scholarly journals Transcription start site profiling uncovers divergent transcription and enhancer-associated RNAs in Drosophila melanogaster

2017 ◽  
Author(s):  
Michael P. Meers ◽  
Karen Adelman ◽  
Robert J. Duronio ◽  
Brian D. Strahl ◽  
Daniel J. McKay ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
Local Density ◽  
Global Analysis ◽  
Regulatory Elements ◽  
Start Site ◽  
Transcription Start ◽  
Vast Number ◽  
Animal Development ◽  
Divergent Transcription

AbstractBackgroundHigh-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors. However, TSS profiling has not been investigated in an orthogonal in vivo setting. Here, we present a comprehensive dataset that links TSS dynamics with nucleosome occupancy and gene expression in the wandering third instar larva, a developmental stage characterized by large-scale shifts in transcriptional programs in preparation for metamorphosis.ResultsThe data recapitulate major regulatory classes of TSSs, based on peak width, promoter-proximal polymerase pausing, and cis-regulatory element density. We confirm the paucity of divergent transcription units in D. melanogaster, but also identify notable exceptions. Furthermore, we identify thousands of novel initiation events occurring at unannotated TSSs that can be classified into functional categories by their local density of histone modifications. Interestingly, a sub-class of these unannotated TSSs overlaps with functionally validated enhancer elements, consistent with a regulatory role for “enhancer RNAs” (eRNAs) in defining transcriptional programs that are important for animal development.ConclusionsHigh-depth TSS mapping is a powerful strategy for identifying and characterizing low-abundance and/or low-stability RNAs. Global analysis of transcription initiation patterns in a developing organism reveals a vast number of novel initiation events that identify potential eRNAs as well as other non-coding transcripts critical for animal development.

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

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
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).

scholarly journals Core Promoter-Dependent TFIIB Conformation and a Role for TFIIB Conformation in Transcription Start Site Selection

2002 ◽  
Vol 22 (19) ◽  
pp. 6697-6705 ◽  
Author(s):  
Jennifer A. Fairley ◽  
Rachel Evans ◽  
Nicola A. Hawkes ◽  
Stefan G. E. Roberts
Keyword(s):  
Transcription Start Site ◽  
Site Selection ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Initiation Site ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
General Transcription Factor ◽  
Selection Of

ABSTRACT The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5′ and 3′ parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.

scholarly journals Cis-regulatory control of the SM50 gene, an early marker of skeletogenic lineage specification in the sea urchin embryo

Development ◽  
1995 ◽  
Vol 121 (7) ◽  
pp. 1957-1970 ◽  
Author(s):  
K.W. Makabe ◽  
C.V. Kirchhamer ◽  
R.J. Britten ◽  
E.H. Davidson
Keyword(s):  
Transcription Start Site ◽  
Sea Urchin ◽  
Regulatory Elements ◽  
Regulatory Control ◽  
Control Element ◽  
Start Site ◽  
Transcription Start ◽  
Sequence Element ◽  
Spatial Control ◽  
Sea Urchin Embryo

The SM50 gene encodes a minor matrix protein of the sea urchin embryo spicule. We carried out a detailed functional analysis of a cis-regulatory region of this gene, extending 440 bp upstream and 120 bp downstream of the transcription start site, that had been shown earlier to confer accurate skeletogenic expression of an injected expression vector. The distal portion of this fragment contains elements controlling amplitude of expression, while the region from −200 to +105 contains spatial control elements that position expression accurately in the skeletogenic lineages of the embryo. A systematic mutagenesis analysis of this region revealed four adjacent regulatory elements, viz two copies of a positively acting sequence (element D) that are positioned just upstream of the transcription start site; an indispensable spatial control element (element C) that is positioned downstream of the start site; and further downstream, a second positively acting sequence (element A). We then constructed a series of synthetic expression constructs. These contained oligonucleotides representing normal and mutated versions of elements D, C, and A, in various combinations. We also changed the promoter of the SM50 gene from a TATA-less to a canonical TATA box form, without any effect on function. Perfect spatial regulation was also produced by a final series of constructs that consisted entirely of heterologous enhancers from the CyIIIa gene, the SV40 early promoter, and synthetic D, C, and A elements. We demonstrate that element C exercises the primary spatial control function of the region we analyzed. We term this a ‘locator’ element. This differs from conventional ‘tissue-specific enhancers’ in that while it is essential for expression, it has no transcriptional activity on its own, and it requires other, separable, positive regulatory elements for activity. In the normal configuration these ancillary positive functions are mediated by elements A and D. Only positively acting control elements were observed in the SM50 regulatory domain throughout this analysis.

scholarly journals A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells

2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Isaac Shamie ◽  
Sascha H Duttke ◽  
Karen J la Cour Karottki ◽  
Claudia Z Han ◽  
Anders H Hansen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Cho Cells ◽  
Genome Engineering ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Silent Genes

Abstract Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

The Regulatory Regions Required for B′ Paramutation and Expression Are Located Far Upstream of the Maize b1 Transcribed Sequences

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 917-930 ◽  
Author(s):  
Maike Stam ◽  
Christiane Belele ◽  
Wusirika Ramakrishna ◽  
Jane E Dorweiler ◽  
Jeffrey L Bennetzen ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Regulatory Elements ◽  
Start Site ◽  
Transcription Start ◽  
Long Distance ◽  
Recombination Mapping ◽  
Heritable Change ◽  
Cis And Trans ◽  
Transcribed Sequences ◽  
Recombination Breakpoints

