Enhanced Promoter Activity Research Articles

Overcoming the Silencing of Doxycycline-Inducible Promoters in hiPSC-derived Cardiomyocytes

Background Human induced pluripotent stem cells (hiPSCs) are pivotal for studying human development, modeling diseases, and advancing regenerative medicine. Effective control of transgene expression is crucial to achieve temporal and quantitative precision in all of these contexts. The doxycycline (dox)-inducible OPTi-OX system, which integrates the Tet-On 3G transactivator and dox-responsive transgene at the hROSA26 and AAVS1 genomic safe harbors (GSHs), respectively, offers a promising solution. Yet, transgene silencing, particularly in hiPSC-derived cardiomyocytes (hiPSC-CMs), limits its utility. Methods To address this, we evaluated strategies to enhance dox-inducible transgene expression. We compared two promoters, TRE3VG and T11, for activity and stability, and investigated the addition of a Ubiquitous Chromatin Opening Element (UCOE) to reduce silencing. We also tested relocating the transgene cassette to the CLYBL GSH, and employed sodium butyrate (SB), a histone deacetylase inhibitor, to restore promoter activity. Transgene expression was assessed via flow cytometry and real-time quantitative PCR. Results TRE3VG exhibited higher activity than T11, but both were prone to silencing. UCOE did not enhance promoter activity in hiPSCs, but modestly reduced silencing in hiPSC-CMs. Targeting the CLYBL locus improved promoter activity compared to AAVS1 in both hiPSCs and hiPSC-CMs. SB restored activity in silenced inducible promoters within hiPSC-CMs, but compromised hiPSC viability. Unexpectedly, Tet-On 3G was silenced in some clones and could not be reactivated by SB. Conclusions These findings underscore the need for integrating multiple strategies, including careful GSH selection, improved cassette design, epigenetic modulation, and clone screening, to develop robust dox-inducible systems that retain functionality during hiPSC differentiation.

The characteristics of CTCF binding sequences contribute to enhancer blocking activity.

While the elements encoding enhancers and promoters have been relatively well studied, the full spectrum of insulator elements which bind the CCCTC binding factor (CTCF), is relatively poorly characterized. This is partly due to the genomic context of CTCF sites greatly influencing their roles and activity. Here we have developed an experimental system to determine the ability of minimal, consistently sized, individual CTCF elements to interpose between enhancers and promoters and thereby reduce gene expression during differentiation. Importantly, each element is tested in the identical location thereby minimising the effect of genomic context. We found no correlation between the ability of CTCF elements to block enhancer-promoter activity with the degree of evolutionary conservation; their resemblance to the consensus core sequences; or the number of CTCF core motifs harboured in the element. Nevertheless, we have shown that the strongest enhancer-promoter blockers include a previously described bound element lying upstream of the CTCF core motif. In addition, we found other uncharacterised DNaseI footprints located close to the core motif that may affect function. We have developed an assay of CTCF sequences which will enable researchers to sub-classify individual CTCF elements in a uniform and unbiased way.

A Single Base Change in the csgD Promoter Resulted in Enhanced Biofilm in Swine-Derived Salmonella Typhimurium.

Pathogenic Salmonella strains causing gastroenteritis typically can colonize and proliferate in the intestines of multiple host species. They retain the ability to form red dry and rough (rdar) biofilms, as seen in Salmonella enterica serovar Typhimurium. Conversely, Salmonella serovar like Typhi, which can cause systemic infections and exhibit host restriction, are rdar-negative. In this study, duck-derived strains and swine-derived strains of S. Typhimurium locate on independent phylogenetic clades and display relative genomic specificity. The duck isolates appear more closely related to human blood isolates and invasive non-typhoidal Salmonella (iNTS), whereas the swine isolates were more distinct. Phenotypically, compared to duck isolates, swine isolates exhibited enhanced biofilm formation that was unaffected by the temperature. The transcriptomic analysis revealed the upregulation of csgDEFG transcription as the direct cause. This upregulation may be mainly attributed to the enhanced promoter activity caused by the G-to-T substitution at position -44 of the csgD promoter. Swine isolates have created biofilm polymorphisms by altering a conserved base present in Salmonella Typhi, iNTS, and most Salmonella Typhimurium (such as duck isolates). This provides a genomic characteristics perspective for understanding Salmonella transmission cycles and evolution.

