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Results for “regulatory element transcripts”

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PubMed1989

In situ detection of transcriptionally active chromatin and genetic regulatory elements in individual viable mammalian cells.

Using a newly developed FACS method for quantifying the expression of the Escherischia coli lacZ reporter gene in viable mammalian cells, we have obtained cloned cell lines in which the expression of lacZ is under the control of native endogenous transcription elements. We infected the murine pre-B cell 70Z/3 with transcriptionally disabled retroviruses containing lacZ and employed the FACS-FDG technique to detect and sort rare lacZ+ cells in which we expect integration is near such endogenous transcription elements. After two rounds of enrichment we obtained a population of cells that was 80-90% positive for lacZ activity. Clones derived from the lacZ+ pool differ from each other with respect to their overall level of lacZ activity as well as in the pattern of lacZ expression among cells within an individual clone. Treatment of these lacZ+ 70Z/3 clones with lipopolysaccharide (LPS; which is known to stimulate differentiation of 70Z/3 from a pre-B cell to an IgM-expressing B cell) greatly decreased lacZ expression in one clone, 7e17. lacZ expression in this clone was 50-100 times lower within 24 hr of LPS addition and coincided with the acquisition of IgM kappa on the surface of 7e17. This suggests that a transcriptionally active domain of chromatin that harbors the lacZ construct is down-regulated during the transition induced by LPS stimulation.

Animals↗

PubMed2006

The Streptomyces NrdR transcriptional regulator is a Zn ribbon/ATP cone protein that binds to the promoter regions of class Ia and class II ribonucleotide reductase operons.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs, either of which is sufficient for vegetative growth. The class Ia RNR is encoded by the nrdAB genes, and the class II RNR is encoded by nrdJ, which is coexpressed with nrdR. We previously showed that the Streptomyces coelicolor nrdR gene encodes a protein, NrdR, which represses transcription of both sets of RNR genes. NrdR is a member of a highly conserved family of proteins that is confined exclusively to prokaryotes. In this report, we describe a physical and biochemical characterization of the S. coelicolor NrdR protein and show that it is a zinc-ATP/dATP-containing protein that binds to the promoter regions of both Streptomyces RNR operons. The NrdR N terminus contains a zinc ribbon motif that is necessary for binding to the upstream regulatory region of both RNR operons. The latter contains two 16-bp direct repeat sequences, termed NrdR boxes, which are located proximal to, or overlap with, the promoter regions. These experiments support the view that NrdR controls the transcription of RNR genes by binding to the NrdR box sequences. We also show that the central NrdR ATP cone domain binds ATP and dATP and that mutations that abolish ATP/dATP binding significantly reduce DNA binding, suggesting that the ATP cone domain may allosterically regulate NrdR binding. We conclude that NrdR is a widely conserved regulator of RNR genes, binding to specific sequence elements in the promoter region and thereby modulating transcription.

Adenosine Triphosphate↗

PubMed2005

The molecular elements that underlie developmental evolution.

Abundant evidence indicates that developmental evolution, the foundation of morphological evolution, is based on changes in gene function. Over the past decade a consensus has developed that transcriptional regulation, acting through enhancer sequences, is the primary level of evolutionarily significant change. Here we propose that other regulatory levels are probably as important as enhancers in developmental evolution. We also explain why these alternative regulatory levels might have been neglected, and briefly discuss ways to test our hypothesis.

Alternative Splicing↗

PubMed2006

The regulatory function of the upstream sequence of the beta-conglycinin alpha subunit gene in seed-specific transcription is associated with the presence of the RY sequence.

beta-conglycinin, a major component of seed-storage proteins in soybean, comprises three subunits: alpha, alpha', and beta. Expression of these genes is spatially regulated in a stringent manner and occurs during seed development. To understand the mechanisms that control expression of the alpha subunit gene, we analyzed the nucleotide sequence of the 2.9-kb region upstream of the gene. The upstream sequence up to -1357 or a series of its 5'-deleted derivatives was fused to the beta-glucuronidase (GUS) gene. These reporter gene constructs were introduced into Arabidopsis thaliana plants via Agrobacterium-mediated gene transfer. Prominent GUS activity was detected in developing seeds of the T3 generation when 245 bp or longer sequences of the upstream region were fused to the GUS gene. We found a clear association of decreased GUS activity with a stepwise deletion of a region containing the RY sequence from the original construct. These results are consistent with the notion that multiple sequence elements including the RY sequences are involved in the seed-specific transcriptional activation of the beta-conglycinin alpha subunit gene in soybean.

