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The role of tetraspanin CD81 in malignant transformation is best studied in colorectal cancer, and it appears that other transcripts beside the fully coding mRNA may also be dysregulated in malignant cells. Recent data from a comprehensive pan‐cancer transcriptome analysis demonstrated differential activity of two alternative CD81 gene promoters in malignant versus nonmalignant gut mucosa. The promoter active in gut mucosa gives rise to transcripts CD81‐203 and CD81‐213, while the promoter active in colon and rectal cancer gives rise to transcripts CD81‐205 and CD81‐215. Our study aimed to explore the biomarker potential of the transcripts from the alternative CD81 gene promoters in colon cancer, as well as to investigate their structure and potential function using in silico tools. The analysis of the transcripts' expression in several colon cell lines cultivated in 2D and 3D and a set of colon cancer and healthy gut mucosa samples by qPCR and RNA sequencing suggested their low expression and stromal origin. Expression patterns in tumor and nontumor tissue along with in silico data suppose that the transcript CD81‐215 may be a noncoding RNA of stromal origin with possible involvement in signaling related to malignant transformation. Significance statement: Elements of the noncoding transcriptome are increasingly being accepted as the factors contributing to the complexity of the genome, but their role remains unexplored in spite of demonstrated relevance for many diseases, including cancer. Noncoding transcripts harbor vast potential for application in personalized medicine, both as prospective therapeutics and therapeutic targets. The differential expression of the transcript CD81‐215 in tumor and nontumor tissue, along with in silico predictions of its structure, localization, and interaction partners, point to the possible involvement of this molecule in colorectal carcinogenesis and its biomarker potential. [ABSTRACT FROM AUTHOR]

Background: Plant species from Rosaceae family are economically important. One of the major environmental factors impacting those species is cold stress. Although several Rosaceae plant genomes have recently been sequenced, there have been very few research conducted on cold upregulated genes and their promoter binding sites. In this study, we used computational approaches to identify and analyse potential cold stress response genes across ten Rosaceae family members. Results: Cold stress upregulated gene data from apple and strawberry were used to identify syntelogs in other Rosaceae species. Gene duplication analysis was carried out to better understand the distribution of these syntelog genes in different Rosaceae members. A total of 11,145 popular abiotic stress transcription factor-binding sites were identified in the upstream region of these potential cold-responsive genes, which were subsequently categorised into distinct transcription factor (TF) classes. MYB classes of transcription factor binding site (TFBS) were abundant, followed by bHLH, WRKY, and AP2/ERF. TFBS patterns in the promoter regions were compared among these species and gene families, found to be quite different even amongst functionally related syntelogs. A case study on important cold stress responsive transcription factor family, AP2/ERF showed less conservation in TFBS patterns in the promoter regions. This indicates that syntelogs from the same group may be comparable at the gene level but not at the level of cis-regulatory elements. Therefore, for such genes from the same family, different repertoire of TFs could be recruited for regulation and expression. Duplication events must have played a significant role in the similarity of TFBS patterns amongst few syntelogs of closely related species. Conclusions: Our study overall suggests that, despite being from the same gene family, different combinations of TFs may play a role in their regulation and expression. The findings of this study will provide information about potential genes involved in the cold stress response, which will aid future functional research of these gene families involved in many important biological processes. [ABSTRACT FROM AUTHOR]

Kalmegh (Andrographis paniculata) spatiotemporally produces medicinally-important ent -labdane-related diterpenoids (ent- LRDs); andrographolide (AD), 14-deoxy-11,12-didehydroandrographolide (DDAD), neoandrographolide (NAD). ApCPS1 and ApCPS2, the ent -copalyl pyrophosphate (ent -CPP)-producing class II diterpene synthases (diTPSs) were identified, but their contributions to ent -CPP precursor supply for ent- LRD biosynthesis were not well understood. Here, we characterized ApCPS4, an additional ent -CPP-forming diTPS. Further, we elucidated in planta function of the ent -CPP-producing diTPSs (ApCPS1,2,4) by integrating transcript-metabolite co-profiles, biochemical analysis and gene functional characterization. ApCPS1,2,4 localized to the plastids, where diterpenoid biosynthesis occurs in plants, but ApCPS1,2,4 transcript expression patterns and ent -LRD contents revealed a strong correlation of ApCPS2 expression and ent -LRD accumulation in kalmegh. ApCPS1,2,4 upstream sequences differentially activated β-glucuronidase (GUS) in Arabidopsis and transiently-transformed kalmegh. Similar to higher expression of ApCPS1 in kalmegh stem, ApCPS1 upstream sequence activated GUS in stem/hypocotyl of Arabidopsis and kalmegh. However, ApCPS2,4 upstream sequences weakly activated GUS expression in Arabidopsis, which was not well correlated with ApCPS2,4 transcript expression in kalmegh tissues. Whereas, ApCPS2,4 upstream sequences could activate GUS expression at a considerable level in kalmegh leaf and roots/calyx, respectively, suggesting the involvement of transcriptional regulator(s) of ApCPS2,4 that might participate in kalmegh-specific diterpenoid pathway. Interestingly, ApCPS2 -silenced kalmegh showed a drastic reduction in AD, DDAD and NAD contents and compromised defense against insect herbivore Spodoptera litura. However, ent -LRD contents and herbivore defense in ApCPS1 or ApCPS4 -silenced plants remained largely unaltered. Overall, these results suggested an important role of ApCPS2 in producing ent -CPP for medicinal ent -LRD biosynthesis and defense against insect herbivore. • This work clarifies the roles of A. paniculata ent -copalyl pyrophosphate synthases in medicinal diterpenoid biosynthesis. • ApCPS4 was biochemically characterized as an ent -copalyl pyrophosphate synthase. • ApCPS1,2,4 localized in the plastids and their upstream sequences differentially activated β-glucuronidase. • ApCPS2 transcript expression strongly correlated with diterpenoid levels in tissues. • ApCPS2 silencing led to a drastic reduction in diterpenoid levels and compromised defense to insect herbivore. [ABSTRACT FROM AUTHOR]

Objective: Odontogenic ameloblast-associated protein (ODAM) is produced by maturation stage ameloblasts and junctional epithelium (JE). The function of ODAM is thought to be involved in the attachment of teeth and JE. To elucidate transcriptional regulation of human ODAM gene in inflamed gingiva, we have analyzed the effects of TNF-α on the expression of ODAM gene in Ca9-22 and Sa3 gingival epithelial cells. Materials and methods: Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10 ng/ml). ODAM mRNA and protein levels were analyzed by qPCR and Western blotting. Luciferase (LUC) analyses were performed using LUC constructs inserted in various lengths of ODAM gene promoter. Gel shift and chromatin immunoprecipitation (ChIP) assays were carried out. Results: TNF-α increased ODAM mRNA and protein levels at 3 to 24 h. TNF-α induced LUC activities of the ODAM gene promoter constructs, and the activities were inhibited by protein kinase A, tyrosine kinase, MEK1/2, PI3-kinase and NF-κB inhibitors. Gel shift and ChIP assays revealed that TNF-α increased CCAAT/enhancer-binding protein (C/EBP) β and Yin Yang1 (YY1) binding to three kinds of C/EBPs and YY1 elements. Conclusion: These results demonstrate that TNF-α stimulates ODAM gene transcription via C/EBPs and YY1 elements in the human ODAM gene promoter. [ABSTRACT FROM AUTHOR]

The estimation of hypermethylation of the cell cycle (RASSF1A, p16/INK4A, p14/ARF) and detoxification (GSTP1) gene promoters was carried out in blood leukocytes of humans exposed to radiation as a result of the Chernobyl accident (98 individuals: accident liquidators, 76 individuals; adult residents of the territories with radionuclide contamination, 135–688 kBq/m2, 22 individuals) depending on their cytogenetic status. The results of multiple regression analysis ("Frequency of aberrations ~ age + number of hypermethylated genes") indicate a correlation of the total level of chromosomal type aberrations with the considered epigenetic disturbances (β = 0.256; р = 0.011), but not with age (β = –0.138; p = 0.165). The frequency of these cytogenetic disorders increases with an increase in the number of methylated loci. Thus, a positive association between the damages to the genome induced by radiation exposure in the range of small and medium doses (long time ago) and hypermethylation of promoters of the genes of the main protective systems of the cells was detected. [ABSTRACT FROM AUTHOR]

