Showing papers on "Upstream activating sequence published in 2001"

A conditional tissue-specific transgene expression system using inducible GAL4

Abstract: In Drosophila, the most widely used system for generating spatially restricted transgene expression is based on the yeast GAL4 protein and its target upstream activating sequence (UAS). To permit temporal as well as spatial control over UAS-transgene expression, we have explored the use of a conditional RU486-dependent GAL4 protein (GeneSwitch) in Drosophila. By using cloned promoter fragments of the embryonic lethal abnormal vision gene or the myosin heavy chain gene, we have expressed GeneSwitch specifically in neurons or muscles and show that its transcriptional activity within the target tissues depends on the presence of the activator RU486 (mifepristone). We used available UAS-reporter lines to demonstrate RU486-dependent tissue-specific transgene expression in larvae. Reporter protein expression could be detected 5 h after systemic application of RU486 by either feeding or “larval bathing.” Transgene expression levels were dose-dependent on RU486 concentration in larval food, with low background expression in the absence of RU486. By using genetically altered ion channels as reporters, we were able to change the physiological properties of larval bodywall muscles in an RU486-dependent fashion. We demonstrate here the applicability of GeneSwitch for conditional tissue-specific expression in Drosophila, and we provide tools to control pre- and postsynaptic expression of transgenes at the larval neuromuscular junction during postembryonic life.

The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4.

Abstract: Previous studies demonstrated that the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex facilitates the binding of TATA-binding protein (TBP) during transcriptional activation of the GAL1 gene of Saccharomyces cerevisiae. TBP binding was shown to require the SAGA components Spt3 and Spt20/Ada5, but not the SAGA component Gcn5. We have now examined whether SAGA is directly required as a coactivator in vivo by using chromatin immunoprecipitation analysis. Our results demonstrate that SAGA is physically recruited in vivo to the upstream activation sequence (UAS) regions of the galactose-inducible GAL genes. This recruitment is dependent on both induction by galactose and the Gal4 activation domain. Furthermore, we demonstrate that another well-characterized activator, Gal4–VP16, also recruits SAGA in vivo. Finally, we provide evidence that a specific interaction between Spt3 and TBP in vivo is important for Gal4 transcriptional activation at a step after SAGA recruitment. These results, taken together with previous studies, demonstrate a dependent pathway for the recruitment of TBP to GAL gene promoters consisting of the recruitment of SAGA by Gal4 and the subsequent recruitment of TBP by SAGA.

SAGA is an essential in vivo target of the yeast acidic activator Gal4p

Abstract: Despite major advances in characterizing the eukaryotic transcriptional machinery, the function of promoter-specific transcriptional activators (activators) is still not understood. For example, in no case have the direct in vivo targets of a transcriptional activator been unambiguously identified, nor has it been resolved whether activators have a single essential target or multiple redundant targets. Here we address these issues for the prototype acidic activator yeast Gal4p. Gal4p binds to the upstream activating sequence (UAS) of GAL1 and several other GAL genes and stimulates transcription in the presence of galactose. Previous studies have shown that GAL1 transcription is dependent on the yeast SAGA (Spt/Ada/GCN5/acetyltransferase) complex. Using formaldehyde-based in vivo cross-linking, we show that the Gal4p activation domain recruits SAGA to the GAL1 UAS. If SAGA is not recruited to the UAS, the preinitiation complex (PIC) fails to assemble at the GAL1 core promoter, and transcription does not occur. SAGA, but not other transcription components, is also recruited by the Gal4p activation domain to a plasmid containing minimal Gal4p-binding sites. Recruitment of SAGA by Gal4p and stimulation of PIC assembly is dependent on several SAGA subunits but not the SAGA histone acetyl-transferase (HAT) GCN5. Based on these and other results, we conclude that SAGA is an essential target of Gal4p that, following recruitment to the UAS, facilitates PIC assembly and transcription.