Antigens, Plant↗

PubMed1990

A novel tumor necrosis factor-responsive transcription factor which recognizes a regulatory element in hemopoietic growth factor genes.

A conserved DNA sequence element, termed cytokine 1 (CK-1), is found in the promoter regions of many hemopoietic growth factor (HGF) genes. Mutational analyses and modification interference experiments show that this sequence specifically binds a nuclear transcription factor, NF-GMa, which is a protein with a molecular mass of 43 kilodaltons. It interacts with different affinities with the CK-1-like sequence from a number of HGF genes, including granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte (G)-CSF, interleukin 3 (IL-3), and IL-5. We show here that the level of NF-GMa binding is induced in embryonic fibroblasts by tumor necrosis factor-alpha (TNF-alpha) treatment and that the CK-1 sequence from the G-CSF gene is a TNF-alpha-responsive enhancer in these cells. The NF-GMa protein is distinct from another TNF-alpha-responsive transcription factor, NF-kappa B, by several criteria. Firstly, several NF-kappa B-binding sites, although having sequence similarity with the CK-1 sequence, cannot compete efficiently for NF-GMa binding to CK-1. Secondly, the CK-1 sequence from both G-CSF and GM-CSF does not respond to phorbol ester treatment as would an NF-kappa B-binding element. These results demonstrate that NF-GMa is a novel transcription factor inducible by TNF-alpha and binds to a common element in HGF gene promoters.

Base Sequence↗

PubMed2006

Cis-motifs upstream of the transcription and translation initiation sites are effectively revealed by their positional disequilibrium in eukaryote genomes using frequency distribution curves.

BACKGROUND: The discovery of cis-regulatory motifs still remains a challenging task even though the number of sequenced genomes is constantly growing. Computational analyses using pattern search algorithms have been valuable in phylogenetic footprinting approaches as have expression profile experiments to predict co-occurring motifs. Surprisingly little is known about the nature of cis-regulatory element (CRE) distribution in promoters. RESULTS: In this paper we used the Motif Mapper open-source collection of visual basic scripts for the analysis of motifs in any aligned set of DNA sequences. We focused on promoter motif distribution curves to identify positional over-representation of DNA motifs. Using differentially aligned datasets from the model species Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and Saccharomyces cerevisiae, we convincingly demonstrated the importance of the position and orientation for motif discovery. Analysis with known CREs and all possible hexanucleotides showed that some functional elements gather close to the transcription and translation initiation sites and that elements other than the TATA-box motif are conserved between eukaryote promoters. While a high background frequency usually decreases the effectiveness of such an enumerative investigation, we improved our analysis by conducting motif distribution maps using large datasets. CONCLUSION: This is the first study to reveal positional over-representation of CREs and promoter motifs in a cross-species approach. CREs and motifs shared between eukaryotic promoters support the observation that an eukaryotic promoter structure has been conserved throughout evolutionary time. Furthermore, with the information on positional enrichment of a motif or a known functional CRE, it is possible to get a more detailed insight into where an element appears to function. This in turn might accelerate the in depth examination of known and yet unknown cis-regulatory sequences in the laboratory.

Algorithms↗

PubMed2006

Identification of genetic variation and putative regulatory regions in bovine CARD15.

