Downstream Promoter Sequences Research Articles

Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing.

RNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapid assay is available for measuring the changes in Pol II pausing as a result of altered promoter sequences. Here, we overcome this hurdle by showing that ChIP-nexus captures the endogenous Pol II pausing on transfected plasmids. Using this reporter-ChIP-nexus assay in Drosophila cells, we show that the pausing stability is influenced by downstream promoter sequences, but that the strongest contribution to Pol II pausing comes from the initiator sequence, in which a single nucleotide, a G at the +2 position, is critical for stable Pol II pausing. These results establish reporter-ChIP-nexus as a valuable tool to analyze Pol II pausing.

Origins of transcriptional transition: balance between upstream and downstream regulatory gene sequences.

ABSTRACTBy measuring individual mRNA production at the single-cell level, we investigated the lac promoter’s transcriptional transition during cell growth phases. In exponential phase, variation in transition rates generates two mixed phenotypes, low and high numbers of mRNAs, by modulating their burst frequency and sizes. Independent activation of the regulatory-gene sequence does not produce bimodal populations at the mRNA level, but bimodal populations are produced when the regulatory gene is activated coordinately with the upstream and downstream region promoter sequence (URS and DRS, respectively). Time-lapse microscopy of mRNAs for lac and a variant lac promoter confirm this observation. Activation of the URS/DRS elements of the promoter reveals a counterplay behavior during cell phases. The promoter transition rate coupled with cell phases determines the mRNA and transcriptional noise. We further show that bias in partitioning of RNA does not lead to phenotypic switching. Our results demonstrate that the balance between the URS and the DRS in transcriptional regulation determines population diversity.

Massively parallel resequencing of the isogenic Drosophila melanogaster strain w1118; iso-2; iso-3 identifies hotspots for mutations in sensory perception genes

We used the Illumina reversible-short sequencing technology to obtain 17-fold average depth (s.d.~8) of ~94% of the euchromatic genome and ~1-5% of the heterochromatin sequence of the Drosophila melogaster isogenic strain w1118; iso-2; iso-3. We show that this strain has a ~9 kb deletion that uncovers the first exon of the white (w) gene, ~4 kb of downstream promoter sequences, and most of the first intron, thus demonstrating that whole-genome sequencing can be used for mutation characterization. We chose this strain because there are thousands of transposon insertion lines and hundreds of isogenic deficiency lines available with this genetic background, such as the Exelixis, Inc., and the DrosDEL collections. We compared our sequence to Release 5 of the finished reference genome sequence which was made from the isogenic strain y1; cn1 bw1 sp1 and identified ~356,614 candidate SNPs in the ~117 Mb unique sequence genome, which represents a substitution rate of ~1/305 nucleotides (~0.30%). The distribution of SNPs is not uniform, but rather there is a ~2-fold increase in SNPs on the autosome arms compared with the X chromosome and a ~7-fold increase when compared to the small 4th chromosome. This is consistent with previous analyses that demonstrated a correlation between recombination frequency and SNP frequency. An unexpected finding was a SNP hotpot in a ~20Mb central region of the 4th chromosome, which might indicate higher than expected recombination frequency in this region of this chromosome. Interestingly, genes involved in sensory perception are enriched in SNP hotspots and genes encoding developmental genes are enriched in SNP coldspots, which suggests that recombination frequencies might be proportional to the evolutionary selection coefficient. There are currently 12 Drosophila species sequenced, and this represents one of many isogenic Drosophila melanogaster genome sequences that are in progress. Because of the dramatic increase in power in using isogenic lines rather than outbred individuals, the SNP information should be valuable as a test bed for understanding genotype-by-environment interactions in human population studies.

ADR1 interacts with a down-stream positive element to activate PS1 transcription

We have identified downstream promoter sequence of the PS1 gene that may be regulated by novel transcription factors. 3' deletion from +178 to +165 had no effect on PS1 transcription. 3' deletion from +178 to +140 decreased promoter activity by 50%. Further 3' deletion from +178 to +114 decreased promoter activity by 80%. Therefore, a crucial element controlling over 80% of the promoter activity in SK-N-SH cell line is located between +114 and +165. Electrophoretic mobility shift assays suggested that zinc finger proteins Sp1 and ADR1 interacted with the PS1 promoter sequence (+114 to +140) and promoter region (+140 to +165) respectively. A three base pair substitution within the core sequence (GGCGGGGA to GGCGactA) of the ADR1 consensus in the element (+140 to +165) that abolished ADR1-DNA interaction, reduced PS1 transcription by 50%. The substitution mutation in the sequence (+114 to +140) that abolished Sp1-DNA interaction had no effect on PS1 expression. These data suggest that a novel mammalian trans-activator protein ADR1 binds to the downstream element (+140 to +165) to activate PS1 transcription.

