Promoter CpG Island Research Articles

Leptin Protein Expression and Promoter Methylation in Ovarian Cancer: A Strong Prognostic Value with Theranostic Promises.

Ovarian cancer (OC) is the deadliest among all gynecological cancers. Epidemiological studies showed that obesity might influence many cancers including OC. One of the key factors that may link obesity and OC is leptin (LEP), known as an adipokine with pleiotropic effects on body homeostasis. This study aims to investigate the expression pattern of LEP, assess the methylation profiles of LEP and their associations with clinicopathological features including survival outcomes of OC patients. The protein expression of LEP was evaluated in 208 samples using both tissue microarray and immunohistochemistry techniques. The methylation profiles of LEP were measured in 63 formalin-fixed, paraffin-embedded tumor tissues by quantitative polymerase chain reaction using a MethyLight assay. Our results showed a significant association of LEP protein overexpression with several clinicopathological variables, mainly tumor subtype, LVI, age of menarche, tumor size and stage (p < 0.04). Kaplan–Meier analysis (using low expression versus high expression as a discriminator) indicated that LEP protein overexpression is a powerful positive prognosticator of both OC recurrence (DFS) and disease-specific survival (DSS) in our OC cohort (log-rank p = 0.01 and p = 0.002, respectively). This implies that patients with high LEP expression profiles live longer with less recurrence rates. Methylation analysis results demonstrated a clear association between no/low LEP protein expression pattern (38%) and LEP promoter CpG island hypermethylation (43%). Results of this study suggest that LEP is a powerful prognosticator of OC recurrence and DSS. LEP expression in OC seems to be regulated by its promoter hypermethylation through gene partial/total silencing. Further multi-institutional studies using larger cohorts are required to demystify the intricate molecular functions of this leptin-driven effects in OC pathophysiology and to accurately assess its theranostic potential and validate its prognostic/predictive power in OC onset, progression towards more effective and personalized management of OC patients.

Investigation of promoter regions, motifs, and CpG islands in the regulation of gene expression in Trametes hirsuta strain 072

BackgroundIn silico analysis of transcription start sites, promoter regions, transcription factors and their binding sites, and CpG islands for the Trametes hirsuta strain 072 genome were performed to understand the regulation mechanisms of gene expression and its genetic variations in the genomes. Therefore, a computational survey was carried out for the Trametes hirsuta strain 072 genome with the open reading frames from the National Center for Biotechnology Information database. Seventeen functional sequences were used to analyze promoter regions and their regulatory elements. ResultThe present study revealed that 94% of Trametes hirsuta strain 072 genes contained more than two TSSs. Among these identified TSSs, a TSS with the highest predictive score was considered to determine a promoter region of the genes. Moreover, a total of five common candidate motifs such as MotI, MotII, MotIII, MotIV, and MotV were identified. Among these motifs, motif IV was investigated as the common promoter motif for 41.17% of genes that serve as binding sites for transcription factors (TFs) involved in the expression regulation of Trametes hirsuta strain 072 genes. Motif IV was also compared to registered motifs in publically available databases to see if they are similar to known regulatory motifs for TF using TOMTOM web server. Hence, it was revealed that MotIV might serve as the binding site mainly for the leucine zipper TF gene family to regulate a gene expression of Trametes hirsuta strain 072. Regarding CpG island determination, it was concluded that there is no CpG island in both promoter and gene body regions of the Trametes hirsuta strain 072 genome. ConclusionsThis study provides a better insight into further molecular characterization which aimed to efficiently exploit a white rot fungus, Trametes hirsuta strain 072, for several biotechnological applications aimed to revitalize a severely contaminated environment.

