TATA Box Region Research Articles

AtSNP_TATAdb: Candidate Molecular Markers of Plant Advantages Related to Single Nucleotide Polymorphisms within Proximal Promoters of Arabidopsis thaliana L.

The mainstream of the post-genome target-assisted breeding in crop plant species includes biofortification such as high-throughput phenotyping along with genome-based selection. Therefore, in this work, we used the Web-service Plant_SNP_TATA_Z-tester, which we have previously developed, to run a uniform in silico analysis of the transcriptional alterations of 54,013 protein-coding transcripts from 32,833 Arabidopsis thaliana L. genes caused by 871,707 SNPs located in the proximal promoter region. The analysis identified 54,993 SNPs as significantly decreasing or increasing gene expression through changes in TATA-binding protein affinity to the promoters. The existence of these SNPs in highly conserved proximal promoters may be explained as intraspecific diversity kept by the stabilizing natural selection. To support this, we hand-annotated papers on some of the Arabidopsis genes possessing these SNPs or on their orthologs in other plant species and demonstrated the effects of changes in these gene expressions on plant vital traits. We integrated in silico estimates of the TBP-promoter affinity in the AtSNP_TATAdb knowledge base and showed their significant correlations with independent in vivo experimental data. These correlations appeared to be robust to variations in statistical criteria, genomic environment of TATA box regions, plants species and growing conditions.

Demethylation in promoter region of severely damaged hepatocytes enhances chemokine receptor CXCR4 gene expression.

The liver is known to possess remarkable regenerative potential, but persistent inflammation or severe acute injury can lead to liver fibrosis and incomplete regeneration, ultimately resulting in liver failure. Recent studies have shown that the axis of two types of CXCL12 receptors, CXCR4 and CXCR7, plays a crucial role in liver fibrosis and regeneration. The present study aimed to investigate the regulatory factors involved in CXCR4 expression in injured liver. Immunohistochemical screening of liver tissue samples collected during liver transplantation revealed a reciprocal expression pattern between CXCR4 and MeCP2. An in vitro system involving cultured cell lines and H2O2 treatment was established to study the impact of oxidative stress on signaling pathways and epigenetic alterations that affect CXCR4 mRNA expression. Operating through distinct signaling pathways, H2O2 treatment induced a dose-dependent increase in CXCR4 expression in both hepatocyte- and intrahepatic cholangiocyte-derived cells. Treatment of the cells with trichostatin and azacytidine modulated CXCR4 expression in hepatocytes by modifying the methylation status of CpG dinucleotides located in a pair of TA repeats adjacent to the TATA box of the CXCR4 gene promoter. Only MeCP2 bound to oligonucleotides representing the TATA box region when the cytosine residues within the sequence were methylated, as revealed by electrophoretic mobility shift assay (EMSA). Methylation-specific PCR analysis of microdissected samples revealed a correlation between the loss of CpG methylation and the upregulation of CXCR4 in injured hepatocytes, replicating the findings from the in vitro study. Besides the conventional MEK/ERK and NF-κB signaling pathways that activate CXCR4 in intrahepatic cholangiocytes, the unique epigenetic modifications observed in hepatocytes might also contribute to a shift in the CXCR4-CXCR7 balance towards CXCR4, leading to irreversible liver injury and fibrosis. This study highlights the importance of epigenetic modifications in regulating CXCR4 expression in liver injury and fibrosis.

IProm-Zea: A two-layer model to identify plant promoters and their types using convolutional neural network

