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Results for “regulatory element transcripts”

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PubMed2000

Tissue-specific regulation of cyclin E transcription during Drosophila melanogaster embryogenesis.

Cyclin E is an essential regulator of S phase entry. We have previously shown that transcriptional regulation of the gene that encodes Drosophila cyclin E, DmcycE, plays an important role in the control of the G(1) to S phase transition during development. We report here the first comprehensive analysis of the transcriptional regulation of a G(1 )phase cell cycle regulatory gene during embryogenesis. Analysis of deficiencies, a genomic transformant and reporter gene constructs revealed that DmcycE transcription is controlled by a large and complex cis-regulatory region containing tissue- and stage-specific components. Separate regulatory elements for transcription in epidermal cells during cell cycles 14-16, central nervous system cells and peripheral nervous system cells were found. An additional cis-regulatory element drives transcription in thoracic epidermal cells that undergo a 17th cell cycle when other epidermal cells have arrested in G(1 )phase prior to terminal differentiation. The complexity of DmcycE transcriptional regulation argues against a model in which DmcycE transcription is regulated simply and solely by G(1) to S phase transcription regulators such as RB, E2F and DP. Rather, our study demonstrates that tissue-specific transcriptional regulatory mechanisms are important components of the control of cyclin E transcription and thus of cell proliferation in metazoans.

Animals↗

PubMed1992

A human binding site for transcription factor USF/MLTF mimics the negative regulatory element of human immunodeficiency virus type 1.

Transcriptional regulation of the proviral form of the human immunodeficiency virus type 1 (HIV-1) is exerted by its 5' long terminal repeat (LTR), which contains recognition sites for several cell factors. By gel retardation and DNase I footprinting experiments we have identified a binding site for a human nuclear protein between nucleotides -152 to -174 upstream of transcription start site, in a region previously recognized as a negative regulator of transcription (negative regulatory element, NRE). The recognized sequence contains the dyad symmetry element CACGTG, which represents a binding motif, very conserved through evolution, present in a putative human DNA replication origin (pB48), in the upstream element of the major late promoter (MLP-UE) of adenovirus, and, as transcriptional element, upstream of many eukaryotic genes. Common binding activities exist in human nuclear extracts for pB48, MLP-UE and the HIV-1 LTR; at least three protein species recognize the LTR sequence, of 44 (corresponding to transcription factor USF/MLTF), 70, and 110 kDa, respectively. Chloramphenicol acetyltransferase assays suggest that the USF/MLTF binding site located in the HIV-1 LTR acts as a negative regulator of transcription, and that it contributes to the overall negative function exerted by the NRE. An oligonucleotide corresponding to another characterized human USF/MLTF binding site can functionally replace part of the activity of the NRE. This negative function is exerted both in presence or absence of tat transactivation, in different cell lines, and after PMA stimulation.

Adenoviruses, Human↗

PubMed2001

Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes.

Roles of sterol regulatory element-binding proteins (SREBPs) have been established as lipid synthetic transcription factors especially for cholesterol and fatty acid synthesis. SREBPs have unique characteristics. Firstly, they are membrane-bound proteins and the N-terminal active portions enter nucleus to activate their target genes after proteolytic cleavage, which requires sterol-sensing molecule, SREBP-activating protein (SCAP) and is crucial for sterol-regulation. Secondly, they bind and activate sterol-regulatory (SREs) containing promoters as well as some E-boxes, which makes SREBPs eligible to regulate a wide range of lipid genes. Finally, three isoforms, SREBP-1a-1c, and have different roles in lipid synthesis. In vivo studies using transgenic and knockout mice suggest that SREBP-1 seems to be involved in energy metabolism including fatty acid and glucose/insulin metabolism, whereas SREBP-2 is specific to cholesterol synthesis. Future studies will be focused on understanding molecular mechanisms sensing cellular sterol and energy states where SREBPs are deeply involved.

Animals↗

PubMed2005

Characterization of putative cis-regulatory elements that control the transcriptional activity of the human Oct4 promoter.