161 Epigenetic Changes in Poultry Due to Reprogramming of the Gut Microbiota at an Early Stage of Embryonic Development

Abstract Epigenetic regulation of the gene expression is an interaction of the external environment with the genetic information. They are potentially heritable changes in the gene expression which does not involve alteration in DNA sequence and can be triggered by microRNA activity and DNA methylation. MicroRNA is fraction of small RNA molecules that have a fundamental impact on gene expression. DNA methylation inhibits DNA transcription by addition of the methyl residues to the cysteine within the CpG islands of the gene promoters. These processes can be modulated by environmental factors, such as intestinal microbiota modification. In poultry, the microbiota can be reprogrammed using in ovo technology at an early stage of embryo development. The intestinal microbiota is therefore stimulated and rearranged by injecting bioactive substances into air chamber of eggs on the day 12 of incubation. We have proved that the administration of lactic acid bacteria strains and galactooligosaccharide in ovo is effective in modulating of the intestinal microbiota. The administration of bioactive compounds has been demonstrated to influence gene expression in immune, intestinal and metabolic tissues. However, it has been noticed that a significant part of genes is silenced. In our experiment after in ovo administration of the substances in different genotypes (chicken broiler and native Polish breed) the range of tissues was collected: liver, caecal tonsils, spleen. By performing the bioinformatic analysis of the expression microarray, silenced genes and active miRNAs were selected. Methylation was analysed using the global and MSP-qPCR method, and analysis of miRNA activity using miRCURY LNA PCR Systems. We confirmed that negative regulation of the gene expression have epigenetic character and its mechanism depends on the genotype and the substance administered in ovo. Epigenetic nature of research is new direction of host-microbiome interaction. Research was financed by grant UMO-2017/25/N/NZ9/01822 funded by National Science Centre (Poland).

Bona Fide Tumor Suppressor Genes Hypermethylated in Melanoma: A Narrative Review.

Loss-of-function events in tumor suppressor genes (TSGs) contribute to the development and progression of cutaneous malignant melanoma (CMM). Epigenetic alterations are the major mechanisms of TSG inactivation, in particular, silencing by promoter CpG-island hypermethylation. TSGs are valuable tools in diagnosis and prognosis and, possibly, in future targeted therapy. The aim of this narrative review is to outline bona fide TSGs affected by promoter CpG-island hypermethylation and their functional role in the progression of CMM. We conducted a systematic literature review to identify studies providing evidence of bona fide TSGs by cell line or animal experiments. We performed a broad first search and a gene-specific second search, supplemented by reference checking. We included studies describing bona fide TSGs in CMM with promoter CpG-island hypermethylation in which inactivating mechanisms were reported. We extracted data about protein role, pathway, experiments conducted to meet the bona fide criteria and hallmarks of cancer acquired by TSG inactivation. A total of 24 studies were included, describing 24 bona fide TSGs silenced by promoter CpG-island hypermethylation in CMM. Their effect on cell proliferation, apoptosis, growth, senescence, angiogenesis, migration, invasion or metastasis is also described. These data give further insight into the role of TSGs in the progression of CMM.

In silico analysis of promoter region and regulatory elements of glucan endo-1,3-beta-glucosidase encoding genes in Solanum tuberosum: cultivar DM 1-3 516 R44

BackgroundPotato (Solanum tuberosum L.) is one of the most important food crops in the world. Pathogens remain as one of the major constraints limiting potato productivity. Thus, understanding of gene regulation mechanism of pathogenesis-related genes such as glucan endo-1,3-beta-glucosidase is a foundation for genetic engineering of potato for disease resistance and reduces the use of fungicides. In the present study, 19 genes were selected and attempts were made through in silico methods to identify and characterize the promoter regions, regulatory elements, and CpG islands of glucan endo-1,3-beta-glucosidase gene in Solanum tuberosum cultivar DM 1-3 516 R44. ResultsThe current analysis revealed that single transcription start sites (TSSs) were present in 12/19 (63.2%) of promoter regions analyzed. The predictive score at a cutoff value of 0.8 for the majority (84.2%) of the promoter regions ranged from 0.90 to 1.00. The locations for 42% of the TSSs were below −500 bp relative to the start codon (ATG). MβGII was identified as the common promoter motif for 94.4% of the genes with an E value of 3.5e−001. The CpG analysis showed low CpG density in the promoter regions of most of the genes except for gene ID102593331 and ID: 102595860. The number of SSRs per gene ranged from 2 to 9 with repeat lengths of 2 to 6 bp. Evolutionary distances ranged from 0.685 to 0.770 (mean = 0.73), demonstrating narrower genetic diversity range. Phylogeny was inferred using the UPGMA method, and gene sequences from different species were found to be clustered together. ConclusionIn silico identified regulatory elements in promoter regions will contribute to our understanding of the regulatory mechanism of glucan endo-1,3-beta-glucosidase genes and provide a promising target for genetic engineering to improve disease resistance in potatoes.

Epigenetic signatures differentiate uterine and soft tissue leiomyosarcoma.