Mutations in caspase recruitment domain 15 (CARD15) are associated with susceptibility to Crohn's disease and Blau Syndrome. We performed comparative analyses of the bovine, murine, and human CARD15 transcripts to elucidate functionality of bovine CARD15 and examine its potential role in bovine disease resistance. Comparative analyses of intronic sequence across seven divergent species were performed to identify putative regulatory element binding motifs. High levels of interspecies conservation in sequence, genomic structure, and protein domains were detected indicating common functionality for CARD15 in cattle, human, and mouse. We identified species-specific regulatory elements in the 5' and 3' untranslated regions, suggesting that modes of regulation may have diverged across species. Thirty-one conserved putative regulatory element binding motifs were identified in the CARD15 intronic sequence of seven species. To assess the extent of genetic diversity within bovine CARD15, 41 individuals from two subspecies were sequenced and screened for polymorphisms. Thirty-six single nucleotide polymorphisms (SNPs) were identified. Finally, 20 subspecies-specific haplotypes were predicted with 7 and 13 unique haplotypes explaining the diversity within B. taurus taurus and B. taurus indicus animals, respectively. Strong evidence for a simple causal relationship between these SNP loci and their haplotypes with Johne's disease was not detected.

3' Untranslated Regions↗

PubMed2004

Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii.

The X-ray structure of native cellobiohydrolase IB (CBH IB) from the filamentous fungus Talaromyces emersonii, PDB 1Q9H, was solved to 2.4 A by molecular replacement. 1Q9H is a glycoprotein that consists of a large, single domain with dimensions of approximately 60 A x 40 A x 50 A and an overall beta-sandwich structure, the characteristic fold of Family 7 glycosyl hydrolases (GH7). It is the first structure of a native glycoprotein and cellulase from this thermophilic eukaryote. The long cellulose-binding tunnel seen in GH7 Cel7A from Trichoderma reesei is conserved in 1Q9H, as are the catalytic residues. As a result of deletions and other changes in loop regions, the binding and catalytic properties of T. emersonii 1Q9H are different. The gene (cel7) encoding CBH IB was isolated from T. emersonii and expressed heterologously with an N-terminal polyHis-tag, in Escherichia coli. The deduced amino acid sequence of cel7 is homologous to fungal cellobiohydrolases in GH7. The recombinant cellobiohydrolase was virtually inactive against methylumberiferyl-cellobioside and chloronitrophenyl-lactoside, but partial activity could be restored after refolding of the urea-denatured enzyme. Profiles of cel7 expression in T. emersonii, investigated by Northern blot analysis, revealed that expression is regulated at the transcriptional level. Putative regulatory element consensus sequences for cellulase transcription factors have been identified in the upstream region of the cel7 genomic sequence.

Amino Acid Sequence↗

PubMed2006

POXO: a web-enabled tool series to discover transcription factor binding sites.

We present POXO, a comprehensive tool series to discover transcription factor binding sites from co-expressed genes (www.bioinfo.biocenter.helsinki.fi/poxo). POXO manages tasks such as functional evaluation and grouping of genes, sequence retrieval, pattern discovery and pattern verification. It also allows users to tailor analytical pipelines from these tools, with single mouse clicks. One typical pipeline of POXO begins by examining the biological functions that a set of co-expressed genes are involved in. In this examination, the functional coherence of the gene set is evaluated and representative functions are associated with the gene set. This examination can also be used to group genes into functionally similar subsets, if several biological processes are affected in the experiment. The next step in the pipeline is then to discover over-represented nucleotide patterns from the upstream sequences of the selected gene sets. This enables to investigate the possibility that the genes are co-regulated by common cis-elements. If over-represented patterns are found, similar ones can then be clustered together and be verified. The performance of POXO is demonstrated by analysing expression data from pathogen treated Arabidopsis thaliana. In this example, POXO detected activated gene sets and suggested transcription factors responsible for their regulation.

Arabidopsis↗

PubMed2006

A MAPT mutation in a regulatory element upstream of exon 10 causes frontotemporal dementia.