A promoter is a short DNA sequence near the start codon, responsible for initiating the transcription of a specific gene in the genome. The accurate recognition of promoters is important for achieving a better understanding of transcriptional regulation. Because of their importance in the process of biological transcriptional regulation, there is an urgent need to develop in silico tools to identify promoters and their types in a timely and accurate manner. A number of prediction methods have been developed in this regard; however, almost all of them are merely used for identifying promoters and their strength or sigma types. The TATA box region in TATA promoter influences the post-transcriptional processes; therefore, in the current study, we developed a two-layer predictor called “iProm-Zea” using the convolutional neural network (CNN) for identify TATA and TATA less promoters. The first layer can be used to identify a given DNA sequence as a promoter or non-promoter. The second layer can be used to identify whether the recognized promoter is the TATA promoter. To find an optimal feature encoding scheme and model, we employed four feature encoding schemes on different machine learning and CNN algorithms, and based on the evaluation results, we selected a one-hot encoding scheme and a CNN model for iProm-Zea. The 5-fold cross validation testing results demonstrated that the constructed predictor showed great potential for identifying promoters and classifying them as TATA and TATA less promoters. Furthermore, we performed cross-species analysis of iProm-Zea to evaluate its performance in other species. Moreover, to make it easier for other experimental scientists to obtain the results they need, we established a freely accessible and user-friendly web server at http://nsclbio.jbnu.ac.kr/tools/iProm-Zea/.

DCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.

SUMMARYBifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

A systemic review of association between UDP glucuronosyltransferase family 1 member A1 (UGT1A1) polymorphisms in Gilbert's syndrome in Sickle Cell Disease

Gilbert's syndrome (GS) is a benign hereditary disorder of bilirubin metabolism due to a mutation in the UDP glucuronosyltransferase family 1 member A1 (UGT1A1) gene which results in hyperbilirubinaemia and related complications mainly cholelithiasis. It can be co-inherited along with sickle cell anaemia, thalassaemias and other haemoglobinopathies including glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis and cystic fibrosis. More than 100 mutations have been reported in UGT1A1 gene and the most common as insertion of extra (TA) nucleotides in the promoter region of TATA box. The more the number of TA repeats, the higher is the bilirubin levels. These mutations result in a 10%–35% reduction in the UGT1A1 enzyme activity resulting in mild to moderate unconjugated hyperbilirubinaemia and related complications. For diagnosis the mode of inheritance is more important than testing in the patients. However; the inheritance pattern of GS differs in ethnicities. For early diagnosis to prevent worsening of the symptoms and for timely management one should be aware of the inheritance pattern in patient. In this systemic analysis we studied the association between complications in GS with the genotypes and complications. It was found that TA7/7 is more significant in GS with sickle cell disease (SCD) group when compared to healthy controls with 2.2% chances of having this genotype in GS with SCD than healthy controls. The significance of having TA7/7 genotype is similar in GS with SCD and α-thalasaemia group. However, there is a high recommendation to carry out multicentre studies and conduct meta-analyses for establishing universal recommendations.

GENOTYPING OF CAMELS OF ALMATY REGION BY CSN2 DAIRY PRODUCTIVITY GENE

Camel milk is highly valued for its medicinal properties and high nutritional value. Camel milk and its products are easily digestible and therefore natural for the human body. Thus, the study of genetic diversity and selection of camels according to preferred genotypes is an important component of camel breeding in Kazakhstan. The β-casein protein, encoded by the CSN2 gene, makes up the largest part of the casein fraction of camel milk and is considered the main gene for the presence of alleles associated with different levels of expression. The g.2126A> G transition is located the TATA box region of the CSN2 gene promoter, with a presumed effect on the transcription factor binding activity, which determines the intensity of this gene expression. In the studied camel populations bred in the Almaty region, the total number of which is 53 animals, the frequency of the G allele was 0.36, while the frequency of the A allele was 0.64. Also, the Hardy-Weinberg equilibrium was equal to 0,006. The level of polymorphism showed a high result in populations 1, 2 and 4, amounting to 100%, while in the third population this indicator was 66.67%. For the studied populations, such statistical indicators were identified as the genotypes occurrence frequency, the observed and effective number of alleles, Nei's gene diversity etc., which give the population genetic characteristics of populations.

IPTT(2 L)-CNN: A Two-Layer Predictor for Identifying Promoters and Their Types in Plant Genomes by Convolutional Neural Network.