Abstract Paramutation is an interaction between alleles that leads to a heritable change in the expression of one allele. In B′/B-I plants, B-I (high transcription) always changes to B′ (low transcription). The new B′ allele retains the low expression state in the next generation and paramutates B-I at a frequency of 100%. Comparisons of the structure and expression of B′ with that of a closely related allele that does not participate in paramutation demonstrated that transcription from the same promoter-proximal sequences is not sufficient for paramutation. Fine-structure recombination mapping localized sequences required for B′ expression and paramutation. The entire 110 kb upstream of the B′ transcription start site was cloned and sequenced and the recombination breakpoints were determined for 12 recombinant alleles. Sequences required for expression and paramutation mapped to distinct regions, 8.5-49 kb and 93-106 kb upstream of the B′ transcription start site, respectively. Sequencing and DNA blot analyses indicate that the B′ region required for paramutation is mostly unique or low copy in the maize genome. These results represent the first example of long-distance regulatory elements in plants and demonstrate that paramutation is mediated by long-distance cis and trans interactions.

scholarly journals Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter-encoded enhancer activities

2018 ◽  
Vol 46 (11) ◽  
pp. 5455-5469 ◽  
Author(s):  
Sarah Rennie ◽  
Maria Dalby ◽  
Marta Lloret-Llinares ◽  
Stylianos Bakoulis ◽  
Christian Dalager Vaagensø ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Core Promoter ◽  
Start Site ◽  
Transcription Start ◽  
Site Analysis ◽  
Divergent Transcription

MOLECULAR CLONING OF THE HUMAN GENE FOR VON WILLEBRAND FACTOR AND IDENTIFICATION OF THE TRANSCRIPTION INITIATION SITE

1987 ◽  
Author(s):  
Corolyn J Collins ◽  
Richard B Levene ◽  
Christina P Ravera ◽  
Marker J Dombalagian ◽  
David M Livingston ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Start Site ◽  
Von Willebrand Factor ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Start Site ◽  
Transcription Start ◽  
Von Willebrand's Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Most patients with von Willebrand's disease appear to have a defect affecting the level of expression of the von Willebrand factor (vWf) gene. Thus, an understanding of the pathogenesis of von Willebrand's disease will require an analysis of the structure and function of the vWf gene in normals and in patients. To begin such analyses, we have screened a human genomic cosmid library with probes obtained from vWf cDNA and isolated a colinear segment spanning ≈175 kb in five overlapping clones. This segment extends ≈25 kb upstream and ≈5 kb downstream of the transcription start and stop sites for vWf mRNA, implying the vWf gene has a length of ≈150 kb. Within one of these clones, the vWf transcription initiation sites have been mapped. A portion of the promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the major transcription start site. Primer extension analysis suggests that sequences contained within the downstream repeat of the transcription start site may be used as minor initiation sites in endothelial cells. Transfection studies are underway to evaluate the role of sequences within this promoter region in gene regulatory activity. Comparative restriction analyses of cloned and chromosomal DNA segments strongly suggests that no major alterations ocurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-expressing endothelial cells and non-expressing white blood cells suggests that no major rearrangements are associated with vWf gene expression. Finally, cross hybridization patterns among seven mammalian species suggests a strong conservation of genomic sequences encoding the plasma portion of vWf, but a lower degree of conservation of sequences encoding the N terminal region of provWf.

scholarly journals Transcription start site profiling uncovers divergent transcription and enhancer-associated RNAs in Drosophila melanogaster

BMC Genomics ◽  
2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Michael P. Meers ◽  
Karen Adelman ◽  
Robert J. Duronio ◽  
Brian D. Strahl ◽  
Daniel J. McKay ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Start Site ◽  
Start Site ◽  
Transcription Start ◽  
Divergent Transcription

scholarly journals RNA Processing of Nitrogenase Transcripts in the Cyanobacterium Anabaena variabilis

2010 ◽  
Vol 192 (13) ◽  
pp. 3311-3320 ◽  
Author(s):  
Justin L. Ungerer ◽  
Brenda S. Pratte ◽  
Teresa Thiel
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
De Novo ◽  
Start Site ◽  
Transcription Start ◽  
Stem Loop ◽  
Upstream Gene ◽  
Genes Encoding ◽  
Nitrogenase Genes ◽  
Cyanobacterium Anabaena

ABSTRACT Little is known about the regulation of nitrogenase genes in cyanobacteria. Transcription of the nifH1 and vnfH genes, encoding dinitrogenase reductases for the heterocyst-specific Mo-nitrogenase and the alternative V-nitrogenase, respectively, was studied by using a lacZ reporter. Despite evidence for a transcription start site just upstream of nifH1 and vnfH, promoter fragments that included these start sites did not drive the transcription of lacZ and, for nifH1, did not drive the expression of nifHDK1. Further analysis using larger regions upstream of nifH1 indicated that a promoter within nifU1 and a promoter upstream of nifB1 both contributed to expression of nifHDK1, with the nifB1 promoter contributing to most of the expression. Similarly, while the region upstream of vnfH, containing the putative transcription start site, did not drive expression of lacZ, the region that included the promoter for the upstream gene, ava4055, did. Characterization of the previously reported nifH1 and vnfH transcriptional start sites by 5′RACE (5′ rapid amplification of cDNA ends) revealed that these 5′ ends resulted from processing of larger transcripts rather than by de novo transcription initiation. The 5′ positions of both the vnfH and nifH1 transcripts lie at the base of a stem-loop structure that may serve to stabilize the nifHDK1 and vnfH specific transcripts compared to the transcripts for other genes in the operons providing the proper stoichiometry for the Nif proteins for nitrogenase synthesis.

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