We report here the genetic analysis of a newly ascertained kindred in which frontotemporal dementia occurs in an apparent autosomal dominant fashion, and in which a novel MAPT gene mutation co-segregates with disease. Sequencing the MAPT gene in affected individuals revealed a change in intron 9. This finding supports earlier studies on the effect of a splice-accepting element in inclusion of exon 10 in the MAPT transcript. This mutation sheds light on a novel mechanism by which over-expression of 4-repeat tau leads to disease. Based on our current findings, we propose a novel mechanism by which intronic mutations can lead to frontotemporal dementia.

Animals↗

PubMed2006

Foxl2 function in ovarian development.

Foxl2 is a forkhead transcription factor essential for proper reproductive function in females. Human patients carrying mutations in the FOXL2 gene display blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), an autosomal dominant disease associated with eyelid defects and premature ovarian failure in females. Recently, animal models for BPES have been developed that in combination with a catalogue of human FOXL2 mutations provide further insight into its molecular function. Mice homozygous mutant for Foxl2 display craniofacial malformations and female infertility. The analysis of the murine phenotype has revealed that Foxl2 is required for granulosa cell function. These ovarian somatic cells surround and nourish the oocyte and play an important role in follicle formation and activation. Mutations upstream of FOXL2 in humans, not affecting the coding sequence itself, have also been shown to cause BPES, which points to the existence of a distant regulatory element necessary for proper gene expression. The same regulatory sequences may be deleted in the goat polled intersex syndrome (PIS), in which FoxL2 expression is severely reduced. Sequence comparison of FoxL2 from several vertebrate species has shown that it is a highly conserved gene involved in ovary development. Thus, the detailed understanding of Foxl2 function and regulation and the identification of its transcriptional targets may open new avenues for the treatment of female infertility in the future.

Abnormalities, Multiple↗

PubMed2005

Functional interaction between two transcription factors involved in the developmental regulation of a small heat stress protein gene promoter.

Hahsp17.6G1 is the promoter of a small heat stress protein (sHSP) from sunflower (Helianthus annuus) that is activated during zygotic embryogenesis, but which does not respond to heat stress. We report here the cloning of a transcription factor (TF), sunflower drought-responsive element binding factor 2 (HaDREB2), by one-hybrid interaction with functional cis-elements in Hahsp17.6G1. We have analyzed the functional interaction between HaDREB2 and a second transcription factor, sunflower heat stress factor A9 (HaHSFA9), which was previously assigned to the regulation of Hahsp17.6G1. HaDREB2 and HaHSFA9 synergistically trans-activate the Hahsp17.6G1 promoter in bombarded sunflower embryos. This synergistic interaction is heat stress factor (HSF) specific and requires the binding of both factors to the promoter. The C-terminal region of HaHSFA9 is sufficient for the HSF specificity. Our results represent an example of a functional interaction between members of the Apetala 2 (HaDREB2) and HSF (HaHSFA9) families of transcription factors. We suggest new roles in zygotic embryogenesis for specific members of the AP2 transcription factor family.

Amino Acid Sequence↗

PubMed2006

Functional classification, genomic organization, putatively cis-acting regulatory elements, and relationship to quantitative trait loci, of sorghum genes with rhizome-enriched expression.