The estimation of hypermethylation of the cell cycle (RASSF1A, p16/INK4A, p14/ARF) and detoxification (GSTP1) gene promoters was carried out in blood leukocytes of humans exposed to radiation as a result of the Chernobyl accident (98 individuals: accident liquidators, 76 individuals; adult residents of the territories with radionuclide contamination, 135–688 kBq/m2, 22 individuals) depending on their cytogenetic status. The results of multiple regression analysis ("Frequency of aberrations ~ age + number of hypermethylated genes") indicate a correlation of the total level of chromosomal type aberrations with the considered epigenetic disturbances (β = 0.256; р = 0.011), but not with age (β = –0.138; p = 0.165). The frequency of these cytogenetic disorders increases with an increase in the number of methylated loci. Thus, a positive association between the damages to the genome induced by radiation exposure in the range of small and medium doses (long time ago) and hypermethylation of promoters of the genes of the main protective systems of the cells was detected. [ABSTRACT FROM AUTHOR]

The estimation of hypermethylation of the cell cycle (RASSF1A, p16/INK4A, p14/ARF) and detoxification (GSTP1) gene promoters was carried out in blood leukocytes of humans exposed to radiation as a result of the Chernobyl accident (98 individuals: accident liquidators, 76 individuals; adult residents of the territories with radionuclide contamination, 135–688 kBq/m2, 22 individuals) depending on their cytogenetic status. The results of multiple regression analysis ("Frequency of aberrations ~ age + number of hypermethylated genes") indicate a correlation of the total level of chromosomal type aberrations with the considered epigenetic disturbances (β = 0.256; р = 0.011), but not with age (β = –0.138; p = 0.165). The frequency of these cytogenetic disorders increases with an increase in the number of methylated loci. Thus, a positive association between the damages to the genome induced by radiation exposure in the range of small and medium doses (long time ago) and hypermethylation of promoters of the genes of the main protective systems of the cells was detected. [ABSTRACT FROM AUTHOR]

Genetic modification of Rhodococcus jostii RHA1 was carried out in order to optimise the production of pyridine-2,4-dicarboxylic acid and pyridine-2,5-dicarboxylic acid bioproducts from lignin or lignocellulose breakdown, via insertion of either the Sphingobium SYK-6 ligAB genes or Paenibacillus praA gene respectively. Insertion of inducible plasmid pTipQC2 expression vector containing either ligAB or praA genes into a ΔpcaHG R. jostii RHA1 gene deletion strain gave 2–threefold higher titres of PDCA production from lignocellulose (200–287 mg/L), compared to plasmid expression in wild-type R. jostii RHA1. The ligAB genes were inserted in place of the chromosomal pcaHG genes encoding protocatechuate 3,4-dioxygenase, under the control of inducible Picl or PnitA promoters, or a constitutive Ptpc5 promoter, producing 2,4-PDCA products using either wheat straw lignocellulose or commercial soda lignin as carbon source. Insertion of Amycolatopsis sp. 75iv2 dyp2 gene on a pTipQC2 expression plasmid led to enhanced titres of 2,4-PDCA products, due to enhanced rate of lignin degradation. Growth in minimal media containing wheat straw lignocellulose led to the production of 2,4-PDCA in 330 mg/L titre in 40 h, with > tenfold enhanced productivity, compared with plasmid-based expression of ligAB genes in wild-type R. jostii RHA1. Production of 2,4-PDCA was also observed using several different polymeric lignins as carbon sources, and a titre of 240 mg/L was observed using a commercially available soda lignin as feedstock. [ABSTRACT FROM AUTHOR]

Objective: Calcitonin receptor-like receptor (CRLR) regulates vasoconstriction and dilatation; the expression increases during hypoxia via activation of hypoxia response element (HRE) in CRLR gene promoter region. Variant in HRE, as well short tandem repeat (STR) variants near HRE in CRLR alters the gene expression. This study focused on a case–control study to investigate the expression of genetic typing CLRL promoter variant in pregnant women with severe preeclampsia and normal pregnancies, we also tried to describe interesting findings of the genetic expression in anemic patients in the severe preeclampsia group. Our aimed to observe the correlation of CRLR gene promoter variant and anemia in severe preeclampsia. Results: There was no nucleotide variant in HRE; CACA box prior to HRE varied in length (15–24); CACA box with length > 20 was used as cut off point. Hb was lower in CACA box length ≥ 21 (10.33 ± 1.57) vs. < 21 (11.01 ± 1.67; p = 0.391). CACA box polymorphism and anemia were correlated in severe preeclampsia (p = 0.005) OR 0.038 (CI 0.003–0.544); not in normal (p = 0.069). [ABSTRACT FROM AUTHOR]

• Peripheral IFN-γ stimulation can promote cognitive and behavioral functions in mice. • STAT1 binds to the target gene of β-catenin promoter (−218∼−204 bp), which plays a positive regulatory effect on β-catenin. • IFN-γ can regulate wnt/β-catenin signaling pathways through STAT1 activation to promote NSCs' proliferation, differentiation. Interferon-gamma (IFN-γ) is critical for central nervous system (CNS) functions and it may be a promising treatment to stimulate CNS regeneration. However, previous studies reported inconsistent results, and the molecular mechanisms remain controversial. Here we show that IFN-γ-treated mice via intraperitoneal injection have elevated IFN-γ level in central hippocampus and superior cognitive behaviors IFN-γ could activates the level of protein expression of Wnt7a, β-catenin, and CyclinD1 in Wnt/β-catenin signaling pathway of mice hippocampus. Functional and mechanism analysis in vitro revealed that IFN-γ promoted the proliferation and differentiation in primary cultured neural stem cells (NSCs). STAT1 was accountable for IFN-γ-induced activation of the β-catenin promoter, and IFN-γ increased the binding affinity of STAT1 to β-catenin promoter based on luciferase activity and chromatin immunoprecipitation. Our results suggest that IFN-γ exerts many effects ranging from cognitive function in vivo to NSC proliferation, self-renewal, and differentiation in vitro. It does so by recruiting STAT1 to the β-catenin promoter, enhancing cis-regulation by STAT1, and ultimately activating Wnt/β-catenin signaling. In this study, we first found that STAT1 was recruited into the promoter of β-catenin to activate β-catenin expression, and this effect was regulated by IFN-γ. It is also discovered firstly that Wnt/β-catenin and JAK/STAT pathways form cross-links through STAT1. Promoting neurogenesis through immune stimulation might be a promising strategy for repairing the diseased/injured CNS. This study provides a scientific basis for immunomodulation to promote nerve regeneration and offer a new therapeutic direction for central nervous system regeneration. [ABSTRACT FROM AUTHOR]

Background: The epoxyeicosatrienoic acids (EETs) have antihypertensive, anti-inflammatory, and organ protective properties and their circulation levels are related to hypertension, diabetes mellitus, cardiovascular diseases, and preeclampsia. Soluble epoxide hydrolase (sEH) catalyses the degradation of EETs to less biologically active dihydroxyeicosatrienoic acids. Here, we sequenced the promoter region of EPHX2 to investigate the association between promoter sequence alterations that we thought to affect the expression levels of the enzyme and preeclampsia (PE). Methods: Nucleotide sequencing of the promoter region of the EPHX2, spanning from position -671 to +30, was performed on 100 pregnant women with PE and, 20 or more weeks pregnant normotensive, healthy women (n = 100). Results: Pregnant women who carry rs4149235, rs4149232, rs73227309, and rs62504268 polymorphisms have 4.4, 2.4, 2.3, and 2.8 times significantly increased risk of PE, respectively. CCGG (OR: 3.11; 95% Cl: 1.12-8.62) and CCCA (OR: 0.45; 95% Cl: 0.36-0.55) haplotypes were associated with an increased and decreased risk of PE, respectively. Conclusions: Four SNPs (rs4149232, rs4149235, rs73227309, and rs62504268) in the promoter region of the EPHX2, and CCGG and CCCA haplotypes of these 4 SNPs were significantly associated with PE. These SNPs in the promoter region may affect sEH expression and thus enzyme activity and may play a role in PE pathogenesis by causing individual differences in EET levels. However, future studies are needed to confirm our findings and examine the effect of these SNPs on the sEH expression and/or enzyme activity. [ABSTRACT FROM AUTHOR]

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability. [ABSTRACT FROM AUTHOR]

