On the organisation of the regulatory region of the zebrafish deltaD gene

Development ◽  
2002 ◽  
Vol 129 (20) ◽  
pp. 4773-4784 ◽  
Author(s):  
Stefan Hans ◽  
José A. Campos-Ortega

deltaD is one of the four zebrafish Delta homologues presently known. Experimental evidence indicates that deltaD participates in a number of important processes during embryogenesis, including early neurogenesis and somitogenesis, whereby the protein it encodes acts as a ligand for members of the Notch receptor family. In accordance with its functional role, deltaD is transcribed in several domains of mesodermal and ectodermal origin during embryogenesis. We have analysed the organisation of the regulatory region of the deltaD gene using fusions to the reporter gene gfp and germline transgenesis. Cis-regulatory sequences are dispersed over a stretch of 12.5 kb of genomic DNA, and are organised in a similar manner to those in the regulatory region of the Delta-like 1 gene of mouse. Germline transformation using a minigene comprising 10.5 kb of this genomic DNA attached to the 3′ end of a full-length cDNA clone rescues the phenotype of embryos homozygous for the amorphic deltaD mutation after eightAR33. Several genomic regions that drive transcription in mesodermal and neuroectodermal domains have been identified. Transcription in all the neural expression domains, with one exception, is controlled by two relatively small genomic regions, which are regulated by the proneural proteins neurogenin 1 and zash1a/b acting as transcriptional activators that bind to so-called E-boxes. Transcriptional control of deltaD by proneural proteins therefore represents a molecular target for the regulatory feedback loop mediated by the Notch pathway in lateral inhibition.

1996 ◽  
Vol 16 (9) ◽  
pp. 5058-5068 ◽  
Author(s):  
M A Shield ◽  
H S Haugen ◽  
C H Clegg ◽  
S D Hauschka

Previous analysis of the muscle creatine kinase (MCK) gene indicated that control elements required for transcription in adult mouse muscle differed from those required in cell culture, suggesting that distinct modes of muscle gene regulation occur in vivo. To examine this further, we measured the activity of MCK transgenes containing E-box and promoter deletions in a variety of striated muscles. Simultaneous mutation of three E boxes in the 1,256-bp MCK 5' region, which abolished transcription in muscle cultures, had strikingly different effects in mice. The mutations abolished transgene expression in cardiac and tongue muscle and caused a reduction in expression in the soleus muscle (a muscle with many slow fibers) but did not affect expression in predominantly fast muscles: quadriceps, abdominals, and extensor digitorum longus. Other regulatory sequences with muscle-type-specific activities were found within the 358-bp 5'-flanking region. This proximal region conferred relatively strong expression in limb and abdominal skeletal muscles but was inactive in cardiac and tongue muscles. However, when the 206-bp 5' enhancer was ligated to the 358-bp region, high levels of tissue-specific expression were restored in all muscle types. These results indicate that E boxes and a proximal regulatory region are differentially required for maximal MCK transgene expression in different striated muscles. The overall results also imply that within skeletal muscles, the steady-state expression of the MCK gene and possibly other muscle genes depends on transcriptional mechanisms that differ between fast and slow fibers as well as between the anatomical and physiological attributes of each specific muscle.


2017 ◽  
Vol 37 (15) ◽  
Author(s):  
Nunciada Salma ◽  
Jun S. Song ◽  
Akinori Kawakami ◽  
Suprabha P. Devi ◽  
Mehdi Khaled ◽  
...  

ABSTRACT Members of the MiT transcription factor family are pivotal regulators of several lineage-selective differentiation programs. We show that two of these, Tfeb and Tfe3, control the regulator of adipogenesis, peroxisome proliferator-activated receptor γ2 (Pparγ2). Knockdown of Tfeb or Tfe3 expression during in vitro adipogenesis causes dramatic downregulation of Pparγ2 expression as well as adipogenesis. Additionally, we found that these factors regulate Pparγ2 in mature adipocytes. Next, we demonstrated that Tfeb and Tfe3 act directly by binding to consensus E-boxes within the Pparγ transcriptional regulatory region. This transcriptional control also exists in vivo, as we discovered that wild-type mice in the fed state increased their expression of Tfe3, Tf3b, and Pparγ in white adipose tissue. Furthermore, Tfe3 knockout (Tfe3KO) mice in the fed state failed to upregulate Pparγ and the adiponectin gene, a Pparγ-dependent gene, confirming the in vivo role for Tfe3. Lastly, we found that blood glucose is elevated and serum adiponectin levels are suppressed in the Tfe3KO mice, indicating that the Tfe3/Tfeb/Pparγ2 axis may contribute to whole-body energy balance. Thus, we offer new insights into the upstream regulation of Pparγ by Tfe3/Tf3b and propose that targeting these transcription factors may offer opportunities to complement existing approaches for the treatment of diseases that have dysregulated energy metabolism.


