TATA Box Motifs Research Articles

DeePromClass: Delineator for Eukaryotic Core Promoters Employing Deep Neural Networks.

Computational promoter identification in eukaryotes is a classical biological problem that should be refurbished with the availability of an avalanche of experimental data and emerging deep learning technologies. The current knowledge indicates that eukaryotic core promoters display multifarious signals such as TATA-Box, Inr element, TCT, and Pause-button, etc., and structural motifs such as G-quadruplexes. In the present study, we combined the power of deep learning with a plethora of promoter motifs to delineate promoter and non-promoters gleaned from the statistical properties of DNA sequence arrangement. To this end, we implemented convolutional neural network (CNN) and long short-term memory (LSTM) recurrent neural network architecture for five model systems with [-100 to +50] segments relative to the transcription start site being the core promoter. Unlike previous state-of-the-art tools, which furnish a binary decision of promoter or non-promoter, we classify a chunk of 151mer sequence into a promoter along with the consensus signal type or a non-promoter. The combined CNN-LSTM model; we call "DeePromClass", achieved testing accuracy of 90.6%, 93.6%, 91.8%, 86.5%, and 84.0% for S. cerevisiae, C. elegans, D. melanogaster, Mus musculus, and Homo sapiens respectively. In total, our tool provides an insightful update on next-generation promoter prediction tools for promoter biologists.

Comparative studies of genomic and epigenetic factors influencing transcriptional variation in two insect species.

Different genes show different levels of expression variability. For example, highly expressed genes tend to exhibit less expression variability. Genes whose promoters have TATA box and initiator motifs tend to have increased expression variability. On the other hand, DNA methylation of transcriptional units, or gene body DNA methylation, is associated with reduced gene expression variability in many species. Interestingly, some insect lineages, most notably Diptera including the canonical model insect Drosophila melanogaster, have lost DNA methylation. Therefore, it is of interest to determine whether genomic features similarly influence gene expression variability in lineages with and without DNA methylation. We analyzed recently generated large-scale data sets in D. melanogaster and honey bee (Apis mellifera) to investigate these questions. Our analysis shows that increased gene expression levels are consistently associated with reduced expression variability in both species, while the presence of TATA box is consistently associated with increased gene expression variability. In contrast, initiator motifs and gene lengths have weak effects limited to some data sets. Importantly, we show that a sequence characteristics indicative of gene body DNA methylation is strongly and negatively associate with gene expression variability in honey bees, while it shows no such association in D. melanogaster. These results suggest the evolutionary loss of DNA methylation in some insect lineages has reshaped the molecular mechanisms concerning the regulation of gene expression variability.

TAF4b transcription networks regulating early oocyte differentiation.

Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.

Exogenous artificial DNA forms chromatin structure with active transcription in yeast

Yeast artificial chromosomes (YACs) are important tools for sequencing, gene cloning, and transferring large quantities of genetic information. However, the structure and activity of YAC chromatin, as well as the unintended impacts of introducing foreign DNA sequences on DNA-associated biochemical events, have not been widely explored. Here, we showed that abundant genetic elements like TATA box and transcription factor-binding motifs occurred unintentionally in a previously reported data-carrying chromosome (dChr). In addition, we used state-of-the-art sequencing technologies to comprehensively profile the genetic, epigenetic, transcriptional, and proteomic characteristics of the exogenous dChr. We found that the data-carrying DNA formed active chromatin with high chromatin accessibility and H3K4 tri-methylation levels. The dChr also displayed highly pervasive transcriptional ability and transcribed hundreds of noncoding RNAs. The results demonstrated that exogenous artificial chromosomes formed chromatin structures and did not remain as naked or loose plasmids. A better understanding of the YAC chromatin nature will improve our ability to design better data-storage chromosomes.

Transcriptional Pausing and Activation at Exons-1 and -2, Respectively, Mediate the MGMT Gene Expression in Human Glioblastoma Cells.