Optimizing enzyme properties to enhance dihydroxyacetone production via methylglyoxal biosensor development

BackgroundDihydroxyacetone (DHA) stands as a crucial chemical material extensively utilized in the cosmetics industry. DHA production through the dephosphorylation of dihydroxyacetone phosphate, an intermediate product of the glycolysis pathway in Escherichia coli, presents a prospective alternative for industrial production. However, insights into the pivotal enzyme, dihydroxyacetone phosphate dephosphorylase (HdpA), remain limited for informed engineering. Consequently, the development of an efficient tool for high-throughput screening of HdpA hypermutants becomes imperative.ResultsThis study introduces a methylglyoxal biosensor, based on the formaldehyde-responding regulator FrmR, for the selection of HdpA. Initial modifications involved the insertion of the FrmR binding site upstream of the −35 region and into the spacer region between the −10 and −35 regions of the constitutive promoter J23110. Although the hybrid promoter retained constitutive expression, expression of FrmR led to complete repression. The addition of 350 μM methylglyoxal promptly alleviated FrmR inhibition, enhancing promoter activity by more than 40-fold. The methylglyoxal biosensor system exhibited a gradual increase in fluorescence intensity with methylglyoxal concentrations ranging from 10 to 500 μM. Notably, the biosensor system responded to methylglyoxal spontaneously converted from added DHA, facilitating the separation of DHA producing and non-producing strains through flow cytometry sorting. Subsequently, the methylglyoxal biosensor was successfully applied to screen a library of HdpA mutants, identifying two strains harboring specific mutants 267G > T and D110G/G151C that showed improved DHA production by 68% and 114%, respectively. Expressing of these two HdpA mutants directly in a DHA-producing strain also increased DHA production from 1.45 to 1.92 and 2.29 g/L, respectively, demonstrating the enhanced enzyme properties of the HdpA mutants.ConclusionsThe methylglyoxal biosensor offers a novel strategy for constructing genetically encoded biosensors and serves as a robust platform for indirectly determining DHA levels by responding to methylglyoxal. This property enables efficiently screening of HdpA hypermutants to enhance DHA production.

Functional Characterization of the MeSSIII-1 Gene and Its Promoter from Cassava.

Soluble starch synthases (SSs) play important roles in the synthesis of cassava starch. However, the expression characteristics of the cassava SSs genes have not been elucidated. In this study, the MeSSIII-1 gene and its promoter, from SC8 cassava cultivars, were respectively isolated by PCR amplification. MeSSIII-1 protein was localized to the chloroplasts. qRT-PCR analysis revealed that the MeSSIII-1 gene was expressed in almost all tissues tested, and the expression in mature leaves was 18.9 times more than that in tuber roots. MeSSIII-1 expression was induced by methyljasmonate (MeJA), abscisic acid (ABA), and ethylene (ET) hormones in cassava. MeSSIII-1 expression patterns were further confirmed in proMeSSIII-1 transgenic cassava. The promoter deletion analysis showed that the -264 bp to -1 bp MeSSIII-1 promoter has basal activity. The range from -1228 bp to -987 bp and -488 bp to -264 bp significantly enhance promoter activity. The regions from -987 bp to -747 bp and -747 bp to -488 bp have repressive activity. These findings will provide an important reference for research on the potential function and transcriptional regulation mechanisms of the MeSSIII-1 gene and for further in-depth exploration of the regulatory network of its internal functional elements.