Leiomyosarcomas (LMS) are diverse, rare, and aggressive mesenchymal soft tissue sarcomas. Epigenetic alterations influence multiple aspects of cancer, however epigenetic profiling of LMS has been limited. The goal of this study was to delineate the molecular landscape of LMS for subtype-specific differences (uterine LMS (ULMS) vs soft tissue LMS (STLMS)) based on integrated analysis of DNA methylation and gene expression to identify potential targets for therapeutic intervention and diagnosis. We identified differentially methylated and differentially expressed genes associated with ULMS and STLMS using DNA methylation and RNA-seq data from primary tumors. Two main clusters were identified through unsupervised hierarchical clustering: ULMS-enriched cluster and STLMS-enriched cluster. The integrated analysis demonstrated 34 genes associated with hypermethylation of the promoter CpG islands and downregulation of gene expression in ULMS or STLMS. In summary, these results indicate that differential DNA methylation and gene expression patterns are associated with ULMS and STLMS. Further studies are needed to delineate the contribution of epigenetic regulation to LMS subtype-specific gene expression and determine the roles of the differentially methylated and differentially expressed genes as potential therapeutic targets or biomarkers.

A CpG island promoter drives the CXXC5 gene expression

CXXC5 is a member of the zinc-finger CXXC family that binds to unmethylated CpG dinucleotides. CXXC5 modulates gene expressions resulting in diverse cellular events mediated by distinct signaling pathways. However, the mechanism responsible for CXXC5 expression remains largely unknown. We found here that of the 14 annotated CXXC5 transcripts with distinct 5′ untranslated regions encoding the same protein, transcript variant 2 with the highest expression level among variants represents the main transcript in cell models. The DNA segment in and at the immediate 5′-sequences of the first exon of variant 2 contains a core promoter within which multiple transcription start sites are present. Residing in a region with high G–C nucleotide content and CpG repeats, the core promoter is unmethylated, deficient in nucleosomes, and associated with active RNA polymerase-II. These findings suggest that a CpG island promoter drives CXXC5 expression. Promoter pull-down revealed the association of various transcription factors (TFs) and transcription co-regulatory proteins, as well as proteins involved in histone/chromatin, DNA, and RNA processing with the core promoter. Of the TFs, we verified that ELF1 and MAZ contribute to CXXC5 expression. Moreover, the first exon of variant 2 may contain a G-quadruplex forming region that could modulate CXXC5 expression.

HOX gene regulation in glioma stem cells, mediators of resistance in breast cancer, and paradoxical therapeutic interventions

Therapy resistance remains a major challenge during the treatment of patients with advanced cancer. Due to the complex nature of the cellular and molecular mechanisms underlying therapy resistance, research efforts to tackle this challenge have focused on diverse aspects: from cancer stem cell (CSC) biology, therapy resistance-specific signatures to the development of new combinatorial therapeutic approaches. Among other articles, the August issue of Molecular Oncology includes several studies that directly or indirectly explore therapy resistance in patients with advanced cancer. In some cancers, therapeutic resistance has been linked to the presence of CSCs. In glioma, for example, glioma stem cells (GSCs) represent a tumor cell subpopulation underlying tumor relapse. Understanding the molecular mechanisms behind GSC gene expression may give new insights on tumorigenesis and tumor relapse. In their recent study, Le Boiteux et al. [[1]] analyzed six patient-derived GSC lines, as well as 70 primary adult diffuse glioma samples to map the mechanisms leading to deregulation of the HOX gene clusters, which have been previously implicated in the oncogenic potential of GSCs. The authors identified widespread reactivation of HOX genes in adult IDH wild-type (IDHwt) diffuse glioma samples and GSCs. HOX gene deregulation was associated with self- and cross-regulatory interactions between HOX transcription factors, but not with genomic rearrangements. Integrative analysis of high-throughput DNA methylation analysis, ChIPseq, and RNAseq data demonstrated that the absence of hypermethylation on canonical and alternative bivalent CpG islands in promoters, as well as loss of the H3K27me3 epigenetic mark, underlies aberrant gene expression of HOX clusters in IDHwt gliomas. Besides providing a comprehensive epigenetic analysis of HOX gene regulation in glioma, the study by Le Boiteux et al. highlights the complexity of HOX gene expression via alternative promoters in cancer. In a different setting, Mohammad Sultan and colleagues employed an in vivo genome-wide shRNA screen to identify potential mediators or biomarkers of paclitaxel resistance in patients with advanced breast cancer [[2]]. The transcriptional repressor BCL6 was identified among the top paclitaxel resistance hits, and BCL6 overexpression was detected in tumors of chemotherapy-resistant patients. To further test the role of BCL6 in paclitaxel resistance, the authors performed BCL6 knockdown and BCL6 inhibitor experiments both in vitro and in vivo, in a mouse model of triple-negative breast cancer. BCL6 targeting in preclinical models enhanced the efficacy of paclitaxel treatment. Thus, BCL6 may serve as a biomarker for drug resistance and may potentially be targeted for the treatment of patients with breast cancer that have developed paclitaxel resistance. In addition to chemotherapy, targeted therapies designed to block mitogenic signaling pathways are often used for the treatment of patients with cancer. However, drug resistance to these therapeutic agents represents a major challenge for the treatment of advanced cancer. In their perspective, Matheus Henrique Dias and Rene Bernards explore the prospects of a ‘paradoxical intervention’ strategy for the treatment of therapy-resistant cancers [[3]]. Such a strategy would involve overactivation, rather than inhibition, of mitotic signaling in cancer cells. In their view, such an approach would result in an overload of the stress response pathways and disruption of cancer cell homeostasis and viability. The authors review related data from several independent experimental models, discuss future perspectives, and explore the potential drawbacks of such a paradoxical intervention strategy for the treatment of patients with cancer.