Background: The therapeutically important DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 [Int. J. Mol. Sci. 2021, 22(5), 2492], we addressed its significance in MGMT transcription. Methods: RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak MGMT-E1 expression in MGMT-proficient SF188 and T98G GBM cells, compared to active transcription of MGMT-E2. In the MGMT-deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT-E2 and MGMT-E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and MGMT-E2, respectively. Conclusions: The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of MGMT transcription in glioma and other cancer types.

SEQUENCING OF THE U6 PROMOTERS IN CASTOR BEANS AND VECTOR CONSTRUCTION FOR CRISPR/Cas9 GENOMIC EDITING ON THEIR BASIS

Castor bean is an important crop in many countries. It is mainly used to obtain the castor oil, which is widely applied in various industries. The rich protein cakes and meals are remains after the oil is pressed. They are promising for use as protein additives in the fodder production. However, it is limited due to the presence of toxins. These are ricin protein and ricinine alkaloid. One of such methods can be genomic editing using the CRISPR/Cas9 system. The technology consists in cutting the target region of the intact DNA by the Cas9 enzyme with the assistance of a short guide RNA fragment. The delivery of the Cas9 and guide RNA genes into the cell of the edited plant is often carried out by plasmid vectors. For efficient synthesis of the guide RNA in such vectors, the promoters of small nuclear RNA genes are usually used. In dicotyledonous plants editing, the promoter of the Arabidopsis U6 gene is most often used. In this work, the amplification, sequencing and analysis of the castor bean U6 promoters were carried out at the first time. The obtained sequences were analyzed and used in the construction of CRISPR/Cas9 vectors for the ricin gene editing. Our aim was to study castor bean U6 promoters with help by bioinformatics and molecular genetic approaches. Zanzibar Green and Gibzonskaya varieties of castor bean plants were used in this work. DNA was isolated from young leaves. The preparation of sequences, alignments and homology level calculation were carried out by GenDoc program (http://www.nrbsc.org/gfx/genedoc/index.html). Primers for amplification of castor bean U6 promoters were designed by Primer3 program (http://bioinfo.ut.ee/primer3-0.4.0/). PCR was performed using a C-100 PCR machine (Bio-Rad Laboratories, Inc., USA). PCR products were separated in 1.5 % agarose gel at 6 B/cm in the Sub-Cell GT electrophoresis camera (Bio-Rad Laboratories, Inc., USA). The amplicons were purified with the GeneJET PCR Purification Kit (Thermo Fisher Scientific, Inc., USA). The purified amplicons were cloned into the pAL2-T vector (Evrogen, Russia) according to protocol of manufacturer. In the CRISPR/Cas9 vector construction, the pRGE31, HindIII and SbfI restriction enzymes (Sibenzyme, Russia) and nucleotides were used. Purification of the digested products was carried out with the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Inc., USA). Bioinformatic search were conducted in the GenBank base, and 12 scaffolds with the castor bean U6 gene were found. Six promoters with intact USE and TATA-box elements of regulation were used in the primer design for amplification with cv. Zanzibar Green and cv. Gibzonskaya DNA matrices. One fragment PCR products were cloned and sequenced. The analysis of the obtained amplicon sequences reveled that promoter regions of two studied varieties were similar. The level of promoter identity from different amplicons ranged within 51-77 %. In comparison of these promoters with ones from other plants, the level of homology was 42-64 %. The promoter sequences with intact USE and TATA-box motifs were used in construction of the CRISPR/Cas9 vectors which can be used for efficient editing of the castor bean genes involved in the ricin and ricinine synthesis.

Genetics and Genomics of SOST: Functional Analysis of Variants and Genomic Regulation in Osteoblasts.