RNA polymerase III and RNA polymerase II promoter complexes are heterochromatin barriers in Saccharomyces cerevisiae

Abstract: The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. Several DNA elements have been identified that act to separate these chromatin domains. We report a detailed characterization of one of these elements, identifying it as a unique tRNA gene possessing the ability to block the spread of silent chromatin in Saccharomyces cerevisiae efficiently. Transcriptional potential of the tRNA gene is critical for barrier activity, as mutations in the tRNA promoter elements, or in extragenic loci that inhibit RNA polymerase III complex assembly, reduce barrier activity. Also, we have reconstituted the Drosophila gypsy element as a heterochromatin barrier in yeast, and have identified other yeast sequences, including the CHA1 upstream activating sequence, that function as barrier elements. Extragenic mutations in the acetyltransferase genes SAS2 and GCN5 also reduce tRNA barrier activity, and tethering of a GAL4/SAS2 fusion creates a robust barrier. We propose that silencing mediated by the Sir proteins competes with barrier element-associated chromatin remodeling activity.

Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences

Abstract: The Oct-4 gene encodes a transcription factor that is specifically expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that differentiate into somatic tissues lose Oct-4 expression. Regulation of Oct-4 gene transcription involves a TATA-less minimal promoter and two upstream elements: the proximal (PE) and distal enhancers (DE). We report here the nucleotide sequence of the 5′ upstream regulatory regions of the human and murine Oct-4 genes. A comparative alignment analysis between these regions and those of the bovine Oct-4 ortholog reveals four conserved regions of homology (CR 1 to 4) between these species (66–94% conservation). The 1A sequence within the mouse PE is located approximately half-way between CR 2 and CR 3. A putative Sp1/Sp3 binding site and the overlapping hormone responsive element (HRE) in CR 1 are identical in all three species. A high number of CCC(A/T)CCC motifs exhibit various levels of homology in these upstream regions. We discuss the importance of these and other sequences and present candidate factors that may bind and regulate Oct-4 gene expression.

Telomere looping permits gene activation by a downstream UAS in yeast

Abstract: In yeast (Saccharomyces cerevisiae), transcriptional activators, such as Gal4 and Gal4–VP16, work ordinarily from sites located in the upstream activating sequence (UAS) positioned about 250 base pairs upstream of the transcription start site1. In contrast to their behaviour in mammalian cells, however, such activators fail to work when positioned at distances greater than ∼600–700 base pairs upstream2, or anywhere downstream3,4 of the gene. Here we show that, in yeast, a gene bearing an enhancer positioned 1–2 kilobases downstream of the gene is activated if the reporter is linked to a telomere, but not if it is positioned at an internal chromosomal locus. These observations are explained by the finding that yeast telomeres form back-folding, or looped, structures. Because yeast telomeric regions resemble the heterochromatin found in higher eukaryotes, these findings might also explain why transcription of some higher eukaryotic genes depends on their location in heterochromatin.

Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer

Abstract: We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS–y–IR hairpin is expressed under the control of different promoter–GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.

The Swi5 activator recruits the Mediator complex to the HO promoter without RNA polymerase II

Abstract: Regulation of HO gene expression in the yeast Saccharomyces cerevisiae is intricately orchestrated by an assortment of gene-specific DNA-binding and non-DNA binding regulators. Binding of the early G1 transcription factor Swi5 to the distal URS1 element of the HO promoter initiates a cascade of events through recruitment of the Swi/Snf and SAGA complexes. In late G1, binding of transcription factor SBF to promoter proximal sequences results in the timely expression of HO. In this work we describe an important additional layer of complexity to the current model by identifying a connection between Swi5 and the Mediator/RNA polymerase II holoenzyme complex. We show that Swi5 recruits Mediator to HO by specific interaction with the Gal11 module of the Mediator complex. Importantly, binding of both the Gal11 and Srb4 mediator components to the upstream region of HO is independent of the SBF factor. Swi/Snf is required for Mediator binding, and genetic suppression experiments suggest that Swi/Snf and Mediator act in the same genetic pathway of HO activation. Experiments examining the kinetics of binding show that Mediator binds to HO promoter elements 1.5 kb upstream of the transcription start site in early G1, but this binding occurs without RNA Pol II. RNA Pol II does not bind to HO until late G1, when HO is actively transcribed, and binding occurs exclusively to the TATA region.