Small molecules able to bind non-canonical G-quadruplex DNA structures (G4) have been recently tested as novel potential agents for the treatment of prostate cancer thanks to their repression of aberrant androgen receptor gene. However, metastatic castration-resistant prostate cancer (mCRPC), a letal form of prostate cancer, is still incurable. Here we tested two naphthalenediimide derivatives, previously reported as multitarget agents, on a couple of relevant mCRPC cell models (DU145 and PC-3). We showed that these compounds interfere with the RAS/MEK/ERK and PI3K/AKT pathways. Interestingly, both these two biological processes depend upon Epidermal Growth Factor Receptor (EGFR) activation. By means of biological and analytical tools we showed that our compounds are efficient inducers of the structural transition of the EGFR promoter towards a G-quadruplex conformation, ultimately leading to a reduction of the receptor production. The overall result is an interesting cytotoxic profile for these two derivatives. Thanks to their activity at different steps, these compounds can open the way to novel therapeutic approaches for mCRPC that could contribute to escape resistance to selective treatments. Image 1 • NDI can target multiple cellular G-quadruplexes. • In NDI-treated mCRPC cells the expression level of EGFR is reduced. • NDI-treatment can help to escape some resistance mechanisms. • Distinct biological outcomes characterize NDI-treated mCRPC cells. • Cell defense programs may impact on NDI biological activity. [ABSTRACT FROM AUTHOR]

Gene promoter methylation is a common epigenetic event, taking place in the early phase of tumorigenesis, which has a great potential as a diagnostic and prognostic cancer biomarker. In this umbrella review, we provide an overview on the association between gene-promoter methylation of protein-coding genes and cancer risk based on currently available meta-analyses data on gene promoter methylation. We searched MEDLINE via PubMed and the Cochrane Database of Systematic Reviews for meta-analyses that examine the association between gene-promoter methylation and cancer, published until January 2019 in English. We used AMSTAR to assess the quality of the included studies and applied a set of pre-specified criteria to evaluate the magnitude of each association. We provide a comprehensive overview of 80 unique combinations between 22 different genes and 18 cancer outcomes, all of which indicated a positive association between promoter hypermethylation and cancer. In total, the 70 meta-analyses produced significant results under a random-effects model with odds ratios that ranged from 1.94 to 26.60, with the summary effect being in favor of the unmethylated group in all cases. Three of the strong evidence associations involve RASSF1 methylation on bladder cancer risk (OR = 18.46; 95% CI: 12.69–26.85; I2 = 0%), MGMT methylation on NSCLC (OR = 4.25; 95% CI: 2.83–6.38; I2 = 22.4%) and RARB methylation on prostate cancer (OR = 6.87; 95% CI: 4.68–10.08; I2 = 0%). Meta-analyses showed a moderate quality, AMSTAR score ranging from 4 to 9 (Mdn = 8; IQR: 7.0 to 8.0). As primary studies and meta-analyses on the subject accumulate, more genetic loci may be found to be highly associated with specific cancer types and hence the biomarker sets will become wider. • Overview of 80 unique combinations between 22 genes and 18 cancer outcomes • All showed a positive association between promoter hypermethylation and cancer. • Strong evidence linked methylation of MGMT , RASSF1 and RARB to various cancers. • Quality of studies was moderate, AMSTAR ranged from 4 to 9 (Mdn = 8; IQR: 7 to 8). [ABSTRACT FROM AUTHOR]

The cohesin protein complex mediates sister chromatid cohesion to ensure accurate chromosome segregation, and also influences gene transcription in higher eukaryotes. Modest deficits in cohesin function that do not alter chromosome segregation cause significant birth defects. The mechanisms by which cohesin participates in gene regulation have been studied in Drosophila , revealing that it is involved in gene activation by transcriptional enhancers and epigenetic gene silencing mediated by Polycomb group proteins. Recent studies reveal that early DNA replication origins are important for determining which genes associate with cohesin, and suggest that cohesin at replication origins is important for establishing both sister chromatid cohesion and enhancer–promoter communication. Cohesin participates in gene activation and Polycomb repressive complex function via multiple mechanisms. Cohesin association with transcriptional enhancers and gene promoters is linked to their proximity to early DNA replication origins. Cohesin association with early replication origins may be important for establishing both sister chromatid cohesion and enhancer–promoter communication. [ABSTRACT FROM AUTHOR]

Highlights • Aberrant DNA methylation patterns are a hallmark of diseases, such as cancer, Alzheimer's disease, and cardiovascular disease, and mathematical analysis of the mechanics of DNA methylation has the potential to give new insights into these mechanisms. • We develop a fast and robust methodology for selecting computational models for DNA methylation. • We demonstrate the effectiveness of the method on recovering parameter values associated with the processes which underpin DNA methylation. • Our work opens the possibility for further biological experimentation based on our finding that the parameters which govern DNA methylation are influenced by gene promoter methylation levels. • Our work showcases the utility of Bayesian analysis and its application to complex biological problems. Abstract Epigenetics is coming to the fore as a key process which underpins health. In particular emerging experimental evidence has associated alterations to DNA methylation status with healthspan and aging. Mammalian DNA methylation status is maintained by an intricate array of biochemical and molecular processes. It can be argued changes to these fundamental cellular processes ultimately drive the formation of aberrant DNA methylation patterns, which are a hallmark of diseases, such as cancer, Alzheimer's disease and cardiovascular disease. In recent years mathematical models have been used as effective tools to help advance our understanding of the dynamics which underpin DNA methylation. In this paper we present linear and nonlinear models which encapsulate the dynamics of the molecular mechanisms which define DNA methylation. Applying a recently developed Bayesian algorithm for parameter estimation and model selection, we are able to estimate distributions of parameters which include nominal parameter values. Using limited noisy observations, the method also identified which methylation model the observations originated from, signaling that our method has practical applications in identifying what models best match the biological data for DNA methylation. [ABSTRACT FROM AUTHOR]

The human (h) placental lactogenic hormone chorionic somatomammotropin (CS) is highly produced during pregnancy and acts as a metabolic adaptor in response to maternal insulin resistance. Maternal obesity can exacerbate this "resistance", and a >75% decrease in CS RNA levels was observed in term placentas from obese vs. lean women. The genes coding for hCS (hCS-A and hCS-B) and placental growth hormone (hGH-V) as well as the hCS-L pseudogene and pituitary growth hormone (GH) gene (hGH-N) are located at a single locus on chromosome 17. Three remote hypersensitive sites (HS III-V) located >28 kb upstream of hGH-N as well as local hCS gene promoter and enhancer regions are implicated in hCS gene expression. A placentaspecific chromosomal architecture, including interaction between HS III-V and hCS but not hGH gene promoters, was detected in placentas from lean women (BMI <25 kg/m2) by using the chromosome conformation capture assay. This architecture was disrupted by prepregnancy maternal obesity (BMI >35 kg/m²), resulting in a predominant interaction between HS III and the hGH-N promoter, which was also observed in nonplacental tissues. This was accompanied by a decrease in hCS levels, which was consistent with reduced RNA polymerase II and CCAAT/enhancer-binding protein-β association with individual hCS promoter and enhancer sequences, respectively. Thus, pre-pregnancy maternal obesity disrupts the placental hGH/CS gene locus chromosomal architecture. However, based on data from obese women who develop GDM, insulin treatment partially recapitulates the chromosomal architecture seen in lean women and positively affects hCS production, presumably facilitating prolactin receptor-related signaling by hCS. [ABSTRACT FROM AUTHOR]

Transcription of vitellogenin ( vtg ) genes are initiated when estradiol-17β (E 2 )-estrogen receptor (ER) complexes bind estrogen response elements (ERE) located in the gene promoter region. Transcriptional regulation of dual vtg subtypes (major salmonid A-type vtg : vtgAs ; minor C-type vtg : vtgC ) by E 2 was investigated under co-expression of a potential major transcriptional factor, erα1 , in cutthroat trout. Two forms of trout vtgAs promoters (1 and 2) and one vtgC promoter were sequenced. These promoters structurally differ based on the number of EREs present. The vtgAs promoter 1 exhibited the highest maximal transcriptional activity by in vitro gene reporter assays. The concentration of E 2 that induces 50% of gene reporter activity (half-maximal effective concentrations, EC 50 ) was similar among all vtg promoters and also to the EC 50 of E 2 administered to induce vtg transcription in vivo . This study revealed a difference in transcriptional properties of multiple vtg promoters for the first time in a salmonid species, providing the basis to understand mechanisms underlying regulation of vitellogenesis via dual vtg gene expression. [ABSTRACT FROM AUTHOR]