Octamer-binding transcription factor-4 (Oct4), a member of the POU domain transcription factors, is crucial for both early embryonic development and the maintenance of stem cell pluripotency. The human Oct4 (hOct4) 5' upstream sequence contains four conserved regions (CR1, 2, 3, 4) that are homologous in the murine. In this study, we constructed a series of deletion mutants of the hOct4 5' upstream region and identified cis-regulatory elements that may be important determinants for the transcriptional activity of the hOct4 promoter. Our studies showed that CR2, 3, and 4 each acted as positive cis-regulatory elements in hOct4 promoter activity. We also newly identified a putative negative cis-acting element located between CR1 and CR2. In addition, the sequence -380/-1 at CR1 that contains a GC box was sufficient to provide the minimal promoter activity. Site-directed mutagenesis and electrophoretic mobility shift assays revealed the GC box located in the -380/-1 region may play a critical role in controlling the transcriptional activity of hOct4 by the direct binding of Sp1 or Sp3 transcription factors to the GC box. An overexpression study showed that Sp1 and Sp3 positively and negatively regulate hOct4 promoter activity. Thus, the hOct4 promoter upstream region contains multiple regulatory elements, one of which, the GC box, may be an important cis-regulatory element that regulates the transcription of the hOct4 promoter by the binding of Sp family transcription factors.

Base Sequence↗

PubMed1994

Assignment of the membrane attachment, DNA binding, and transcriptional activation domains of sterol regulatory element-binding protein-1 (SREBP-1).

Transcription of the low density lipoprotein receptor gene and other sterol-regulated genes is stimulated by sterol regulatory element-binding protein-1 (SREBP-1), a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) transcription factor. Human SREBP-1 is synthesized as an 1147-amino acid precursor that is attached intrinsically to membranes of the nuclear envelope and endoplasmic reticulum. In sterol-depleted cells the precursor is cleaved to generate an NH2-terminal fragment that enters the nucleus and activates transcription by binding to sterol regulatory element-1 (SRE-1). Sterols prevent transcriptional activation by blocking the proteolytic cleavage. In the current studies, performed with hamster SREBP-1, we used mutational analysis to localize the transcriptional activation domain to an acidic NH2-terminal sequence. Deletion of this sequence converted SREBP-1 from an activator to an inhibitor of transcription. DNA binding was assigned to the basic region of the bHLH-ZIP domain. Binding was abolished by substitution of 3 amino acids that were previously implicated in DNA binding by Max, another bHLH-ZIP protein. The membrane attachment domain was localized to two hydrophobic regions at residues 477-497 and 536-556. Truncation of SREBP-1 prior to these regions gave rise to an NH2-terminal fragment that was soluble and entered the nucleus. This fragment was more than 30-fold more active than full-length SREBP-1 in stimulating transcription of an SRE-1 containing reporter gene in transfected cells. Deletion of the hydrophobic sequences (delta 476-556) yielded a protein that appeared cytosolic by immunofluorescence microscopy but failed to enter the nucleus readily, apparently because of inhibition by sequences in the remaining COOH-terminal domain. This study provides a picture of the domain structure of SREBP-1 and further elucidates the mechanism by which it adjusts gene transcription to maintain cholesterol homeostasis in animal cells.

Amino Acid Sequence↗

PubMed2003

Evidence implicating Ku antigen as a structural factor in RNA polymerase II-mediated transcription.

Ku antigen is an abundant nuclear protein with multiple functions that depend mainly on Ku's prolific and highly verstatile interactions with DNA. We have shown previously that the direct binding of Ku in vitro to negative regulatory element 1 (NRE1), a transcriptional regulatory element in the long terminal repeat of mouse mammary tumour virus, correlates with the regulation of viral transcription by Ku. In this study, we have sought to explore the interaction of Ku with NRE1 in vivo in yeast one-hybrid experiments. Unexpectedly, we observed that human Ku70 carrying a transcriptional activation domain from the yeast Gal4 protein induced transcription of yeast reporter genes pleiotrophically, independent of NRE1, promoter, reporter gene and chromosomal location. Ku80 with the same activation domain had no effect on transcription when expressed alone, but reconstituted activation when co-expressed with native human Ku70. The requirements for transcriptional activation by Ku-Gal4 activation domain proteins correlated with previous descriptions of the requirements for DNA sequence-independent DNA binding by Ku, but were distinct from determinants for DNA-end binding by a truncated Ku heterodimer determined recently by crystallography. These results suggest a preferential targeting of Ku to transcriptionally active chromatin that indicate a possible function for Ku within the RNA polymerase II holoenzyme.

Animals↗

PubMed1988

Interaction of a common cellular transcription factor, ATF, with regulatory elements in both E1a- and cyclic AMP-inducible promoters.