Downstream promoter sequence of an Indian isolate of Rice tungro bacilliform virus alters tissue-specific expression in host rice and acts differentially in heterologous system

An Indian isolate of Rice tungro bacilliform virus from West Bengal (RTBV-WB) showed significant nucleotide differences in its putative promoter region when compared with a previously characterized isolate from Philippines. The transcription start site of RTBV-WB was mapped followed by assessing the activity and tissue-specificity of the full-length (FL) promoter (-231 to +645) and several of its upstream and downstream deletions by studying the expression of beta-Glucuronidase (GUS) reporter gene in transgenic rice (Oryza sativa L. subsp. indica) plants at various stages of development. In addition to the expected vascular-specific expression pattern, studied by histochemical staining, GUS enzymatic assay and northern and RT-PCR analysis, two novel patterns were revealed in some of the downstream deleted versions; a non-expressing type, representing no expression at any stage in any tissue and constitutive type, representing constitutive expression at all stages in most tissues. This indicated the presence of previously unreported positive and negative cis-regulatory elements in the downstream region. The negative element and a putative enhancer region in the upstream region specifically bound to rice nuclear proteins in vitro. The FL and its deletion derivatives were also active in heterologous systems like tobacco (Nicotiana tabacum) and wheat (Triticum durum). Expression patterns in tobacco were different from those observed in rice suggesting the importance of upstream elements in those systems and host-specific regulation of the promoter in diverse organisms. Thus, the RTBV-WB FL promoter and its derivatives contain an array of cis-elements, which control constitutive or tissue- and development-specific gene expression in a combinatorial fashion.

Thalassaemia mutations within the 5'UTR of the human beta-globin gene disrupt transcription.

The mechanisms by which mutations within the 5' untranslated region (UTR) of the human beta-globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four 'silent' mutations in the human beta-globin 5'UTR, namely: +10(-T), +22(G --> A), +33(C --> G) and +(40-43)(-AAAC), which are present in patients with beta-thalassaemia intermedia. Expression of these genes under the control of the beta-globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61.6%, 68%, 85.2% and 70.6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3' end processing or mRNA stability. Nuclear run-on experiments demonstrated that mutations +10(-T) and +33(C --> G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G --> A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post-transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment.

Sequence-dependent solution structure and motions of 13 TATA/TBP (TATA-box binding protein) complexes.

The TATA element is a well-known example of a DNA promoter sequence recognized by the TATA box binding protein (TBP) through its intrinsic motion and deformability. Although TBP recognizes the TATA element octamer unusually (through the minor groove, which lacks the distinctive features of the major groove), single base-pair replacements alter transcriptional activity. Recent crystallographic experiments have suggested that TATA/TBP complexes differing by a single base pair retain substantial structural similarity despite their functional differences in activating transcription. To investigate the subtle role of sequence-dependent motion within the TATA element and certain aspects of its effect on assembly of the transcriptional complex, we examine 5-ns dynamics trajectories of 13 variant TATA/TBP complexes differing from each other by a single base pair. They include the wild-type (WT) adenovirus 2 major late promoter (AdMLP) TATA element, TATAAAAG (the octamer specifies positions -31 to -24 with respect to the transcription initiation site), and the variants A31 (i.e., AATAAAAG), T30, A29, C29, G28, T28, T27, G26, T26, C25, T25, and T24. Our simulated TATA/TBP complexes develop sequence-dependent structure and motion trends that may lead to favorable orientations for high-activity variants (with respect to binding TFIIA, TFIIB, and other transcription factors), while conversely, accelerate dissociation of low-activity TATA/TBP complexes. The motions that promote favorable geometries for preinitiation complexes include small rotations between TBP's N- and C-terminal domains, sense strand DNA backbone "slithering," and rotations in TBP's H2 and H2' helices. Low-activity variants tend to translate the H1 and H1' helices and withdraw the intercalating phenylalanines. These cumulative DNA and protein motions lead to a spatial spread of complex orientations up to 4 A; this is associated with an overall bend of the variant TATA/TBP complexes that spans 93 degrees to 110 degrees (107 degrees for the crystal reference). Taken together, our analyses imply larger differences when these local structural and bending changes are extended to longer DNA (upstream and downstream) and suggest that specific local TATA/TBP motions (e.g., shifts in TBP helices and TATA bases and backbone) play a role in modulating the formation and maintenance of the transcription initiation complex.