The present study aimed to explore the effects of Helicobacter pylori (H. pylori) infection on the expression of transcription factor GATA binding protein 3 (GATA-3) and connexin 32 (Cx32) in cultured gastric mucosa cells, and their association with each other. GES-1 cells were co-cultured with East Asian type cytotoxin-associated gene A+ H. pylori in the H. pylori group, and without H. pylori culture in the control group. Additionally, Mongolian gerbils were gavaged with H. pylori, and later the gastric antrum tissues were collected. The GATA-3 and Cx32 mRNA and protein expression levels were detected by a reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The scratch labeling fluorescent dye tracer (SLDT) technique was used to detect the gap junctional intercellular communication (GJIC) function. GATA-3 small interfering RNA (siRNA) was transfected into BGC823 cells and its effect on Cx32 expression levels was detected. The impact of GATA-3 on Cx32 promoter transcriptional activity was detected using a dual luciferase reporter assay. The results revealed that H. pylori infection increased GATA-3 expression and decreased Cx32 expression in GES-1 cells and in animal gastric tissues compared with their respective controls, whilst in BGC823 cells, GATA-3 siRNA increased Cx32 expression compared with the control. In the SLDT experiment of GES-1 cells with H. pylori infection, the fluorescent dye was primarily limited to a single cell row close to the scratch, and only a limited amount of dye passing to the second cell row, indicating that the GJIC function was substantially reduced or absent compared with the control group, where the fluorescence dye transferred to the neighboring cells of 3-4 rows, indicating a stronger GJIC function comparatively. GATA-3 inhibited the expression of the luciferase reporter gene, compared with the controls, suggesting that GATA-3 inhibited the expression of Cx32 by binding to Cx32 promoter sites. These results indicated that H. pylori-increased GATA-3 expression, which downregulated Cx32 expression, may serve an important function in gastric carcinogenesis, and GATA-3 siRNA may serve a function in the prevention and treatment of gastric cancer. [ABSTRACT FROM AUTHOR]

Although it is difficult to detect αvβ6 integrin (αvβ6) in normal epithelia cells, its expression is upregulated during wound healing and carcinogenesis. Overexpression of αvβ6 has been demonstrated in epithelial cell carcinomas, such as adenocarcinoma of the colon and ovary. However, the expression of αvβ6 has not been reported in hepatocellular carcinoma (HCC). We previously indicated that LPA may induce αvβ6-mediated TGF-β1 signaling mechanisms during the pathogenesis of lung injury and fibrosis. In addition, transforming growth factor-β1 (TGF-β1) and lysophosphatidic acid (LPA) have been demonstrated to participate in the progression of HCC. In the present study, we hypothesized that TGF-β1 and LPA would serve a key role in the subunit integrin β6 (Itgβ6) transcriptional regulatory mechanism in HCC. It was identified that human HCC tissues and Hep-3B cells expressed Itgβ6. Treatment of Hep-3B with TGF-β1 or LPA increased the expression of Itgβ6. Furthermore, truncation experiments indicated a positive regulatory region at −326 to −157 bp of the Itgβ6 promoter. TGF-β1 and LPA increased transcriptional activation at this regulatory region. To the best of our knowledge, the present study was the first to demonstrate Itgβ6 expression in HCC, and the data indicate that TGF-β1 and LPA regulate Itgβ6 expression through the Itgβ6 gene promoter, which is an important factor in the development of HCC. [ABSTRACT FROM AUTHOR]

Objective: Amelotin (AMTN) is an enamel protein that is localized in the basal lamina of ameloblasts in their maturation stage and the internal basal lamina of junctional epithelium (JE) and it is suggested that AMTN could be involved in the dentogingival attachment. To elucidate the transcriptional regulation of human AMTN gene in inflamed gingiva, we have analyzed the effect of tumor necrosis factor-α (TNF-α) on the expression of AMTN gene in Ca9-22 and Sa3 human gingival epithelial cells.Materials and methods: Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10 ng/ml). AMTN mRNA and protein levels were measured by real-time PCR and Western blotting. Transient transfection analyses were completed using the various lengths of human AMTN gene promoter constructs with or without TNF-α. Gel mobility shift and chromatin immunoprecipitation assays were performed to investigate the transcription factors bindings to the human AMTN gene promoter by TNF-α.Results: TNF-α (10 ng/ml) increased AMTN mRNA and protein levels after 12 h. TNF-α induced luciferase activities of human AMTN gene promoter constructs (− 211AMTN, − 353AMTN, and − 501AMTN). TNF-α-induced luciferase activities were partially inhibited in the mutation − 353AMTN constructs that included 3-bp mutations in CCAAT enhancer-binding protein 1 (C/EBP1), C/EBP2 and Ying Yang 1 (YY1) elements. Transcriptional activities induced by TNF-α were inhibited by protein kinase A, Src-tyrosine kinase, MEK1/2, p38 kinase, NF-κB, and PI3-kinase inhibitors. Gel shift assays showed that TNF-α increased nuclear proteins binding to two types of C/EBP elements (C/EBP1 and C/EBP2) and YY1 element. The results of the chromatin immunoprecipitation assays showed that C/EBPβ binding to C/EBP1 and C/EBP2, and YY1 binding to YY1 were increased by TNF-α.Conclusions: These findings demonstrated that TNF-α stimulates AMTN gene transcription in human gingival epithelial cells via C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter. [ABSTRACT FROM AUTHOR]

Follicular dendritic cell‐secreted protein (FDC‐SP) is a secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium. To elucidate the transcriptional regulation of the human FDC‐SP gene by tumor necrosis factor‐α (TNF‐α), we conducted real‐time PCR, Western blotting, transient transfection analyses with chimeric constructs of the FDC‐SP gene promoter linked to a luciferase reporter gene, gel mobility shift and chromatin immunoprecipitation assays using Ca9‐22 gingival epithelial cells. TNF‐α (10 ng/ml) induced FDC‐SP mRNA and protein levels at 3 hr and reached maximum at 12 hr. In transient transfection assays, TNF‐α (12 hr) increased the LUC activities of constructs between −116FDCSP and −948FDCSP including the human FDC‐SP gene promoter. Transcriptional stimulations by TNF‐α were partially inhibited in the −345FDCSP constructs that included 3‐bp mutations in the YY1, GATA, CCAAT enhancer‐binding protein 2 (C/EBP2) and C/EBP3. Transcriptional activities induced by TNF‐α were inhibited by tyrosine kinase, MEK1/2 and phosphoinositide 3‐kinase inhibitors. The results of ChIP assays showed that YY1, GATA and C/EBPβ transcription factors interacted with the YY1, GATA, C/EBP2 and C/EBP3 elements that were increased by TNF‐α. These studies show that TNF‐α stimulates human FDC‐SP gene transcription by targeting YY1, GATA, C/EBP2 and C/EBP3 in the FDC‐SP gene promoter. [ABSTRACT FROM AUTHOR]

Bone sialoprotein (BSP) is a glycoprotein associated with mineralized tissues. In this study, we investigated the regulation of Bsp transcription by calcium hydroxide [Ca(OH)2] in rat osteosarcoma‐derived osteoblast‐like ROS 17/2.8 cells and stromal bone marrow cells. Application of Ca(OH)2 (0.4 mM) increased the levels of runt‐related transcription factor 2 (Runx2) and BspmRNAs at 3 and 6 h and the level of BSP protein at 12 h. Transient transfection analyses were performed using chimeric constructs encompassing different regions of the rat Bsp gene promoter ligated to a luciferase reporter gene. It was found that Ca(OH)2 increased the luciferase activities of the pLUC3 and pLUC4 constructs. Introduction of 2‐bp mutations to the luciferase construct showed that the effects of Ca(OH)2 were mediated by cAMP response‐element (CRE) and fibroblast growth factor 2 (FGF2) response element (FRE). Luciferase activities induced by Ca(OH)2 were blocked by protein kinase C (PKC), protein kinase A (PKA), phosphoinositide‐3‐kinase (PI3‐K), and extracellular signal‐regulated kinase 1/2 (ERK1/2) inhibitors and by calcium‐sensing receptor (CASR) antagonists. Gel‐shift analyses showed that Ca(OH)2 increased binding of nuclear protein to CRE and FRE. Dexamethasone‐induced mineralization in stromal bone marrow cells was abrogated by CASR antagonists. These studies demonstrate that Ca(OH)2 regulates Bsp transcription via the CASR by targeting CRE and FRE in the rat Bsp gene promoter. [ABSTRACT FROM AUTHOR]

Cellular DNA topoisomerases (topo I and topo II) are highly conserved enzymes that regulate the topology of DNA during normal genome transactions, such as DNA transcription and replication. In budding yeast, topo I is dispensable whereas topo II is essential, suggesting fundamental and exclusive roles for topo II, which might include the functions of the topo IIa and topo IIb isoforms found in mammalian cells. In this review, we discuss major findings of the structure and chromosomal organization of genes regulated by topo II in budding yeast. Experimental data was derived from short (10 min) and long term (120 min) responses to topo II inactivation in top-2 ts mutants. First, we discuss how short term responses reveal a subset of yeast genes that are regulated by topo II depending on their promoter architecture. These short term responses also uncovered topo II regulation of transcription across multi-gene clusters, plausibly by common DNA topology management. Finally, we examine the effects of deactivated topo II on the elongation of RNA transcripts. Each study provides an insight into the particular chromatin structure that interacts with the activity of topo II. These findings are of notable clinical interest as numerous anti-cancer therapies interfere with topo II activity. [ABSTRACT FROM AUTHOR]