Novel critical role of a human Mediator complex for basal RNA polymerase II transcription

Abstract: Human Mediator complexes have been described as important bridging factors that enhance the effect of activators in purified systems and in chromatin. Here we report a novel basal function of a human Mediator complex. A monoclonal antibody was generated that depleted the majority of Mediator components from crude cell extracts. The removal of human Mediator abolished transcription by RNA polymerase II. This was observed on all genes tested, on TATA-containing and TATA-less promoters, both in the presence and absence of activators. To identify the relevant complex a combined biochemical and immunopurification protocol was applied. Two variants termed Mediator and basal Mediator were functionally and structurally distinguished. Basal Mediator function relies on additional constraints, which is reflected in the observation that it is essential in crude but not in purified systems. We conclude that basal Mediator is a novel general transcription factor of RNA polymerase II.

Transcriptional activation: risky business

Abstract: Transcriptional regulation is all about getting RNA polymerase to the right place on the gene at the right time and making sure that it is competent to conduct transcription. Traditional views of this process place most of their emphasis on the events that precede initiation of transcription. We imagine a promoter-bound transcriptional activator (or collection of activators) recruiting components of the basal transcriptional machinery to the DNA, eventually leading to the recruitment of RNA polymerase II and the onset of gene transcription. Although these events play a crucial role in regulating gene expression, they are only half the story. Correct regulation of transcription requires that polymerase not only initiates when and where it should, but that it stops initiating when no longer appropriate. But how are the signals from transcriptional activators, telling RNA polymerase to fire, terminated? Is this process governed by chance, with activators simply falling off the promoter at a certain frequency? Or is there some more direct mechanism, whereby activators are aggressively limited from uncontrolled promoter activation? A new article by suggests the latter may be true, and provides a mechanism for how a component of the basal transcription machinery can mark the activators it has encountered, sentencing them to an early death or banishing them from the nucleus. The ability of the basal transcriptional apparatus to mark activators provides an efficient way to limit activator function and ensures that continuing transcription initiation at a promoter is coupled to the continuing synthesis and activation of transcriptional activators.

Expression of the REB transcriptional activator in rice grains improves the yield of recombinant proteins whose genes are controlled by a Reb-responsive promoter.

Abstract: The gene encoding the rice transcription factor, REB (rice endosperm bZIP) was cloned from a bacterial artificial chromosome library of rice. The cloned 6,227-bp-long Reb gene is composed of six exons and five introns and is flanked by a 1.2-kb 5′ promoter and a 1.2-kb 3′ terminator region. The function of the Reb gene was explored by a transient assay by using a rice immature endosperm system. The effector constructs containing the native gene or fusion genes linking Reb to the rice actin ( Act ) or globulin ( Glb ) gene promoters and the reporter gene construct Glb-β-glucuronidase (GUS) were used in this study. When these effector constructs were cotransferred with the reporter uid A gene encoding GUS under the control of the Glb promoter into immature rice endosperm cells, the Glb promoter was activated. The transient GUS expression was 2.0 to 2.5-fold higher with the effector construct than without. When the upstream activation sequence containing the GCCACGT(A/C)AG motifs of the Glb promoter was deleted, the activation by REB was abolished. On the other hand, a gain-of-function experiment showed that inserting the upstream activation sequence into the glutelin-1 ( Gt1 ) promoter made it responsive to activation by REB. When cotransformed with Reb gene, mature transgenic rice grains containing the human lysozyme gene driven by the Glb promoter produced 3.7-fold more lysozyme. Accumulation of recombinant lysozyme in mature seed ranged from 30.57 to 279.61 μg⋅mg −1 total soluble protein in individual transformants from 30 independent transformation events. Thus, our results show that REB is not only a transcriptional activator, it can also be used to increase the expression of recombinant protein in transgenic rice grains.

