Repressor Region Research Articles

Epigenetic mutation load is weakly correlated with epigenetic age acceleration.

DNA methylation (DNAm) age estimators are widely used to study aging-related conditions. It is not yet known whether DNAm age is associated with the accumulation of stochastic epigenetic mutations (SEMs), which reflect dysfunctions of the epigenetic maintenance system. Here, we defined epigenetic mutation load (EML) as the total number of SEMs per individual. We assessed associations between EML and DNAm age acceleration estimators using biweight midcorrelations in four population-based studies (total n = 6,388). EML was not only positively associated with chronological age (meta r = 0.171), but also with four measures of epigenetic age acceleration: the Horvath pan tissue clock, intrinsic epigenetic age acceleration, the Hannum clock, and the GrimAge clock (meta-analysis correlation ranging from r = 0.109 to 0.179). We further conducted pathway enrichment analyses for each participant’s SEMs. The enrichment result demonstrated the stochasticity of epigenetic mutations, meanwhile implicated several pathways: signaling, neurogenesis, neurotransmitter, glucocorticoid, and circadian rhythm pathways may contribute to faster DNAm age acceleration. Finally, investigating genomic-region specific EML, we found that EMLs located within regions of transcriptional repression (TSS1500, TSS200, and 1stExon) were associated with faster age acceleration. Overall, our findings suggest a role for the accumulation of epigenetic mutations in the aging process.

Chromatin as a key consumer in the metabolite economy.

In eukaryotes, chromatin remodeling and post-translational modifications (PTMs) shape the local chromatin landscape to establish permissive and repressive regions within the genome, orchestrating transcription, replication, and DNA repair in concert with other epigenetic mechanisms. Though cellular nutrient signaling encompasses a huge number of pathways, recent attention has turned to the hypothesis that the metabolic state of the cell is communicated to the genome through the type and concentration of metabolites in the nucleus that are cofactors for chromatin-modifying enzymes. Importantly, both epigenetic and metabolic dysregulation are hallmarks of a range of diseases, and this metabolism-chromatin axis may yield a well of new therapeutic targets. In this Perspective, we highlight emerging themes in the inter-regulation of the genome and metabolism via chromatin, including nonenzymatic histone modifications arising from chemically reactive metabolites, the expansion of PTM diversity from cofactor-promiscuous chromatin-modifying enzymes, and evidence for the existence and importance of subnucleocytoplasmic metabolite pools.

Establishment of immortalised cell lines derived from female Shiba goat KNDy and GnRH neurones.

Kisspeptin plays a critical role in governing gonadotrophin-releasing hormone (GnRH)/gonadotrophin secretion and subsequent reproductive function in mammals. The hypothalamic arcuate nucleus (ARC) kisspeptin neurones, which co-express neurokinin B (NKB) and dynorphin A (Dyn) and are referred to as KNDy neurones, are considered to be involved in GnRH generation. The present study aimed to establish cell lines derived from goat KNDy and GnRH neurones. Primary-cultured cells of female Shiba goat foetal hypothalamic ARC and preoptic area (POA) tissues were immortalised with the infection of lentivirus containing the simian virus 40 large T-antigen gene. Clones of the immortalised cells were selected by the gene expression of a neuronal marker, and then the neurone-derived cell clones were further selected by the gene expression of KNDy or GnRH neurone markers. As a result, we obtained a KNDy neurone cell line (GA28) from the ARC, as well as two GnRH neurone cell lines (GP11 and GP31) from the POA. Immunocytochemistry revealed the expression of kisspeptin, NKB and Dyn in GA28 cells, as well as GnRH in GP11 and GP31 cells. GnRH secretion from GP11 and GP31 cells into the media was confirmed by an enzyme immunoassay. Moreover, kisspeptin challenge increased intracellular Ca2+ levels in subsets of both GP11 and GP31 cells. Kisspeptin mRNA expression in GA28 cells, which expressed the oestrogen receptor alpha gene, was significantly reduced by 17β-oestradiol treatment. Furthermore, the transcriptional core promoter and repressive regions of the goat NKB gene were detected using GA28 cells. In conclusion, we have established goat KNDy and GnRH neurone cell lines that could be used to analyse molecular and cellular mechanisms regulating KNDy and GnRH neurones in vitro, facilitating the clarification of reproductive neuroendocrine mechanisms in ruminants.

Multifaceted genome-wide study identifies novel regulatory loci in SLC22A11 and ZNF45 for body mass index in Indians.