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase capable of hydrolysing a variety of esters and is considered to be a candidate gene affecting fat deposition traits. Gene expression profiles of HSL were analysed in various adipose tissues of cattle, and the effect of HSL on lipid metabolism genes was analysed by a PCR array. Novel polymorphisms were identified within the HSL regulatory domain by sequencing, and the relationship between HSL variants and fat deposition traits was analysed. HSL mRNA was highly expressed in the subcutaneous and visceral fat of cattle. CPT1B/CPT1C and other lipocatabolic genes were upregulated, and lipogenesis-related genes ( FASN , LPL and ACOT12 ) were downregulated by HSL overexpression in BFFs. Five novel variants in the HSL functional domain were significantly associated with fat deposition traits, including FCR, LBT, MFW and fatty acid composition. HSL plays a pivotal role in the regulation of lipolysis and fatty acid biosynthesis in beef cattle. [ABSTRACT FROM AUTHOR]

The Receptor for Activated C Kinase 1 (RACK1) is a scaffold protein for different kinases and membrane receptors. RACK1 can shuttle proteins to their sites of action, facilitate cross-talk among distinct signaling pathways or recruit other signaling proteins into the complexes. Therefore, it is a key mediator of various pathways and is involved in various biological events including development, immune response, brain activity and cancer. Because of its importance, it is of extreme significance to understand the transcriptional mechanisms governing its expression. The identification of regulatory elements in the promoter of RACK1 shed some light on its transcriptional modulation in physiological and pathological context. Literature data support the existence of a complex hormonal balance, between glucocorticoids and androgens, in the control of RACK1 expression due to specific and complex interactions on the RACK1 promoter. These and other informations suggest that a better understanding of RACK1 transcriptional regulation is essential to unravel its role. Furthermore, the modulation of its expression in physiological or pathological conditions may be of interest in different context, such as aging and cancer. [ABSTRACT FROM AUTHOR]

Studies on the etiology of essential hypertension (EH) have demonstrated that chronic inflammation contributes to the onset and development of elevated blood pressure. Toll-like receptors (TLRs), important immune receptors, serve a role in chronic inflammation and are associated with EH. In the present study, 96 patients with EH, and 96 age- and sex-matched healthy controls were recruited, and eight cytosine-phosphate-guanine (CpG) dinucleotides (CpG1-8) were analyzed using bisulfite pyrosequencing technology. It was observed that the methylation levels of all of the eight CpG dinucleotides were decreased in the EH group compared with the control group; however, only CpG1 (2.83±1.34 vs. 3.44±1.75; P=0.009), CpG6 (3.58±3.64 vs. 8.30±4.13; P<0.001) and CpG8 (8.91±5.32 vs. 11.33±3.87; P<0.001) were significantly different, as demonstrated by paired t-test analysis. In addition, logistic regression analysis demonstrated that CpG6 hypomethylation was a risk factor of EH (odds ratio=1.10; adjusted P=0.009), and CpG6 methylation level was observed to be negatively correlated with systolic blood pressure (r=-0.304; P<0.001) and diastolic blood pressure (r=-0.329; P<0.001). Additionally, receiver operating characteristic curve analysis demonstrated that a methylation level of 7.5% for CpG6 (area under the curve, 0.834; P<0.001) was an appropriate threshold value to predict the risk of EH. With generalized multifactor dimensionality reduction, a potential gene-gene interaction between CpG6 and CpG8 (P=0.001), and gene-environment interactions between smoking, alcohol consumption, CpG6, CpG7 and CpG8 (P=0.011), were observed. In conclusion, the results of the present study demonstrated that hypomethylation of the TLR2 promoter, particularly CpG6, was associated with the risk of EH in this population. Additionally, a gene-gene interaction between CpG6 and CpG8, and interactions between environmental factors, including smoking and alcohol consumption, and CpG6, CpG7 and CpG8, may be associated with the risk of EH. [ABSTRACT FROM AUTHOR]

Background G-rich sequences undergo unique structural equilibria to form G-quadruplexes (G4) both in vitro and in cell systems. Several pathologies emerged to be directly related to G4 occurrence at defined genomic portions. Additionally, G-rich sequences are significantly represented around transcription start sites (TSS) thus leading to the hypothesis of a gene regulatory function for G4. Thus, the tuning of G4 formation has been proposed as a new powerful tool to regulate gene expression to treat related pathologies. However, up-to date this approach did not provide any new really efficient treatment. Scope of review Here, we summarize the most recent advances on the correlation between the structural features of G4 in human promoters and the role these systems physiologically exert. In particular we focus on the effect of G4 localization among cell compartments and along the promoters in correlation with protein interaction networks and epigenetic state. Finally the intrinsic structural features of G4 at promoters are discussed to unveil the contribution of different G4 structural modules in this complex architecture. Major conclusions It emerges that G4s play several roles in the intriguing and complex mechanism of gene expression, being able to produce opposite effects on the same target. This reflects the occurrence of a highly variegate network of several components working simultaneously. General significance The resulting picture is still fuzzy but some points of strength are definitely emerging, which prompts all of us to strengthen our efforts in view of a selective control of gene expression through G4 modulation. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. [ABSTRACT FROM AUTHOR]

In vertebrates, estrogens and estrogen mimicking chemicals modulate gene expression mainly through a genomic pathway mediated by the estrogen receptors (ERs). Although the existence of an ER orthologue in the mollusc genome has been known for some time, its role in estrogen signalling has yet to be deciphered. This is largely due to its constitutive (ligand-independent) activation and a limited mechanistic understanding of its regulation. To fill this knowledge gap, we cloned and characterised an ER cDNA ( sgER ) and the 5 ′ -flanking region of the gene from the Sydney rock oyster Saccostrea glomerata . The sgER cDNA is predicted to encode a 477-amino acid protein that contains a DNA-binding domain (DBD) and a ligand-binding domain (LBD) typically conserved among both vertebrate and invertebrate ERs. A comparison of the sgER LBD sequence with those of other ligand-dependent ERs revealed that the sgER LBD is variable at several conserved residues known to be critical for ligand binding and receptor activation. Ligand binding assays using fluorescent-labelled E2 and purified sgER protein confirmed that sgER is devoid of estrogen binding. In silico analysis of the sgER 5 ′ -flanking sequence indicated the presence of three putative estrogen responsive element (ERE) half-sites and several putative sites for ER-interacting transcription factors, suggesting that the sgER promoter may be autoregulated by its own gene product. sgER mRNA is ubiquitously expressed in adult oyster tissues, with the highest expression found in the ovary. Ovarian expression of sgER mRNA was significantly upregulated following in vitro and in vivo exposure to 17β-estradiol (E2). Notably, the activation of sgER expression by E2 in vitro was abolished by the specific ER antagonist ICI 182, 780. To determine whether sgER expression is epigenetically regulated, the in vivo DNA methylation status of the putative proximal promoter in ovarian tissues was assessed using bisulfite genomic sequencing. The results showed that the promoter is predominantly hypomethylated (with 0–3.3% methylcytosines) regardless of sgER mRNA levels. Overall, our investigations suggest that the estrogen responsiveness of sgER is regulated by a novel ligand-dependent receptor, presumably via a non-genomic pathway(s) of estrogen signalling. [ABSTRACT FROM AUTHOR]

Purpose: Downregulation of miR- 125b- 1 is associated with poor prognosis in breast cancer patients. In this work we investigated the effect of histone modifications on the regulation of this gene promoter. Methods and results: We evaluated the enrichment of two histone modifications involved in gene repression, H3K9me3 and H3K27me3, on the miR- 125b- 1 promoter in two breast cancer cell lines, MCF7 (luminal A subtype) and MDA-MB-231 (triple-negative subtype), compared to the non-transformed breast cell line MCF10A. H3K27me3 and H3K9me3 were enriched in MCF7 and MDA-MB-231 cells, respectively. Next, we used an EZH2 inhibitor to examine the reactivation of miR- 125b- 1 in MCF7 cells and evaluated the transcriptional levels of pri-miR-125b-1 and mature miR-125b by qRT-PCR. pri-miRNA and mature miRNA transcripts were both increased after treatment of MCF7 cells with the EZH2 inhibitor, whereas no effect on miR- 125b- 1 expression levels was observed in MDA-MB-231 and MCF10A cells. We subsequently evaluated the effect of miR- 125b- 1 reactivation on the expression and protein levels of BAK1, a target of miR-125b. We observed 60 and 70 % decreases in the expression and protein levels of BAK1, respectively, compared to cells that were not treated with the EZH2 inhibitor. We over-expressed KDM4B/JMJD2B to reactivate this miRNA, resulting in a three-fold increase in miR-125b expression compared with the same cell line without KDM4B/JMJD2B over-expression. Conclusion: The miR- 125b- 1 is repressed by different epigenetic mechanisms depending on the breast cancer subtype and that miR- 125b- 1 reactivation specifically eliminates the effect of repressive histone modifications on the expression of an pro-apoptotic target. [ABSTRACT FROM AUTHOR]