A promoter is a short DNA sequence near to the start codon, responsible for initiating transcription of a specific gene in genome. The accurate recognition of promoters has great significance for a better understanding of the transcriptional regulation. Because of their importance in the process of biological transcriptional regulation, there is an urgent need to develop in silico tools to identify promoters and their types timely and accurately. A number of prediction methods had been developed in this regard; however, almost all of them were merely used for identifying promoters and their strength or sigma types. Owing to that TATA box region in TATA promoter that influences posttranscriptional processes, in the current study, we developed a two-layer predictor called iPTT(2L)-CNN by using the convolutional neural network (CNN) for identifying TATA and TATA-less promoters. The first layer can be used to identify a given DNA sequence as a promoter or nonpromoter. The second layer is used to identify whether the recognized promoter is TATA promoter or not. The 5-fold crossvalidation and independent testing results demonstrate that the constructed predictor is promising for identifying promoter and classifying TATA and TATA-less promoter. Furthermore, to make it easier for most experimental scientists get the results they need, a user-friendly web server has been established at http://www.jci-bioinfo.cn/iPPT(2L)-CNN.

Aspirin enhances cisplatin sensitivity of resistant non-small cell lung carcinoma stem-like cells by targeting mTOR-Akt axis to repress migration

Conventional chemotherapeutic regimens are unable to prevent metastasis of non-small cell lung carcinoma (NSCLC) thereby leaving cancer incurable. Cancer stem cells (CSCs) are considered to be the origin of this therapeutic limitation. In the present study we report that the migration potential of NSCLCs is linked to its CSC content. While cisplatin alone fails to inhibit the migration of CSC-enriched NSCLC spheroids, in a combination with non-steroidal anti inflammatory drug (NSAID) aspirin retards the same. A search for the underlying mechanism revealed that aspirin pre-treatment abrogates p300 binding both at TATA-box and initiator (INR) regions of mTOR promoter of CSCs, thereby impeding RNA polymerase II binding at those sites and repressing mTOR gene transcription. As a consequence of mTOR down-regulation, Akt is deactivated via dephosphorylation at Ser473 residue thereby activating Gsk3β that in turn causes destabilization of Snail and β-catenin, thus reverting epithelial to mesenchymal transition (EMT). However, alone aspirin fails to hinder migration since it does not inhibit the Integrin/Fak pathway, which is highly activated in NSCLC stem cells. On the other hand, in aspirin pre-treated CSCs, cisplatin stalls migration by hindering the integrin pathway. These results signify the efficacy of aspirin in sensitizing NSCLC stem cells towards the anti-migration effect of cisplatin. Cumulatively, our findings raise the possibility that aspirin might emerge as a promising drug in combinatorial therapy with the existing chemotherapeutic agents that fail to impede migration of NSCLC stem cells otherwise. This may consequently lead to the advancement of remedial outcome for the metastatic NSCLCs.

Characterization of the copper-sensing transcriptional regulator CopR from the hyperthermophilic archeaon Thermococcus onnurineus NA1.

A putative copper ion-sensing transcriptional regulator CopR (TON_0836) from Thermococcus onnurineus NA1 was characterized. The CopR protein consists of a winged helix-turn-helix DNA-binding domain in the amino-terminal region and a TRASH domain that is assumed to be involved in metal ion-sensing in the carboxyl-terminal region. The CopR protein was most strongly bound to a region between its own gene promoter and a counter directional promoter region for copper efflux system CopA. When the divalent metals such as nickel, cobalt, copper, and iron were present, the CopR protein was dissociated from the target promoters on electrophoretic mobility shift assay (EMSA). The highest sensible ion is copper which affected protein releasing under 10µM concentrations. CopR recognizes a significant upstream region of TATA box on CopR own promoter and acts as a transcriptional repressor in an in vitro transcription assay. Through site-directed mutagenesis of the DNA-binding domain, R34M mutant protein completely lost the DNA-binding activity on target promoter. When the conserved cysteine residues in C144XXC147 motif 1 of the TRASH domain were mutated into glycine, the double cysteine residue mutant protein alone lost the copper-binding activity. Therefore, CopR is a copper-sensing transcriptional regulator and acts as a repressor for autoregulation and for a putative copper efflux system CopA of T. onnurineus NA1.