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

BANP opens chromatin and activates CpG-island-regulated genes.

The majority of gene transcripts generated by RNA polymeraseII in mammalian genomes initiate at CpG island (CGI) promoters1,2, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type3-5. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters6-8. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.

Interrelationship Of c-Myc, Micro RNA 29 and 34 Families, DNA methyltransferases and Methylation In Acute Myeloid Leukemia

BackgroundMicro RNAs (miRs) are small non-coding RNAs of 19-25 bases in length having the ability to modulate the expression of other genes. MiRs are frequently involved in carcinogenesis and its expression analysis to predict the phenotype of malignancies. Transcriptional silencing of tumor suppressor genes in cancer cells is often associated with hypermethylation of the promoter CpG islands. Three different DNMTs play major roles in establishing and maintaining DNA methylation patterns. It has been demonstrated that miR-29 family down-regulate DNMTs expression and several tumor suppressor miRs are hypermethylated and consequently down-regulated in cancer cells. Many studies have evaluated miR expression, epigenetics and the prognosis of acute myeloid leukemia (AML), but interrelationships between expression and methylation of miRs and DNMTs expression in AML patients' samples has not been fully elucidated. MethodsBM samples obtained from 54 of AML patients are subjected to the study after informed consent. Control BM samples are obtained from12 patients suffering from lymphoma. This study was approved by IRB of Gunma University Hospital. DNA, RNA and miR were extracted from BM mononuclear cells. The expression levels of miR 29a, 29b, 29c, 34a, 34b, 34c and mRNAs of DNMT1, 3A, 3B, primary miR (pri-miR) 29a/b-1, pri-miR 34a, 34b/c, c-myc, p21 were quantified by real time PCR with either Taqman-probe or SYBR green. Methylation specific PCR (MSP) was carried out with bisulfite converted DNA to see methylation status of for miR 34a, 34b/c. ResultsThe expression levels of miR29a, 29b, 29c, pri-miR29a/b-1, 34a, 34b, 34b star and 34c were significantly reduced in cells of AML (median 0.55, 0.19, 0.34, 50.98, 213.40, 1.61, 260.09 and 288.49 respectively) than of control (3.08, 4.78, 3.57, 2082.49, 472.68, 28.99 and 1781.33,) (p<0.001). Contrary, the expression levels of pri-miR34a and miR 34a star, were not significantly different between the AML and control samples. The promoter of miR34a and miR34b/c have CpG island which are often hyper-methylated thus we examined methylation of these miRs using MSP. We found that the promoters of miR34a and miR34b/c are more frequently methylated in AML than in control (38% vs 11%, p=0.005, 69% vs 17%, p<0.001). These results implied that miR expressions are epigenetically regulated through DNA methylation in AML. MiR34a, miR34b/c and pri-miR34a levels were not significantly different in between methylated and un-methylated group of AML, even though the miRs tended to be lower in methylated group. When the correlation between DNMTs and miRs expression levels were analyzed in AML, DNMTs expression tended to be negatively correlated with miR 34 family expression, but significant negative correlation were observed in DNMT1 and miR34b (r=-0.365, p=0.014) DNMT3B and miR34b, c (r=-0.556, p=0.017, r=-0.494, p=0.037, respectively). MiR34 family transcription is up-regulated by TP53. We analyzed the correlation between miR34 family and well-known TP53 target p21, but there was no significant correlation. C-myc is also known to suppress miR29 family which is negative regulator of DNMTs. C-myc level is significantly higher in AML than control (0.019 vs. 0.007, p=0.001) and miR29 family is lower in AML, but the correlation between c-myc and miR29 expressions in AML did not reach statistical significance. C-myc is positively correlated with DNMT1 and 3A expressions (r=0.753, r=0.610, p<0.001). There were significant positive correlations among miRs expressions in AML samples; miR29a-29b r=0.717, 29a-29c r=0.739, 29a-34a r=0.568, p<0.001, 29a-34b r=0.309, p=0.029, miR29b-29c r=0.893, 29b-34a r=0.596, 29b-34b r=0.621, 29b-34c r=0.518, 29c-34a r=0.646, 29c-34b r=0.655, 29c-34c r=0.500, p<0.001. When the cell lines were treated with 5 deoxy-azacytidine, miR34a, b, c expression levels increased after the treatment, suggesting that these miRs expression was regulated at least in part by promoter hypermethylation. ConclusionWe found significant reduction of miRs expression in AML and the reduction in part associated with hypermethylation. Direct correlations in between miR expression, methylation and DNMT could not be shown in this study, however, we somehow showed interrelationships between c-myc and DNMTs, miR29 and miR34 family. C-myc, miR29, DNMT, methylation, miR34 axis may play an important role in AML development and progression. Disclosures:No relevant conflicts of interest to declare.