SOST encodes the sclerostin protein, which acts as a key extracellular inhibitor of the canonical Wnt pathway in bone, playing a crucial role in skeletal development and bone homeostasis. The objective of this work was to assess the functionality of two variants previously identified (the rare variant rs570754792 and the missense variant p.Val10Ile) and to investigate the physical interactors of the SOST proximal promoter region in bone cells. Through a promoter luciferase reporter assay we show that the minor allele of rs570754792, a variant located in the extended TATA box motif, displays a significant decrease in promoter activity. Likewise, through western blot studies of extracellular and intracellular sclerostin, we observe a reduced expression of the p.Val10Ile mutant protein. Finally, using a circular chromosome conformation capture assay (4C-seq) in 3 bone cell types (MSC, hFOB, Saos-2), we have detected physical interactions between the SOST proximal promoter and the ECR5 enhancer, several additional enhancers located between EVT4 and MEOX1 and a distant region containing exon 18 of DHX8. In conclusion, SOST presents functional regulatory and missense variants that affect its expression and displays physical contacts with far reaching genomic sequences, which may play a role in its regulation within bone cells.

Unexplored regulatory sequences of divergently paired GLA and HNRNPH2 loci pertinent to Fabry disease in human kidney and skin cells: Presence of an active bidirectional promoter.

Fabry disease (FD) is a rare hereditary disorder characterized by a wide range of symptoms caused by a variety of mutations in the galactosidase α (GLA) gene. The heterogeneous nuclear ribonucleoprotein (HNRNPH2) gene is divergently paired with GLA on chromosome X and is thought to be implicated in FD. However, insufficient information is available on the regulatory mechanisms associated with the expression of HNRNPH2 and the GLA loci. Therefore, the current study performed bioinformatics analyses to assess the GLA and HNRNPH2 loci and investigate the regulatory mechanisms involved in the expression of each gene. The regulatory mechanisms underlying GLA and HNRNPH2 were revealed. The expression of each gene was associated with a bidirectional promoter (BDP) characterized by the absence of TATA box motifs and the presence of specific transcription factor binding sites (TFBSs) and a CpG Island (CGI). The nuclear run-on transcription assay confirmed the activity of BDP GLA and HNRNPH2 transcription in 293T. Methylation-specific PCR analysis demonstrated a statistically significant variation in the DNA methylation pattern of BDP in several cell lines, including human adult epidermal keratinocytes (AEKs), human renal glomerular endothelial cells, human renal epithelial cells and 293T cells. The highest observed significance was demonstrated in AEKs (P<0.05). The results of the chromatin-immunoprecipitation assay using 293T cells identified specific TFBS motifs for Yin Yang 1 and nuclear respiratory factor 1 transcription factors in BDPs. The National Center for Biotechnology Information-single nucleotide polymorphism database revealed pathogenic variants in the BDP sequence. Additionally, a previously reported variant associated with a severe heterozygous female case of GLA FD was mapped in BDP. The results of the present study suggested that the expression of the divergent paired loci, GLA and HNRNPH2, were controlled by BDP. Mutations in BDP may also serve a role in FD and may explain clinical disease diversity.

Abstract 4331: Potential role of the splicing factor SF3B1 in epigenetic regulation and activation of p53 signaling