Variability in the UDP-glucuronosyltransferase 1A1 (UGT1A1) promotor in a HIV-infected Portuguese population

Introduction: Dolutegravir is an HIV-1 integrase inhibitor (INI) recently approved for the treatment of HIV infection and is metabolized by UGT1A1 [1]. This protein is responsible for the glucuronidation of many compounds, and some functional polymorphisms in the UGT1A1 gene results in a decrease of enzyme activity [2]. The polymorphism (rs8175347) is associated with thymine-adenine (TA) repeats located in the TATA box in the promoter region of the UGT1A1 gene. The increasing number of TA repeats is inversely associated with UGT1A1 transcription, with 6 repeats (allele*1) being associated with normal UGT1A1 activity phenotype. 7 and 8 TA repeats (alleles *28 and *37, respectively) with low UGT1A1 activity phenotype [1]. Knowledge of the frequency distribution of functional polymorphisms in a HIV-infected population may improve therapeutic options. The present study aims to identify the frequency of UGT1A1 (rs8175347) gene polymorphism in a population of Portuguese HIV-infected patients and compare with other ethnic groups. Materials and methods: The study includes 74 Portuguese HIV-infected adult patients receiving ARV therapy. The informed consent and the study protocol were approved by ethics committee. Genotyping the (TA) insertion in the TATA box region of UGT1A1 gene was performed by PCR amplification followed by direct sequencing. The allelic and genotype frequencies were tested for Hardy-Weinberg equilibrium by goodness off fit χ2 test with a 95% CI, using The R Project for Statistical Computing. Obtained frequencies were then compared with 219 unrelated individuals collected from the database Pharmgkb using a χ2 test. Results: All frequencies in our study group were in Hardy-Weinberg equilibrium (p = 0.4016). In this study the allelic frequencies for UGT1A1 *1, *28 and * 37 alleles were 61.49%, 30.41% and 8.11%, respectively, being significantly different from African, Asian and European (p < 0.001) ethnic groups. The genotype frequencies for UGT1A 1*1/*1, *1/*28, *1/*37, *28/*28 and *28/*37 were 32.43%, 44.59%, 13.51%, 6.76% and 2.70%, respectively. In our sample a total of 67.56% patients have UGT1A1 * 28 or * 37 alleles which was significantly associated with lower UGT1A1 activity phenotype. Discussion and conclusions: Obtained UGT1A1*28 and *37 frequencies were similar to the ones observed in African population and different from those of European and Asian origin. Patients with UGT1A1*28 and *37 alleles in our study may be at risk of dolutegravir therapeutic failure, consequently drug monitoring is recommended to guide dose adjustment. Our study contributes for personalized medicine optimization, indicating the individual differences in genetic characteristics.

Insights into an Extensively Fragmented Eukaryotic Genome: De Novo Genome Sequencing of the Multinuclear Ciliate Uroleptopsis citrina.

Ciliated protists are a large group of single-celled eukaryotes with separate germline and somatic nuclei in each cell. The somatic genome is developed from the zygotic nucleus through a series of chromosomal rearrangements, including fragmentation, DNA elimination, de novo telomere addition, and DNA amplification. This unique feature makes them perfect models for research in genome biology and evolution. However, genomic research of ciliates has been limited to a few species, owing to problems with DNA contamination and obstacles in cultivation. Here, we introduce a method combining telomere-primer PCR amplification and high-throughput sequencing, which can reduce DNA contamination and obtain genomic data efficiently. Based on this method, we report a draft somatic genome of a multimacronuclear ciliate, Uroleptopsis citrina. 1) The telomeric sequence in U. citrina is confirmed to be C4A4C4A4C4 by directly blunt-end cloning. 2) Genomic analysis of the resulting chromosomes shows a “one-gene one-chromosome” pattern, with a small number of multiple-gene chromosomes. 3) Amino acid usage is analyzed, and reassignment of stop codons is confirmed. 4) Chromosomal analysis shows an obvious asymmetrical GC skew and high bias between A and T in the subtelomeric regions of the sense-strand, with the detection of an 11-bp high AT motif region in the 3′ subtelomeric region. 5) The subtelomeric sequence also has an obvious 40 nt strand oscillation of nucleotide ratio. 6) In the 5′ subtelomeric region of the coding strand, the distribution of potential TATA-box regions is illustrated, which accumulate between 30 and 50 nt. This work provides a valuable reference for genomic research and furthers our understanding of the dynamic nature of unicellular eukaryotic genomes.