Obesity, a risk factor for multiple diseases (e.g. diabetes, hypertension, cancers) originates through complex interactions between genes and prevailing environment (food habit and lifestyle) that varies across populations. Indians exhibit a unique obesity phenotype with high abdominal adiposity for a given body weight compared to matched white populations suggesting presence of population-specific genetic and environmental factors influencing obesity. However, Indian population-specific genetic contributors for obesity have not been explored yet. Therefore, to identify potential genetic contributors, we performed a two-staged genome-wide association study (GWAS) for body mass index (BMI), a common measure to evaluate obesity in 5973 Indian adults and the lead findings were further replicated in 1286 Indian adolescents. Our study revealed novel association of variants-rs6913677 in BAI3 gene (p = 1.08 × 10-8) and rs2078267 in SLC22A11 gene (p = 4.62 × 10-8) at GWAS significance, and of rs8100011 in ZNF45 gene (p = 1.04 × 10-7) with near GWAS significance. As genetic loci may dictate the phenotype through modulation of epigenetic processes, we overlapped genetic data of identified signals with their DNA methylation patterns in 236 Indian individuals and performed methylation quantitative trait loci (meth-QTL) analysis. Further, functional roles of discovered variants and underlying genes were speculated using publicly available gene regulatory databases (ENCODE, JASPAR, GeneHancer, GTEx). The identified variants in BAI3 and SLC22A11 genes were found to dictate methylation patterns at unique CpGs harboring critical cis-regulatory elements. Further, BAI3, SLC22A11 and ZNF45 variants were located in repressive chromatin, active enhancer, and active chromatin regions, respectively, in human subcutaneous adipose tissue in ENCODE database. Additionally, these genomic regions represented potential binding sites for key transcription factors implicated in obesity and/or metabolic disorders. Interestingly, GTEx portal identify rs8100011 as a robust cis-expression quantitative trait locus (cis-eQTL) in subcutaneous adipose tissue (p = 1.6 × 10-7), and ZNF45 gene expression in skeletal muscle of Indian subjects showed an inverse correlation with BMI indicating its possible role in obesity. In conclusion, our study discovered 3 novel population-specific functional genetic variants (rs6913677, rs2078267, rs8100011) in 2 novel (SLC22A11 and ZNF45) and 1 earlier reported gene (BAI3) for BMI in Indians. Our study decodes key genomic loci underlying obesity phenotype in Indians that may serve as prospective drug targets in future.

Overexpression of a maize BR transcription factor ZmBZR1 in Arabidopsis enlarges organ and seed size of the transgenic plants

In plants, the organ size is one of the most important features and regulated by an elaborate developmental program involving both internal and external signals. The steroidal hormone brassinosteroid (BR) plays an important role in regulating the organ size. BRASSINAZOLE RESISTANT 1 (BZR1) is one of important transcription factors that regulate organ size in BR signal pathway in Arabidopsis. The function of BZR1 on organ size is well characterized in Arabidopsis, but poorly understood in maize (Zea mays). To understand the mechanism of intrinsic organ size regulated by BZR1 during organogenesis, we identified the maize BZR1 and examined its function in Arabidopsis. Overexpression of ZmBZR1 displayed phenotypes of enlarged cotyledons, rosette leaves, floral organ and seed size in Arabidopsis. The cells in rosette leaves as well as other organs in transgenic ZmBZR1 lines were dramatically larger and longer than those in Col-0. ChIP and RNA-seq analysis showed ZmBZR1 can directly bind to the promoter region of organ size related genes, Germination Repression and Cell Expansion receptor-like kinase (GRACE) and KIP-RELATED PROTEIN6 (KRP6) to regulate their expression, suggesting ZmBZR1 is required for the progressive increase in cells during Arabidopsis development. Collectively, our findings provide significant insights into the mechanisms underlying regulation of organ size mediated by maize BZR1.

A novel understanding of postoperative complications: In vitro study of the impact of propofol on epigenetic modifications in cholinergic genes

BackgroundPropofol is a widely used anaesthetic drug with advantageous operating conditions and recovery profile. However, propofol could have long term effects on neuronal cells and is associated with post-operative delirium (POD). In this context, one of the contributing factors to the pathogenesis of POD is a reduction of cholinesterase activity. Accordingly, we investigated the effects of propofol on the methylation, expression and activity of cholinergic genes and proteins in an in-vitro model.ResultsWe found that propofol indeed reduced the activity of AChE / BChE in our in-vitro model, without affecting the protein levels. Furthermore, we could show that propofol reduced the methylation of a repressor region of the CHRNA7 gene without changing the secretion of pro–or anti-inflammatory cytokines. Lastly, propofol changed the expression patterns of genes responsible for maintaining the epigenetic status of the cell and accordingly reduced the tri-methylation of H3 K27.ConclusionIn conclusion we found a possible functional link between propofol treatment and POD, due to a reduced cholinergic activity. In addition to this, propofol changed the expression of different maintenance genes of the epigenome that also affected histone methylation. Thus, propofol treatment may also induce strong, long lasting changes in the brain by potentially altering the epigenetic landscape.