Human aging is associated with epigenetic modification of the genome. DNA methylation at cytosines appears currently as the best characterised modification that occurs during the mammalian lifetime. Such methylation changes at regulatory region can provide insights to track contributor age for criminal investigation. The EpiTect Methyl II PCR system (QIAGEN) was used to compare methylation levels of CpG islands in the promoter regions of a number of age related genes, of which four successfully showed changes across the lifespan (NPTX2, KCNQ1DN, GRIA2 and TRIM58). This technique is based on the detection of remaining input genome after digestion with a methylation-sensitive restriction enzyme. This study examined DNA specimens from 80 female subjects of various ages (18-91 years) obtained from blood, using primers designed to flank the studied gene loci. The data obtained from DNA methylation quantification showed successful discrimination among volunteered ages. Overall, the difference between predicted and real age was about 11 years and absolute mean differences (AMD) was only 7.2 years error. We suggest the EpiTect system can be used as fast and simple innovative tool in future forensic age estimation. [ABSTRACT FROM AUTHOR]

The gene encoding macrophage migration inhibitory factor (MIF) has been proposed as candidate tuberculosis (TB) susceptibility gene. In order to elucidate whether MIF gene variants are associated with susceptibility to retreatment cases of TB, and prevent drug-resistant TB prevalence, we conducted a study based on paired human population data. MIF − 173 G/C single nucleotide polymorphisms (rs755622) were genotyped using polymerase chain reaction-restriction fragment length polymorphism. MIF levels were detected with enzyme-linked immunosorbent assay. Association analysis of polymorphism to TB showed that distribution of MIF − 173 genotypes (GC + CC) was significantly higher in total cases of TB than in the controls. Statistically significant differences of frequencies for MIF − 173 (GG vs. GC + CC) were demonstrated when comparing total cases of TB, new cases of TB, and retreatment cases of TB to controls, respectively. In contrast, the frequencies of MIF − 173 (GG vs. GC + CC) demonstrated no difference between new cases of TB and retreatment cases of TB. Association analysis of MIF protein concentrations to TB indicated that MIF concentration is significantly higher in total cases of TB, new cases of TB, and retreatment cases of TB than in controls ( P < 0.01). In summary, our results demonstrated that MIF gene − 173 G/C single nucleotide polymorphisms implicate in genetic susceptibility to TB, and GC + CC of MIF − 173 site increases the risk of TB. We also found that no correlation between − 173 G/C single nucleotide polymorphism and retreatment cases of TB in Yunnan Province population of China. [ABSTRACT FROM AUTHOR]

Osteoblasts respond to many growth factors including IGF-I and TGF-β, which themselves are sensitive to other bone growth regulators. Here we show that IGF-I gene promoter activity in prostaglandin E2 (PGE2) induced osteoblasts is suppressed by dihydrotestosterone (DHT) through an essential C/EBP response element (RE) in exon 1 of the igf1 gene. Inhibition by DHT fails to occur when the androgen receptor (AR) gene is mutated within its DNA binding domain. Correspondingly, DHT activated AR inhibits gene transactivation by C/EBPδ, and transgenic C/EBPδ expression inhibits AR activity. Inhibition by DHT persists when upstream Smad and Runx REs in the IGF-I gene promoter are mutated. TGF-β also enhances IGF-I gene promoter activity, although modestly relative to PGE2, and independently of the C/EBP, Smad, or Runx REs. Still, DHT suppresses TGF-β induced IGF-I promoter activity, but not its effects on DNA or collagen synthesis. Notably, DHT suppresses plasminogen activator inhibitor gene promoter activity, but synergistically increases Smad dependent gene promoter activity in TGF-β induced cells, which are differentially sensitive to AR mutations and the AR co-regulator ARA55. Finally, although the PGE2 sensitive C/EBP RE in the igf1 gene is not essential for basal TGF-β induction, C/EBPδ activity through this site is potently enhanced by TGF-β. Thus DHT suppresses the PGE2 and TGF-β induced IGF-I gene promoter and differentiates other aspects of TGF-β activity in osteoblasts. Our results extend the complex interactions among local and systemic bone growth regulators to DHT, and predict complications from anabolic steroid use in other DHT sensitive tissues. [ABSTRACT FROM AUTHOR]

The detection of gene promoter methylation plays increasing roles in personalized medicine. In this study, an improved gene promoter methylation assay based on fluorescence polarization in 5'-nuclease reaction was developed. The novel assay offered a homogeneous annealing and cleavage reaction fully integrated with PCR which used a probe labeled with fluorescence without quencher to obtain the decreased fluorescence polarization values. In this platform, gene promoter methylated and unmethylated alleles were detected simultaneously in a tube. O 6 -methylguanine-DNA methyltransferase gene promoter methylation in 103 glioma tissue samples and epidermal growth factor receptor gene promoter methylation in 116 primary non-small-cell lung carcinoma tissue samples were detected by the novel assay and sequencing, absolute quantitative analysis of methylated allele in parallel. The accuracy of the results measured by the improved fluorescence polarization assay was evaluated using the paired-samples t test. No significant difference was found ( P > 0.05). Therefore, the improved fluorescence polarization assay in 5'-nuclease reaction demonstrated a homogeneous, reliable and cost-effective method for gene promoter methylation analysis in clinic. That would provide a scientific basis for applying a reasonable therapeutic regimen in future treatment. [ABSTRACT FROM AUTHOR]

Heat shock protein 90α (Hsp90α), encoded by the HSP90AA1 gene, is the stress inducible isoform of the molecular chaperone Hsp90. Hsp90α is regulated differently and has different functions when compared to the constitutively expressed Hsp90β isoform, despite high amino acid sequence identity between the two proteins. These differences are likely due to variations in nucleotide sequence within non-coding regions, which allows for specific regulation through interaction with particular transcription factors, and to subtle changes in amino acid sequence that allow for unique post-translational modifications. This article will specifically focus on the expression, function and regulation of Hsp90α. [ABSTRACT FROM AUTHOR]

The development of the nervous system is a temporally and spatially coordinated process that relies on the proper regulation of the genes involved. Neurotrophins and their receptors are directly responsible for the survival and differentiation of sensory and sympathetic neurons; however, it is not fully understood how genes encoding Trk neurotrophin receptors are regulated. Here, we show that rat Bex3 protein specifically regulates TrkA expression by acting at the trkA gene promoter level. Bex3 dimerization and shuttling to the nucleus regulate the transcription of the trkA promoter under basal conditions and also enhance nerve growth factor (NGF)-mediated trkA promoter activation. Moreover, qChIP assays indicate that Bex3 associates with the trkA promoter within a 150 bp sequence, immediately upstream from the transcription start site, which is sufficient to mediate the effects of Bex3. Consequently, the downregulation of Bex3 using shRNA increases neuronal apoptosis in NGF-dependent sensory neurons deprived of NGF and compromises PC12 cell differentiation in response to NGF. Our results support an important role for Bex3 in the regulation of TrkA expression and in NGF-mediated functions through modulation of the trkA promoter. [ABSTRACT FROM AUTHOR]

We have previously reported that synthetic dsRNA can activate p21 expression by targeting the p21 promoter, thereby suppressing the proliferation of human bladder cancer cells. As complementarity between dsRNA and its target sequences is necessary for RNA activation, miRNAs may also trigger p21 expression through the same mechanism. Here, the expression levels of three miRNAs (miR‐370, miR‐1180 and miR‐1236) decreased in bladder cancer tissues compared to healthy controls and the levels of these mRNAs positively correlated with p21 mRNA levels. The three miRNAs induced nuclear p21 expression through p21‐promoter binding. Overexpression of the three miRNAs inhibited the proliferation of bladder cancer cells mainly by regulating p21. Therefore, these miRNAs could be candidates for anti‐cancer drugs.miR‐370, miR‐1180 and miR‐1236 were lower expression in bladder cancer. miRNAs levels positively correlate p21 mRNA levels in bladder cancer tissues. miRNAs induce nuclear p21 expression in human bladder cancer cells. miRNAs upregulate p21 by directly targeting its promoter. miRNAs inhibit bladder cancer cells by upregulating p21. [ABSTRACT FROM AUTHOR]