Differential expression of PPP1R12A transcripts, including those harbouring alternatively spliced micro-exons, in placentae from complicated pregnancies

IntroductionPlacenta-associated pregnancy complications, including pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are conditions postulated to originate from initial failure of placentation, leading to clinical sequelae indicative of endothelial dysfunction. Vascular smooth muscle aberrations have also been implicated in the pathogenesis of both disorders via smooth muscle contractility and relaxation mediated by Myosin Light Chain Phosphatase (MLCP) and the oppositional contractile action of Myosin Light Chain Kinase. PPP1R12A is a constituent part of the MLCP complex responsible for dephosphorylation of myosin fibrils. We hypothesize that alternative splicing of micro-exons result in isoforms lacking the functional leucine zipper (LZ) domain which may give those cells expressing these alternative transcripts a tendency towards contraction and vasoconstriction. MethodsExpression was determined by qRT-PCR. Epigenetic profiling consisted of bisulphite-based DNA methylation analysis and ChIP for underlying histone modifications. ResultsWe identified several novel transcripts with alternative micro-exon inclusion that would produce LZ- PPP1R12A protein. qRT-PCR revealed some isoforms, including the PPP1R12A canonical transcript, are differentially expressed in placenta biopsies from PE and IUGR samples compared to uncomplicated pregnancies. DiscussionWe propose that upregulation of PPP1R12A expression in complicated pregnancies may be due to enhanced promoter activity leading to increased transcription as a response to physiological stress in the placenta, which we show is independent of promoter DNA methylation.

Exploring non-coding genetic variability in ACE2: Functional annotation and in vitro validation of regulatory variants

The surge in human whole-genome sequencing data has facilitated the study of non-coding region variations, yet understanding their biological significance remains a challenge. We used a computational workflow to assess the regulatory potential of non-coding variants, with a particular focus on the Angiotensin Converting Enzyme 2 (ACE2) gene. This gene is crucial in physiological processes and serves as the entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 19 (COVID-19). In our analysis, using data from the gnomAD population database and functional annotation, we identified 17 significant Single Nucleotide Variants (SNVs) in ACE2, particularly in its enhancers, promoters, and 3′ untranslated regions (UTRs). We found preliminary evidence supporting the regulatory impact of some of these variants on ACE2 expression. Our detailed examination of two SNVs, rs147718775 and rs140394675, in the ACE2 promoter revealed that these co-occurring SNVs, when mutated, significantly enhance promoter activity, suggesting a possible increase in specific ACE2 isoform expression. This method proves effective in identifying and interpreting impactful non-coding variants, aiding in further studies and enhancing understanding of molecular bases of monogenic and complex traits.

Enhancer-promoter activation by the Kaposi sarcoma-associated herpesvirus episome maintenance protein LANA

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV. Genome-wide analyses demonstrate increased levels of enhancer RNA transcription as well as activating chromatin marks at LANA-bound enhancers. 3D genome conformation analyses identified genes critical for latency and tumorigenesis as targets of LANA-occupied enhancers, and LANA depletion results in their downregulation. These findings reveal a mechanism in enhancer-gene coordination and describe a role through which the main KSHV tethering protein regulates essential gene expression in PEL.

RsPDR8, a member of ABCG subfamily, plays a positive role in regulating cadmium efflux and tolerance in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) is one of the most vital root vegetable crops worldwide. Cadmium (Cd), a non-essential and toxic heavy metal, can dramatically restrict radish taproot quality and safety. Although the Peiotrpic Drug Resistance (PDR) genes play crucial roles in heavy metal accumulation and transport in plants, the systematic identification and functional characterization of RsPDRs remain largely unexplored in radish. Herein, a total of 19 RsPDR genes were identified from the radish genome. A few RsPDRs, including RsPDR1, RsPDR8 and RsPDR12, showed significant differential expression under Cd and lead (Pb) stress in the ‘NAU-YH’ genotype. Interestingly, the plasma membrane-localized RsPDR8 exhibited significantly up-regulated expression and enhanced promoter activity under Cd exposure. Ectopic expression of RsPDR8 conferred Cd tolerance via reducing Cd accumulation in yeast cells. Moreover, the transient transformation of RsPDR8 revealed that it positively regulated Cd tolerance by promoting ROS scavenging and enhancing membrane permeability in radish. In addition, overexpression of RsPDR8 increased root elongation but deceased Cd accumulation compared with the WT plants in Arabidopsis, demonstrating that it could play a positive role in mediating Cd efflux and tolerance in plants. Together, these results would facilitate deciphering the molecular mechanism underlying RsPDR8-mediated Cd tolerance and detoxification in radish.