LncRNAs with miRNAs in regulation of gastric, liver, and colorectal cancers: updates in recent years.

Long noncoding RNA (lncRNA) is a kind of RNAi molecule composed of hundreds to thousands of nucleotides. There are several major types of functional lncRNAs which participate in some important cellular pathways. LncRNA-RNA interaction controls mRNA translation and degradation or serves as a microRNA (miRNA) sponge for silencing. LncRNA-protein interaction regulates protein activity in transcriptional activation and silencing. LncRNA guide, decoy, and scaffold regulate transcription regulators of enhancer or repressor region of the coding genes for alteration of expression. LncRNA plays a role in cellular responses including the following activities: regulation of chromatin structural modification and gene expression for epigenetic and cell function control, promotion of hematopoiesis and maturation of immunity, cell programming in stem cell and somatic cell development, modulation of pathogen infection, switching glycolysis and lipid metabolism, and initiation of autoimmune diseases. LncRNA, together with miRNA, are considered the critical elements in cancer development. It has been demonstrated that tumorigenesis could be driven by homeostatic imbalance of lncRNA/miRNA/cancer regulatory factors resulting in biochemical and physiological alterations inside the cells. Cancer-driven lncRNAs with other cellular RNAs, epigenetic modulators, or protein effectors may change gene expression level and affect the viability, immortality, and motility of the cells that facilitate cancer cell cycle rearrangement, angiogenesis, proliferation, and metastasis. Molecular medicine will be the future trend for development. LncRNA/miRNA could be one of the potential candidates in this category. Continuous studies in lncRNA functional discrepancy between cancer cells and normal cells and regional and rational genetic differences of lncRNA profiles are critical for clinical research which is beneficial for clinical practice.

Expression of Pichia pastoris tRNAProCCG and its function

Translocation ribonucleic acid (tRNA) is one of the important components in protein synthesis. In order to explore the effect of the changes of tRNAs corresponding to rare codons (rarity tRNAs) on the expression of exogenous genes, the co-expression system of rare tRNA gene and exogenous gene in Pichia pastoris was constructed. The expression of GFP in P. pastoris can be greatly reduced when a repressor region composed of four continuous proline rare codon CCG was added into the GFP gene. The expression amount of the repressed GFP could be increased about 4.9% when tRNAProCCG gene was cointegrated to the 3' of the repressed GFP gene through pPIC9K to the genome of P. pastoris GS115. Meanwhile, the expression amount of the repressed GFP increased about 12.5% by integrating the repressed GFP gene and tRNAProCCG gene to the genome of P. pastoris GS115 through pPIC9K and pFLDα, respectively. Using the same method, NFATc3T-GFP fusion gene and tRNAProCCG gene were co-expressed in P. pastoris GS115 resulting in 21.3% increased of the expression amount of NFATc3T-GFP fusion protein. In conclusion, tRNAProCCG gene has been confirmed to be a kind of rare tRNAs in P. pastoris GS115. Through co-expression of tRNAProCCG gene and heterologous genes which containing the continuous rare codon CCG, the expression of the repressed heterologous genes could be increased significantly. Furthermore, this co-expression system would contribute to screening and determining the other rare tRNAs.

