scholarly journals Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo

1989 ◽  
Vol 9 (11) ◽  
pp. 4994-5002
Author(s):  
M C Blake ◽  
J C Azizkhan
Keyword(s):  
Dihydrofolate Reductase ◽  
Protein Interactions ◽  
Gene Promoter ◽  
Dhfr Gene ◽  
Recognition Sequence ◽  
Nuclear Extracts ◽  
Efficient Expression ◽  
Transcription Factor E2f

The dihydrofolate reductase (DHFR) gene encodes an enzyme important for metabolism and cell growth. We have found multiple DNA-protein interactions within the hamster DHFR gene promoter in vitro. These interactions occur over the consensus binding sites for two eucaryotic transcription factors. Sp1 and E2F. The DHFR E2F consensus site possesses a dyad symmetry and is unique in its location immediately 3' to the major transcription start site. The interaction of E2F with the DHFR promoter has been detected in HeLa nuclear extracts, confirmed by using partially purified E2F, and characterized by both enzymatic and chemical assays of the DNA-protein interaction. A mutation of the E2F recognition sequence which abolishes E2F binding to the DHFR promoter results in a two- to fivefold decrease of in vitro transcriptional activity and a fivefold reduction of DHFR promoter activity in transient-expression assays. Thus, the interaction of E2F with the DHFR promoter is required for efficient expression of the DHFR gene.

scholarly journals Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo.

1989 ◽  
Vol 9 (11) ◽  
pp. 4994-5002 ◽  
Author(s):  
M C Blake ◽  
J C Azizkhan
Keyword(s):  
Dihydrofolate Reductase ◽  
Protein Interactions ◽  
Gene Promoter ◽  
Dhfr Gene ◽  
Recognition Sequence ◽  
Nuclear Extracts ◽  
Efficient Expression ◽  
Transcription Factor E2f

The dihydrofolate reductase (DHFR) gene encodes an enzyme important for metabolism and cell growth. We have found multiple DNA-protein interactions within the hamster DHFR gene promoter in vitro. These interactions occur over the consensus binding sites for two eucaryotic transcription factors. Sp1 and E2F. The DHFR E2F consensus site possesses a dyad symmetry and is unique in its location immediately 3' to the major transcription start site. The interaction of E2F with the DHFR promoter has been detected in HeLa nuclear extracts, confirmed by using partially purified E2F, and characterized by both enzymatic and chemical assays of the DNA-protein interaction. A mutation of the E2F recognition sequence which abolishes E2F binding to the DHFR promoter results in a two- to fivefold decrease of in vitro transcriptional activity and a fivefold reduction of DHFR promoter activity in transient-expression assays. Thus, the interaction of E2F with the DHFR promoter is required for efficient expression of the DHFR gene.

RNA binding by Sxl proteins in vitro and in vivo

1994 ◽  
Vol 14 (7) ◽  
pp. 4975-4990
Author(s):  
M E Samuels ◽  
D Bopp ◽  
R A Colvin ◽  
R F Roscigno ◽  
M A Garcia-Blanco ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Rna Binding ◽  
Polytene Chromosomes ◽  
Regulatory Sequences ◽  
Acceptor Site ◽  
Nuclear Extracts ◽  
Preferential Binding

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

scholarly journals Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter.

1990 ◽  
Vol 10 (12) ◽  
pp. 6632-6641 ◽  
Author(s):  
M C Blake ◽  
R C Jambou ◽  
A G Swick ◽  
J W Kahn ◽  
J C Azizkhan
Keyword(s):  
Dihydrofolate Reductase ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Start Site ◽  
Transcriptional Initiation ◽  
Reductase Gene ◽  
Gc Box

Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.

scholarly journals In vitro transcription and delimitation of promoter elements of the murine dihydrofolate reductase gene.

1986 ◽  
Vol 6 (7) ◽  
pp. 2392-2401 ◽  
Author(s):  
P J Farnham ◽  
R T Schimke
Keyword(s):  
Dihydrofolate Reductase ◽  
In Vitro Transcription ◽  
S Phase ◽  
Specific Factor ◽  
Or Efficiency ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Reductase Gene

We have developed an in vitro transcription system for the murine dihydrofolate reductase gene. Although transcription in vitro from a linearized template was initiated at the same start sites as in vivo, the correct ratios were more closely approximated when a supercoiled template was used. In addition, whereas the dihydrofolate reductase promoter functions bidirectionally in vivo, the initiation signals directed unidirectional transcription in this in vitro system. The dihydrofolate reductase gene does not have a typical TATA box, but has four GGGCGG hexanucleotides within 300 base pairs 5' of the AUG codon. Deletion analysis suggested that, although sequences surrounding each of the GC boxes could specify initiation approximately 40 to 50 nucleotides downstream, three of the four GC boxes could be removed without changing the accuracy or efficiency of initiation at the major in vivo site. The dihydrofolate reductase promoter initiated transcription very rapidly in vitro, with transcripts visible by 1 min and almost maximal by 2 min at 30 degrees C with no preincubation. Nuclear extracts prepared from cells blocked in the S phase by aphidicolin or from adenovirus-infected cells at 16 h postinfection had enhanced dihydrofolate reductase transcriptional activity. This increased in vitro transcription mimicked the increase in dihydrofolate reductase mRNA seen in S-phase cells and suggested the presence of a cell-cycle-specific factor(s) which stimulated transcription from the dihydrofolate reductase gene.

scholarly journals RNA binding by Sxl proteins in vitro and in vivo.