Zhx2 maintains islet β-cell mass and function by transcriptionally regulating Pax6

Emerging evidence shows that pancreatic β-cell function and quality are key determinants in the progression of type 2 diabetes (T2D). The transcription factor zinc finger homeobox 2 (Zhx2) is involved in proliferation and development of multiple cells. However, the exact role of Zhx2 in β-cells and T2D remains completely unknown. Here, we report that Zhx2 orchestrates β-cell mass and function by regulating paired box protein pax-6 (Pax6). We found that β-cell-specific knockout Zhx2 (Zhx2BKO) mice showed a decrease in β-cell proliferation and glucose homeostasis. Under prediabetic and diabetic conditions, we discovered glucose intolerance in both Zhx2BKO-HFD mice and Zhx2BKO-db/db mice, with reduced β-cell mass and insulin secretion. Mechanistically, we demonstrated that Zhx2 targeted the Pax6 promoter region (-1740∼-1563; -862∼-559; -251∼+75), enhanced promoter activity. Overall, Zhx2 maintains β-cell function by transcriptionally regulating Pax6, which provides a therapeutic target for diabetes intervention.

A progestin regulates the prostaglandin pathway in the neuroendocrine system in female mudskipper Boleophthalmus pectinirostris

Sex hormones regulate the reproductive cycle through brain-pituitary axis, but the molecular mechanism is still enigmatic. In the reproductive season, the mudskipper Boleophthalmus pectinirostris possesses a semilunar periodicity spawning rhythm, which coincides with the semilunar periodicity variations in 17α-hydroxyprogesterone, the precursor of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), a sexual progestin in teleosts. In the present study, we investigated in vitro the brain transcriptional differences between DHP-treated tissues and control groups using RNA-seq. Differential expression analysis revealed that 2700 genes significantly differentially expressed, including 1532 up-regulated and 1168 down-regulated genes. The majority of prostaglandin pathway-related genes were dramatically up-regulated, especially the prostaglandin receptor 6 (ptger6). Tissue distribution analysis revealed that ptger6 gene was ubiquitously expressed. In situ hybridization results showed that ptger6, nuclear progestin receptor (pgr), and DHP-induced c-fos mRNA were co-expressed in the ventral telencephalic area, the ventral nucleus of ventral telencephalic area, the anterior part of parvocellular preoptic nucleus, the magnocellular part of magnocellular preoptic nucleus, the ventral zone of periventricular hypothalamus, the anterior tubercular nucleus, the periventricular nucleus of posterior tuberculum, and the torus longitudinalis. DHP significantly enhanced promoter activities of ptger6 via Pgr. Together, this study suggested that DHP regulates the prostaglandin pathway in the neuroendocrine system of teleost fish.

Emergence of an erythroid cell-specific regulatory region in ABO intron 1 attributable to A- or B-antigen expression on erythrocytes in Hominoidea

A- and B-antigens are present on red blood cells (RBCs) as well as other cells and secretions in Hominoidea including humans and apes such as chimpanzees and gibbons, whereas expression of these antigens on RBCs is subtle in monkeys such as Japanese macaques. Previous studies have indicated that H-antigen expression has not completely developed on RBCs in monkeys. Such antigen expression requires the presence of H-antigen and A- or B-transferase expression in cells of erythroid lineage, although whether or not ABO gene regulation is associated with the difference of A- or B-antigen expression between Hominoidea and monkeys has not been examined. Since it has been suggested that ABO expression on human erythrocytes is dependent upon an erythroid cell-specific regulatory region or the + 5.8-kb site in intron 1, we compared the sequences of ABO intron 1 among non-human primates, and demonstrated the presence of sites orthologous to the + 5.8-kb site in chimpanzees and gibbons, and their absence in Japanese macaques. In addition, luciferase assays revealed that the former orthologues enhanced promoter activity, whereas the corresponding site in the latter did not. These results suggested that the A- or B-antigens on RBCs might be ascribed to emergence of the + 5.8-kb site or the corresponding regions in ABO through genetic evolution.