68 - VARIOUS RELATIONSHIPS BETWEEN DNA METHYLATION AND GENE EXPRESSION IN DIFFERENT TISSUES AND AGES

Background Traditionally, DNA methylation is described as a mechanism that represses gene expression by blocking the binding of transcription factors at promoter regions. However, many studies suggest that DNA methylation variation makes a number of diverse contributions to gene expression and its complex underlying mechanisms. Methods To uncover the relationship and mechanism between gene expression and DNA methylation, we tested gene and CpG Pairs (GCPs) for the correlation between gene expression and methylation across different tissues and ages. We collected tissues that have both methylation data and matched expression data from various databases and publications. After filtering samples, we retained 502 adult Prefrontal Cortices (PFC), 43 Lymphoblastoid Cell Llines (LCL), 75 livers, 1201 monocytes, as well as 247 developmental PFCs across the lifespan. To analyze the methylation data, we used the ChAMP package for filtering. For the expression data from different platforms, we used different values as the expression measure. ComBat and SVA were used to control batch effects and other effects for both expression and methylation data. To remove the less variable methylation, we also filtered out 50% methylation probes with small median absolute deviation. For each region, we defined pairs of methylation CpG site and gene expression as CpG sites located within the 10kb flanking region of the corresponding gene or probe. We used Spearman's rank correlations test to assess the correlation of methylation-expression pairs. Results Besides, we performed the False Discovery Rate (FDR) to correct for the multiple hypothesis testing. In the adult PFC region, 230,087 GCPs were analyzed for the Spearman correlation between gene expression and CpG site methylation, and only 8,828 GCPs were significantly correlated after FDR correction. Of these, 5,530 GCPs were negatively correlated and 3,298 GCPs were positively correlated. The developmental PFC and other tissues also exhibited a small proportion of significant GCPs, including 544/174,911 in the LCL, 2,281/210,025 in the liver, 5,134/46,831 in the monocyte and 9,729/134,690 in the developmental PFC. Though thousands of significant GCPs have been identified in different tissues, only 37 of them were consistent in at least three tissues; three of them were shared in all tissues. Additionally, there were 442 negatively and 153 positively correlated GCPs shared between the adult PFC and the developmental PFC. For the CpGs of significant GCPs, we explored the epigenetic state of these CpGs using the Roadmap 15-core state annotation. Negatively correlated CpGs were enriched in active regions containing Active TSS, Flanking Active TSS, and Enhancers, while positive ones were enriched in repressive regions such as Bivalent Enhancer and Repressed PolyComb. Discussion For the genes of significant GCPs, we performed the GO term enrichment analysis using WebGestalt. We found that genes in correlated GCPs in developmental PFC were enriched for neuron projection development (p= 3.96e-12, FDR= 6.11e-09), neuron differentiation (p= 1.08e-11, FDR= 9.25e-09) and synapse part (p= 0e+00, FDR= 0e+00). In the adult PFC data, genes were also enriched for neuron part (p= 2.93e-13, FDR= 3.02e-11), distinct from the GO term referring to genes in the developmental PFC. These results imply that DNA methylation has a special regulated function in cell specification and development.

Characterization and functional analyses of the human HTR1A gene: 5\u2019 regulatory region modulates gene expression in vitro

BackgroundThe serotonin neurotransmitter (5-HT) and its receptors have important roles in neuropsychiatric disorders such as schizophrenia. The aim of this study was to investigate the functional sequences of the 5′ regulation region of the human HTR1A gene to explore the effects on the expression of the 5-HT1A receptor.MethodsFourteen recombinant pGL3-basic vectors containing deletion fragments of the HTR1A gene regulatory region were transfected with HEK-293 and SK-N-SH cells. The relative chemiluminescence intensities of different length fragments were analyzed. The JASPAR software was used for the prediction of transcription factors.ResultsIn the HEK-293 cells, the relative chemiluminescence intensity of the − 1649 bp to − 1550 bp (ATG + 1) fragment was significantly different. Two inhibitory activity regions were found in the − 1409 bp to − 1381 bp and − 1196 bp to − 1124 bp fragments, which might be bound to the GATA or SOX10 transcription factors as predicted by the JASPAR software. In addition, the fragments located from − 1124 bp to − 1064 bp and from − 908 bp to − 722 bp up-regulated protein expression. Only the sequence from − 1550 bp to − 1409 bp demonstrated a difference in luciferase expression in the both cell lines. According to the results of the 5’-UTR truncated vectors, there was a repression region at the distal end of the 5’-UTR, an enhancer region might be present at the proximal end of the transcription start site.ConclusionsAlthough the functional sequences of the HTR1A gene regulatory region were confirmed, the regulatory factors and functional components require further investigation.

An Epigenetic Mitotic Score Tracks the Proliferative History and Capacity of CLL Samples at Diagnosis and Is Associated with Clinical Outcome