Abstract To develop novel and improved cancer therapies, we aim to target the epigenome, which is often deregulated in cancer. We identified the splicing factor SF3B1 as a potential epigenetic regulator in a whole genome siRNA screen for reactivation of aberrantly silenced genes alone or in synergy with the DNMT inhibitor Decitabine (DAC). The screen was done in YB5 colon cancer cells which contain a methylated and silenced CMV promoter driving GFP expression, and confirmed in a similar model in HCT116, another colon cancer cell line. SF3B1 has been studied in splicing, but little is known about its effects on gene regulation outside of this function, prompting us to further investigate this. We validated the screen findings by siRNA (siSF3B1) and inhibition of SF3B1 with Pladienolide B (PB). We then performed RNA-Seq and Reduced Representation Bisulfite Sequencing (RRBS) in cells treated with siSF3B1 and DAC, and we performed ChIP-Seq to assess SF3B1 binding. By flow cytometry, we measured a ~7% and ~5% induction of GFP in YB5 cells after siSF3B1 and DAC, respectively. The combination treatment caused a synergistic increase to ~15% GFP positive cells (p &amp;lt; 0.001). siSF3B1 led to 423 up and 338 downregulated genes, while DAC induced 430 up and 135 downregulated genes. With the combination, there was activation of 1190 genes and downregulation of 904 genes (fold change &amp;gt; 2, FDR &amp;lt; 0.1). There were 695, 119, 1584 genes alternatively spliced following siSF3B1, DAC, and the combination treatment, respectively, but there was no significant overlap with the regulated genes (p &amp;lt; 0.05), suggesting a distinct mechanism for gene expression regulation. There were two major subsets of siSF3B1 upregulated genes. A set of genes had low promoter methylation (0-20% methylation) and were p53 activation targets such as CDKN1A and GADD45A. The other set of genes had high promoter methylation (80-100% methylation) and promoters containing TATA-box motifs. RRBS showed global DNA hypomethylation after DAC treatment as expected, with an average methylation of 76.2% compared to 84.7% methylation in siControl cells. Further demethylation occurred in the combination treated cells, which had an average methylation of 72.7% (p&amp;lt;0.001). ChIP-Seq showed differential binding of SF3B1 at the transcription start sites (TSSs) of genes based on their expression. SF3B1 was depleted at TSSs of expressed genes and enriched at nonexpressed genes, resembling the binding pattern of histone H3. Finally, SF3B1 inhibition with PB alone and with DAC also induced reactivation of gene expression and altered DNA methylation. Together, these findings suggest that SF3B1 may be playing a role in gene regulation outside of its role in splicing. In cancer, SF3B1 transcriptional target genes are potentially tumor suppressor genes and upon knockdown or inhibition of SF3B1, their expression is reactivated leading to antitumor effects. Citation Format: Sandra Deliard, Yasuyuki Okamoto, Jozef Madzo, Somnath Pandey, Jaroslav Jelinek, Jean-Pierre Issa. Potential role of the splicing factor SF3B1 in epigenetic regulation and activation of p53 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4331.

Variation of gene expression in plants is influenced by gene architecture and structural properties of promoters.

In higher eukaryotes, gene architecture and structural properties of promoters have emerged as significant factors influencing variation in number of transcripts (expression level) and specificity of gene expression in a tissue (expression breadth), which eventually shape the phenotype. In this study, transcriptome data of different tissue types at various developmental stages of A. thaliana, O. sativa, S. bicolor and Z. mays have been used to understand the relationship between properties of gene components and its expression. Our findings indicate that in plants, among all gene architecture and structural properties of promoters, compactness of genes in terms of intron content is significantly linked to gene expression level and breadth, whereas in human an exactly opposite scenario is seen. In plants, for the first time we have carried out a quantitative estimation of effect of a particular trait on expression level and breadth, by using multiple regression analysis and it confirms that intron content of primary transcript (as %) is a powerful determinant of expression breadth. Similarly, further regression analysis revealed that among structural properties of the promoters, stability is negatively linked to expression breadth, while DNase1 sensitivity strongly governs gene expression breadth in monocots and gene expression level in dicots. In addition, promoter regions of tissue specific genes are found to be enriched with TATA box and Y-patch motifs. Finally, multi copy orthologous genes in plants are found to be longer, highly regulated and tissue specific.