Genomic and genetic dissection of an archaeal regulon

Abstract: The extremely halophilic archaeon Halobacterium sp. NRC-1 can grow phototrophically by means of light-driven proton pumping by bacteriorhodopsin in the purple membrane. Here, we show by genetic analysis of the wild type, and insertion and double-frame shift mutants of Bat that this transcriptional regulator coordinates synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen. Analysis of the complete Halobacterium sp. NRC-1 genome sequence showed that the regulatory site, upstream activator sequence (UAS), the putative binding site for Bat upstream of the bacterio-opsin gene (bop), is also present upstream to the other Bat-regulated genes. The transcription regulator Bat contains a photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type helix–turn–helix DNA binding motif. We also provide evidence for involvement of the PAS/PAC domain of Bat in redox-sensing activity by genetic analysis of a purple membrane overproducer. Five additional Bat-like putative regulatory genes were found, which together are likely to be responsible for orchestrating the complex response of this archaeon to light and oxygen. Similarities of the bop-like UAS and transcription factors in diverse organisms, including a plant and a γ-proteobacterium, suggest an ancient origin for this regulon capable of coordinating light and oxygen responses in the three major branches of the evolutionary tree of life. Finally, sensitivity of four of five regulon genes to DNA supercoiling is demonstrated and correlated to presence of alternating purine–pyrimidine sequences (RY boxes) near the regulated promoters.

A positive regulator of mitosis, Sok2, functions as a negative regulator of meiosis in Saccharomyces cerevisiae.

Abstract: The choice between meiosis and alternative developmental pathways in budding yeast depends on the expression and activity of transcriptional activator Ime1. The transcription of IME1 is repressed in the presence of glucose, and a low basal level of IME1 RNA is observed in vegetative cultures with acetate as the sole carbon source. IREu, a 32-bp element in the IME1 promoter, exhibits upstream activation sequence activity depending on Msn2 and -4 and the presence of acetate. We show that in the presence of glucose IREu functions as a negative element and that Sok2 mediates this repression activity. We show that Sok2 associates with Msn2. Sok2 functions as a general repressor whose availability and activity depend on glucose. The activity of Sok2 as a repressor depends on phosphorylation of T598 by protein kinase A (PKA). Relief of repression of Sok2 depends on both the N-terminal domain of Sok2 and Ime1. In the absence of glucose and the presence of Ime1 Sok2 is converted to a weak activator. Overexpression of Sok2 or mild expression of Sok2 with its N-terminal domain deleted leads to a decrease in sporulation. Previously it was reported that overexpression of Sok2 suppresses the growth defect resulting from a temperature-sensitive PKA; thus Sok2 has a positive role in mitosis. We show that Candida albicans Efg1, a homolog of Sok2, complements sok2Δ in repressing IREu. Our results demonstrate that Sok2, a positive regulator of mitosis, and Efg1, a positive regulator of filamentation, function as negative regulators of meiosis. We suggest that cells use the same regulators with opposing effects to ensure that meiosis will be an alternative to mitosis.

Promoter sequences regulated by the calcineurin-activated transcription factor Crz1 in the yeast ENA1 gene.

Abstract: In Saccharomyces cerevisiae the transcription of the ENA1 gene is modulated by multiple transduction pathways that respond to osmotic, ionic and nutrient stresses. We have investigated the molecular mechanisms involved in ENA1 induction by the calcium-calcineurin-activated transcription factor Crz1/Tcn1. We found in the ENA1 promoter a calcium-responsive, Crz1-dependent upstream activating region (UASENA1) located between –713 bp and –826 bp relative to the translation start. This region contains two separate control elements: the upstream element (5′-GAATGGCTG-3′) between –813 and –821 binds Crz1p with lower affinity and mostly contributes to basal ENA1 expression, whereas the downstream element (5′-GGGTGGCTG-3′) between –727 and –719 binds Crz1p with higher affinity and is a major determinant of the induction response to calcium.

Differential Regulation of Hepatitis B Virus Gene Expression by the Sp1 Transcription Factor

Abstract: The expression of hepatitis B virus (HBV) genes is regulated by a number of transcription factors. One such factor, Sp1, has two binding sites in the core promoter and one in its upstream regulatory element, which is also known as the ENII enhancer. In this study, we have analyzed the effects of these three Sp1 binding sites on the expression of HBV genes. Our results indicate that both Sp1 binding sites in the core promoter are important for the transcription of the core RNA and the precore RNA. Moreover, while the downstream Sp1 site (the Sp1-1 site) in the core promoter did not affect the transcription of the S gene and the X gene, the upstream Sp1 site (the Sp1-2 site) in the core promoter was found to negatively regulate the transcription of the S gene and the X gene, as removal of the latter led to enhancement of transcription of these two genes. The Sp1 binding site in the ENII enhancer (the Sp1-3 site) positively regulates the expression of all of the HBV genes, as its removal by mutation suppressed the expression of all of the HBV genes. However, the suppressive effect of the Sp1-3 site mutation on the expression of the S gene and the X gene was abolished if the two Sp1 sites in the core promoter were also mutated. These results indicate that Sp1 can serve both as a positive regulator and as a negative regulator for the expression of HBV genes. This dual activity may be important for the differential regulation of HBV gene expression.