Abstract 2107: Transcription factor repertoire basis for epigenetic and consensus molecular subtypes of colorectal cancer

Abstract Colorectal cancer (CRC) is classified into distinct subtypes based on genetic, epigenetic and/or transcriptional subtypes. The underlying mechanisms that drive these subtypes are unknown. By integrating whole genome DNA methylation, gene expression, chromatin patterns, and enhancer elements in CRCs, we show that expression of large numbers of transcriptional factors (TFs) is deregulated in CRCs and these expression patterns drive epigenetic and transcriptional subtypes of CRCs. The first key result emerging is a new understanding of important DNA methylation patterns central to classifying CRCs. Of these, different degrees of cancer associated promoter methylation have been recognized that suppress expression, and/or inducibility of important tumor suppressor genes. Stratified by high to low degrees of hypermethylation, this pattern is termed CpG-island promoter DNA methylation phenotypes or CIMP. We now tightly link CIMP to a cancer-specific hypermethylation at enhancer regulatory elements we term, CpG- enhancer methylation phenotype (CEMP). CIMP and CEMP are driven by decreased expression of multiple TFs with binding sites in these regulatory elements, and the involved TFs are frequently identical between the two regions. In contrast, a different set of TF factors are downregulated in non-CIMP tumors, again in conjunction with methylation patterns linking promoter and enhancer target sites for the involved TFs. Further, enhancer regulatory elements that are hypomethylated in cancer relative to normal tissue are associated with increased expression of another distinct set of TFs with target sites in the corresponding enhancers. Importantly, the TF expression patterns, and the above CIMP, CEMP, and hypomethylation patterns, are established early in the pre-malignant adenomas. Importantly, pan-cancer analyses showed that the above CIMP and CEMP subtypes occur across important cancer types in addition to CRC, again in parallel with marked downregulation of TFs that have target sites in the methylated regulatory elements. All of the above newly defined patterns also provide a basis for the transcriptome-based consensus molecular subtypes (CMS) of CRC. Overall, our studies may provide key understanding of how cancers, and their initiation and progression, are diseases which at the end of the day depend on abnormal patterns of TF expression, and how these relate to methylation alterations at target enhancer and promoter regions. Citation Format: Yuba Bhandari, Rachael Powers, Sehej Parmar, Vinod Krishna, Kurtis E. Bachman, Steve Baylin, Hariharan Easwaran. Transcription factor repertoire basis for epigenetic and consensus molecular subtypes of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2107.

