scholarly journals Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene.

1992 ◽  
Vol 12 (8) ◽  
pp. 3614-3627 ◽  
Author(s):  
P G Traber ◽  
G D Wu ◽  
W Wang
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Nuclear Proteins ◽  
Intestinal Cell ◽  
Specific Gene ◽  
In Vitro Mutagenesis ◽  
Primary Mouse

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.

Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene

1992 ◽  
Vol 12 (8) ◽  
pp. 3614-3627
Author(s):  
P G Traber ◽  
G D Wu ◽  
W Wang
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Nuclear Proteins ◽  
Intestinal Cell ◽  
Specific Gene ◽  
In Vitro Mutagenesis ◽  
Primary Mouse

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.

scholarly journals Phosphorylation of nuclear and DNA-binding proteins in proliferating and quiescent mammalian cells

1981 ◽  
Vol 194 (1) ◽  
pp. 193-207 ◽  
Author(s):  
E W Gerner ◽  
M Costa ◽  
D K Holmes ◽  
B E Magun
Keyword(s):  
Molecular Weight ◽  
Cell Growth ◽  
Dna Binding ◽  
Cyclic Amp ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Dna Binding Proteins ◽  
Exponential Phase ◽  
Nuclear Proteins ◽  
Dependent Growth

The dependence of cell proliferation on nuclear protein phosphorylation was studied with exponential-phase and stationary-phase cultures of Chinese-hamster ovary cells. Nuclear proteins were fractionated, according to their DNA-binding affinities, by using sequential extractions of isolated nuclei with increasing concentrations of NaCl. When viable whole cells were labelled with H332PO4, phosphorylation of nuclear proteins was found to be lower in quiescent cells than in proliferating cells. Phosphorylation of nuclear proteins soluble in 0.30M-NaCl (less than 50% of these proteins bind to DNA) was greater than for those proteins soluble in higher salt concentrations (80-100% of these proteins bind to DNA). Cyclic AMP enhanced the phosphorylation of nuclear proteins soluble in 0.3 m-NaCl by 40-50%, and this stimulation was independent of cell growth. Cyclic AMP also increased the phosphorylation of nuclear proteins soluble in 0.6M-NaCl and 2.0M-NaCl by 40-50% in exponential-phase cultures, but not in stationary-phase cultures. Several examples of specific phosphorylation in response to cyclic AMP were observed, including a 35000-mol.wt. protein in the 0.30 M-NaCl-soluble fraction and several proteins larger than 100000 molecular weight within this fraction. A major peptide of molecular weight approx. 31000 extracted with 0.6M-NaCl was also phosphorylated. Its phosphorylation was independent of cyclic AMP in exponential-phase cultures, and it was not phosphorylated in plateau-phase cells. These changes in cell-growth-dependent phosphorylation occurred in the absence of any apparent qualitative changes in the nuclear protein molecular-weight distributions. These data demonstrate that (1) phosphorylation of nuclear proteins is dependent on the culture's proliferative status, (2) both cyclic AMP-dependent and cyclic AMP-independent specific phosphorylation occurs, and (3) the cyclic AMP-dependent growth-independent phosphorylation that occurs does not appear to be a modification of DNA-binding proteins, whereas the cyclic AMP-dependent growth-dependent phosphorylation does involve modification of DNA binding proteins.

Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

scholarly journals Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements.

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953 ◽  
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

Capturing nuclear sequence-specific DNA-binding proteins by using simian virus 40-derived minichromosomes

1988 ◽  
Vol 8 (2) ◽  
pp. 982-987
Author(s):  
U Nir ◽  
E Fodor ◽  
W J Rutter
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Binding Proteins ◽  
Negative Regulation ◽  
P Element ◽  
Enhancer Function ◽  
Sv40 Dna

We have used recombinant simian virus 40 (SV40) minichromosomes to retrieve sequence-specific DNA-binding proteins derived from the cell nucleus of COS-7 cells. We showed that the transcription factors AP-1 and Sp1 are stably bound to the SV40 DNA late in viral infection. Under similar conditions, minichromosomes carrying the rat insulin (rINS1) enhancer, which is under negative regulation in COS-7 cells, bound two proteins which mapped to distinct regions of the rINS1 enhancer. The SV40 P element competed for one of these proteins which bound to the region from -198 to -230. This factor may be related to AP-1. The other factor selectively bound a regulatory element in the region from -92 to -124 of the insulin enhancer. These proteins may play a role in regulating the rINS1 enhancer function.

Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones

1993 ◽  
Vol 13 (12) ◽  
pp. 7303-7310
Author(s):  
S Altiok ◽  
B Groner
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Mammary Epithelial Cells ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Mammary Epithelial ◽  
Nuclear Proteins ◽  
Single Stranded Dna ◽  
Dna Binding Activity ◽  
Beta Casein

Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA.

scholarly journals A Fast Signal–Induced Activation of Poly(Adp-Ribose) Polymerase

2000 ◽  
Vol 150 (2) ◽  
pp. 293-308 ◽  
Author(s):  
S. Homburg ◽  
L. Visochek ◽  
N. Moran ◽  
F. Dantzer ◽  
E. Priel ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Membrane ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Dna Binding Proteins ◽  
Alternative Mechanism ◽  
Dna Breaks ◽  
Downstream Target ◽  
Inositol 1,4,5 Trisphosphate ◽  
Fast Activation

We present the first evidence for a fast activation of the nuclear protein poly(ADP-ribose) polymerase (PARP) by signals evoked in the cell membrane, constituting a novel mode of signaling to the cell nucleus. PARP, an abundant, highly conserved, chromatin-bound protein found only in eukaryotes, exclusively catalyzes polyADP-ribosylation of DNA-binding proteins, thereby modulating their activity. Activation of PARP, reportedly induced by formation of DNA breaks, is involved in DNA transcription, replication, and repair. Our findings demonstrate an alternative mechanism: a fast activation of PARP, evoked by inositol 1,4,5,-trisphosphate–Ca2+ mobilization, that does not involve DNA breaks. These findings identify PARP as a novel downstream target of phospholipase C, and unveil a novel fast signal–induced modification of DNA-binding proteins by polyADP-ribosylation.

Transcriptional regulation of temperature-induced remodeling of muscle bioenergetics in goldfish

2012 ◽  
Vol 303 (2) ◽  
pp. R150-R158 ◽  
Author(s):  
Katharina Bremer ◽  
Christopher T. Monk ◽  
Brendon J. Gurd ◽  
Christopher D. Moyes
Keyword(s):  
Dna Binding ◽  
Mitochondrial Biogenesis ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Transcriptional Response ◽  
Dna Binding Proteins ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Protein Patterns ◽  
Peroxisome Proliferator Activated Receptor

Central to mammalian mitochondrial biogenesis is the transcriptional master regulator peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), and a network of DNA-binding proteins it coactivates. We explored the role of this pathway in muscle mitochondrial biogenesis in response to thermal acclimation in goldfish ( Carassius auratus ). We investigated the transcriptional response of PGC-1α, PGC-1β, and their antagonist the nuclear receptor interacting protein 1 ( RIP140), as well as the mRNA and protein patterns of DNA-binding proteins that bind PGC-1, including nuclear respiratory factors (NRF) 1 and 2, retinoid X receptor α (RXRα), estrogen-related receptor α (ERRα), thyroid receptor α-1 (TRα-1), PPARα, and PPARβ/δ, and the host cell factor 1 (HCF1), which links PGC-1 and NRF-2. Cold-acclimated (4°C) fish had higher COX activities (4.5-fold) and COX4-1 mRNA levels (3.5-fold per total RNA; 6.5-fold per gram tissue) than warm-acclimated (32°C) fish. The transcription factor patterns were profoundly influenced by changes in RNA per gram tissue (2-fold higher in cold fish) and nuclear protein content (2-fold higher in warm fish). In cold-acclimated fish, mRNA per gram tissue was elevated for PGC-1β, RIP140, NRF-1, HCF1, NRF-2α, NRF-2β-2, ERRα, PPAR β/δ, and RXRα, but other transcriptional regulators either did not change ( PGC-1α, PPARα) or even decreased ( TRα-1). Nuclear protein levels in cold-acclimated fish were higher only for NRF-1; other proteins were either unaffected (NRF-2α, ERRα) or decreased (NRF-2β1/2, TRα, RXRα). Collectively, these data support the role for NRF-1 in regulating cold-induced mitochondrial biogenesis in goldfish, with effects mediated by PGC-1β, rather than PGC-1α.

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