Background: DNA methylation has been extensively described to be related with gene regulation. However, recent data indicate that DNA methylation changes in repressed regions accumulate in every cell division, and thus, they can reflect the proliferative history of the cell. The evaluation of this epigenetic feature in cancer cells may provide information regarding the tumor cell biological evolution and may assist to predict the outcome of the patients.Aims: The global aim of this study was to develop a DNA methylation-based proliferative history score and determine its possible prognostic value in chronic lymphocytic leukemia (CLL).Methods: We used 450k methylation arrays from normal B cells subpopulations and CLLs at diagnosis. To analyze DNA methylation changes specifically in leukemic cells, we performed stringent data quality checks and corrections including FACS sorting and in silico purification of DNA methylation. Thus, we retained 67 normal B cells and 477 CLLs, comprising 140 naive-like CLLs (nCLL), 106 intermediate CLLs (iCLL) and 231 memory-like CLLs (mCLL) based on a recent epigenetic classification of cellular origin. We mapped CpGs falling in repressive regions using ChIP-Seq data of 6 histone marks from healthy B cell subpopulations and CLL patients. With the methylation levels of CpGs in repressive chromatin, we built one score per each sample which we termed Epigenetic Mitotic History (epiMH, ranging from 0 to 1). The epiMH was integrated with whole genome sequencing (WGS) and microarray expression data of the very same CLL patients (n=126). Finally, we analyzed whether epiMH was related to clinical behavior.Results: We showed that epiMH is widely variable in normal B cells, with more advanced differentiation stages displaying the highest epiMH, presumably due to their greater accumulated proliferative history. Then, we calculated the epiMH in CLLs with different cellular origins, namely, nCLL, iCLL and mCLL, and we correlated it with different biological features. Using mutational signatures from WGS data of the same samples, we identified significant correlations of epiMH with signatures 1, 5 and 9, that have been reported to various cell division related mutational processes. Next, differential gene expression analysis between CLLs with high epiMH as compared to those with low epiMH revealed an enrichment in gene signatures related to active proliferation. Collectively, these results support the concept that the epiMH may reflect the past and present proliferative history of the cell. Finally, we postulated that the epiMH may be related with future proliferative capacity and clinical behavior. To test this, we analyzed the clinical impact of epiMH in patients from each epigenetic subgroup. An univariate analysis with epiMH as quantitative variable revealed that the higher the epiMH the worse clinical behavior. Additionally, we performed a multivariate Cox regression model for both overall survival (OS) and time to first treatment (TTT) including epiMH together with other well established prognostic factors for CLL, including age, epigenetic subgroups and the number of driver alterations. We showed that epiMH was an independent significant variable for both OS (HR=1.40, pvalue=0.03) and TTT (HR=1.41, pvalue<0.001) (HRs corresponding to 0.1 epiMH increments).Conclusions: We have developed a proliferative history score (named epiMH) for CLL based on DNA methylation dynamics in repressed chromatin. Our data suggest that the epiMH of patients within each CLL epigenetic subgroup at diagnosis may reflect their proliferative history and capacity, and represents an independent prognostic factor. DisclosuresNo relevant conflicts of interest to declare.

Non-canonical Drosophila X chromosome dosage compensation and repressive topologically associated domains

BackgroundIn animals with XY sex chromosomes, X-linked genes from a single X chromosome in males are imbalanced relative to autosomal genes. To minimize the impact of genic imbalance in male Drosophila, there is a dosage compensation complex (MSL) that equilibrates X-linked gene expression with the autosomes. There are other potential contributions to dosage compensation. Hemizygous autosomal genes located in repressive chromatin domains are often derepressed. If this homolog-dependent repression occurs on the X, which has no pairing partner, then derepression could contribute to male dosage compensation.ResultsWe asked whether different chromatin states or topological associations correlate with X chromosome dosage compensation, especially in regions with little MSL occupancy. Our analyses demonstrated that male X chromosome genes that are located in repressive chromatin states are depleted of MSL occupancy; however, they show dosage compensation. The genes in these repressive regions were also less sensitive to knockdown of MSL components.ConclusionsOur results suggest that this non-canonical dosage compensation is due to the same transacting derepression that occurs on autosomes. This mechanism would facilitate immediate compensation during the evolution of sex chromosomes from autosomes. This mechanism is similar to that of C. elegans, where enhanced recruitment of X chromosomes to the nuclear lamina dampens X chromosome expression as part of the dosage compensation response in XX individuals.

Interactions Between Regulatory Variants in CYP7A1 (Cholesterol 7α-Hydroxylase) Promoter and Enhancer Regions Regulate CYP7A1 Expression.