Molecular Genetics of Esophageal Cancer : Indian Perspective

Background: RIZ1 is one of the tumor-suppressor genes that is silenced in many human cancers. Change in RIZ1 expression has not been reported in ESCC patients. Therefore, the aim of this study was to investigate the role of RIZ1 in ESCC in the Indian population. Methods: Twelve esophageal squamous-cell carcinoma (ESCC) patients in stage IV and 12 healthy individuals were used in this study. Tissue sampling was taken from individuals and total RNA was isolated and then cDNA was synthesized using PCR. RIZ1 primers were then designed, and RIZ1 expression was quantified by qRT-PCR. Mapping of CpG islands in RIZ1 promoter was performed using bioinformatics tools. The promoter methylation status of this gene was studied using u methylation-specific PCR (MSP). T-student test was used to analyze the data.Results: Decreased RIZ1 expression was observed in ESCC compared with healthy controls. The results showed a relatively higher density of CpG dinucleotides in the RIZ1 promoter. No significant difference in promoter methylation was observed in blood and tissue samples.Conclusion: The study showed a significant down-regulation of RIZ-1 gene in the blood and tissue samples of ESCC patients that did not related to the altered promoter methylation.

DNA methylation mediated RSPO2 to promote follicular development in mammals

In female mammals, the proliferation, apoptosis, and estradiol-17β (E2) secretion of granulosa cells (GCs) have come to decide the fate of follicles. DNA methylation and RSPO2 gene of Wnt signaling pathway have been reported to involve in the survival of GCs and follicular development. However, the molecular mechanisms for how DNA methylation regulates the expression of RSPO2 and participates in the follicular development are not clear. In this study, we found that the mRNA and protein levels of RSPO2 significantly increased during follicular development, but the DNA methylation level of RSPO2 promoter decreased gradually. Inhibition of DNA methylation or DNMT1 knockdown could decrease the methylation level of CpG island (CGI) in RSPO2 promoter and upregulate the expression level of RSPO2 in porcine GCs. The hypomethylation of −758/−749 and −563/−553 regions in RSPO2 promoter facilitated the occupancy of transcription factor E2F1 and promoted the transcriptional activity of RSPO2. Moreover, RSPO2 promoted the proliferation of GCs with increasing the expression level of PCNA, CDK1, and CCND1 and promoted the E2 secretion of GCs with increasing the expression level of CYP19A1 and HSD17B1 and inhibited the apoptosis of GCs with decreasing the expression level of Caspase3, cleaved Caspase3, cleaved Caspase8, cleaved Caspase9, cleaved PARP, and BAX. In addition, RSPO2 knockdown promoted the apoptosis of GCs, blocked the development of follicles, and delayed the onset of puberty with decreasing the expression level of Wnt signaling pathway-related genes (LGR4 and CTNNB1) in vivo. Taken together, the hypomethylation of −758/−749 and −563/−553 regions in RSPO2 promoter facilitated the occupancy of E2F1 and enhanced the transcription of RSPO2, which further promoted the proliferation and E2 secretion of GCs, inhibited the apoptosis of GCs, and ultimately ameliorated the development of follicles through Wnt signaling pathway. This study will provide useful information for further exploration on DNA-methylation-mediated RSPO2 pathway during follicular development.

In silico analysis of promoter region and regulatory elements of mitogenome co-expressed trn gene clusters encoding for bio-pesticide in entomopathogenic fungus, Metarhizium anisopliae: strain ME1