ERF transcription factor PpRAP2.12 activates PpVIN2 expression in peach fruit and reduces tolerance to cold stress

The vacuolar invertase gene PpVIN2 negatively regulates cold resistance in peach fruit by promoting sucrose degradation. However, the regulatory mechanisms that control PpVIN2 transcription are not yet understood. In this study, we identified an ethylene-responsive transcription factor (ERF) in a yeast one-hybrid library screen using the PpVIN2 promoter (PpVIN2pro) as bait. ERF PpRAP2.12 directly binds to the ACCGAC cis-element in the PpVIN2pro region, resulting in enhanced promoter activity. In peach fruit, transient overexpression of PpRAP2.12 increases PpVIN2 expression and vacuolar invertase (VIN) activity and decreases sucrose levels. Ectopic expression of PpRAP2.12 in tobacco reduces tolerance to cold stress, resulting in a lower survival rate, higher NtVIN expression and VIN enzymatic activity, and elevated production of reactive oxygen species. Together, these findings suggest that PpRAP2.12 binds to PpVIN2pro and positively regulates its activity to promote sucrose degradation. PpRAP2.12 thus functions as a negative regulator of plant defenses against chilling stress, and may provide a target for mitigating chilling injury in peach fruit.

Hypoxia and TNF-α Synergistically Induce Expression of IL-6 and IL-8 in Human Fibroblast-like Synoviocytes via Enhancing TAK1/NF-κB/HIF-1α Signaling.

Hypoxia and increased levels of inflammatory cytokines in the joints are characteristics of rheumatoid arthritis (RA). However, the effects of hypoxia and tumor necrosis factor-α (TNF-α) on interleukin (IL)-6 and IL-8 production on fibroblast-like synoviocytes (FLSs) remain to be clarified. This study aimed to explore how hypoxia and TNF-α affect the expression of IL-6 and IL-8 in human FLSs isolated from RA patients. Hypoxia or TNF-α treatment alone significantly increased the expression and promoter activity of IL-6, IL-8, and hypoxia-inducible factor-1α (HIF-1α). Treatment of hypoxic FLSs with TNF-α further significantly elevated the expression of these cytokines and enhanced promoter activity of HIF-1α, which was abrogated by treatment with the HIF-1α inhibitor YC-1. Similarly, TNF-α alone elevated the phosphorylation and promoter activity of nuclear factor-κBp65 (NF-κBp65) in the FLSs. These effects were further enhanced by the combined treatment of hypoxia and TNFα but were attenuated by the NF-κB inhibitor BAY11-7082. NF-κB-p65 inhibition decreased the effect of TNF-α on HIF-1α upregulation in the FLSs in response to hypoxia. The combination of hypoxia and TNF-α also significantly upregulated transforming growth factor-β-activated kinase 1 (TAK1) expression, and silencing TAK1 dramatically decreased NF-κB-p65, HIF-1α, IL-6, and IL-8 expression under the same conditions. Our results indicate that hypoxia and TNF-α synergistically increase IL-6 and IL-8 expression in human FLSs via enhancing TAK1/NF-κB/HIF-1α signaling.