Rhizomes are organs of fundamental importance to plant competitiveness and invasiveness. We have identified genes expressed at substantially higher levels in rhizomes than other plant parts, and explored their functional categorization, genomic organization, regulatory motifs, and association with quantitative trait loci (QTLs) conferring rhizomatousness. The finding that genes with rhizome-enriched expression are distributed across a wide range of functional categories suggests some degree of specialization of individual members of many gene families in rhizomatous plants. A disproportionate share of genes with rhizome-enriched expression was implicated in secondary and hormone metabolism, and abiotic stimuli and development. A high frequency of unknown-function genes reflects our still limited knowledge of this plant organ. A putative oligosaccharyl transferase showed the highest degree of rhizome-specific expression, with several transcriptional or regulatory protein complex factors also showing high (but lesser) degrees of specificity. Inferred by the upstream sequences of their putative rice (Oryza sativa) homologs, sorghum (Sorghum bicolor) genes that were relatively highly expressed in rhizome tip tissues were enriched for cis-element motifs, including the pyrimidine box, TATCCA box, and CAREs box, implicating the gibberellins in regulation of many rhizome-specific genes. From cDNA clones showing rhizome-enriched expression, expressed sequence tags forming 455 contigs were plotted on the rice genome and aligned to QTL likelihood intervals for ratooning and rhizomatous traits in rice and sorghum. Highly expressed rhizome genes were somewhat enriched in QTL likelihood intervals for rhizomatousness or ratooning, with specific candidates including some of the most rhizome-specific genes. Some rhizomatousness and ratooning QTLs were shown to be potentially related to one another as a result of ancient duplication, suggesting long-term functional conservation of the underlying genes. Insight into genes and pathways that influence rhizome growth set the stage for genetic and/or exogenous manipulation of rhizomatousness, and for further dissection of the molecular evolution of rhizomatousness.

Chromosome Mapping↗

PubMed2006

Identification of a dehydration and ABA-responsive promoter regulon and isolation of corresponding DNA binding proteins for the group 4 LEA gene CpC2 from C. plantagineum.

The resurrection plant Craterostigma plantagineum (Scrophulariaceae) is used as a model system to investigate the molecular and biochemical basis of desiccation tolerance. Genes which contribute to desiccation tolerance are expressed during dehydration of this plant. One of the dehydration-induced genes is CpC2, a group 4 LEA gene. The CpC2 promoter was analysed and a core promoter region (CPR) was identified which is critical for the responsiveness of the gene to dehydration and the plant hormone ABA. The CPR motif contains two ABA-response elements (ABRE) and a binding site for HDZIP transcription factors. A yeast one-hybrid screen was performed to isolate CPR binding proteins. This resulted in the isolation of a bZIP transcription factor (CpbZIP1) and three highly conserved CpHistone H3 proteins. Two of these CpHistone H3 proteins are constitutively expressed histone H3 variants which are suggested to be involved in gene regulation via histone modification. The CpbZIP1 belongs to the group S of bZIP genes which possess long 5'-UTRs with a putative regulatory function. A second very similar bZIP clone, CpbZIP2, was isolated which contains a conserved small upstream open reading frame (uORF) within the 5'-leader sequence. A possible regulatory role of the uORF is discussed.

Abscisic Acid↗

PubMed1993

Novel regulation of an MHC class I gene response to interferon-gamma.

The ability of IFN-gamma to increase the expression of MHC class I gene products is likely to enhance cytolytic T lymphocyte recognition of viral pathogens and tumor cells. The murine lymphoma AKR SL3-cl.F AZR (SL3-cl.F) responds aberrantly to treatment with interferon-gamma such that H-2Dk surface expression is augmented, but H-2Kk expression remains at constitutive levels. Somatic cell fusions have been used to demonstrate that the lesion responsible for this phenotype is cis-dominant, implicating a primary lesion within the SL3-cl.F H-2Kk gene. In this communication, we have used PCR to analyze the nucleotide sequence in regions of the SL3-cl.F H-2Kk promoter known to contain interferon-responsive enhancer elements. Comparison of the SL3-cl.F H-2Kk sequences to known consensus elements revealed complete identity. In order to identify the lesion responsible for the SL3-cl.F phenotype, two H-2Kk genomic clones were independently isolated from SL3-cl.F. Each clone exists as a 10.5-kbp EcoRI fragment containing the entire structural gene. The site of transcription initiation is at the center of this fragment; therefore, all regulatory elements within 5 kbp of the transcript start site which could alter steady-state message levels are included. Interestingly, IFN-gamma-augmented expression of the H-2Kk gene was restored following DNA-mediated transfection of either of these clones into fibroblast cell lines and the parental cell line SL3-cl.F. Because isolation of these clones required passage of the DNA through a prokaryotic host, which alters the pattern of DNA methylation, there was the possibility that demethylation was responsible for the newly acquired IFN-gamma-responsive phenotype. Treatment of SL3-cl.F with 5-azacytidine, which inhibits de novo methylation, did not restore IFN-gamma-augmented expression, however, thus excluding H-2Kk specific methylation as a potential mechanism. Collectively, these data demonstrate that the alteration responsible for the phenotype observed in SL3-cl.F does not involve known transcriptional regulatory elements. Potential mechanisms which might account for the mutant phenotype are discussed.

