Degradation of constitutive transcription factors during apoptosis in rat thymocytes

1994 ◽  
Vol 72 (11-12) ◽  
pp. 639-648 ◽  
Author(s):  
Ian de Belle ◽  
Lucia Testolin ◽  
Siyaram Pandey ◽  
Christine Carson ◽  
P. Roy Walker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Significant Loss ◽  
Dna Binding Activity ◽  
The Stability ◽  
Induced Apoptosis

Dexamethasone (Dex) accelerates the rate of apoptosis in thymocytes by a process thought to require gene expression. Among the genes implicated in the regulation of this phenomenon are the immediate early genes such as c-fos and c-jun, whose expression is modulated by a complement of preexisting transcription factors. We have analyzed the DNA-binding activity of these constitutive transcription factors during Dex-induced apoptosis in thymocytes to assess their functionality. We observed a progressive loss of the DNA-binding proteins in parallel with the appearance of the characteristic morphological and biochemical features of apoptosis. At the same time we have found a general increase in the nuclear proteolytic activity concomitant with a significant loss of the nuclear nonhistone chromosomal proteins. Indeed, cotreatment of thymocytes with the nonspecific serine protease inhibitor phenyl-methylsulphonyl fluoride was able to partially protect the stability of the DNA-binding proteins and alter the expression of the c-fos and c-jun genes but did not inhibit apoptosis. Our results suggest that the action of a protease(s) is responsible for the degradation of constitutive transcription factors during Dex-induced apoptosis, rendering the death pathway irreversible.Key words: apoptosis, thymocytes, proteolysis, transcription factors, gene expression.

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 Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

2006 ◽  
Vol 71 (2) ◽  
pp. 135-141 ◽  
Author(s):  
Marija Mojsin ◽  
Jelena Djurovic ◽  
Isidora Petrovic ◽  
Aleksandar Krstic ◽  
Danijela Drakulic ◽  
...  
Keyword(s):  
Dna Binding ◽  
Magnetic Separation ◽  
Magnetic Particles ◽  
Binding Proteins ◽  
Rapid Identification ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
High Yield ◽  
Dna Binding Activity ◽  
Putative Binding Site

In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box) within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

scholarly journals 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.

Analysis of epidermal proteins for DNA-binding activity

1987 ◽  
Vol 7 (8) ◽  
pp. 645-651
Author(s):  
Prakash C. Supakar ◽  
Renuka Misra ◽  
Gopal M. Bhatnagar
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Soluble Extract ◽  
Newborn Rat ◽  
Molecular Weights ◽  
Dna Binding Activity ◽  
Cellulose Column

A group of DNA-binding proteins from the soluble extract of newborn rat epidermis have been separated by chromatography using DNA-cellulose columns. The electrophoretogram of the DNA-binding proteins eluted from a single stranded DNA-cellulose column shows five major proteins of molecular weights ranging between 25K to 40K. Both the epidermal protein filaggrin and most keratins, except two high molecular weight keratins, do not show in vitro DNA-binding activity.

scholarly journals On exploring effects of coevolving residues on DNA binding specificity of transcription factors

2021 ◽  
Author(s):  
Yizhao Luan ◽  
Zhi Xie
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Specificity ◽  
Binding Activity ◽  
Dna Interactions ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Binding Activity ◽  
Dna Binding Specificity ◽  
The Stability

Transcription factors (TFs) regulate gene expression by specifically binding to DNA targets. Many factors have been revealed to influence TF-DNA binding specificity. Coevolution of residues in proteins occurs due to a common evolutionary history. However, it is unclear how coevolving residues in TFs contribute to DNA binding specificity. Here, we systematically analyzed TF-DNA interactions from high-throughput experiments for seven TF families, including Homeobox, HLH, bZIP_1, Ets, HMG_box, zf-C4 and Zn_clus TFs. Based on TF-DNA interactions, we detected TF subclass determining sites (TSDSs) defining the heterogeneity of DNA binding preference for each TF family. We showed that the TSDSs were more likely to be coevolving with TSDSs than with non-TSDSs, particularly for Homeobox, HLH, Ets, bZIP_1 and HMG_box TF families. Mutation of the highly coevolving residues could significantly reduce the stability of TF-DNA complex. The distant residues from the DNA interface also contributed to TF-DNA binding activity. Overall, our study gave evidence of the functional importance of coevolved residues in refining transcriptional regulation and provided clues to the application of engineered DNA-binding domains and protein.

scholarly journals Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication

2008 ◽  
Vol 36 (10) ◽  
pp. 3341-3353 ◽  
Author(s):  
Paul Peixoto ◽  
Yang Liu ◽  
Sabine Depauw ◽  
Marie-Paule Hildebrand ◽  
David W. Boykin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Activity ◽  
Direct Inhibition ◽  
Selective Binding ◽  
Dna Binding Activity

Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1

1991 ◽  
Vol 11 (3) ◽  
pp. 1547-1552
Author(s):  
D Leshkowitz ◽  
M D Walker
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Activity ◽  
Insulin Gene ◽  
Hybrid Cells ◽  
Dna Binding Activity ◽  
Insulin Producing Cells ◽  
Insulin Gene Expression ◽  
Insulinoma Cells

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

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.

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