MiRNAs regulate oxidative stress related genes via binding to the 3\u2032 UTR and TATA-box regions: a new hypothesis for cataract pathogenesis

BackgroundAge-related cataracts are related to oxidative stress. However, the genome-wide screening of cataract related oxidative stress related genes are not thoroughly investigated. Our study aims to identify cataract regulated miRNA target genes that are related to oxidative stress and to propose a new possible mechanism for cataract formation.MethodsMicroarrays were used to determine the mRNA expression profiles of both transparent and cataractous lenses. The results were analyzed by significance analyses performed by the microarray software, and bioinformatics analysis was further conducted using Molecular Annotation System. The Eukaryotic Promoter Database (EPD) was used to retrieve promoter sequences and identify TATA-box motifs. Online resource miRWalk was exploited to screen for validated miRNAs targeting mRNAs related to oxidative stress. RNAhybrid online tool was applied to predict the binding between significantly regulated miRNAs in cataract lenses and target mRNAs.ResultsOxidative stress pathway was significantly regulated in cataractous lens samples. Pro-oxidative genes were half up-regulated (11/20), with a small number of genes down-regulated (4/20) and the rest of them with no significant change (5/20). Anti-oxidative genes were partly up-regulated (17/69) and partly down-regulated (17/69). Four down-regulated miRNAs (has-miR-1207-5p, has-miR-124-3p, has-miR-204-3p, has-miR-204-5p) were found to target 3′ UTR of pro-oxidative genes and could also bind to the TATA-box regions of anti-oxidative genes (with the exception of has-miR-204-3p), whilst two up-regulated miRNAs (has-miR-222-3p, has-miR-378a-3p) were found to target 3′ UTR of anti-oxidative genes and could simultaneously bind to the TATA-box regions of pro-oxidative genes.ConclusionsWe propose for the first time a hypothesis that cataract regulated miRNAs could contribute to cataract formation not only by targeting 3′ UTR but also by targeting TATA-box region of oxidative stress related genes. This results in the subsequent elevation of pro-oxidative genes and inhibition of anti-oxidative genes. This miRNA-TATA-box/3′ UTR-gene-regulation network may contribute to cataract pathogenesis.

PD-001 - Interference of PG2 TATA BOX region with the serum PG2 level in gastric cancer

PD-001 - Interference of PG2 TATA BOX region with the serum PG2 level in gastric cancer

Frequency of the UGT1A1*28 Polymorphism in a Romanian Cohort of Gilbert Syndrome Individuals.

Gilbert syndrome (GS) is characterized by unconjugated hyperbilirubinemia without liver disease or overt hemolysis and it is found in 3-10% of the general population. Inherited hyperbilirubinaemia is attributable to a reduced UGT1A1 activity. The UGT1A1 promoter (TA) repeats variants are documented of being involved in abnormally elevated bilirubin levels. The aim of the present study is to analyze the impact of UGT1A1 promoter variants on bilirubin levels in Romanian patients clinically supected with GS. The study group included 897 subjects: 292 GS patients and 605 healthy controls. Genomic DNA was extracted from the peripheral blood leukocytes. All individuals were screened for the presence of the (TA) insertion in the TATA box region of UGT1A1 gene by PCR amplification. This case-control study was conducted at the Department of Medical Genetics, Synevo, Romania. UGT1A1*28 (7TA) revealed the highest frequency (61.87%) of all individuals, while the UGT1A1*1 (6TA) allele was found in 36.79%. We identified two other variants of the UGT1A1 gene, depending on the number of TA repeats in the promoter: 5TA (0.61%) and 8TA (0.72%). The (TA)7/7 homozygous genotype was identified in 32.33% of all individuals, while the (TA)6/7 heterozygous genotype was the most prevalent (57.64%). The wild type (TA)6/6 was identified in 7.36% of the whole cohort. Because other polymorphisms have been associated with GS, the absence of the UGT1A1*28 allele does not rule out this condition. The results suggest that in the Romanian population there is a strong correlation between the UGT1A1*28 polymorphism and hyperbilirubinemia in patients with GS.