3T3 Cells↗

PubMed2006

A recombination silencer that specifies heterochromatin positioning and ikaros association in the immunoglobulin kappa locus.

Allelic exclusion ensures that individual B lymphocytes produce only one kind of antibody molecule. Previous studies have shown that allelic exclusion of the mouse Igkappa locus occurs by the combination of monoallelic silencing and a low level of monoallelic activation for rearrangement combined with a negative feedback loop blocking additional functional rearrangements. Using yeast artificial chromosome-based single-copy isotransgenic mice, we have identified a cis-acting element that negatively regulates rearrangement in this locus, specifically in B cells. The element, termed Sis, resides in the V-J intervening sequence. Sis specifies the targeting of Igkappa transgenes in pre-B and B cells to centromeric heterochromatin and associates with Ikaros, a repressor protein that also colocalizes with centromeric heterochromatin. Significantly, these are hallmarks of silenced endogenous germline Igkappa genes in B cells. These results lead us to propose that Sis participates in the monoallelic silencing aspect of allelic exclusion regulation.

Animals↗

PubMed2006

Cis-regulatory elements in the Accord retrotransposon result in tissue-specific expression of the Drosophila melanogaster insecticide resistance gene Cyp6g1.

Transposable elements are a major mutation source and powerful agents of adaptive change. Some transposable element insertions in genomes increase to a high frequency because of the selective advantage the mutant phenotype provides. Cyp6g1-mediated insecticide resistance in Drosophila melanogaster is due to the upregulation of the cytochrome P450 gene Cyp6g1, leading to the resistance to a variety of insecticide classes. The upregulation of Cyp6g1 is correlated with the presence of the long terminal repeat (LTR) of an Accord retrotransposon inserted 291bp upstream of the Cyp6g1 transcription start site. This resistant allele (DDT-R) is currently at a high frequency in D. melanogaster populations around the world. Here, we characterize the spatial expression of Cyp6g1 in insecticide-resistant and -susceptible strains. We show that the Accord LTR insertion is indeed the resistance-associated mutation and demonstrate that the Accord LTR carries regulatory sequences that increase the expression of Cyp6g1 in tissues important for detoxification, the midgut, Malpighian tubules, and the fat body. This study provides a significant example of how changes in tissue-specific gene expression caused by transposable-element insertions can contribute to adaptation.

Adaptation, Biological↗

PubMed1993

Expression and structural analysis of a teleost homolog of a mammalian zona pellucida gene.

Gene expression in oviparous vertebrates during vitellogenesis is hormonally regulated. Our laboratory has characterized a unique gene (wf female), which is seasonally expressed in the liver of the female winter flounder Pseudopleuronectes americanus. The wf female mRNA is coexpressed with vitellogenin mRNA and reaches a high level during vitellogenesis. The wf female gene is 2554 base pairs in length and encodes a putative protein of 509 amino acids. The gene consists of eight exons separated by seven introns of different sizes. Within exon 1, there are six PQQ-rich repeats. Four of them encode a putative (PQQ)1PKY polypeptide, similar to the repeats found in the extracellular domains of other proteins. Exons 2-7 share homology with the zona pellucida protein genes rc55 of rabbit and zp-2 of mouse, and the positions of intron boundaries are conserved in the wf female and mouse zp-2 genes. In addition, the transcriptional regulatory cis elements (estrogen response element, CCAAT and TATAAA boxes), as found in vertebrate genes, are also conserved for the wf female promoter and mapped upstream from the initiation site of the wf female primary transcript.

Amino Acid Sequence↗