CYP7A1 (cholesterol 7α-hydroxylase) catalyzes the rate-limiting step in bile acid biosynthesis from cholesterol-a main pathway for cholesterol removal from the body. CYP7A1 single-nucleotide polymorphisms (SNPs) are associated with total cholesterol and LDL (low-density lipoprotein) levels, risk of cardiovascular diseases, and other phenotypes; however, results are inconsistent, and causative variants remain uncertain, except for a frequent promoter SNP (rs3808607). We used chromatin conformation capture (4C assay), chromatin immunoprecipitation qPCR assay in hepatocytes, and CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing in hepatocellular carcinoma cell line cells to identify regulatory regions for CYP7A1. We then screened for SNPs located in regulatory regions, testing effects on reporter gene assays and on hepatic CYP7A1 expression by measuring allelic mRNA expression imbalance. 4C assays showed several regions interacting with CYP7A1 promoter. CRISPR-mediated genome editing in hepatocellular carcinoma cell line cells revealed a novel CYP7A1 enhancer and a repressor region, located >10 kb downstream of the CYP7A1 promoter. SNP screening with an allelic mRNA expression imbalance in human livers and reporter gene assays identified a frequent functional SNP (rs9297994) located in the downstream CYP7A1 enhancer region. SNP rs9297994 is in high linkage disequilibrium with promoter SNP rs3808607 but has opposite effects on CYP7A1 mRNA expression. Their combined effects using a 2-SNP model robustly associate with hepatic CYP7A1 mRNA expression, ranging >2 orders of magnitude. Moreover, only the 2-SNP model, but not each SNP alone, is significantly associated with LDL levels, risk of coronary artery disease, statin response, and diabetes mellitus in several clinical cohorts, including CATHGEN (Catheterization Genetics) and Framingham. Two interacting regulatory SNPs modulate CYP7A1 expression and are associated with risk of coronary artery disease and diabetes mellitus.

Buried in PEAT-discovery of a new silencing complex with opposing activities.

Posttranslational histone modifications serve critical roles in gene regulation by determining the functional status of chromatin. Histone‐modifying enzymes often work in large multiprotein complexes. A paper in this issue of The EMBO Journal describes a new chromatin‐modifying complex called PEAT that acts via histone deacetylation. The PEAT complex is involved in heterochromatin formation and gene repression but also appears to have a locus‐specific activating role, possibly through promoting histone acetylation.

The Evf2 Ultraconserved Enhancer lncRNA Functionally and Spatially Organizes Megabase Distant Genes in the Developing Forebrain

Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5'UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3' end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3'-activator and 5'UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility.

UBE2C Directly Targeted by mirR-548-5p Increases the Cellular Growth and Invasive Abilities of Cancer Cells Interacting with the EMT Marker Protein ZEB1/2 in NSCLC

Background: Recent evidence indicates that miR-548-5p dysregulation plays a vital role in tumor progression. Aberrant miR-5485p expression may be linked to tumorigenesis since miRNAs play important roles in various cellular processes and cancer metastasis. UBE2C is a type of ubiquitination enzyme catalyzing the degradation of proteins into smaller polypeptides and amino acids. UBE2C participates in carcinogenesis by regulating the cell cycle, apoptosis, metastasis and transcriptional processes. However, the molecular mechanism of UBE2C as a growth factor regulating the tumorigenesis and metastasis of lung cancer is not clearly. EMT progression is mediated by the EMT-inducing transcriptional factors ZEB1/2, which is an important characteristic for cancer metastasis. What molecules regulate the expression of ZEB1/2 and then regulate the EMT in the lung cancer is also not clearly. We found that miR-548-5p, UBE2C, ZEB1/2 have the similar functions in tumor progression and metastasis. However, the relationship between miR-548-5p, UBE2C and ZBE1/2 in the tumorigenesis and development of lung cancer is still unknown. Methods: the mRNA expression of miR-548-5p, UBE2C was analyzed by RT-PCR assay. The protein level of UBE2C and ZEB1/2 was analyzed by western blot and Immunofluorescent staining. The cellular proliferation was detected by CCK8 and MTT. The cell migration and invasion growth were analyzed by wound healing assay and transwell assay. The activities of promoter and gene transcription were analyzed by luciferase reporter assay. Chromatin immunoprecipitation detected the combining ability of UBE2C and 5'UTRZEB1/ 2. Results: In our study, we found that miR-548-5p was significantly downregulated in lung cancer tissue specimens. Our objective was to elucidate the functions, direct target genes, and molecular mechanisms of miR-548-5p in lung cancer tissue. We found that miR-548-5p downregulated the expression of its direct substrate, 4,5-ubiquitin-conjugating enzyme E2C (UBE2C). UBE2C is a proto-oncogene that is activated in lung cancer. However, its role in lung cancer metastasis is unclear. We found here that UBE2C expression was higher in non-small-cell lung cancer (NSCLC) than in the normal tissue adjacent to it and was associated with increased cell proliferation and invasion. UBE2C enhanced NSCLC progression and metastasis by arresting the cell cycle and inhibiting apoptosis. We used reverse transcription (RT)-PCR assay to determine miR548-5p and UBE2C expression in lung cancer patients. Overexpression of miR-548-5p and UBE2C were established in lung cancer cells. Methyl thiazolyl tetrazolium (MTT)- and colony formation assays were performed to evaluate their effects on cell proliferation. We also performed cell migration- and invasion assays on lung cancer cells overexpressing miR548-5p and under expressing UBE2C. The results indicated that the aforementioned treatments significantly suppressed lung cancer cell proliferation, migration, and invasion. Moreover, luciferase reporter and Chromatin immunoprecipitation assay indicated that miR-548-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. Using RT-PCR and western blot (WB), we also assessed epithelial-mesenchymal transition (EMT). We found that UBE2C knockdown downregulated the expression of the mesenchymal marker vimentin. In contrast, the epithelial marker E-cadherin was upregulated. Therefore, UBE2C promoted EMT. Bioinformatics assays coupled with WB and luciferase assays revealed that UBE2C directly binds to the 5′-untranslated region of the E-cadherin repressor, ZEB1/2 and promotes EMT in lung cancer cells. Conclusion: the miR-548-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. UBE2C is an oncogene promoting EMT in lung cancer cells by directly targeting the 5'-UTR of E-cadherin repressor ZEB1/2. The miR-548-5p, UBE2C, and ZEB1/2 constitute the miR548-5p-UBE2C-ZEB1/2 signal axis which enhances cancer cell invasiveness by directly interacting with the EMT maker protein. We believe that the miR-548-5p-UBE2C-ZEB1/2 signal axis may be a suitable diagnostic marker and a potential target in lung cancer therapy Funding: This work was supported by the Science and Technology Development Foundation of Yantai (2015ZH082), Natural Science Foundation of Shandong Province (ZR2018QH004, ZR2016HB55, ZR2017PH067, ZR2017MH125 and ZR2015PH004), and Research Foundation of Binzhou Medical University (BY2015KYQD25 and BY2015KJ14). Declaration of Interest: The authors declare no competing financial interests. Ethical Approval: The experimental protocol was approved by the Research Ethics Committee of Binzhou Medical University, China (No. 2017-016-01 for human lung cancer specimen and No. 2017-009-09 for mouse experiments in vivo) and the written informed consent was obtained from all subjects. Informed consent was obtained from all individual participants included in the study. All patients were staged based on the criteria of the 7th Edition of the AJCC Cancer Staging Manual: Stomach (2010)