Contribution of downstream promoter elements to transcriptional regulation of the rice tungro bacilliform virus promoter.

Downstream sequences influence activity of the rice tungro bacilliform virus (RTBV) promoter in protoplasts derived from cultured rice cells. We previously identified a DNA element located between positions +50 and +90 relative to the transcription start site to which rice nuclear proteins bind. In this study, using DNA UV crosslinking assays, we show that two rice nuclear proteins bind specifically to this DNA element. We demonstrate that the DNA element enhances RTBV promoter activity in a copy number-dependent manner when transferred to a position upstream of the promoter. In addition, using electrophoretic mobility shift assays, we show that at least two novel nuclear proteins from rice cell suspension cultures bind to a subregion (from +50 to +59) of the DNA element and that a protein from rice root, but not shoot, nuclear extracts interacts with a perfect palindromic sequence motif located within the sequence +45 to +59. Furthermore, a position-dependent GAGA motif, present in three copies within downstream promoter sequences from +1 to +50, is involved in the regulation of RTBV promoter activity.

Downstream Promoter Sequences Facilitate the Formation of a Specific Transcription Factor IID-Promoter Complex Topology Required for Efficient Transcription from the Megalin/Low Density Lipoprotein Receptor-related Protein 2 Promoter

Megalin/low density lipoprotein receptor-related protein 2 (LRP-2) is an endocytic receptor expressed in highly specialized cell types such as parathyroid cells and epithelia of the kidney. Previous experiments identified a nonconsensus TATA element, with the sequence TAGAAAA, as crucial for accurate and efficient transcription from the LRP-2 promoter. Here we show that, in addition to the TAGA element, promoter sequences downstream of the transcription start site contribute significantly to transcription both in vitro and in transfected cells. Deletion and point mutational analyses reveal that the promoter region located between positions +5 and +11 (sequence TTTTGGC) is of particular importance. Complementation experiments in nuclear extracts lacking transcription factor IID (TFIID) activity show that TATA-binding protein-associated factors of TFIID are essential for the function of LRP-2 downstream promoter sequences. Interestingly, DNase I footprinting studies show that the downstream region between positions +5 and +11 does not significantly affect overall TFIID affinity to the promoter but that it profoundly affects the topology of the TFIID x promoter complex not only downstream of the transcription start site, but in particular in the TATA box region. Our observations suggest a model for a novel downstream sequence function, in which TATA-binding protein-associated factor-promoter interactions downstream of the transcription start site modulate TFIID-DNA interactions in the TATA box region.

RNA elements required for RNA recombination function as replication enhancers in vitro and in vivo in a plus-strand RNA virus.

RNA replication requires cis-acting elements to recruit the viral RNA-dependent RNA polymerase (RdRp) and facilitate de novo initiation of complementary strand synthesis. Hairpins that are hot spots for recombination in the genomic RNA of turnip crinkle virus (TCV) and satellite (sat)-RNA C, a parasitic RNA associated with TCV infections, stimulate RNA synthesis 10-fold from a downstream promoter sequence in an in vitro assay using partially purified TCV RdRp. Artificial hairpins had an inhibitory effect on transcription. RNA accumulation in single cells was enhanced 5- to 10-fold when the natural stem-loop structures were inserted into a poorly accumulating sat-RNA. The effect of the stem-loop structures on RNA replication was additive, with insertion of three stem-loop RNA elements increasing sat-RNA accumulation to the greatest extent (25-fold). These stem-loop structures do not influence the stability of the RNAs in vivo, but may serve to recruit the RdRp to the template.

