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 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.

scholarly journals Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells

2003 ◽  
Vol 372 (3) ◽  
pp. 831-839 ◽  
Author(s):  
Elisabetta LAMBERTINI ◽  
Letizia PENOLAZZI ◽  
Silvia GIORDANO ◽  
Laura DEL SENNO ◽  
Roberta PIVA
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcription Start Site ◽  
Thyroid Hormone Receptor ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Start Site ◽  
Mcf7 Cells ◽  
Transcription Start ◽  
Specific Element

(O)estrogen receptor-α (ERα), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERα gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at −117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of γ-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PFa and PFb, that do not present binding sites for known transcription factors and that are involved in a strong DNA–protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERα gene expression in bone. These data provide evidence for different promoter usage of the ERα gene through the recruitment of tissue-specific transcription activators and co-regulators.

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.

Cloning and characterization of the groE heat-shock operon of the marine bacterium Vibrio harveyi

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1483-1492 ◽  
Author(s):  
Dorota Kuchanny-Ardigò ◽  
Barbara Lipińska
Keyword(s):  
Heat Shock ◽  
Transcription Start Site ◽  
Vibrio Harveyi ◽  
Regulatory Elements ◽  
Northern Hybridization ◽  
Dependent Manner ◽  
Start Site ◽  
Transcription Start ◽  
Transcriptional Regulatory Elements ◽  
E Coli

The DNA region of the Vibrio harveyi chromosome containing the heat-shock genes groES and groEL was cloned, and the genes were sequenced. These genes are arranged in the chromosome in the order groES–groEL. Northern hybridization experiments with RNA from V. harveyi and a DNA probe carrying both groES and groEL genes showed a single, heat-inducible transcript of approximately 2200 nt, indicating that these genes form an operon. Primer extension analysis revealed a strong, heat-inducible transcription start site 59 nt upstream of groES, preceded by a sequence typical for the Escherichia coli heat-shock promoters recognized by the σ 32 factor, and a weak transcription start site 25 nt upstream the groES gene, preceded by a sequence typical for σ 70 promoters. Transcription from the latter promoter occurred only at low temperatures. The V. harveyi groE operon cloned in a plasmid in E. coli cells was transcribed in a σ 32-dependent manner; the transcript size and the σ 32-dependent transcription start site were as in V. harveyi cells. Comparison of V. harveyi groE transcription regulation with the other well-characterized groE operons of the γ subdivision of proteobacteria (those of E. coli and Pseudomonas aeruginosa) indicates a high conservation of the transcriptional regulatory elements among these bacteria, with two promoters, σ 32 and σ 70, involved in the regulation. The ability of the cloned groESL genes to complement E. coli groE mutants was tested: V. harveyi groES restored a thermoresistant phenotype to groES bacteria and enabled λ phage to grow in the mutant cells. V. harveyi groEL did not abolish thermosensitivity of groEL bacteria but it complemented the groEL mutant with respect to growth of λ phage. The results suggest that the GroEL chaperone may be more species-specific than the GroES co-chaperone.

The Role of XPB/Ssl2 dsDNA Translocase Processivity in Transcription Start-site Scanning

2021 ◽  
pp. 166813
Author(s):  
Eric J. Tomko ◽  
Olivia Luyties ◽  
Jenna K. Rimel ◽  
Chi-Lin Tsai ◽  
Jill O. Fuss ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Start Site ◽  
Transcription Start

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).

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