1994 ◽  
Vol 14 (7) ◽  
pp. 4975-4990 ◽  
Author(s):  
M E Samuels ◽  
D Bopp ◽  
R A Colvin ◽  
R F Roscigno ◽  
M A Garcia-Blanco ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Rna Binding ◽  
Polytene Chromosomes ◽  
Regulatory Sequences ◽  
Acceptor Site ◽  
Nuclear Extracts ◽  
Preferential Binding

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

scholarly journals Mechanism of action of a repressor of dioxin-dependent induction of Cyp1a1 gene transcription.

1992 ◽  
Vol 12 (5) ◽  
pp. 2115-2123 ◽  
Author(s):  
A J Watson ◽  
K I Weir-Brown ◽  
R M Bannister ◽  
F F Chu ◽  
S Reisz-Porszasz ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
High Salt ◽  
In Vitro Assays ◽  
Ah Receptor ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Gel Mobility Shift

A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.

scholarly journals Nucleosomes accelerate transcription factor dissociation

2013 ◽  
Vol 42 (5) ◽  
pp. 3017-3027 ◽  
Author(s):  
Yi Luo ◽  
Justin A. North ◽  
Sean D. Rose ◽  
Michael G. Poirier
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Protein Complexes ◽  
High Specificity ◽  
Dissociation Rate ◽  
Duplex Dna ◽  
Recognition Sequence ◽  
Nucleosome Arrays

AbstractTranscription factors (TF) bind DNA-target sites within promoters to activate gene expression. TFs target their DNA-recognition sequences with high specificity by binding with resident times of up to hours in vitro. However, in vivo TFs can exchange on the order of seconds. The factors that regulate TF dynamics in vivo and increase dissociation rates by orders of magnitude are not known. We investigated TF binding and dissociation dynamics at their recognition sequence within duplex DNA, single nucleosomes and short nucleosome arrays with single molecule total internal reflection fluorescence (smTIRF) microscopy. We find that the rate of TF dissociation from its site within either nucleosomes or nucleosome arrays is increased by 1000-fold relative to duplex DNA. Our results suggest that TF binding within chromatin could be responsible for the dramatic increase in TF exchange in vivo. Furthermore, these studies demonstrate that nucleosomes regulate DNA–protein interactions not only by preventing DNA–protein binding but by dramatically increasing the dissociation rate of protein complexes from their DNA-binding sites.

scholarly journals Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter

1990 ◽  
Vol 10 (12) ◽  
pp. 6632-6641
Author(s):  
M C Blake ◽  
R C Jambou ◽  
A G Swick ◽  
J W Kahn ◽  
J C Azizkhan
Keyword(s):  
Dihydrofolate Reductase ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Start Site ◽  
Transcriptional Initiation ◽  
Reductase Gene ◽  
Gc Box

Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.

Mechanism of action of a repressor of dioxin-dependent induction of Cyp1a1 gene transcription

1992 ◽  
Vol 12 (5) ◽  
pp. 2115-2123
Author(s):  
A J Watson ◽  
K I Weir-Brown ◽  
R M Bannister ◽  
F F Chu ◽  
S Reisz-Porszasz ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
High Salt ◽  
In Vitro Assays ◽  
Ah Receptor ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Gel Mobility Shift

A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.

In vitro transcription and delimitation of promoter elements of the murine dihydrofolate reductase gene

1986 ◽  
Vol 6 (7) ◽  
pp. 2392-2401
Author(s):  
P J Farnham ◽  
R T Schimke
Keyword(s):  
Dihydrofolate Reductase ◽  
In Vitro Transcription ◽  
S Phase ◽  
Specific Factor ◽  
Or Efficiency ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Reductase Gene

We have developed an in vitro transcription system for the murine dihydrofolate reductase gene. Although transcription in vitro from a linearized template was initiated at the same start sites as in vivo, the correct ratios were more closely approximated when a supercoiled template was used. In addition, whereas the dihydrofolate reductase promoter functions bidirectionally in vivo, the initiation signals directed unidirectional transcription in this in vitro system. The dihydrofolate reductase gene does not have a typical TATA box, but has four GGGCGG hexanucleotides within 300 base pairs 5' of the AUG codon. Deletion analysis suggested that, although sequences surrounding each of the GC boxes could specify initiation approximately 40 to 50 nucleotides downstream, three of the four GC boxes could be removed without changing the accuracy or efficiency of initiation at the major in vivo site. The dihydrofolate reductase promoter initiated transcription very rapidly in vitro, with transcripts visible by 1 min and almost maximal by 2 min at 30 degrees C with no preincubation. Nuclear extracts prepared from cells blocked in the S phase by aphidicolin or from adenovirus-infected cells at 16 h postinfection had enhanced dihydrofolate reductase transcriptional activity. This increased in vitro transcription mimicked the increase in dihydrofolate reductase mRNA seen in S-phase cells and suggested the presence of a cell-cycle-specific factor(s) which stimulated transcription from the dihydrofolate reductase gene.

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