scholarly journals A protocol for rapid degradation of endogenous transcription factors in mammalian cells and identification of direct regulatory targets

2021 ◽  
Vol 2 (2) ◽  
pp. 100530
Author(s):  
Hillary M. Layden ◽  
Nicholas A. Eleuteri ◽  
Scott W. Hiebert ◽  
Kristy R. Stengel
Keyword(s):  
Transcription Factors ◽  
Mammalian Cells ◽  
Rapid Degradation

scholarly journals DNA-binding specificity of GATA family transcription factors.

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

scholarly journals Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells

1994 ◽  
Vol 14 (8) ◽  
pp. 5309-5317
Author(s):  
S P Murphy ◽  
J J Gorzowski ◽  
K D Sarge ◽  
B Phillips
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Embryonal Carcinoma ◽  
Embryonic Stem ◽  
Binding Activity ◽  
Hsp70 Promoter ◽  
Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

Oxygen sensing and molecular adaptation to hypoxia

1996 ◽  
Vol 76 (3) ◽  
pp. 839-885 ◽  
Author(s):  
H. F. Bunn ◽  
R. O. Poyton
Keyword(s):  
Transcription Factors ◽  
Mammalian Cells ◽  
Critical Role ◽  
Hypoxia Inducible Factor ◽  
Signal Transduction Pathway ◽  
Adaptation To Hypoxia ◽  
Adaptive Responses ◽  
Expression Of Genes ◽  
Activation Of Transcription ◽  
Activation Of Oxygen

This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.

MAP kinases and the adaptive response to hypertonicity: functional preservation from yeast to mammals

2004 ◽  
Vol 287 (6) ◽  
pp. F1102-F1110 ◽  
Author(s):  
David Sheikh-Hamad ◽  
Michael C. Gustin
Keyword(s):  
Transcription Factors ◽  
Signaling Pathways ◽  
Adaptive Response ◽  
Mammalian Cells ◽  
Map Kinases ◽  
Current Knowledge ◽  
Cis Elements ◽  
P38 Kinase ◽  
Functional Preservation ◽  
Multiple Signaling

The adaptation to hypertonicity in mammalian cells is driven by multiple signaling pathways that include p38 kinase, Fyn, the catalytic subunit of PKA, ATM, and JNK2. In addition to the well-characterized tonicity enhancer (TonE)-TonE binding protein interaction, other transcription factors (and their respective cis elements) can potentially respond to hypertonicity. This review summarizes the current knowledge about the signaling pathways that regulate the adaptive response to osmotic stress and discusses new insights from yeast that could be relevant to the osmostress response in mammals.

Transfection of Na,K-ATPase α-subunit: regulation of enzyme abundance

1995 ◽  
Vol 73 (5-6) ◽  
pp. 261-268 ◽  
Author(s):  
Nabila M. Shanbaky ◽  
Thomas A. Pressley
Keyword(s):  
Gene Transfer ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Partial Replacement ◽  
Protein Abundance ◽  
Kidney Cells ◽  
Dna Encoding ◽  
Α Subunit ◽  
Rapid Degradation ◽  
Transfected Cells

DNA-mediated gene transfer into mammalian cells was used as a model for investigating the regulation of Na,K-ATPase abundance. Complementary DNA encoding the catalytic α1-subunit from rat was introduced into ouabain-sensitive monkey kidney cells, and transfectants were selected by their ability to survive in normally cytotoxic concentrations of ouabain. The overall specific activity of Na,K-ATPase in the membranes of transfectants was not significantly different from that in control cells, suggesting that there was a partial replacement, rather than an addition, of introduced α1 for the endogenous subunit in the functional enzyme. Immunoblotting with specific antibodies confirmed the similarities in overall α abundance between control and transfected cells. Hybridization analysis of total RNA, however, revealed a higher abundance of the mRNA encoding total α1 in transfected cells. The results suggest that endogenous and introduced α-subunit compete for a limited amount of β, with rapid degradation of unassembled subunits.Key words: DNA-mediated gene transfer, immunoblots, protein abundance, subunit assembly.

scholarly journals Binding of transcription factors creates hot spots for UV photoproducts in vivo

1992 ◽  
Vol 12 (4) ◽  
pp. 1798-1804
Author(s):  
G P Pfeifer ◽  
R Drouin ◽  
A D Riggs ◽  
G P Holmquist
Keyword(s):  
Transcription Factors ◽  
Hot Spots ◽  
Mammalian Cells ◽  
Phosphoglycerate Kinase ◽  
Promoter Sequence ◽  
Mean Value ◽  
Naked Dna ◽  
Dna Photoproducts ◽  
Uv Photoproducts

Cyclobutane dipyrimidines and less than mean value of 6-4 dipyrimidines are the two major classes of mutagenic DNA photoproducts produced by UV irradiation of cells. We developed a method to map cyclobutane dipyrimidines at the DNA sequence level in mammalian cells. The frequency of this class of photoproducts was determined at every dipyrimidine along the human phosphoglycerate kinase-1 (PGK1) promoter sequence and was compared to the UV-induced frequency distribution of mean value of 6-4 dipyrimidines. After irradiation of living cells containing active or inactive PGK1 genes, enzymatic or chemical cleavage at UV photoproducts, and amplification by ligation-mediated polymerase chain reaction, photofootprints were seen in all regions which bind transcription factors and appear as DNase I footprints. Photoproduct frequency within transcription factor binding sites was suppressed or enhanced relative to inactive genes or naked DNA with enhancements of up to 30-fold. Since photoproducts are mutagenic, this indicates that photoproduct (mutation?) hot spots may be tissue specific in mammals.

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