We have previously identified a cellular transcription factor, ATF, which is involved in the expression of multiple adenovirus E1a-inducible genes. Here we show that ATF also binds to regulatory elements in cellular cAMP-inducible promoters. In these cellular promoters, ATF binds to a consensus sequence that has been previously shown to be necessary and in some instances sufficient for cAMP-inducible transcription. Furthermore, we show that binding of ATF to these promoter elements is required for the constitutive in vitro transcriptional activity of the cAMP-inducible somatostatin gene. Taken together, our results suggest that a common cellular transcription factor, ATF, can be regulated in vivo by two apparently unrelated inducing agents: the adenovirus E1a protein and cAMP.

Activating Transcription Factors↗

PubMed2006

Phosphorylation and ubiquitination of the transcription factor sterol regulatory element-binding protein-1 in response to DNA binding.

Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors control cholesterol and lipid metabolism and play critical roles during adipocyte differentiation. The transcription factor SREBP1 is degraded by the ubiquitin-proteasome system following phosphorylation of Thr426 and Ser430 in its phosphodegron. We now demonstrate that the glycogen synthase kinase (GSK)-3beta-dependent phosphorylation of these residues in SREBP1 is enhanced in response to specific DNA binding. DNA binding enhances the direct interaction between the C-terminal domain of SREBP1 and GSK-3beta. Accordingly, we demonstrate that GSK-3beta is recruited to the promoters of SREBP target genes in vivo. As a result of the phosphorylation of Thr426 and Ser430, the ubiquitin ligase Fbw7 is recruited to SREBP molecules associated with target promoters. Using a reconstituted ubiquitination system, we demonstrate that Fbw7-mediated ubiquitination of SREBP1 is dependent on its DNA binding activity. Thus, DNA binding could provide a mechanistic link between the phosphorylation, ubiquitination, and degradation of active transcription factors.

Cell Line↗

PubMed1999

Transcription initiation at the TATA-less spliced leader RNA gene promoter requires at least two DNA-binding proteins and a tripartite architecture that includes an initiator element.

Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the "universal" transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m(7)G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical double-stranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (+1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence-TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II.

Animals↗

PubMed2005

Potential autoregulation of transcription factor PU.1 by an upstream regulatory element.

Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb -14 site confers lineage-specific reporter gene expression in vivo. This kb -14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.

Animals↗

PubMed2001

Unsaturated fatty acid-mediated decreases in sterol regulatory element-mediated gene transcription are linked to cellular sphingolipid metabolism.

A major physiological feedback mechanism of cholesterol in transcription of a number of lipid metabolism-related genes is mediated by sterol regulatory elements (SREs) and their binding proteins (SREBPs). Polyunsaturated free fatty acids alone, as well as synergistically with sterols, decrease SRE-mediated gene expression up to 80% in a dose-dependent manner by decreasing levels of the active transcription factor SREBP. We investigated potential mechanisms for this effect. We hypothesized that free fatty acids reduce SREBP-mediated gene transcription by increasing intracellular cholesterol content through the hydrolysis of cellular sphingomyelin, which has a high affinity for free cholesterol. We also questioned whether the lipid second messenger ceramide, a product of sphingomyelin hydrolysis, can decrease SRE-mediated gene transcription. First we investigated the effect of fatty acids on sphingomyelin hydrolysis. Incubation of [(3)H]choline-labeled cells with unsaturated (but not saturated) fatty acids induced hydrolysis of [(3)H]choline-labeled sphingomyelin. Also, incubation of cell extracts from fatty acid-treated cells with [(3)H]sphingomyelin increased generation of [(3)H]ceramide compared with control cells in vitro. We found that addition of ceramide analogs alone and additively with fatty acids decreased SRE expression and that ceramide analogs reduced levels of the transcriptionally active forms of SREBP-1 and SREBP-2. Increasing intracellular ceramide levels by exogenous sphingomyelinase or inhibition of ceramidase decreased SRE-mediated gene expression. None of the above conditions induced apoptosis. Incubation with U18666A, a compound that inhibits intracellular cholesterol movement, increased SRE-mediated gene transcription. C(2)-ceramide abrogated the effect of U18666A on SRE-mediated gene transcription, suggesting cholesterol-independent regulation of SREBP. We provide evidence that sphingomyelin hydrolysis and intermediates of sphingomyelin metabolism (in addition to cholesterol and fatty acids) contribute to regulation of SRE-mediated gene transcription.

Animals↗

PubMed1990

Isolation and characterization of a Drosophila hydei histone DNA repeat unit.