MicroRNA-30a Suppresses the Activation of Hepatic Stellate Cells by Inhibiting Epithelial-to-Mesenchymal Transition

Background/Aims: The activation of hepatic stellate cells (HSCs) is considered as a pivotal event in liver fibrosis and epithelial-mesenchymal transition (EMT) process has been reported to be involved in HSC activation. It is known that microRNAs (miRNAs) play a pro-fibrotic or anti-fibrotic role in HSC activation. Recently, emerging studies show that miR-30a is down-regulated in human cancers and over-expression of miR-30a inhibits tumor growth and invasion via suppressing EMT process. However, whether miR-30a could regulate EMT process in HSC activation is still unclear. Methods: miR-30a expression was quantified using real-time PCR in carbon tetrachloride (CCl₄)-induced rat liver fibrosis, activated HSCs and patients with cirrhosis. Roles of miR-30a in liver fibrosis in vivo and in vitro were also analyzed. Luciferase activity assays were performed to examine the binding of miR-30a to the 3′-untranslated region of snail family transcriptional repressor 1 (Snai1). Results: miR-30a was down-regulated in human cirrhotic tissues. In CCl₄ rats, reduced miR-30a was found in fibrotic liver tissues as well as isolated HSCs. There was a significant reduction in miR-30a in primary HSCs during culture days. miR-30a over-expression resulted in the suppression of CCl₄-induced liver fibrosis. Restoration of miR-30a led to the inhibition of HSC activation including cell proliferation, α-SMA and collagen expression. Notably, miR-30a inhibited EMT process, with a reduction in TGF-β1 and Vimentin as well as an increase in GFAP and E-cadherin. miR-30a induced a significant reduction in Snai1 protein expression when compared with the control. Interestingly, Snail protein expression was increased during liver fibrosis, indicating that there may be a negative correlation between miR-30a level and Snai1 protein expression. Further studies demonstrated that Snai1 was a target of miR-30a. Conclusion: Our results suggest that miR-30a inhibits EMT process, at least in part, via reduction of Snai1, leading to the suppression of HSC activation in liver fibrosis.