P.08.10: Interference of PG2 Tata Box Region with the Serum PG2 Level in Gastric Cancer

P.08.10: Interference of PG2 Tata Box Region with the Serum PG2 Level in Gastric Cancer

HIV-1 Infection-Induced Suppression of the Let-7i/IL-2 Axis Contributes to CD4(+) T Cell Death.

The mechanisms underlying HIV-1-mediated CD4+ T cell depletion are highly complicated. Interleukin-2 (IL-2) is a key cytokine that maintains the survival and proliferation of activated CD4+ T cells. IL-2 levels are disturbed during HIV-1 infection, but the underlying mechanism(s) requires further investigation. We have reported that cellular microRNA (miRNA) let-7i upregulates IL-2 expression by targeting the promoter TATA-box region, which functions as a positive regulator. In this study, we found that HIV-1 infection decreases the expression of let-7i in CD4+ T cells by attenuating its promoter activity. The reduced let-7i miRNA expression led to a decline in IL-2 levels. A let-7i mimic increased IL-2 expression and subsequently enhanced the resistance of CD4+ T cells to HIV-1-induced apoptosis. By contrast, the blockage of let-7i with a specific inhibitor resulted in elevated CD4+ T cell apoptosis during HIV-1 infection. Furthermore, by knocking down the expression of IL-2, we found that the let-7i-mediated CD4+ T cell resistance to apoptosis during HIV-1 infection was dependent on IL-2 signaling rather than an alternative CD95-mediated cell-death pathway. Taken together, our findings reveal a novel pathway for HIV-1-induced dysregulation of IL-2 cytokines and depletion of CD4+ T-lymphocytes.

147. Transcription Factor Binding Site in Plasmid Regulates Persistence of Transgene Expression in Mouse Liver

Persistent expression of transgene is most desirable for treatment of genetic diseases. It is primarily controlled by genetic elements in the promoter region where transcription factors bind. The objective of this study is to assess the role of the transcription factor binding sites (TFBS) present in the most commonly used CMV and albumin promoters containing the promoter core sequence and several TFBS for ATF1, NFκB, SRF, AP1, Sp1, NF1α, CEBPA, HNF4α, and HNF1 in determining persistency of transgene expression in mice. Secreted embryonic alkaline phosphatase (SEAP) and interleukin 10 (IL-10) were used as a reporter to monitor transgene expression. Variousdeletion mutation plasmids at the promoter region were made to examine the function of each of the TFBS in regulating reporter gene expression. Similarly, a series of new plasmids containing only one TFBS inserted at the 5’ end of the TATA-box region was made to assess its unique activity in affecting transgene expression. Our results showed that TFBSs are essential for effective transgene expression in mouse liver after a hydrodynamic tail vein gene delivery. Plasmid with a TATA-box with all TFBS deleted resulted in minimal level of gene product, 700-fold lower than the original plasmids. Reporter gene expression appears transient reaching the background level in less than 2 weeks. The more TFBS the plasmid has, the higher level of gene product was seen. Plasmid containing a single TFBS either NFκB, ATF1, CEBPA, HNF4α, or HNF1 exhibits persistent transgene expression for more than 2 months, at which animals injected with plasmids containing NFkB, ATF1, CEBPA, HNF4α, or HNF1 exhibited gene product level 60, 130, 1020, 2250, and 2076 folds higher than that of animals injected with plasmids without TFBS, respectively. Mechanism study employing real time PCR, Western Blotting, and ChIP reveals involvement of histone acetylation and the binding of acetylated histones to plasmid DNA sequences in TFBS-mediated enhancement in persistency of reporter gene expression. TFBSs for HNF4α appear most potent in recruiting H4AC and TBP proteins to the plasmids. Our results suggest that TFBSs for NFkB, ATF1, CEBPA, HNF4α, and HNF1 are important elements to include into the promoter for achieving longer-term gene expression. In addition, histone acetylation on plasmid is a probable pathway that TFBS regulate the duration of transgene expression.