Multiple downstream promoter modules regulate the transcription of the Drosophila melanogaster I, doc and F elements

The basal promoters of three Drosophila long interspersed nuclear elements (LINEs), the I factor and the F and Doc elements, have the same architecture. In each, transcription is directed by an initiator which is faithfully and efficiently recognized only when flanked 3′ by a DNA segment ∼20 bp in length called the B region. The B regions of the three promoters are interchangeable and have a complex structure, comprising three functionally distinct elements: de1, de2 and de3. While de2 is relatively conserved, fitting the consensus RGACGTGY, de1 and de3 vary among the three promoters. At different levels, each downstream element is able to ensure accurate recognition of the initiator. The de2 domain stimulates transcription of the F, I and Doc promoters to the same extent. In contrast, the I de1 domain stimulates transcription much more efficiently than the corresponding domains of the F and Doc elements. The finding that de2 is selectively required in order to detect full activity of enhancer sequences found in the F element suggests that de1 and de2 interact with different proteins. The B regions can be replaced by and synergize with a TATA element, can functionally substitute for downstream promoter sequences in the Drosophila hsp70 gene, and significantly activate the mouse terminal deoxynucleotidyl transferase initiator. Our data suggest that the B regions stimulate transcription by providing sites of interaction for the TFIID complex. Sequences homologous to the de1 to de3 array are found downstream from the transcription start site(s) both in TATA-less and TATA-containing promoters.

Evidence for estrogen receptor-mediated regulation of human gonadotropin-releasing hormone promoter activity in human placental cells

Two fragments of the human gonadotropin-releasing hormone (GnRH) promoter, one containing 0.6 kb of the downstream promoter sequence (H-1) and another 1.8 kb fragment (H-2) containing the upstream promoter region with a deletion of the downstream promoter sequence, were fused to a promoterless luciferase (Luc) reporter construct and transfected into the human placental (JEG) cells. JEG cells transfected with both constructs showed insignificant changes in luciferase activity in response to estradiol. However, cotransfection of H-2-Luc construct with a vector expressing a human estrogen receptor (ER) cDNA results in dose-dependent decreases in luciferase activity in response to estradiol. This ER mediated down-regulation of promoter activity was retained in constructs with the GnRH promoter deleted to position 548 bp 5′ to the upstream transcription start site. Further deletion of upstream promoter sequence to 169 bp reversed the estrogen responsiveness from inhibitory to stimulatory. Thus, this study demonstrated that the upstream GnRH promoter region can be regulated by estrogen in transfected JEG cells.

Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription.

TFIIA is thought to play an important role in transcriptional regulation in higher eukaryotes, but its precise function is unclear. A human cDNA encoding a protein with 45% identity to the small subunit of yeast TFIIA has been isolated. TFIIA activity could be reconstituted by the mixing of recombinant large (alpha beta) and small (gamma) subunits. TFIIA-depleted HeLa nuclear extracts were used to demonstrate that TFIIA is essential for basal and activated transcription by several distinct classes of activators. Recombinant TFIIA functioned in transcriptional activation whether expressed as a dimer (alpha beta+gamma) or as a trimer (alpha+beta+gamma), which closely resembles the native form. Yeast TFIIA also functioned in transcriptional activation, and the human gamma subunit was functionally interchangeable with TOA2, its yeast homolog. Recombinant TFIIA mediated the stimulation of TFIID binding to the TATA region and downstream promoter sequences by the Zta transcriptional activator. Significantly, we found that TFIIA bound directly to Zta in an activation domain-dependent manner. One consequence of the TFIIA-mediated interaction between Zta and TFIID was the formation of a promoter-bound complex resistant to TATA oligonucleotide competition. These results demonstrate that TFIIA is an evolutionarily conserved general factor critical for activator-regulated transcription.

A sequence-specific single-strand DNA binding protein that contacts repressor sequences in the human GM-CSF promoter.

NF-GMb is a nuclear factor that binds to the proximal promoter of the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene. NF-GMb has a subunit molecular weight of 22 kDa, is constitutively expressed in embryonic fibroblasts and binds to sequences within the adjacent CK-1 and CK-2 elements (CK-1/CK-2 region), located at approximately -100 in the GM-CSF gene promoter. These elements are conserved in haemopoietic growth factor (HGF) genes. NF-GMb binding requires the presence of repeated 5'CAGG3' sequences that overlap the binding sites for positive activators. Surprisingly, NF-GMb was found to bind solely to single-strand DNA, namely the non-coding strand of the GM-CSF CK-1/CK-2 region. NF-GMb may belong to a family of single-strand DNA binding (ssdb) proteins that have 5'CAGG3' sequences within their binding sites. Functional analysis of the proximal GM-CSF promoter revealed that sequences in the -114 to -79 region of the promoter containing the NF-GMb binding sites had no intrinsic activity in fibroblasts but could, however, repress tumour necrosis factor-alpha (TNF-alpha) inducible expression directed by downstream promoter sequences (-65 to -31). Subsequent mutation analysis showed that sequences involved in repression correlated with those required for NF-GMb binding.