Aim Promoter-targeted small activating RNAs (saRNAs) have been shown to be able to induce target gene expression, a mechanism known as RNA activation (RNAa). The present study tested whether saRNA can induce the overexpression of TRPV5 in human cells derived from the kidney and subsequently manipulate cell calcium uptake. Main methods Three saRNAs complementary to the TRPV5 promoter were synthesized and transfected into cells. TRPV5 expression at the RNA and protein levels was analyzed by quantitative real-time PCR and Western blotting respectively. For functional study, transcellular Ca 2 + transportation was tested by fura-2 analysis. Dihydrotestosterone (DHT), a suppressor of cellular calcium transportation, was administered to challenge the activating effect of selected saRNA. Key findings One of these synthesized saRNAs, ds-2939, significantly induced the expression of TRPV5 at both mRNA and protein levels. Fura-2 analysis revealed that the intracellular Ca 2 + concentration was elevated by ds-2939. DHT treatment reduced transmembrane Ca 2 + transport, which was partially antagonized by ds-2939. Significance Our results suggest that a saRNA targeting TRPV5 promoter can be utilized to manipulate the transmembrane Ca 2 + transport by upregulating the expression of TRPV5 and may serve as an alternative for the treatment of Ca 2 + balance-related diseases. [ABSTRACT FROM AUTHOR]

The inhibitor of differentiation Id2, a protein lacking the basic DNA-binding domain, is involved in the modulation of a number of biological processes. The molecular mechanisms explaining Id2 pleiotropic functions are poorly understood. Id2 and E2F4 are known to bind simultaneously to c-myc promoter. To study whether Id2 plays a global role on transcriptional regulation, we performed in vivo genome-wide ChIP/chip experiments for Id2 and E2F4 in adult mouse liver. An Id2-containing complex was bound to a common sequence downstream from the TSS on a subset of 442 E2F4 target genes mainly related to cell development and chromatin structure. We found a positive correlation between Id2 protein levels and the expression of E2F4/Id2 targets in fetal and adult liver. Id2 protein stability increased in fetal liver by interaction with USP1 de-ubiquitinating enzyme, which was induced during development. In adult liver, USP1 and Id2 levels dramatically decreased. In differentiated liver tissue, when Id2 concentration was low, E2F4/Id2 was bound to the same region as paused Pol II and target genes remained transcriptionally inactive. Conversely, in fetal liver when Id2 levels were increased, Id2 and Pol II were released from gene promoters and target genes up-regulated. During liver regeneration after partial hepatectomy, we obtained the same results as in fetal liver. Our results suggest that Id2 might be part of a reversible development-related program involved in the paused-ON/OFF state of Pol II on selected genes that would remain responsive to specific stimuli. [ABSTRACT FROM AUTHOR]

Background Mutations in BRCA1/2 genes are involved in the pathogenesis of breast and ovarian cancer. Inactivation of these genes can also be mediated by hypermethylation of CpGs in the promoter regions. Aim of this study was to analyse the clinical impact of BRCA1 promoter gene methylation status in a homogenous cohort of high-grade serous ovarian cancer (HGSOC) patients. Methods The cohort included 257 primary HGSOC patients treated by cytoreduction and platinum-based chemotherapy. DNA was extracted from fresh frozen tissue samples. BRCA1 gene promoter methylation rate was assessed using polymerase chain reaction (PCR). Results 14.8% of patients presented hypermethylation within a selected region of the BRCA1 promoter. The rate of hypermethylation was significantly higher in younger patients (20.8% hypermethylation in the age group > 58years versus 8.7% hypermethylation in the age group >58years; p=0.008). Optimal tumour debulking could be reached in 63% of patients, without significant differences in the extent of residual disease with respect to the methylation status. No impact of BRCA1 gene promoter methylation status on progression free- and overall-survival rates was found. No significant differences within BRCA1 promoter methylation status between primary and metastatic tissue could be observed. These results on BRCA1 promoter methylation status were also confirmed in a subgroup of 107 patients found negative for BRCA1 exon 11 mutations. Conclusions Our data suggest that BRCA1 methylation determines the earlier onset of HGSOC. Furthermore our study supports the idea that BRCAness is not only due to mutations but also to epigenetic changes in BRCA1 promoter gene. [ABSTRACT FROM AUTHOR]

Abstract: Background: The thioredoxin system maintains redox balance through the action of thioredoxin and thioredoxin reductase. Thioredoxin regulates the activity of various substrates, including those that function to counteract cellular oxidative stress. These include the peroxiredoxins, methionine sulfoxide reductase A and specific transcription factors. Of particular relevance is Redox Factor-1, which in turn activates other redox-regulated transcription factors. Scope of review: Experimentally defined transcription factor binding sites in the human thioredoxin and thioredoxin reductase gene promoters together with promoters of the major thioredoxin system substrates involved in regulating cellular redox status are discussed. An in silico approach was used to identify potential putative binding sites for these transcription factors in all of these promoters. Major conclusions: Our analysis reveals that many redox gene promoters contain the same transcription factor binding sites. Several of these transcription factors are in turn redox regulated. The ARE is present in several of these promoters and is bound by Nrf2 during various oxidative stress stimuli to upregulate gene expression. Other transcription factors also bind to these promoters during the same oxidative stress stimuli, with this redundancy supporting the importance of the antioxidant response. Putative transcription factor sites were identified in silico, which in combination with specific regulatory knowledge for that gene promoter may inform future experiments. General significance: Redox proteins are involved in many cellular signalling pathways and aberrant expression can lead to disease or other pathological conditions. Therefore understanding how their expression is regulated is relevant for developing therapeutic agents that target these pathways. [Copyright &y& Elsevier]

Ubiquitous eukaryotic non-coding circular RNAs are involved in numerous co- and post-transcriptional regulatory mechanisms. Recently, we reported full-length intronic circular RNAs (flicRNAs) in Entamoeba histolytica, with 3′ss–5′ss ligation points and 5′ss GU-rich elements essential for their biogenesis and their suggested role in transcription regulation. Here, we explored how flicRNAs impact gene expression regulation. Using CLIP assays, followed by qRT-PCR, we identified that the RabX13 control flicRNA and virulence-associated flicRNAs were bound to the HA-tagged RNA Pol II C-terminus domain in E. histolytica transformants. The U2 snRNA was also present in such complexes, indicating that they belonged to transcription initiation/elongation complexes. Correspondingly, inhibition of the second step of splicing using boric acid reduced flicRNA formation and modified the expression of their parental genes and non-related genes. flicRNAs were also recovered from chromatin immunoprecipitation eluates, indicating that the flicRNA-Pol II complex was formed in the promoter of their cognate genes. Finally, two flicRNAs were found to be cytosolic, whose functions remain to be uncovered. Here, we provide novel evidence of the role of flicRNAs in gene expression regulation in cis, apparently in a widespread fashion, as an element bound to the RNA polymerase II transcription initiation complex, in E. histolytica. [ABSTRACT FROM AUTHOR]

Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea ( Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule. [ABSTRACT FROM AUTHOR]

Highlights: [•] PMA/ionomycin activates TNFSF15 transcription in human monocytes. [•] A minimal −74/26 promoter fragment mediates a 30-fold increase in transactivation. [•] AP1/c-jun binds to the TNFSF15 5′UTR and mutation of this site inhibits TNFSF15 promoter activity. [•] NFκB binds to TNFSF15 −210bp region, although mutation of this site enhances promoter activity. [•] Global inhibition of AP1 or NFκB pathways downregulates TL1A mRNA and protein expression in human monocytes. [ABSTRACT FROM AUTHOR]

Multidrug resistance (MDR), one of the main reasons for diminishing efficacy of prolonged chemotherapy, is frequently caused by the elevated expression of the ABCB1/MDR1 gene encoding PGP (P-glycoprotein). EAPP (E2F Associated PhosphoProtein) is a frequently overexpressed protein in human tumor cells. It inhibits apoptosis in a p21-dependent manner. We show here that EAPP stimulates the MDR1 promoter resulting in higher PGP levels. Independently of EAPP, E2F1 also increases the activity of the MDR1 promoter. Co-expression of pRb inhibits E2F1-, but not EAPP-dependent promoter activation. The upregulation of PGP might contribute to the survival of tumor cells during chemotherapy and worsen the prognosis for the patient. [ABSTRACT FROM AUTHOR]