Histone genes in D. hydei are organized in tandemly repeated clusters., accomodating in total 120-140 repeat units. We cloned one of the repeat units and analysed the nucleotide sequence. The repeat unit has a size of 5.1 x 10(3) base-pairs and contains one copy of each of the genes coding for the core histones and one copy coding for the histone H1. In the promoter regions of the genes we identified the presumptive cap sites and TATA boxes. Two additional sequence elements are shared by all five Drosophila hydei histone genes in the cluster. The sequence CCCTCT/G1 is found in the region upstream of the presumptive CAP sites. The sequence element AGTGAA occurs downstream of the presumptive cap sites and is, in contrast to the promoter element, also seen in the histone genes of Drosophila melanogaster. Cell-cycle dependent regulation of transcription of the Drosophila histone genes may be different from that in other eukaryotes since sequence elements involved in the regulation of cell-cycle dependent transcription are absent. Also other regulatory elements for transcription differ from those of other genes. The highly conserved H1-specific promoter sequence AAACACA and the H2B specific promoter sequence ATTTGCAT, which are involved in the cell-cycle dependent transcription of those histone genes in eukaryotes, are missing in the Drosophila genes. However at the 3' end of the genes the palindrome and the purine-rich region, both conserved sequence elements in histone genes of eukaryotes, are present. The spacer regions show a simple sequence organization. The silent site substitution rate between the coding regions of the D. hydei and D. melanogaster histone genes is at least 1.5 times higher for Drosophila than for sea urchin histone genes.

Amino Acid Sequence↗

PubMed1996

Two distinct regulatory elements control quail cdc2 transcription: possible involvement in the control of retinoblast differentiation.

It is a characteristic of the central nervous system of higher eukaryotes that neurons, after an initial proliferation phase, remain postmitotic for their whole life span. In the developing quail neuroretina, most retinoblasts become postmitotic after 7-8 days of incubation. They also cease to express cdc2, which is presumably necessary to allow retinoblasts to definitively leave the cell cycle. The molecular mechanisms monitoring cdc2 expression during differentiation remain partly understood. To further study the control of cdc2 transcription in avian cells, we have cloned the quail cdc2 promoter. Two functional regulatory elements have been characterized. One of them contains an E2F-binding site. Human E2F-1 was found to transactivate the quail cdc2 promoter very efficiently in avian and human cells. Gel retardation experiments are presented, suggesting that E2F, in association with different partners, is a major regulatory of cdc2 transcription during the development of the neuroretina. Our data also indicate that another transcription factor binds to the octamer CAGGTGGC located 115 nucleotides above the main transcription start site. This motif is thus another important regulatory element participating in the control of cdc2 expression.

Amino Acid Sequence↗

PubMed2004

Differential regulation of sterol regulatory element-binding protein 1c transcriptional activity by insulin and liver X receptor during liver development.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors involved in the synthesis of cholesterol and fatty acids. In adults, the isoform SREBP-1c is the predominant transcript in the liver of fed animals, and it activates triglyceride production from glucose when diet is enriched in carbohydrates. Studies have shown that SREBP-1c expression is dependent on insulin but also on the availability of oxysterols, ligands of the nuclear liver X receptor (LXR). The aim of this study was to investigate the regulation of the hepatic SREBP-1c expression in vivo in situations where drastic nutritional and hormonal changes occur, from the gestation to the weaning period. In this paper, we report the discovery of LXR-independent SREBP-1c transcriptional activity during late gestation. In utero insulin injection prior to the natural rise in insulin in late gestation triggers SREBP-1c mRNA elevation, nuclear SREBP-1c binding activity, and expression of its target genes independently of LXR transactivation. On the other hand, during suckling, we observed strong SREBP-1c mRNA expression despite very low plasma insulin, an expression that may be due to LXR transactivation. In contrast to insulin, LXR is not sufficient to trigger nuclear SREBP-1c binding activity and target gene induction. This could be due to the concomitant induction of INSIG-2a by LXR and subsequent retention of SREBP-1c in the endoplasmic reticulum.

Animals↗

PubMed2001

Cloning and characterization of the human stearoyl-CoA desaturase gene promoter: transcriptional activation by sterol regulatory element binding protein and repression by polyunsaturated fatty acids and cholesterol.

