A 5′ Splice Site Enhances the Recruitment of Basal Transcription Initiation Factors In Vivo

Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys–Drash syndrome and pre-eclampsia). Administration of recombinant VEGF165b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGFxxxb isoforms to the pro-angiogenic VEGFxxx isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGFxxxb isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology.

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SPT15, the gene encoding the yeast TATA binding factor TFIID, is required for normal transcription initiation in vivo

Cell ◽

10.1016/0092-8674(89)90516-3 ◽

1989 ◽

Vol 58 (6) ◽

pp. 1183-1191 ◽

Cited By ~ 106

Author(s):

David M. Eisenmann ◽

Catherine Dollard ◽

Fred Winston

Keyword(s):

Transcription Initiation ◽

Gene Encoding ◽

Binding Factor

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Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603271113 ◽

2016 ◽

Vol 113 (21) ◽

pp. E2899-E2905 ◽

Cited By ~ 23

Author(s):

Irina O. Vvedenskaya ◽

Hanif Vahedian-Movahed ◽

Yuanchao Zhang ◽

Deanne M. Taylor ◽

Richard H. Ebright ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Start Site ◽

Transcription Initiation ◽

Specific Protein ◽

Start Site ◽

Transcription Start ◽

Transcription Bubble ◽

Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

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Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2832-2839.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2832-2839

Author(s):

A S Ponticelli ◽

K Struhl

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Initiation Site ◽

Activator Proteins ◽

Nuclear Extracts ◽

Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

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Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3841-3849.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3841-3849

Author(s):

B Zenzie-Gregory ◽

A Khachi ◽

I P Garraway ◽

S T Smale

Keyword(s):

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Upstream Region ◽

Promoter Strength ◽

Recognition Sequence ◽

Specific Recognition ◽

Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

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Heat shock factor can activate transcription while bound to nucleosomal DNA in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.189-199.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 189-199

Author(s):

D S Pederson ◽

T Fidrych

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Transcription Initiation ◽

Heat Shock Factor ◽

Micrococcal Nuclease ◽

Nucleosome Assembly ◽

Heat Shock Element ◽

Yeast Saccharomyces Cerevisiae ◽

Nucleosomal Dna

After each round of replication, new transcription initiation complexes must assemble on promoter DNA. This process may compete with packaging of the same promoter sequences into nucleosomes. To elucidate interactions between regulatory transcription factors and nucleosomes on newly replicated DNA, we asked whether heat shock factor (HSF) could be made to bind to nucleosomal DNA in vivo. A heat shock element (HSE) was embedded at either of two different sites within a DNA segment that directs the formation of a stable, positioned nucleosome. The resulting DNA segments were coupled to a reporter gene and transfected into the yeast Saccharomyces cerevisiae. Transcription from these two plasmid constructions after induction by heat shock was similar in amount to that from a control plasmid in which HSF binds to nucleosome-free DNA. High-resolution genomic footprint mapping of DNase I and micrococcal nuclease cleavage sites indicated that the HSE in these two plasmids was, nevertheless, packaged in a nucleosome. The inclusion of HSE sequences within (but relatively close to the edge of) the nucleosome did not alter the position of the nucleosome which formed with the parental DNA fragment. Genomic footprint analyses also suggested that the HSE-containing nucleosome was unchanged by the induction of transcription. Quantitative comparisons with control plasmids ruled out the possibility that HSF was bound only to a small fraction of molecules that might have escaped nucleosome assembly. Analysis of the helical orientation of HSE DNA in the nucleosome indicated that HSF contacted DNA residues that faced outward from the histone octamer. We discuss the significance of these results with regard to the role of nucleosomes in inhibiting transcription and the normal occurrence of nucleosome-free regions in promoters.

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Eukaryotic initiation factors and protein synthesis after resistance exercise in rats

Journal of Applied Physiology ◽

10.1152/jappl.2000.88.3.1036 ◽

2000 ◽

Vol 88 (3) ◽

pp. 1036-1042 ◽

Cited By ~ 26

Author(s):

Peter A. Farrell ◽

Jazmir M. Hernandez ◽

Mark J. Fedele ◽

Thomas C. Vary ◽

Scot R. Kimball ◽

...