Molecular dissection of genes and promoters involved in glycyrrhizin biosynthesis revealed phytohormone induced modulation in Glycyrrhiza glabra L

The study reports cloning and characterization of complete biosynthetic gene cluster committed to glycyrrhizin biosynthesis along with their corresponding promoter regions from Glycyrrhiza glabra. The identified genes namely, β-amyrin synthase, β-amyrin-11-oxidase, 11-oxo-beta-amyrin 30-oxidase and UDP-dependent glucosyltransferase, were hetrologously expressed in Nicotiana benthamiana for functional validation. The phyto-hormone, naphthalene acetic acid was shown to prompt maximum up regulation (1.3–14.0 folds) of all the genes, followed by gibberellic acid (0.001–10.0 folds) and abscisic acid (0.2–7.7 folds) treatments. The promoter-GUS fusion constructs infiltrated leaves of the identified genes exhibited enhanced promoter activity of β-amyrin synthase (3.9 & 3.0 folds) and 11-oxo-beta-amyrin 30-oxidase (3.6 & 3.2 folds) under the GA3 and NAA treatments, respectively as compared to their respective untreated controls. The transcriptional control of the three phytohormones studied could be correlated to the cis-responsive elements present in the upstream regions of the individual genes. The study provided an insight into the intricate interaction between hormone-responsive motifs with the corresponding co-expression of the glycyrrhizin biosynthetic pathway genes. The study will help in understanding the phytohormones-mediated regulation of glycyrrhizin biosynthesis and its modulation in the plant.

Independent Cis-Regulatory Modules within the Herpes Simplex Virus 1 Infected Cell Protein 0 (ICP0) Promoter Are Transactivated by Krüppel-like Factor 15 and Glucocorticoid Receptor.

A corticosteroid antagonist impairs Herpes Simplex Virus 1 (HSV-1) productive infection and explant-induced reactivation from latency, suggesting corticosteroids and the glucocorticoid receptor (GR) mediate certain aspects of these complex virus–host interactions. GR-hormone complexes regulate transcription positively and negatively, in part, by binding GR response elements (GREs). Recent studies revealed infected cell protein 0 (ICP0), ICP4, and ICP27 promoter/cis-regulatory modules (CRMs) are cooperatively transactivated by GR and Krüppel-like factor 15 (KLF15), which forms a feed-forward transcription loop. We hypothesized the ICP0 promoter contains independent CRMs that are transactivated by GR, KLF15, and the synthetic corticosteroid dexamethasone (DEX). This hypothesis is based on the finding that the ICP0 promoter contains multiple transcription factor binding sites, and GR and KLF15 cooperatively transactivate the full-length ICP0 promoter. ICP0 promoter sequences spanning −800 to −635 (fragment A) were efficiently transactivated by GR, KLF15, and DEX in monkey kidney cells (Vero), whereas GR and DEX significantly enhanced promoter activity in mouse neuroblastoma cells (Neuro-2A). Furthermore, ICP0 fragment B (−458 to −635) was efficiently transactivated by GR, KLF15, and DEX in Vero cells, but not Neuro-2A cells. Finally, fragment D (−232 to −24) was transactivated significantly in Vero cells by GR, KLF15, and DEX, whereas KLF15 and DEX were sufficient for transactivation in Neuro-2A cells. Collectively, these studies revealed efficient transactivation of three independent CRMs within the ICP0 promoter by GR, KLF15, and/or DEX. Finally, GC-rich sequences containing specificity protein 1 (Sp1) binding sites were essential for transactivation.

Thrombin Induces COX-2 and PGE2 Expression via PAR1/PKCalpha/MAPK-Dependent NF-kappaB Activation in Human Tracheal Smooth Muscle Cells.

