmed that SASH3 may be a biomarker for the prognosis and diagnosis of human cancer.