Abstract: Staphylococcal nuclease domain-containing protein 1 (SND1), also called Tudor-SN, is required for many biological events ranging from gene expression to cell growth regulation. Promoter regulation of SND1 gene and its molecular mechanism have remained elusive to date. In this work, we have identified SND1 as a new target gene for NF-κB, Sp1 and NF-Y transcription factors. We isolated and characterized a 3808 bp sequence corresponding to the human SND1 gene promoter (GenBank ID: EF690304). It lacks the typical TATA-box element and contains a CpG island with several Sp1 binding sites at the 3′ end, and a highly conserved 300 bp segment with two inverted CCAAT boxes that bind NF-Y, in addition to NF-κB sites and other cis-regulatory elements. Electrophoretic mobility shift assays and chromatin immunoprecipitation experiments confirmed the ability of SND1 promoter to bind NF-κB, Sp1 and NF-Y in vitro and in vivo. Deletion analysis of the 5′-flanking region by luciferase reporter assays, showed the minimum promoter activity 112 base-pair upstream from the transcription start site, and an enhancer region between −112 and −274 bp responsible for the maximal transcriptional activity of the promoter. Site-directed mutagenesis of the CCAAT and GC boxes and the NF-κB elements within the proximal region substantially reduced SND1 promoter activity. Proinflammatory cytokine TNF-α caused an increase of SND1 promoter activity that is mediated, at least in part, via NF-κB as mutation in the NF-κB sites impaired the promoter stimulation. We provide for the first time the characterization of the human SND1 promoter activity and establish a transcriptional network associated to the key transcription factors NF-κB, Sp1 and NF-Y that operates in the control of the SND1 gene expression. [Copyright &y& Elsevier]

Single-nucleotide polymorphisms (SNP) in the coding sequence of cytochrome p450 (CYP3A28) have been associated with milk yield and composition, and calving traits in cows. In this study, we aimed to determine whether (i) the CYP3A28 regulatory region was polymorphic and (ii) SNP genotype, forage type, body condition and their interactions affect cow productivity. Primers for CYP3A28 promoter were designed to amplify a 483-bp segment by PCR. Amplicon sequences revealed seven SNP (T-318C, T-113A, C-189T, T-78G, A6G, G17A and T21C) in Brahman (38 cows), Brahman x Angus reciprocal crosses (47 cows) and crossbreds (98 cows). Angus cows ( n = 41) appeared to be fixed at those SNP locations. Genotype and forage {endophyte-infected tall fescue [KY+; Lolium arundinaceum (Schreb.) S. J. Darbyshire] vs. bermudagrass [ Cynodon dactylon (L.) Pers.]} effects on lifetime (8-years) calving rate, and calf weaning weights and heights were determined in Herd 1 (126 cows); genotype and BC (low vs. moderate) effects on calving date and calving percent were determined in Herd 2 (98 cows). Four SNP (T-318C, T-113A, A06G and T21C) appeared to be related to cattle productivity, CC cows at T-318C having a lower (p < 0.05) lifetime calving rate than TC or TT cows (65%, 85% and 81% respectively). Cows that grazed KY+ and were TT at T-318C produced calves that tended (p < 0.07) to weigh less than their contemporaries. Moreover, calves of TT cows were shorter (p < 0.05) at weaning than calves of CC or TC cows. In Herd 2, moderate-BC cows that were TT or AA at T-318C, T-113A, T-78G, A6G and T21C had greater (p < 0.05) calving rates (74-80%) than heterozygous cows (46-60%), and low-BC cows that were AA at G17A calved at least 6 days earlier (p < 0.05) than heterozygous cows. Our findings suggest that SNP in the CYP3A28 regulatory region of Brahman-influenced cows are associated with cattle productivity. [ABSTRACT FROM AUTHOR]

BACKGROUND Deleted in AZoospermia-like (DAZL) is an autosomal homologue of Y chromosome-linked DAZ gene located on chromosome 3p24. DAZL is only expressed in the gonads and is critical to germ cell development in different species. However, the regulation of DAZL has not been explored. METHODS Reporter assays, electrophoretic mobility shift assays, supershift assays and bisulfate sequencing were used to identify the core promoter region of DAZL. Sequence analysis was used to identify single-nucleotide polymorphisms (SNPs) in the promoter region. A total of 337 infertile men with abnormal semen parameters and 203 fertile men with normal semen parameters were subjected to sequence analysis of the DAZL promoter region. RESULTS The DAZL gene core promoter is located 1 kb upstream of the transcription start site. Three SNPs (−792G>A, −669A>C and −309T>C) were identified in our population. Of these three SNPs, −792G>A was more prevalent in the infertile men (P= 0.0005). Quantitative analysis revealed that genotypes of −792G>A had effects on sperm concentration (P= 0.0025) and motility (P= 1.5 × 10–7). The G to A substitution was associated with decreased binding of the nuclear respiratory factor-1 (NRF-1) to the promoter region and decreased reporter gene activity. CONCLUSION We have identified the core promoter of the human DAZL gene. We also provide preliminary evidence for the role of a novel SNP of the DAZL gene promoter in human spermatogenic failure. [ABSTRACT FROM AUTHOR]

Abstract: MS4a4B is a novel member of the membrane-spanning 4-domain family, subfamily A (MS4A) specifically expressed in mouse T cells. We have shown previously that expression of MS4a4B in T cells is upregulated upon T cell activation, suggesting that MS4a4B may play a functional role in regulation of T cell responses. However, little is known about the mechanisms that regulate MS4a4B gene expression. In this study, we explored the potential mechanism underlying TCR-stimulation-induced expression of MS4a4B by promoter analysis. We cloned 2495bp of 5′-flanking region upstream of the MS4a4B start code and inserted the DNA fragment into pGL4.20 reporter plasmid. To analyze promoter activity of the cloned DNA fragment, we transiently transfected EL4 thymoma cells and the T32 cell line with reporter plasmids. Expression of reporter gene was determined by dual-luciferase assay. Potential activator- and repressor-binding sites were analyzed by serial length of 5′-deletion. We have identified at least two potential activator binding regions and two potential repressor binding regions. The activator binding sites have been localized to two fragments, which are a 442-base pair region (region A) positioned from −1176 to −735, and a 119-base pair region (region B) positioned −188 to −70 respectively. MatInspector analysis showed that region A contains the consensus binding motif of the AP-1 family of transcription factors. Machinery analysis showed that nuclear proteins extracted from anti-CD3/anti-CD28-activated primary T cells specifically bind to the AP-1 binding element. In contrast, blockade by AP-1 inhibitor in culture decreased MS4a4B expression in T cells. Our data demonstrate that TCR-stimulation induces transactivation of AP-1 transcription factor, which subsequently binds to the MS4a4B promoter and upregulates expression of MS4a4B in activated T cells. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Panax notoginseng (Burk.) F.H. Chen has been used as a health product and natural remedy in traditional medicine for cardiovascular diseases for more than 1000 years in Asia, including China, Japan, and Korea. Panax notoginseng saponins (PNS) are the major effective ingredients extracted from Panax notoginseng. Aim of the study: The purpose of this study was to investigate whether Panax notoginseng saponins (PNS) attenuated atherosclerosis by inducing liver X receptor alpha (LXRα) expression and to elucidate the mechanisms responsible for the effects. Materials and methods: The AS rats were treated once daily with PNS (100mg/kg, i.p.), and pathological changes in the aorta were observed using Sudan IV staining. The expression of LXRα in the aortic wall was measured by Western blot analysis. THP-1 macrophages were cultured with PNS in the presence or absence of geranylgeranyl pyrophosphate ammonium salt (GGPP), an LXRα antagonist. The expression of LXRα and its target genes ATP-binding cassette A1 and G1 (ABCA1, ABCG1) were determined by qRT-PCR. The transcriptional activation of the LXRα gene promoter was analyzed by a reporter assay. The NF-κB DNA binding activity and the expression of interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1) was evaluated respectively by Trans-AM NF-κB ELISA and ELISA in THP-1 macrophages that were stimulated with LPS after treatment with PNS and GGPP. Results: PNS treatment alleviated the typical pathological changes associated with atherosclerosis in rats. The expression of LXRα was increased in rat aortas after treatment with PNS. In vitro, PNS increased LXRα mRNA levels in THP-1 macrophages. The reporter assays showed that PNS enhanced transcriptional activation of the LXRα gene promoter and led to the upregulation of ABCA1 and ABCG1 expression. This upregulation could be reversed by treatment with GGPP. Additionally, PNS inhibited NF-κB DNA binding activity and reduced secretion of IL-6 and MCP-1 in LPS-stimulated THP-1 macrophages. These effects could be reversed by GGPP. Conclusions: The results indicated that the PNS-mediated attenuation of AS may, at least partly, due to LXRα uprergulation. The mechanisms of action included enhancement transcriptional activation of the LXRα gene promoter by PNS and subsequent upregulation of ABCA1 and ABCG1 and inhibition of NF-κB DNA binding activity. [Copyright &y& Elsevier]