Stearoyl-CoA desaturase (SCD) is a microsomal enzyme required for the biosynthesis of oleate (C18:1) and palmitoleate (C16:1) which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides and cholesterol esters. Previously the full-length human skin cDNA was sequenced and the exon and intron structure of the single functional SCD gene determined. Here we report on the cloning and characterization of the promoter region of the human SCD gene. The human promoter structure is very similar to that of the mouse SCD1 isoform and contains conserved regulatory sequences for the binding of several transcription factors including the sterol regulatory element binding protein (SREBP), CCAAT enhancer binding protein-alpha (C/EBPalpha) and nuclear factor-1 (NF-1) that have been shown to transactivate the transcription of the mouse SCD1 gene. Polyunsaturated fatty acids and cholesterol decreased the SCD promoter-luciferase activity when transiently transfected into HepG2 cells. The decrease in promoter activity correlated with decreases in endogenous SCD mRNA and protein levels. Cotransfection experiment in HepG2 cells showed transactivation of the SCD promoter-luciferase activity by an expression vector containing SREBP-1a and 1c. Our studies indicate that the transcription of the human SCD gene is repressed by polyunsaturated fatty acids and cholesterol and that SREBP plays a role in the transcriptional activation of this gene.

Animals↗

PubMed2003

Regulation of the CDP-choline pathway by sterol regulatory element binding proteins involves transcriptional and post-transcriptional mechanisms.

The synthesis of phosphatidylcholine (PtdCho) by the CDP-choline pathway is under the control of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). Sterol regulatory element binding proteins (SREBPs) have been proposed to regulate CCT at the transcriptional level, or via the synthesis of lipid activators or substrates of the CDP-choline pathway. To assess the contributions of these two mechanisms, we examined CCTalpha expression and PtdCho synthesis by the CDP-choline pathway in cholesterol and fatty acid auxotrophic CHO M19 cells inducibly expressing constitutively active nuclear forms of SREBP1a or SREBP2. Induction of either SREBP resulted in increased expression of mRNAs for sterol-regulated genes, elevated fatty acid and cholesterol synthesis (>10-50-fold) and increased PtdCho synthesis (2-fold). CCTalpha mRNA was increased 2-fold by enforced expression of SREBP1a or SREBP2. The resultant increase in CCTalpha protein and activity (2-fold) was restricted primarily to the soluble fraction of cells, and increased CCTalpha activity in vivo was not detected. Inhibition of the synthesis of fatty acids or their CoA esters by cerulenin or triacsin C respectively following SREBP induction effectively blocked the accompanying elevation in PtdCho synthesis. Thus PtdCho synthesis was driven by increased synthesis of fatty acids or a product thereof. These data show that transcriptional activation of CCTalpha is modest relative to that of other SREBP-regulated genes, and that stimulation of PtdCho synthesis by SREBPs in CHO cells is due primarily to increased fatty acid synthesis.

Acyl Coenzyme A↗

PubMed2024

Cis-regulatory control of transcriptional timing and noise in response to estrogen.

Cis-regulatory elements control transcription levels, temporal dynamics, and cell-cell variation or transcriptional noise. However, the combination of regulatory features that control these different attributes is not fully understood. Here, we used single-cell RNA-seq during an estrogen treatment time course and machine learning to identify predictors of expression timing and noise. We found that genes with multiple active enhancers exhibit faster temporal responses. We verified this finding by showing that manipulation of enhancer activity changes the temporal response of estrogen target genes. Analysis of transcriptional noise uncovered a relationship between promoter and enhancer activity, with active promoters associated with low noise and active enhancers linked to high noise. Finally, we observed that co-expression across single cells is an emergent property associated with chromatin looping, timing, and noise. Overall, our results indicate a fundamental tradeoff between a gene's ability to quickly respond to incoming signals and maintain low variation across cells.

Humans↗

PubMed2006

Evolution of candidate transcriptional regulatory motifs since the human-chimpanzee divergence.

BACKGROUND: Despite the recent completion of the chimpanzee genome project, few functionally significant sequence differences between humans and chimpanzees have thus far been identified. Alteration in transcriptional regulatory mechanisms represents an important platform for evolutionary change, suggesting that a significant proportion of functional human-chimpanzee sequence differences may affect regulatory elements. RESULTS: To explore this hypothesis, we performed genome-wide identification of conserved candidate transcription-factor binding sites that have evolved since the divergence of humans and chimpanzees. Analysis of candidate transcription-factor binding sites conserved between mouse and chimpanzee yet absent in human indicated that loss of candidate transcription-factor binding sites in the human lineage was not random but instead correlated with the biologic functions of associated genes. CONCLUSION: Our data support the notion that changes in transcriptional regulation have contributed to the recent evolution of humans. Moreover, genes associated with mutated candidate transcription-factor binding sites highlight potential pathways underlying human-chimpanzee divergence.

Animals↗