2004 ◽  
Vol 24 (13) ◽  
pp. 5923-5936 ◽  
Author(s):  
Jung-whan Kim ◽  
Karen I. Zeller ◽  
Yunyue Wang ◽  
Anil G. Jegga ◽  
Bruce J. Aronow ◽  
...  

ABSTRACT Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpression enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes directly regulated by Myc is not known, we chose Myc as a model transcription factor to determine whether it binds target glycolytic genes that have conserved canonical Myc binding sites or E boxes (5′-CACGTG-3′). Conserved canonical E boxes in ENO1, HK2, and LDHA occur in 31- to 111-bp islands with high interspecies sequence identity (>65%). Trafac analysis revealed another region in ENO1 that corresponds to a murine region with a noncanonical E box. Myc bound all these conserved regions well in the human P493-6 B lymphocytes. We also determined whether Myc could bind nonconserved canonical E boxes found in the remaining human glycolytic genes. Myc bound PFKM, but it did not significantly bind GPI, PGK1, and PKM2. Binding to BPGM, PGAM2, and PKLR was not detected. Both GAPD and TPI1 do not have conserved E boxes but are induced and bound by Myc through regions with noncanonical E boxes. Our results indicate that Myc binds well to conserved canonical E boxes, but not nonconserved E boxes. However, the binding of Myc to unpredicted genomic regions with noncanonical E boxes reveals a limitation of phylogenetic footprinting. In aggregate, these observations indicate that Myc is an important regulator of glycolytic genes, suggesting that MYC plays a key role in a switch to glycolytic metabolism during cell proliferation or tumorigenesis.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Somayyeh Sedaghatjoo ◽  
Monika K. Forster ◽  
Ludwig Niessen ◽  
Petr Karlovsky ◽  
Berta Killermann ◽  
...  

AbstractTilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.


Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 521
Author(s):  
Catia Giovannini ◽  
Francesca Fornari ◽  
Fabio Piscaglia ◽  
Laura Gramantieri

The Notch family includes evolutionary conserved genes that encode for single-pass transmembrane receptors involved in stem cell maintenance, development and cell fate determination of many cell lineages. Upon activation by different ligands, and depending on the cell type, Notch signaling plays pleomorphic roles in hepatocellular carcinoma (HCC) affecting neoplastic growth, invasion capability and stem like properties. A specific knowledge of the deregulated expression of each Notch receptor and ligand, coupled with resultant phenotypic changes, is still lacking in HCC. Therefore, while interfering with Notch signaling might represent a promising therapeutic approach, the complexity of Notch/ligands interactions and the variable consequences of their modulations raises concerns when performed in undefined molecular background. The gamma-secretase inhibitors (GSIs), representing the most utilized approach for Notch inhibition in clinical trials, are characterized by important adverse effects due to the non-specific nature of GSIs themselves and to the lack of molecular criteria guiding patient selection. In this review, we briefly summarize the mechanisms involved in Notch pathway activation in HCC supporting the development of alternatives to the γ-secretase pan-inhibitor for HCC therapy.


1988 ◽  
Vol 8 (4) ◽  
pp. 1821-1825
Author(s):  
K A Kelley ◽  
J W Chamberlain ◽  
J A Nolan ◽  
A L Horwich ◽  
F Kalousek ◽  
...  

In an attempt to use mouse metallothionein-I (mMT-I) regulatory sequences to direct expression of human ornithine transcarbamylase in the liver of transgenic animals, fusion genes joining either 1.6 kilobases or 185 base pairs of the mMT-I regulatory region to the human ornithine transcarbamylase protein-coding sequence were used to produce transgenic mice. In mice carrying the fusion gene with 1.6 kilobases of the mMT-I 5'-flanking sequences, transgene expression was observed in a wide range of tissues, but, unexpectedly, expression in liver was never observed. Surprisingly, in mice carrying the fusion gene regulated by only 185 base pairs of the mMT-I 5'-flanking sequences, the transgene was expressed exclusively in male germ cells during the tetraploid, pachytene stage of meiosis.