Heterogeneous pattern of DNA methylation in developmentally important genes correlates with its chromatin conformation

BackgroundDNA methylation is a major epigenetic modification, playing a crucial role in the development and differentiation of higher organisms. DNA methylation is also known to regulate transcription by gene repression. Various developmental genes such as c-mos, HoxB5, Sox11, and Sry show tissue-specific gene expression that was shown to be regulated by promoter DNA methylation. The aim of the present study is to investigate the establishment of chromatin marks (active or repressive) in relation to heterogeneous methylation in the promoter regions of these developmentally important genes.ResultsChromatin-immunoprecipitation (ChIP) assays were performed to immuno-precipitate chromatin by antibodies against both active (H3K4me3) and repressive (H3K9me3) chromatin regions. The analysis of ChIP results showed that both the percentage input and fold enrichment of activated chromatin was higher in tissues expressing the respective genes as compared to the tissues not expressing the same set of genes. This was true for all the genes selected for the study (c-mos, HoxB5, Sox11, and Sry). These findings illustrate that inconsistent DNA methylation patterns (sporadic, mosaic and heterogeneous) may also influence gene regulation, thereby resulting in the modulation of chromatin conformation.ConclusionsThese findings illustrate that various patterns of DNA methylation (asynchronous, mosaic and heterogeneous) correlates with chromatin modification, resulting in the gene regulation.

DNA methylation promotes paired box 2 expression via myeloid zinc finger 1 in endometrial cancer.

This work investigated the role of paired box 2 (PAX2) in endometrial cancer and its epigenetic regulation mechanism. Endometrial cancer tissues and cell lines exhibited increased PAX2 expression compared with hyperplasia, normal endometrium and endometrial epithelial cells. Knock-down of PAX2 resulted in reduced cell viability, invasion and migration, and PAX2 overexpression caused the opposite effects. Increased methylation of the PAX2 promoter was observed in both cancer tissues and cell lines and was positively correlated with PAX2 expression. After 5-Aza-CdR treatment, PAX2 mRNA and protein were down-regulated, and PAX2 methylation was decreased. Deletion analysis confirmed that a repressive transcriptional regulatory region of the PAX2 promoter coincided with the hypermethylated region identified in MassARRAY analysis. Binding sites of myeloid zinc finger 1 (MZF1) are predicted in the defined region. Knock-down of MZF1 up-regulated the transcriptional activity and protein level of PAX2 after 5-Aza-CdR treatment, which indicated that MZF1 may act as a repressive transcription factor when the PAX2 promoter is unmethylated. In conclusion, PAX2 is involved in the carcinogenesis of endometrial cancer by stimulating cell growth and promoting cell motility. The overexpression of PAX2 in endometrial cancer is regulated by promoter hypermethylation and the transcription factor MZF1.

Non-neoplastic Fallopian Tube Epithelium Carrying Gene Mutations of a Novel SOX2 Repressor Region is Soil of High-grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) has a high mortality rate because the disease is asymptomatic in early stages and is resistant to treatments. A novel modality for diagnosis in the early stages of carcinogenesis is needed. In this issue of EBioMedicine, Hellner et al. present novel genetic aspects of small lesions of HGSOC (Hellner et al., 2016). They performed screening by LFR WGS technology for small HGSOC lesions from a patient who underwent treatment and found 750 genomic mutations. Gene ontology analysis revealed that the gene mutations are related to stem cell differentiation, and the authors finally found 6 novel non-coding mutations (BB1 - BB6) in proximity of sex determining region Y-box2 (SOX2), a key driver of stem cell differentiation (Takahashi and Yamanaka, 2006). Deep genetic analysis revealed that the BB5 region is commonly mutated in HGSOC tissues. A reporter assay and genome engineering using CRISPR/Cas9 system revealed that the BB5 region is a novel repressor of the SOX2 gene and that mutation in the BB5 region induces protein expression of SOX2. Mutations in the BB5 region were detected in non-neoplastic fallopian tube epithelium (FTE) of HGSOC cases and also in normal FTE of BRCA1 or BRCA2 mutation carriers who are at high risk for HGSOC. Surprisingly, immunohistochemical analysis of BB5 mutation carriers revealed that SOX2 protein is broadly expressed in even normal FTEs, indicating that non-neoplastic FTEs with BB5 mutation propagated clonally. SOX2 protein is expressed in earlier stages than TP53 protein expression indicating overexpression of SOX2 protein is earlier event than TP53 gene mutation. Interestingly, SOX2 protein expression decreased after the establishment of an ovarian cancer lesion. These results suggest that mutation in the SOX2 BB5 region is an early carcinogenesis driver mutation in HGSOCs and that detection of SOX2 protein is a promising biological marker for prediction of HGSOC.