Co-expression and regulation of photorespiratory genes in Arabidopsis thaliana: A bioinformatic approach

Being a pathway tightly linked to photosynthesis, photorespiration is regulated by light, but also by both nutrients and metabolites on transcriptional level. However, only little is known about the signals and how they are integrated on promoter level to coordinate the whole pathway. Using a bioinformatic approach we analyzed the co-expression patterns of photorespiratory genes, the predicted cis-regulatory elements in their 5′ upstream regions and the existence of introns in their 5′UTRs. We found that there are groups of photorespiratory genes that are strongly co-expressed among each other. The analysis showed a high co-expression between photorespiration and ammonia re-fixation. However, a strong co-regulation between two genes, like GDCH1 and GDCH2, did not necessarily mean that these genes share common cis-element in their 5′ upstream regions. TATA-box, MYB1AT, and MYB4 binding site motifs occurred in 16 out of 20 genes. Furthermore, photorespiratory genes are subjected to alternative splicing. We discuss the presence of cis-elements in the context of both stress responses and development. A genome wide analysis of Arabidopsis 5′UTRs revealed that 5′UTRs introns are overrepresented in photorespiratory genes. Promoter:gusA studies indicated that photorespiratory gene expression is also regulated by intron-mediated enhancement (IME). As already shown for glutamate: glyoxylate aminotransferase, transcript abundance of serine: glyoxylate aminotransferase was affected by IME on mRNA level. IME of gene expression of glycolate oxidase was shown to act on translational level.

A Cellular MicroRNA Facilitates Regulatory T Lymphocyte Development by Targeting the FOXP3 Promoter TATA-Box Motif.

The CD4+CD25+FOXP3+ regulatory T cells (Tregs) mediate immunological self-tolerance and suppress various immune responses. FOXP3 is a key transcriptional factor for the generation and development of Tregs. Its expression is regulated by various cytokines including TGF-β, IL-2, and IL-10. It is important to further identify the regulatory factors for Tregs. Given that many microRNAs (miRNAs) could specifically interact with the core promoter region and specifically enhance the transcription of many target genes, we searched for any possible miRNA(s) targeting the core promoter region of the FOXP3 gene. We found that miR-4281, an miRNA specifically expressed in hominids, can potently and specifically upregulate FOXP3 expression by directly interacting with the TATA-box motif in the human FOXP3 promoter. Consequently, miR-4281 significantly accelerated the differentiation of human naive cells to induced Tregs (iTregs) that possess immune suppressor functions and weaken the development of graft-versus-host disease in a humanized mouse model. Interestingly, iTregs induced by the combination of TGF-β, IL-2, and chemically synthesized miR-4281 were more stable and functional than those induced by TGF-β and IL-2 alone. Moreover, we found that the IL-2/STAT5 signal transduction upregulates FOXP3 expression not only through the classical pathway, but also by enhancing the expression of the miR-4281 precursor gene (SNCB) and, correspondingly, miR-4281. This study reveals a novel mechanism regulating FOXP3 expression and human iTreg development and, therefore, offers a new therapeutic target to manipulate immunosuppressive system.

Pichia pastoris Alcohol Oxidase 1 (AOX1) Core Promoter Engineering by High Resolution Systematic Mutagenesis.

Unravelling the core promoter sequence-function relationship is fundamental for engineering transcription initiation and thereby a feasible "tuning knob" for fine-tuning expression in synthetic biology and metabolic engineering applications. Here a systematic replacement studies of the core promoter and 5' untranslated region (5'UTR) of the exceptionally strong and tightly methanol regulated Komagataella phaffii (syn. Pichia pastoris) alcohol oxidase 1 (AOX1) promoter at unprecedented resolution is performed. Adjacent triplets of the 200 bp long core promoter are mutated at a time by changing the wild-type sequence into cytosine or adenine triplets, resulting in 130 variants that are cloned upstream of an eGFP reporter gene providing a library for expression fine-tuning. Mutations in the TATA box motif, regions downstream of the transcription start site or next to the start codon in the 5'UTR had a significant effect on the eGFP fluorescence. Surprisingly, mutations in most other regions are tolerated, indicating that yeast core promoters can show a high tolerance toward small mutations, supporting regulatory models of degenerate motifs, or redundant design. The authors exploited these neutral core promoter positions, not affecting expression, to introduce extrinsic sequence elements such as cloning sites (allowing targeted core promoter/5'UTR modifications) and bacterial promoters (applicable in multi host vectors).