Yeast chromatin structure and regulation of GAL gene expression.

Abstract: Yeast genomic DNA is covered by nucleosome cores spaced by short, discrete length linkers. The short linkers, reinforced by novel histone properties, create a number of unique and dynamic nucleosome structural features in vivo: permanent unpeeling of DNA from the ends of the core, an inability to bind even full 147 bp core DNA lengths, and facility to undergo a conformational transition that resembles the changes found in active chromatin. These features probably explain how yeast can maintain most of its genome in a transcribable state and avoid large-scale packaging away of inactive genes. The GAL genes provide a closely regulated system in which to study gene-specific chromatin structure. GAL structural genes are inactive without galactose but are highly transcribed in its presence; the expression patterns of the regulatory genes can account for many of the features of GAL structural gene control. In the inactive state, GAL genes demonstrate a characteristic promoter chromosomal organization; the major upstream activation sequence (UASG) elements lie in open, hypersensitive regions, whereas the TATA and transcription start sites are in nucleosomes. This organization helps implement gene regulation in this state and may benefit the organism. Induction of GAL expression triggers Gal4p-dependent upstream nucleosome disruption. Disruption is transient and can readily be reversed by a Gal80p-dependent nucleosome deposition process. Both are sensitive to the metabolic state of the cell. Induction triggers different kinds of nucleosome changes on the coding sequences, perhaps reflecting the differing roles of nucleosomes on coding versus promoter regions. GAL gene activation is a complex process involving multiple Gal4p activities, numerous positive and negative cofactors, and the histone tails. DNA bending and chromosomal architecture of the promoter regions may also play a role in GAL regulation. Regulator-mediated competition between nucleosomes and the TATA binding protein complex for the TATA region is probably a central aspect of GAL regulation and a focal point for the numerous factors and processes that contribute to it.

Analysis of core promoter sequences located downstream from the TATA element in the hsp70 promoter from Drosophila melanogaster.

Abstract: TFIID recognizes multiple sequence elements in the hsp70 promoter of Drosophila. Here, we investigate the function of sequences downstream from the TATA element. A mutation in the initiator was identified that caused an eightfold reduction in binding of TFIID and a fourfold reduction in transcription in vitro. Another mutation in the +24 to +29 region was somewhat less inhibitory, but a mutation in the +14 to +19 region had essentially no effect. The normal promoter and the mutants in the initiator and the +24 to +29 region were transformed into flies by P element-mediated transformation. The initiator mutation reduced expression an average of twofold in adult flies, whereas the mutation in the +24 to +29 region had essentially no effect. In contrast, a promoter combining the two mutations was expressed an average of sixfold less than the wild type. The results suggest that the initiator and the +24 to +29 region could serve overlapping functions in vivo. Protein-DNA cross-linking was used to identify which subunits of TFIID contact the +24 to +29 region and the initiator. No specific subunits were found to cross-link to the +24 to +29 region. In contrast, the initiator cross-linked exclusively to dTAF230. Remarkably, dTAF230 cross-links approximately 10 times more efficiently to the nontranscribed strand than to the transcribed strand at the initiator.

A novel upstream RNA polymerase III promoter element becomes essential when the chromatin structure of the yeast U6 RNA gene is altered.

Abstract: The Saccharomyces cerevisiae U6 RNA gene, SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription of SNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position -30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele of SNR6 with decreased spacing between the A and B blocks, snr6-Delta42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Delta42 is much more sensitive to mutations in a (dT-dA)(7) tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Delta42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription of SNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)(7) tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.

Sp1 is a key regulator of the PDGF beta-receptor transcription.