BackgroundPest control strategies almost entirely rely on chemical insecticides, which cause environmental problems such as biosphere deterioration and emergence of resistant pests. Bio-pesticide is an alternative approach, which uses organisms such as entomopathogenic fungi, Metarhizium anisopliae, to control pests. Screening such potential organism at a molecular level and understanding their gene regulation mechanism is an important approach to reduce emergence of pesticide resistance and worsening of the biosphere. Understanding promoter regions which play a pivotal role in gene regulation is crucial. In particular, identification of the promoter regions in M. anisopliae Strain ME1 remains poorly understood. To our knowledge, the mitogenome trn gene clusters of M. anisopliae Strain ME1 were not characterized. Here, we used machine learning approach to identify and characterize the promoter regions, regulatory elements, and CpG island densities of 15 protein coding genes of entomopathogenic fungi, M. anisolpliae Strain ME1. ResultsThe current analysis revealed multiple transcription start sites (TSS) for all utilized sequences, except for promoter region genes of Pro-cob and Pro-nad5. With reference to the start codon (ATG), 85.3% of TSS was located above – 500 bp. Based on the standard predictive score at cut off value of 0.8a, the current study revealed 54.7% of predictive score greater than or equal from 0.9 promoter prediction score. Expectation maximization algorithm output identified five candidate motifs. Nonetheless, of all candidate motifs, MtrnI was revealed as the common promoter region motif with a value of 76.9% both in terms of size of binding sites and with an E value of 9.1E−054. Accordingly, we perceived that MtrnI serve as the binding site for tryptophan cluster with P value 0.0044 and C4 type zinc fingers functions as the binding site to regulate gene expression of M. anisopliae Strain ME1. The analysis revealed that mitogenome trn gene clusters of M. anisopliae Strain ME1 showed homologues evolutionary ancestor supported with a bootstrap value of 100%. ConclusionIdentified common candidate motifs and binding transcription factors through in silico approach are likely expected to contribute for better understanding of gene expression and strain improvement of M. anisopliae Strain ME1 for its bio-pesticides role.

The miR-455-3p/HDAC2 axis plays a pivotal role in the progression and reversal of liver fibrosis and is regulated by epigenetics.

Histone deacetylases (HDACs), especially HDAC2, play a role in alleviating liver fibrosis; however, the specific upstream regulation mechanism is unknown. Herein, TargetScan was used to predict the potential upstream targets of HDAC2, and the role of miR-455-3p was explored. The dual luciferase reporter assay showed that miR-455-3p binds to the 3' UTR of HDAC2 mRNA. Additionally, miR-455-3p was downregulated in the liver tissues of patients with cirrhosis and mice with liver fibrosis, as well as in primary HSCs isolated from fibrotic mouse livers and TGF-β-treated LX-2 cells. In contrast, it is highly expressed in the reversal stage of hepatic fibrosis and MDI-cultured LX-2 cells. Our functional analyses showed that miR-455-3p overexpression facilitated apoptosis and reduced the expression of pro-fibrotic markers and the proliferation of activated LX-2 cells. On the contrary, miR-455-3p inhibition converted inactivated LX-2 cells into activated, proliferative, fibrogenic cells. Interestingly, restoration of HDAC2 expression partially blocked the function of miR-455-3p. Downregulated miR-455-3p expression can be restored by DNA methyltransferases in activated LX-2 cells. Methylation-specific PCR, bisulfite sequencing PCR, and chromatin immunoprecipitation assays indicated that the methylation level of miR-455-3p promoter CpG islands was elevated in TGF-β-treated LX-2 cells and that miR-455-3p was downregulated in activated LX-2 cells by DNA hypermethylation, which is mediated by DNMT3b and DNMT1. In conclusion, miR-455-3p acts as a liver fibrosis suppressor by targeting HDAC2, and its deficiency further aggravates the reversal phase of fibrosis. Thus, the epigenetics mediated miR-455-3p/HDAC2 axis may serve as a novel potential therapeutic target for clinical treatment of hepatic fibrosis.

A new approach to decode DNA methylome and genomic variants simultaneously from double strand bisulfite sequencing.

Genetic and epigenetic contributions to various diseases and biological processes have been well-recognized. However, simultaneous identification of single-nucleotide variants (SNVs) and DNA methylation levels from traditional bisulfite sequencing data is still challenging. Here, we develop double strand bisulfite sequencing (DSBS) for genome-wide accurate identification of SNVs and DNA methylation simultaneously at a single-base resolution by using one dataset. Locking Watson and Crick strand together by hairpin adapter followed by bisulfite treatment and massive parallel sequencing, DSBS simultaneously sequences the bisulfite-converted Watson and Crick strand in one paired-end read, eliminating the strand bias of bisulfite sequencing data. Mutual correction of read1 and read2 can estimate the amplification and sequencing errors, and enables our developed computational pipeline, DSBS Analyzer (https://github.com/tianguolangzi/DSBS), to accurately identify SNV and DNA methylation. Additionally, using DSBS, we provide a genome-wide hemimethylation landscape in the human cells, and reveal that the density of DNA hemimethylation sites in promoter region and CpG island is lower than that in other genomic regions. The cost-effective new approach, which decodes DNA methylome and genomic variants simultaneously, will facilitate more comprehensive studies on numerous diseases and biological processes driven by both genetic and epigenetic variations.

