The rat albumin promoter is composed of six distinct positive elements within 130 nucleotides

No fewer than six different positive regulatory elements concentrated within 130 base pairs constitute the rat albumin promoter, which drives highly tissue specific transcription in rat hepatoma cells in culture. Inactivation of each element led to a decrease in transcriptional efficiency: from upstream to downstream, 3- to 4-fold for distal elements III and II, 15-fold for distal element I, and 50-fold for the CCAAT box and the proximal element (PE). Three of these elements, distal elements III and II and, more crucially, the PE, were found to be involved in the tissue-specific character of transcription, with an additional negative regulation possibly superimposed at the level of the PE. Finally, our mapping of these regulatory elements in vivo entirely coincided with footprint data obtained in vitro, thereby allowing the tentative assignment of specific factors to the effects observed in vivo.

Download Full-text

SMAD1 and SMAD5 Expression Is Coordinately Regulated by FLI1 and GATA2 during Endothelial Development

Molecular and Cellular Biology ◽

10.1128/mcb.00239-15 ◽

2015 ◽

Vol 35 (12) ◽

pp. 2165-2172 ◽

Cited By ~ 6

Author(s):

Jonathon Marks-Bluth ◽

Anchit Khanna ◽

Vashe Chandrakanthan ◽

Julie Thoms ◽

Thomas Bee ◽

...

Keyword(s):

Endothelial Cells ◽

Selective Advantage ◽

Regulatory Elements ◽

Bmp Signaling ◽

Tissue Specific ◽

Morphogenetic Protein ◽

Smad Signaling Pathway ◽

Endothelial Development

The bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin,in vitroreporter assay, andin vivotransgenic data to identify and validateSmad1+63 and theSmad5promoter as tissue-specificcis-regulatory elements that are active in the developing endothelium. The activity of these elements in the endothelium was dependent on highly conserved ETS, GATA, and E-box motifs, and chromatin immunoprecipitation showed high levels of enrichment of FLI1, GATA2, and SCL at these sites in endothelial cell lines and E11 dorsal aortasin vivo. Knockdown of FLI1 and GATA2 but not SCL reduced the expression of SMAD1 and SMAD5 in endothelial cellsin vitro. In contrast, CD31+cKit−endothelial cells harvested from embryonic day 9 (E9) aorta-gonad-mesonephros (AGM) regions of GATA2 null embryos showed reducedSmad1but notSmad5transcript levels. This is suggestive of a degree ofin vivoselection where, in the case of reduced SMAD1 levels, endothelial cells with more robust SMAD5 expression have a selective advantage.

Download Full-text

HNF1alpha is involved in tissue-specific regulation of CFTR gene expression

Biochemical Journal ◽

10.1042/bj20031157 ◽

2004 ◽

Vol 378 (3) ◽

pp. 909-918 ◽

Cited By ~ 44

Author(s):

Nathalie MOUCHEL ◽

Sytse A. HENSTRA ◽

Victoria A. McCARTHY ◽

Sarah H. WILLIAMS ◽

Marios PHYLACTIDES ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Tissue Specificity ◽

Regulatory Elements ◽

Upstream Region ◽

Mrna Levels ◽

Tissue Specific ◽

Specific Regulation

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex pattern of expression with tissue-specific and temporal regulation. However, the genetic elements and transcription factors that control CFTR expression are largely unidentified. The CFTR promoter does not confer tissue specificity on gene expression, suggesting that there are regulatory elements outside the upstream region. Analysis of potential regulatory elements defined as DNase 1-hypersensitive sites within introns of the gene revealed multiple predicted binding sites for the HNF1α (hepatocyte nuclear factor 1α) transcription factor. HNF1α, which is expressed in many of the same epithelial cell types as CFTR and shows similar differentiation-dependent changes in gene expression, bound to these sites in vitro. Overexpression of heterologous HNF1α augmented CFTR transcription in vivo. In contrast, antisense inhibition of HNF1α transcription decreased the CFTR mRNA levels. Hnf1α knockout mice showed lower levels of CFTR mRNA in their small intestine in comparison with wild-type mice. This is the first report of a transcription factor, which confers tissue specificity on the expression of this important disease-associated gene.