2019 ◽  
Author(s):  
Patrick C. Fiaux ◽  
Hsiuyi V. Chen ◽  
Aaron R. Chen ◽  
Poshen B. Chen ◽  
Graham McVicker

AbstractCRISPR screens are a powerful new technology for the identification of genome sequences that affect cellular phenotypes such as gene expression, survival, and proliferation. By tiling single-guide RNA (sgRNA) target sites across large genomic regions, CRISPR screens have the potential to systematically discovery novel functional sequences, however, a lack of purpose-built analysis tools limits the effectiveness of this approach. Here we describe RELICS, a Bayesian hierarchical model for the discovery of functional sequences from tiling CRISPR screens. RELICS considers the overlapping effects of multiple nearby functional sequences, accounts for the ‘area of effect’ surrounding sgRNA target sites, models overdispersion in sgRNA counts, combines information across multiple pools, and estimates the number of functional sequences supported by the data. In simulations, RELICS outperforms existing methods and provides higher resolution predictions. We apply RELICS to published CRISPR interference and CRISPR activation screens and predict novel regulatory sequences, several of which we experimentally validate. In summary, RELICS is a powerful new analysis method for tiling CRISPR screens that enables the discovery of functional sequences with unprecedented resolution and accuracy.


2021 ◽  
Vol 30 (1) ◽  
pp. 95-103
Author(s):  
Mohammad Shamimul Alam ◽  
Israt Jahan ◽  
Sadniman Rahman ◽  
Hawa Jahan ◽  
Kaniz Fatema

Tilapia is a hardy fish which can survive in water bodies polluted with heavy metals. Metal resistance is conferred by higher expression of metallothionein gene (mt) in many organisms. Level, time and tissue-specificity of gene expression is regulated through transcription factor binding sites (TFBS) which may be present in the upstream, downstream, or even in the introns of a gene. So, as a candidate regulatory region, the 5’upstream sequence of mt gene in three tilapia species, Oreochromis aureus, O. niloticus and O. mossambicus was studied. The targeted region was PCR-amplified and then sequenced using a pair of custom-designed primer. A total of only 2.7% variation was found in the sequenced genomic region among the three species. Metal-related TFBS were predicted from these sequences. A total of twenty eight TFBS were found in O. aureus and twenty nine in O. mossambicus and O. niloticus. The number of metalrelated TFBS predicted in the targeted sequence was significantly higher compared to that found in randomly selected other genomic regions of same size from O. niloticus genome. Thus, the results suggest the presence of putative regulatory elements in the targeted upstream region which might have important role in the regulation of mt gene function. Dhaka Univ. J. Biol. Sci. 30(1): 95-103, 2021 (January)


1990 ◽  
Vol 10 (8) ◽  
pp. 3965-3978
Author(s):  
K A Won ◽  
H Baumann

Expression of the rat alpha 1-acid glycoprotein gene is stimulated by interleukin-1 (IL-1) and interleukin-6 (IL-6) and is synergistically enhanced by the combination of the two. The distal regulatory element (DRE), a 142-base-pair (bp) sequence located 5 kilobase pairs upstream of the transcriptional start site, appears to be crucial for this cytokine response. The cytokine-specific regulatory sequences within the DRE have been identified by inserting individual DRE subregions, selected combinations of these, or a few linker mutated fragments into a plasmid containing an enhancerless simian virus 40 promoter linked to the chloramphenicol acetyltransferase gene. The regulatory activity was determined in transiently transfected human and rat hepatoma cells. The IL-1 response region was confined to the 5'-most 62 bp of the DRE, and its function seemed to depend on at least two separate components. The same region was also responsive to phorbol ester treatment. The IL-6 regulatory function was dependent on a 54-bp sequence located within the 3' half of the DRE. When the IL-1 response region was recombined with the IL-6 regulatory region of the DRE or with IL-6 response elements of other plasma protein genes, a strong cooperative action by IL-1 and IL-6 was achieved. The functional DRE sequences were recognized by nuclear proteins extracted from rat liver and hepatoma cells. However, no cytokine-inducible binding activity was detectable, which suggests that transcriptional regulation through the DRE might be controlled by posttranslational modification of constitutively bound trans-acting factors.


1989 ◽  
Vol 9 (5) ◽  
pp. 2224-2227
Author(s):  
R A Rippe ◽  
S I Lorenzen ◽  
D A Brenner ◽  
M Breindl

We have identified two blocks of regulatory sequences located in the 5'-flanking region and the first intron of the mouse alpha 1 type I collagen (COL1A1) gene. Both blocks were found to contain positive as well as negative regulatory elements. Sequences located within 222 base pairs upstream of the transcription start site showed a strong stimulatory effect on the COL1A1 promoter and were sufficient for tissue-specific regulation of the COL1A1 gene. The combined upstream and intron regulatory sequences showed a marked inhibition of COL1A1 promoter activity in fibroblasts. This finding suggests that additional, more remote regulatory sequences may be required for establishing the high level of activity of the endogenous COL1A1 gene in fibroblastoid cells.


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