The inflammation of the airway and lung could be triggered by upregulation cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) induced by various proinflammatory factors. COX-2 induction by thrombin has been shown to play a vital role in various inflammatory diseases. However, in human tracheal smooth muscle cells (HTSMCs), how thrombin enhanced the levels of COX-2/PGE2 is not completely characterized. Thus, in this study, the levels of COX-2 expression and PGE2 synthesis induced by thrombin were determined by Western blot, promoter-reporter assay, real-time PCR, and ELISA kit. The various signaling components involved in the thrombin-mediated responses were differentiated by transfection with siRNAs and selective pharmacological inhibitors. The role of NF-κB was assessed by a chromatin immunoprecipitation (ChIP) assay, immunofluorescent staining, as well as Western blot. Our results verified that thrombin markedly triggered PGE2 secretion via COX-2 upregulation which were diminished by the inhibitor of thrombin (PPACK), PAR1 (SCH79797), Gi/o protein (GPA2), Gq protein (GPA2A), PKCα (Gö6976), p38 MAPK (SB202190), JNK1/2 (SP600125), MEK1/2 (U0126), or NF-κB (helenalin) and transfection with siRNA of PAR1, Gqα, Giα, PKCα, JNK2, p38, p42, or p65. Moreover, thrombin induced PAR1-dependent PKCα phosphorylation in HTSMCs. We also observed that thrombin induced p38 MAPK, JNK1/2, and p42/p44 MAPK activation through a PAR1/PKCα pathway. Thrombin promoted phosphorylation of NF-κB p65, leading to nuclear translocation and binding to the COX-2 promoter element to enhance promoter activity, which was reduced by Gö6976, SP600125, SB202190, or U0126. These findings supported that COX-2/PGE2 expression triggered by thrombin was engaged in PAR1/Gq or Gi/o/PKCα/MAPK-dependent NF-κB activation in HTSMCs.

Combined effects of cigarette smoking, DNA methyltransferase 3B genetic polymorphism, and DNA damage on lung cancer

BackgroundSmoking increases DNA methylation and DNA damage, and DNA damage acts as a vital cause of tumor development. The DNA methyltransferase 3B (DNMT3B) enhances promoter activity and methylation of tumor suppressor genes. Tea polyphenols may inhibit DNMT activity. We designed a case-control study to evaluate the combined effects of smoking, green tea consumption, DNMT3B − 149 polymorphism, and DNA damage on lung cancer occurrence.MethodsQuestionnaires were administered to obtain demographic characteristics, life styles, and family histories of lung cancer from 190 primary lung cancer cases and 380 healthy controls. Genotypes and cellular DNA damage were determined by polymerase chain reaction and comet assay, respectively.ResultsThe mean DNA tail moment for lung cancer cases was significantly higher than that for healthy controls. Compared to nonsmokers carrying the DNMT3B − 149 CT genotype, smokers carrying the TT genotype had a greater lung cancer risk (odds ratio [OR]: 2.83, 95% confidence interval [CI]: 1.62–4.93). DNA damage levels were divided by the tertile of the healthy controls’ values. Compared to nonsmokers with low DNA damage, smokers with moderate DNA damage (OR: 2.37, 95% CI: 1.54–3.63) and smokers with high DNA damage (OR: 3.97, 95% CI: 2.63–5.98) had elevated lung cancer risks. Interaction between smoking and DNA damage significantly affected lung cancer risk.ConclusionsOur study suggested that the DNMT3B − 149 TT genotype, which has higher promoter activity, can increase the lung cancer risk elicited by smoking, and DNA damage may further promote smoking related lung cancer development.

Topological isolation of developmental regulators in mammalian genomes

Precise control of mammalian gene expression is facilitated through epigenetic mechanisms and nuclear organization. In particular, insulated chromosome structures are important for regulatory control, but the phenotypic consequences of their boundary disruption on developmental processes are complex and remain insufficiently understood. Here, we generated deeply sequenced Hi-C data for human pluripotent stem cells (hPSCs) that allowed us to identify CTCF loop domains that have highly conserved boundary CTCF sites and show a notable enrichment of individual developmental regulators. Importantly, perturbation of such a boundary in hPSCs interfered with proper differentiation through deregulated distal enhancer-promoter activity. Finally, we found that germline variations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Taken together, our findings highlight the importance of developmental gene isolation through chromosomal folding structures as a mechanism to ensure their proper expression.