Keyword(s):

Protein Synthesis ◽

Resistance Exercise ◽

Translational Control ◽

Sprague Dawley ◽

Initiation Factors ◽

Eukaryotic Initiation Factors ◽

Sprague Dawley Rats ◽

Arterial Plasma ◽

Response To Exercise

Translational control of protein synthesis depends on numerous eukaryotic initiation factors (eIFs) and we have previously shown ( Am. J. Physiol. Endocrinol. Metab. 276: E721–E727, 1999) that increases in one factor, eIF2B, are associated with increases in rates of protein synthesis after resistance exercise in rats. In the present study we investigated whether the eIF4E family of initiation factors is also involved with an anabolic response to exercise. Male Sprague-Dawley rats either remained sedentary ( n = 6) or performed acute resistance exercise ( n = 6), and rates of protein synthesis were assessed in vivo 16 h after the last session of resistance exercise. eIF4E complexed to eIF4G (eIF4E ⋅ eIF4G), eIF4E binding protein 1 (4E-BP1) complexed to eIF4E, and phosphorylation state of eIF4E and 4E-BP1 (γ-form) were assessed in gastrocnemius. Rates of protein synthesis were higher in exercised rats compared with sedentary rats [205 ± 8 (SE) vs. 164 ± 5.5 nmol phenylalanine incorporated ⋅ g muscle−1 ⋅ h−1, respectively; P < 0.05]. Arterial plasma insulin concentrations were not different between the two groups. A trend ( P = 0.09) for an increase in eIF4E ⋅ eIF4G with exercise was noted; however, no statistically significant differences were observed in any of the components of the eIF4E family in response to resistance exercise. These new data, along with our previous report on eIF2B, suggest that the regulation of peptide chain initiation after exercise is more dependent on eIF2B than on the eIF4E system.

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Stimulated initiation of mitogen-activated protein kinase phosphatase-1 (MKP-1) gene transcription involves the synergistic action of multiple cis-acting elements in the proximal promoter

Biochemical Journal ◽

10.1042/bj20031022 ◽

2004 ◽

Vol 378 (2) ◽

pp. 473-484 ◽

Cited By ~ 43

Author(s):

Stephan RYSER ◽

Abbas MASSIHA ◽

Isabelle PIUZ ◽

Werner SCHLEGEL

Keyword(s):

Gene Transcription ◽

Transcription Initiation ◽

Site Directed Mutagenesis ◽

Mitogen Activated Protein Kinase ◽

Proximal Promoter ◽

In Vivo Studies ◽

Luciferase Reporter ◽

Basal Transcription ◽

Band Shift ◽

Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs) are inactivated by a dual specificity phosphatase, MAPK phosphatase-1 (MKP-1). MKP-1 is transcribed as an immediate early response gene (IEG) following various stimuli. In the pituitary cell line GH4C1, MKP-1 gene transcription is strongly induced by thyrotropin-releasing hormone (TRH) as well as by epidermal growth factor (EGF) as a consequence of activated MAPK/extracellular-signal-regulated kinase (ERK) signalling. Intriguingly, reporter gene analysis with the MKP-1 promoter showed strong basal transcription, but only limited induction by TRH and EGF. Site-directed mutagenesis of the reporter construct combined with band-shift and in vivo studies revealed that part of the constitutive activity of the MKP-1 promoter resides in two GC boxes bound by Sp1 and Sp3 transcription factors in the minimal promoter. Basal transcription of transiently transfected luciferase reporter can be initiated by either of the two GC boxes or also by either of the two cAMP/Ca2+ responsive elements or by the E-box present in the proximal promoter. On the other hand, when analysed by stable transfection, the five responsive elements are acting in synergy to transactivate the MKP-1 proximal promoter. We show in this study that the MKP-1 promoter can function as a constitutive promoter or as a rapid and transient sensor for the activation state of MAPKs/ERKs. This dual mode of transcription initiation may have different consequences for the control of a block to elongation situated in the first exon of the MKP-1 gene, as described previously [Ryser, Tortola, van Haasteren, Muda, Li and Schlegel (2001) J. Biol. Chem. 276, 33319–33327].

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