Association of methylation level and transcript level in TRAF5 gene with ankylosing spondylitis: a case-control study.

To explore the association between methylation level and transcript level of TNF receptor-associated factor 5 (TRAF5) gene with ankylosing spondylitis (AS) in Chinese Han population. Methylation and mRNA expression level of the TRAF5 gene were tested in 98 patients and 98 healthy controls. Among the 21 CpG sites, methylation levels at eight sites were significantly different between AS patients and healthy controls. However, only three sites remained significantly different after the correction by the Benjamini-Hochberg method. Compared with controls, the CpG island of TRAF5 gene promoter was highly methylated in AS patients, and the relative mRNA expression level of TRAF5 was significantly reduced in AS patients. And the mRNA level was negatively correlated with the methylation level of TRAF5 gene in AS patients (rs = -0.453, P < 0.001). Subgroup analyses indicated that there was no significant difference in the level of methylation between groups of different status of HLA-B27 and medications in AS patients. Multiple linear regression showed that disease-modifying antirheumatic drugs could reduce methylation levels of AS patients after adjusting for the effects of other drugs. In conclusion, the hypermethylation of the TRAF5 might contribute to the pathogenesis of AS, but many open questions remain.

Aberrant hypermethylation induced downregulation of antisense lncRNA STXBP5-AS1 and its sense gene STXBP5 correlate with tumorigenesis of glioma

AimsThe expression of antisense lncRNA STXBP5-AS1 and its sense gene STXBP5 were found to be downregulated in glioma by RNA sequencing; however, the function and mechanism of both two genes in the development of glioma have not been studied. Materials and methodsQRT-PCR and western blot were used to determine the transcriptional and translational levels of moleculars. MSP and BSP assays were used to evaluate the methylation status of promoter CpG island. MTT, EdU, flow cytometry, and transwell assays were used to reveal biological effects. The in vivo mice model was used to validate the role of target genes in tumorigenesis. Key findingsThe mRNA and protein expression of STXBP5 was significantly downregulated in glioma tissues and positively correlated with prognosis. STXBP5-AS1 was downregulated in glioma cells and tissues, and associated with tumor size and clinical stages. Both of two genes were significantly restored in cells treatment with 5-Aza. The promoter CpG island of STXBP5/STXBP5-AS1 was hypermethylated in glioma cells, but partially methylated in NHA cells. We found that promoter methylation frequency was significantly higher in glioma tissues. Functionally, overexpression of STXBP5 and STXBP5-AS1 inhibited cell proliferation, migration, and invasion and promoted apoptosis in vitro, whereas depletion of STXBP5 and STXBP5-AS1 showed opposite effects. Both the mRNA and protein expression of STXBP5 were positively regulated by STXBP5-AS1. Ectopic expression of STXBP5 and STXBP5-AS1 suppressed tumor formation in vivo. SignificanceOur findings suggested that epigenetically silenced STXBP5-AS1 and STXBP5 might act as novel tumor suppressors of glioma.

Widespread overexpression from the four DNA hypermethylated HOX clusters in aggressive (IDHwt) glioma is associated with H3K27me3 depletion and alternative promoter usage.

In human, the 39 coding HOX genes and 18 referenced noncoding antisense transcripts are arranged in four genomic clusters named HOXA, B, C, and D. This highly conserved family belongs to the homeobox class of genes that encode transcription factors required for normal development. Therefore, HOX gene deregulation might contribute to the development of many cancer types. Here, we study HOX gene deregulation in adult glioma, a common type of primary brain tumor. We performed extensive molecular analysis of tumor samples, classified according to their isocitrate dehydrogenase (IDH1) gene mutation status, and of glioma stem cells. We found widespread expression of sense and antisense HOX transcripts only in aggressive (IDHwt) glioma samples, although the four HOX clusters displayed DNA hypermethylation. Integrative analysis of expression, DNA methylation, and histone modification signatures along the clusters revealed that HOX gene upregulation relies on canonical and alternative bivalent CpG island promoters that escape hypermethylation. H3K27me3 loss at these promoters emerges as the main cause of widespread HOX gene upregulation in IDHwt glioma cell lines and tumors. Our study provides the first comprehensive description of the epigenetic changes at HOX clusters and their contribution to the transcriptional changes observed in adult glioma. It also identified putative ‘master’ HOX proteins that might contribute to the tumorigenic potential of glioma stem cells.