Defective Rab1b Related Megakaryocytic ER-to-Golgi Transport in RUNX1 Haplodeficiency

RUNX1, a major hematopoietic transcription factor, plays a critical role in megakaryopoiesis, megakaryocytic (MK) maturation and platelet production. Patients with RUNX1 haplodeficiency have thrombocytopenia, abnormal platelet function and impaired granule secretion. Defective dense and/or -alpha granules are a major finding in these patients. We have reported a patient with inherited thrombocytopenia, decreased granules, and abnormal platelet function (Gabbeta et al, Blood 87: 1368-76, 1996) with a heterozygous RUNX1 mutation (c.508+1G>1) (Sun et al, Blood 103: 948-54, 2004). Platelet expression profiling revealed down-regulation of several genes including ALOX12, MYL9, PRKCQ, PF4, and PLDN (Sun et al J Thromb Haemost 5: 146-154, 2007) and we have shown these to be direct RUNX1 transcriptional targets. In addition, a small G protein, RAB1B, was significantly decreased on expression profiling of the patient platelets relative to control subjects (fold change: 0.32, p < 0.025; confirmed by qPCR). With real-time PCR platelet RAB1B mRNA was decreased by 50% compared to healthy 5 subjects. Immunofluorescence studies showed that Rab1b protein was decreased in patient platelets compared to that of normal subjects.RAB GTPases are key players in vesicle trafficking, granule targeting of proteins, and secretion. Rab1b has been implicated in ER-to-Golgi transport in COS cells, but cell biological roles of RAB1B in MK cells are unexplored. We addressed the hypothesis that RAB1B is a transcriptional target of RUNX1 and regulates ER-to-Golgi transport in MK cells. In silico analysis of the RAB1B promoter region (-1/-785 bp from the ATG) revealed 4 RUNX1 consensus sites: site-1 (-440/-435), site-2 (-622/-617), site-3 (-660/-655) and site-4 (-770/-765). Following phorbol 12-myristate 13-acetate treatment of human erythroleukemia (HEL) cells to induce MK transformation, chromatin immunoprecipitation (ChIP) revealed RUNX1 binding to RAB1B sites 1 and 4 but not to other sites. Electrophoretic mobility shift assays using HEL cell proteins showed RUNX1 binding to these sites. In luciferase reporter assays, mutation individually of sites 1 and 4 reduced the promoter activity by 90%, compared to that of full-length promoter. RUNX1 over-expression enhanced RAB1B promoter activity by 2-fold and RAB1B protein by ~30%. Enhancement of promoter activity was abrogated by mutations of sites 1 and 4, indicating that these sites were functional. RUNX1 siRNA reduced RAB1B promoter activity and RAB1B protein.We studied vesicle trafficking from ER-to-Golgi in HEL cells using vesicular stomatitis virus G envelope glycoprotein (VSVG) tagged with enhanced green fluorescence protein (EGFP) and GalT on siRNA downregulation of RUNX1 or RAB1B . (Presley JF et al, Nature 389: 81-85, 1997). VSVG-GFP colocalized with GalT in the Golgi in control siRNA-transfected cells, whereas in RUNX1 knockdown cells, VSVG was not colocalized with GalT; moreover, Golgi structures were disrupted. Similar results were obtained in RAB1B downregulation by siRNA. These effects of RUNX1 knockdown were reversed by RAB1B overexpression. Together these results suggest that RAB1B downregulation may be the primary cause of the impaired ER-to-Golgi transport by RUNX1 downregulation.Conclusions: These studies show that RAB1B is a key regulator of vesicle trafficking in the secretory and endocytic pathway in megakaryocytes and platelets. This is the first evidence that RAB1B is a direct transcriptional target of RUNX1 and provides a mechanism for RAB1B down regulation in our RUNX1 haplodeficient patient. One of the hallmarks of RUNX1 haplodeficiency is defective granule formation and secretion. We propose RAB1B reduction of ER-to-Golgi transport contributes to defects in granule formation and secretion in patients with RUNX1 deficiency. DisclosuresNo relevant conflicts of interest to declare.