RNA Polymerase III Accurately Initiates Transcription from RNA Polymerase II Promoters in Vitro

In eukaryotes, there are three major RNA polymerases (Pol) in the nucleus, which are commonly described as transcribing non-overlapping subsets of genes. Structural studies have highlighted a conserved core shared among all three transcription systems. Initiation of human Pol III from TATA box-containing Pol II promoters under conditions with impaired Pol II transcription activity have been described previously. RNA polymerase III and Pol II were found to co-localize at the promoters of the c-myc gene and the RPPH1 sRNA in vivo. Here, I report that Pol III can, like Pol II, initiate transcription from most tested Pol II core promoters when assayed with crude human nuclear extracts (HSK, SNF, or Dignam). Both polymerases often initiate from the same transcription start site, and depend on a TATA box or AT-rich region but not the downstream promoter element (DPE) or the motif ten element (MTE). Moderate (∼2-fold) changes in the ratio of DNA template to nuclear extract were sufficient to change Pol II-mediated transcription to a mixture of Pol II- and Pol III-, or to a solely Pol III-dependent initiation of transcription from Pol II promoters. Polymerase specificity is thus not fixed but a variable that depends on the properties of the promoter and the transcription conditions. These findings provide functional evidence for a close similarity between the Pol II and Pol III transcription complexes, and additionally explain previous controversies in the literature.

Cloning Ubiquitin gene with promoter region from Zea mays L.

Ubiquitin is a protein in eukaryote cells and its sequence is highly conserved. The protein has been involved in many cellular processes, such as protein turnover, chromatin structure, cell cycle control, DNA repair and response to heat shock and other stresses. The gene codes for this protein (Ubiquitin gene) and its promoter have been cloned. The function of the gene and its promoter from many plants like rice, maize and gladiolus plants has been studied. Ubiquitin promoter plays an important role in gene expression in monocot. In this article, we present the results on cloning the gene coding for Ubiquitin protein (Ubi gene) and its promoter from maize line H240 for genetic engineering studies in maize. The data obtained showed that the promoter region, transcription region and TATA box region of cloned Ubi gene from maize line H240 have high level of homology comparing to orthologous sequenes in plant genome. The cloned Ubi gene was submited to the GenBank with assession number JX947345.1. The results of this study will, considerablely, contribute to the study and application of Ubiquitin promoter in gene transfer of monocot, in general, and maize, in particular.

A novel HIV-1-encoded microRNA enhances its viral replication by targeting the TATA box region

BackgroundA lot of microRNAs (miRNAs) derived from viral genomes have been identified. Many of them play various important roles in virus replication and virus-host interaction. Cellular miRNAs have been shown to participate in the regulation of HIV-1 viral replication, while the role of viral-encoded miRNAs in this process is largely unknown.ResultsIn this report, through a strategy combining computational prediction and deep sequencing, we identified a novel HIV-1-encoded miRNA, miR-H3. MiR-H3 locates in the mRNA region encoding the active center of reverse transcriptase (RT) and exhibits high sequence conservation among different subtypes of HIV-1 viruses. Overexpression of miR-H3 increases viral production and the mutations in miR-H3 sequence significantly impair the viral replication of wildtype HIV-1 viruses, suggesting that it is a replication-enhancing miRNA. MiR-H3 upregulates HIV-1 RNA transcription and protein expression. A serial deletion assay suggests that miR-H3 targets HIV-1 5′ LTR and upregulates the promoter activity. It interacts with the TATA box in HIV-1 5′ LTR and sequence-specifically activates the viral transcription. In addition, chemically-synthesized small RNAs targeting HIV-1 TATA box activate HIV-1 production from resting CD4+ T cells isolated from HIV-1-infected patients on suppressive highly active antiretroviral therapy (HAART).ConclusionsWe have identified a novel HIV-1-encoded miRNA which specifically enhances viral production and provide a specific method to activate HIV-1 latency.