Transactivation functions facilitate the disruption of chromatin structure by estrogen receptor derivatives in vivo.

The activation of gene transcription by nuclear receptors is invariably associated with alterations in chromatin structure at hormone-responsive elements of target genes. To identify the molecular functions underlying receptor-mediated chromatin structure alterations we have evaluated the effects of DNA binding and transactivation of estrogen receptor derivatives on the promoter chromatin structure of estrogen-responsive reporter minichromosomes in Saccharomyces cerevisiae. We report here that the DNase I-hypersensitive chromatin structure at the promoter region is not simply a consequence of estrogen receptor binding to estrogen-responsive elements but is greatly enhanced by transactivation functions. These chromatin structure alterations are dependent on the presence of more than one estrogen-responsive element as well as downstream promoter sequences and appear to be correlated with transcriptional competence of the promoter. Our results imply that a disruption of chromatin structure at promoters is associated with the establishment of active transcription complexes. Since RNA polymerase cannot initiate transcription on nucleosomal DNA in vitro (Lorch, Y., Lapointe, J.W., and Kornberg, R.D. (1987) Cell 49, 203-210) this local disruption of chromatin structure may represent a nucleosome-free window, allowing initiation to occur in vivo.

Downstream deletion analysis of the lac promoter.

We have generated a series of deletions in the downstream region of the lac promoter. The promoter activities of these mutations were compared by measuring the levels of beta-galactosidase gene expression in vivo. Our results show that deletion of downstream lac promoter sequences changes the promoter strength only two- to threefold. The effects of these deletions on transcription initiation site location were studied through primer extension assay of in vivo mRNAs. We found that the transcription start sites are primarily chosen as an approximate distance from the -10 region of the lac promoter; however, starts are sometimes manifested at a GAATT(C) sequence, which is identical to the wild-type preferred start site. lac promoter P2 and a newly identified promoter, P3, are transcribed in vivo at low levels. Catabolite activator protein complexed with cyclic AMP represses P2 and P3 expression in vivo. The secondary catabolite activator protein binding site plays at most a modest role in catabolite repression in vivo.

Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.

Sequences Far Downstream from the Classical tRNA Promoter Elements Bind RNA Polymerase III Transcription Factors

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.

Detection of Multidrug Resistance (MDR1) Gene RNA Expression in Human Tumors by a Sensitive Ribonuclease Protection Assay

The human MDR1 gene encoding P‐glycoprotein, an energy‐dependent drug‐efflux pump, was initially isolated from a multidrug‐resistant KB carcinoma cell. When a 3 kb genomlc sequence isolated from normal human tissue including the major downstream promoter and the first and second exons of the MDR1 gene was compared to the equivalent fragment from KB cells, the MDR1 gene from KB carcinoma cells was found to have a point mutation in the first exon. Although this mutation does not affect the downstream promoter sequence or the coding sequence of the MDR1 gene, it creates a single base mismatch between the 5′ KB genomic fragment previously used for RNase protection analysis of MDR1 RNA expression in normal tissues and thereby reduces the sensitivity of this assay. Using the DNA fragment from normal tissues rather than KB cells, we have reanalyzed MDR1 mRNA levels in 12 renal carcinomas and 4 colon adenocarcinomas. By this RNase protection assay, MDR1 RNA levels are as high in these tumors as in the multidrug‐resistant cell line, KB‐8–5. The ribonuclease protection assay indicated that the major downstream promoter was mainly used in these clinical samples including two samples of RNA from metastatic renal cancer. This assay appears to be a very sensitive and specific assay for detecting MDR1 mRNA levels and mRNA initiation sites in clinical samples.

Identification of a downstream sequence and binding protein that regulate adenovirus major late promoter transcription in vitro.

Gel electrophoresis mobility shift and DNase I footprint assays detect a cellular nuclear protein in extracts made from uninfected human cells which binds to a downstream promoter sequence (DPS) in the human adenovirus 2 major late promoter. By DNase I footprint and mutation analyses, we have determined that this new regulatory element extends from positions +146 to +165 (relative to the cap site at position +1). We show by UV cross-linking that a 40-kDa polypeptide specifically binds to this region. Mutations within the DPS which decrease protein binding by 80-90% also cause a 2.5-3-fold decrease in in vitro major late promoter transcription efficiency. Alteration of the template in the 5'-flanking region of the DPS does not affect nuclear protein binding or transcription efficiency. Interestingly, a T----G transversion at position +160 which increases protein binding also impairs promoter activity.