Download Full-text

The Radiation-Induced Regenerative Response of Adult Tissue-Specific Stem Cells: Models and Signaling Pathways

Cancers ◽

10.3390/cancers13040855 ◽

2021 ◽

Vol 13 (4) ◽

pp. 855

Author(s):

Paola Serrano Martinez ◽

Lorena Giuranno ◽

Marc Vooijs ◽

Robert P. Coppes

Keyword(s):

Signaling Pathways ◽

Progenitor Cells ◽

Tissue Regeneration ◽

Normal Tissue ◽

Cellular Response ◽

Adult Tissue ◽

Tissue Specific ◽

Radiation Induced

Radiotherapy is involved in the treatment of many cancers, but damage induced to the surrounding normal tissue is often inevitable. Evidence suggests that the maintenance of homeostasis and regeneration of the normal tissue is driven by specific adult tissue stem/progenitor cells. These tasks involve the input from several signaling pathways. Irradiation also targets these stem/progenitor cells, triggering a cellular response aimed at achieving tissue regeneration. Here we discuss the currently used in vitro and in vivo models and the involved specific tissue stem/progenitor cell signaling pathways to study the response to irradiation. The combination of the use of complex in vitro models that offer high in vivo resemblance and lineage tracing models, which address organ complexity constitute potential tools for the study of the stem/progenitor cellular response post-irradiation. The Notch, Wnt, Hippo, Hedgehog, and autophagy signaling pathways have been found as crucial for driving stem/progenitor radiation-induced tissue regeneration. We review how these signaling pathways drive the response of solid tissue-specific stem/progenitor cells to radiotherapy and the used models to address this.

Download Full-text

Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

Biochemical Journal ◽

10.1042/bj3130745 ◽

1996 ◽

Vol 313 (3) ◽

pp. 745-752 ◽

Cited By ~ 5

Author(s):

Françoise LEVAVASSEUR ◽

Jocelyne LIÉTARD ◽

Kohei OGAWA ◽

Nathalie THÉRET ◽

Peter D. BURBELO ◽

...

Keyword(s):

Transcriptional Activation ◽

Hepatoma Cell ◽

Primary Cultures ◽

Regulatory Elements ◽

Hepatoma Cells ◽

Basement Membranes ◽

Major Protein ◽

Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

Download Full-text

Force generation by protein–DNA co-condensation

Nature Physics ◽

10.1038/s41567-021-01285-1 ◽

2021 ◽

Cited By ~ 1

Author(s):

Thomas Quail ◽

Stefan Golfier ◽

Maria Elsner ◽

Keisuke Ishihara ◽

Vasanthanarayan Murugesan ◽

...

Keyword(s):

Optical Tweezers ◽

Self Assembly ◽

Spider Silk ◽

Regulatory Elements ◽

Heterochromatin Formation ◽

First Order Phase Transition ◽

Dna Strand ◽

First Order Phase

AbstractInteractions between liquids and surfaces generate forces1,2 that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water3, modulation of the material properties of spider silk4 and self-assembly of microstructures5. Recent studies have shown that cells assemble biomolecular condensates via phase separation6. In the nucleus, these condensates are thought to drive transcription7, heterochromatin formation8, nucleolus assembly9 and DNA repair10. Here we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein–DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism is a possible general regulatory principle for chromatin organization that may be relevant in vivo.

Download Full-text

Considerations when investigating lncRNA function in vivo

eLife ◽

10.7554/elife.03058 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 222

Author(s):

Andrew R Bassett ◽

Asifa Akhtar ◽

Denise P Barlow ◽

Adrian P Bird ◽

Neil Brockdorff ◽

...

Keyword(s):

Normal Cell ◽

Regulatory Elements ◽

Genomic Locus ◽

Cellular Processes ◽

Cell Processes ◽

Non Coding Rnas ◽

Per Se ◽

Dna Regulatory Elements

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.

Download Full-text

Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4557-4560.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4557-4560

Author(s):

O Bakker ◽

J N Philipsen ◽

B C Hennis ◽

G Ab

Keyword(s):

Dimethyl Sulfate ◽

Upstream Region ◽

Nuclear Factor ◽

Base Pairs ◽

Exonuclease Iii ◽

Factor I ◽

Nuclear Factor I ◽

Vitellogenin Gene

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.

Download Full-text

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4548-4557.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4548-4557

Author(s):

J Hirsh ◽

B A Morgan ◽

S B Scholnick

Keyword(s):

Drosophila Melanogaster ◽

Germ Line ◽

Regulatory Elements ◽

P Element ◽

Developmental Expression ◽

Upstream Region ◽

Regulatory Sequences ◽

Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

Download Full-text

A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

10.1101/2020.07.27.223008 ◽

2020 ◽

Author(s):

Yueyang Wang ◽

Alan Y. Hsu ◽

Eric M. Walton ◽

Ramizah Syahirah ◽

Tianqi Wang ◽

...

Keyword(s):

Cell Motility ◽

Neutrophil Migration ◽

Transgenic Fish ◽

Subcellular Location ◽

Vector System ◽

Specific Gene ◽

Tissue Specific ◽

Biological Studies

AbstractTissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

Download Full-text