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.

Genome-wide comparative analysis of LEAFY promoter sequence in angiosperms

Regulation of the flowering mechanism is influenced by many environmental factors. Dissecting the regulatory processes upstream of the LFY (LEAFY) gene will help us to understand the molecular mechanisms of floral induction. In total, 53 LFY sequences were identified in 37 species. Among the 53 selected LFY promoters and after eliminating the short sequences, 47 LFY promoters were analyzed. Comparative genome studies for LFY promoters among plants showed that TATA-box existed in all herbaceous plants. The 1345-bp promoter sequence upstream to hickory LFY gene was cloned and analyzed, together with functional studies. The result of sequence alignment showed that the region of the hickory LFY promoter has only two conserved auxin response elements (AuxRE), whereas other plants had four. The positions of AuxRE in hickory and walnut were the same, but they were different from the positions from other plants. Furthermore the sequence analysis showed that the promoter have TATA-box and CAAT-box motifs. Deletion analysis of these motifs did not block β-glucuronidase (GUS) activity during the transient expression assay, suggesting that it may be a TATA-less promoter. Low temperature and light significantly induced the full-length promoter to increase about two folds of the GUS enzymatic activity, suggesting these environmental factors induced flowering in hickory.

Molecular cloning, polymorphism, and functional activity of the bovine and water buffalo Mx2 gene promoter region.

BackgroundBovine Mx2 gene sequences were already reported, but further information about the gene properties is not yet available. The objective of the current study was to elucidate the structural properties of the bovine Mx2 gene mainly the promoter region and its possible functional role. If available, such information would help in assessing the functional properties of the gene, which was reported to confer antiviral action against recombinant VSV.ResultsExaminations on the bovine genomic BAC clone—confirmed to contain the Mx2 gene—revealed 883-bp sequences. A computer scan unequivocally identified a 788-bp promoter region containing a typical TATA box, three ISREs and other promoter-specific motifs. Comparative analysis of nine bovine genomic DNA samples showed 19 nucleotide substitutions suggesting the existence of five different genotypes in the promoter region. The water buffalo Mx2 promoter region was determined by using primers based on the bovine Mx2 promoter region disclosing 893-bp, with 56 substitutions, two insertions, 9 and 1 nt at two different sites. A functional analysis of the putative ISRE indicated that ISRE played a synergetic role in the activation of bovine Mx2 gene transcription.ConclusionBovine and water buffalo Mx2 promoter region was identified disclosing, the conserved ISRE, located in the proximal end of the promoter region like other members of the antiviral family, suggesting functional activity under interferon stimulation.

Affinity and competition for TBP are molecular determinants of gene expression noise.

Cell-to-cell variation in gene expression levels (noise) generates phenotypic diversity and is an important phenomenon in evolution, development and disease. TATA-box binding protein (TBP) is an essential factor that is required at virtually every eukaryotic promoter to initiate transcription. While the presence of a TATA-box motif in the promoter has been strongly linked with noise, the molecular mechanism driving this relationship is less well understood. Through an integrated analysis of multiple large-scale data sets, computer simulation and experimental validation in yeast, we provide molecular insights into how noise arises as an emergent property of variable binding affinity of TBP for different promoter sequences, competition between interaction partners to bind the same surface on TBP (to either promote or disrupt transcription initiation) and variable residence times of TBP complexes at a promoter. These determinants may be fine-tuned under different conditions and during evolution to modulate eukaryotic gene expression noise.

Widespread and evolutionary analysis of a MITE family Monkey King in Brassicaceae.