Abstract: The mouse PDGF β-receptor promoter is tightly controlled by NF-Y that binds to a CCAAT box located upstream of the initiation site [1, 2]. In this report, we show that Sp1 plays an essential role in the PDGF β-receptor transcription. Within the upstream GC rich area there are two Sp1 binding sites located in close proximity to the CCAAT box. Deletion of the GC rich region resulted in a 50% decrease of the transcriptional activity of the promoter, and a complete loss of its responsiveness to over-expression of Sp1. There was an additive effect between NF-Y and Sp1 in reporter activity when they were co-transfected together with the promoter-reporter construct. Furthermore, transfection of NF-Y failed to enhance transcriptional activity when the Sp1 binding sites were deleted from the promoter, suggesting an important role for Sp1 in this NF-Y controlled transcription. We have recently reported that c-Myc represses PDGF β-receptor transcription through its interference with the transactivation activity of NF-Y [3]. In the case of p21waf1/cip1 transcription, c-Myc was shown to repress its transcription by sequestering Sp1 [4]. However, we could not find any effect of Sp1 in the c-Myc-mediated repression on the PDGF β-receptor promoter, since the deletion of Sp1 binding sites could not attenuate the repression by c-Myc on the promoter activity.

Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription

Abstract: Human annexin A5 is a ubiquitous protein implicated in diverse signal transduction processes associated with cell growth and differentiation, and its gene regulation is an important component of this function. Promoter transcriptional activity was determined for a wide 5' portion of the human annexin A5 gene, from bp -1275 to +79 relative to the most 5' of several discrete transcription start points. Transfection experiments carried out in HeLa cells identified the segment from bp -202 to +79 as the minimal promoter conferring optimal transcriptional activity. Two canonical Sp1 sites in the immediate 5' flanking region of a CpG island were required for significant transcription. Strong repressive activity in the distal promoter region between bp -717 to -1153 was attributed to the presence of an endogenous retroviral long terminal repeat, homologous with long terminal repeat 47B. The downstream sequence from bp position +31 to +79 in untranslated exon 1 was also essential for transcription, as its deletion from any of the plasmid constructs abolished activity in transfection assays. Electrophoretic mobility-shift assays, Southwestern-blot analysis and affinity chromatography were used to identify a protein doublet of relative molecular mass 35 kDa that bound an octanucleotide palindromic sequence in exon 1. The DNA cis-element resembled an E-box, but did not bind higher molecular mass transcription factors, such as upstream stimulatory factor or activator protein 4. The discovery of a downstream element crucial for annexin A5 gene transcription, and its interaction with a potentially novel transcription factor or complex, may provide a clue to understanding the initiation of transcription by TATA-less, multiple start site promoters.

Multiple Transcription Factors Regulate the Inducible Expression of the Human Complement Receptor 2 Promoter

Abstract: Complement receptor 2 (CR2) is regulated at the transcriptional level, but the promoter elements and the transcription factors that bind to them and contribute to its regulation are unknown. After documenting that PMA and cAMP induced the activity of the CR2 promoter by 10-fold, we conducted promoter truncation and mutagenesis experiments, in conjunction with shift assays, to determine the functionally important regions of the promoter and the proteins that bind to them. We identified two regions, separated by approximately 900 nucleotides, which together were responsible for inducible promoter activity. Mutagenesis of single promoter elements demonstrated a functional upstream stimulatory factor/E box in the TATA box-proximal region and three equally important, closely spaced, CREB/AP-1 half-sites in the upstream promoter region. The cAMP response element-binding protein (CREB)/AP-1 half-sites bound in vitro Jun and CREB that are induced by protein kinases A and/or C. The 900-nucleotide segment stretching between the above two regions had no functional impact on the induced transcription, and its deletion increased the promoter activity. Finally, a region upstream of the distal site had a repressor activity on CR2 transcription. Moreover, IL-4 induced binding of CREB and AP-1 to the upstream promoter elements and resulted in increased CR2 surface protein expression. These studies have characterized regions of the CR2 promoter and the transcription factors that bind to them and are crucial to induced CR2 expression. Our studies may provide insights to novel approaches to modulate B cell function by regulating CR2 gene transcription.