BackgroundMiniature inverted repeat transposable elements (MITEs) are important components of eukaryotic genomes, with hundreds of families and many copies, which may play important roles in gene regulation and genome evolution. However, few studies have investigated the molecular mechanisms involved. In our previous study, a Tourist-like MITE, Monkey King, was identified from the promoter region of a flowering time gene, BnFLC.A10, in Brassica napus. Based on this MITE, the characteristics and potential roles on gene regulation of the MITE family were analyzed in Brassicaceae.ResultsThe characteristics of the Tourist-like MITE family Monkey King in Brassicaceae, including its distribution, copies and insertion sites in the genomes of major Brassicaceae species were analyzed in this study. Monkey King was actively amplified in Brassica after divergence from Arabidopsis, which was indicated by the prompt increase in copy number and by phylogenetic analysis. The genomic variations caused by Monkey King insertions, both intra- and inter-species in Brassica, were traced by PCR amplification. Genomic sequence analysis showed that most complete Monkey King elements are located in gene-rich regions, less than 3kb from genes, in both the B. rapa and A. thaliana genomes. Sixty-seven Brassica expressed sequence tags carrying Monkey King fragments were also identified from the NCBI database. Bisulfite sequencing identified specific DNA methylation of cytosine residues in the Monkey King sequence. A fragment containing putative TATA-box motifs in the MITE sequence could bind with nuclear protein(s) extracted from leaves of B. napus plants. A Monkey King-related microRNA, bna-miR6031, was identified in the microRNA database. In transgenic A. thaliana, when the Monkey King element was inserted upstream of 35S promoter, the promoter activity was weakened.ConclusionMonkey King, a Brassicaceae Tourist-like MITE family, has amplified relatively recently and has induced intra- and inter-species genomic variations in Brassica. Monkey King elements are most abundant in the vicinity of genes and may have a substantial effect on genome-wide gene regulation in Brassicaceae. Monkey King insertions potentially regulate gene expression and genome evolution through epigenetic modification and new regulatory motif production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0490-9) contains supplementary material, which is available to authorized users.

The TATA-box motif and its impact on transcriptional gene regulation by miRNAs.

Emerging evidence suggests that components of the small RNAs pathway interact with chromatin to regulate nuclear events, such as gene transcription. However, it has recently been reported that in some cases, gene transcription regulation by cellular miRNAs can occur via targeting the TATA-box motif without altering epigenetic modifications. This observation supports the notion that multiple mechanisms of miRNA-based transcriptional regulation exist, enhancing our understanding of the complexity of small RNA-mediated gene regulatory pathways. Here, we remark that miRNA-mediated transcriptional modulation, through the TATA-box motif, may be a synergistic approach for transcriptional control.

ISOLATION OF Β-ACTIN PROMOTER DERIVED FROM WALKING CATFISH (CLARIAS BATRACHUS)

Fish growth improvement as economic traits can be solved through fish transgenic production. Growth hormone gene is inserted into transgenic vector construction to over-express fish growth. The promoter as a part of the expression vector has an important role in its regulation. The use of promoter which is derived from mammalian or virus (such as CMV/ cytomegalovirus) in the expression vector, in specific goal as food material, has customer resistant rather than a promoter which is derived from in- sibling species. Beside of it, transgene expression level when using in-sibling promoter showed higher than using mammalian or viral promoter. The β-actin promoter is screened from walking catfish pituitary genome DNA using primers: pBA-cy-F (5’- GTGWGTGACGCYGGACCAAATC-3’) as forward primer and pBA-cy-R (5’- CCATRTCRTCCCAGTTGGTSACAAT-3’) as reverse primer, produced an amplicon of 1,7 kb in length. Sequence analysis using TF BindTM indicated transcription factor elements: TATA box, CCAAT box, enhancer (CAAT), and CarGG (CAAATGG) motif. This result showed that promoter which is obtained from this research is useful in construction of all catfish growth hormone vector expression catfish transgenic production. Keywords: β-actin promoter, growth hormone, expression vector, walking catfish (Clarias batrachus)