scholarly journals Effect of the nuclear factors EmBP1 and viviparous1 on the transcription of the Em gene in HeLa nuclear extracts.

1997 ◽  
Vol 9 (10) ◽  
pp. 1791-1803 ◽  
Author(s):  
M A Razik ◽  
R S Quatrano
Keyword(s):  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Em Gene

Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene

1988 ◽  
Vol 8 (10) ◽  
pp. 4362-4369
Author(s):  
Y Li ◽  
R F Shen ◽  
S Y Tsai ◽  
S L Woo
Keyword(s):  
Rat Liver ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Flanking Sequence ◽  
Tissue Specific ◽  
Hepatic Expression ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Alpha 1 Antitrypsin

The human alpha-1-antitrypsin (AAT) gene is expressed in the liver, and its deficiency causes pulmonary emphysema. We have demonstrated that its 5'-flanking region contains cis-acting elements capable of directing proper transcription in the presence of rat liver nuclear extract. The in vitro transcription system is tissue-specific in that the AAT promoter is functional in nuclear extracts prepared from the liver but not from HeLa cells. Experiments in which rat liver and HeLa nuclear extracts were mixed suggested the presence of a specific activator(s) in hepatocytes rather than a repressor(s) in nonproducing cells. Two protected regions were detected in the promoter by DNase I footprinting analysis with rat liver nuclear extracts. Region one spanned -78 to -52 and region two spanned -125 to -100 in the 5'-flanking sequence of the gene. By gel retardation assays with synthetic oligonucleotides, at least two distinct liver nuclear factors were identified, HNF-1 and HNF-2 (hepatocyte nuclear factors), which bound specifically to the first and second region, respectively. We present evidence that HNF-1 and HNF-2 are positively acting, tissue-specific transcription factors that regulate hepatic expression of the human AAT gene.

scholarly journals Yeast and Human RNA Polymerase II Elongation Complexes: Evidence for Functional Differences and Postinitiation Recruitment of Factors

2003 ◽  
Vol 2 (2) ◽  
pp. 318-327 ◽  
Author(s):  
Timothy S. Pardee ◽  
Mohamed A. Ghazy ◽  
Alfred S. Ponticelli
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Exonuclease Activity ◽  
Nascent Transcript ◽  
Dna Templates ◽  
Functional Differences ◽  
Nuclear Extracts ◽  
Nuclear Factors

ABSTRACT Immobilized DNA templates, glycerol gradient centrifugation, and native gel analysis were utilized to isolate and compare functional RNA polymerase II (RNAPII) elongation complexes from Saccharomyces cerevisiae and human cell nuclear extracts. Yeast elongation complexes blocked by incorporation of 3′-O-methyl-GTP into the nascent transcript exhibited a sedimentation coefficient of 35S, were less tightly associated to the template than their human counterparts, and displayed no detectable 3′-5′ exonuclease activity on the associated transcript. In contrast, blocked human elongation complexes were more tightly bound to the template, and multiple forms were identified, with the largest exhibiting a sedimentation coefficient of 60S. Analysis of the associated transcripts revealed that a subset of the human elongation complexes exhibited strong 3′-5′ exonuclease activity. Although isolated human preinitiation complexes were competent for efficient transcription, their ability to generate 60S elongation complexes was strikingly impaired. These findings demonstrate functional and size differences between S. cerevisiae and human RNAPII elongation complexes and support the view that the formation of mature elongation complexes involves recruitment of nuclear factors after the initiation of transcription.

scholarly journals Identification of distal silencing elements in the murine interferon-A11 gene promoter

1996 ◽  
Vol 317 (3) ◽  
pp. 697-706 ◽  
Author(s):  
Pascal ROFFET ◽  
Sébastien LOPEZ ◽  
Sébastien NAVARRO ◽  
Marie-Thérèse BANDU ◽  
Colette COULOMBEL ◽  
...  
Keyword(s):  
Transcriptional Response ◽  
Gene Promoter ◽  
Inhibitory Effect ◽  
Transient Transfection Assay ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Multigenic Family ◽  
Responsive Element ◽  
Related Proteins ◽  
Distal Promoter

The murine interferon-A11 (Mu IFN-A11) gene is a member of the IFN-A multigenic family. In mouse L929 cells, the weak response of the gene's promoter to viral induction is due to a combination of both a point mutation in the virus responsive element (VRE) and the presence of negatively regulating sequences surrounding the VRE. In the distal part of the promoter, the negatively acting E1E2 sequence was delimited. This sequence displays an inhibitory effect in either orientation or position on the inducibility of a virus-responsive heterologous promoter. It selectively represses VRE-dependent transcription but is not able to reduce the transcriptional activity of a VRE-lacking promoter. In a transient transfection assay, an E1E2-containing DNA competitor was able to derepress the native Mu IFN-A11 promoter. Specific nuclear factors bind to this sequence; thus the binding of trans-regulators participates in the repression of the Mu IFN-A11 gene. The E1E2 sequence contains an IFN regulatory factor (IRF)-binding site. Recombinant IRF2 binds this sequence and anti-IRF2 antibodies supershift a major complex formed with nuclear extracts. The protein composing the complex is 50 kDa in size, indicating the presence of IRF2 or antigenically related proteins in the complex. The Mu IFN-A11 gene is the first example within the murine IFN-A family, in which a distal promoter element has been identified that can negatively modulate the transcriptional response to viral induction.

A G-string positive cis-regulatory element in the LpS1 promoter binds two distinct nuclear factors distributed non-uniformly in Lytechinus pictus embryos

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1345-1355 ◽  
Author(s):  
M. Xiang ◽  
S.Y. Lu ◽  
M. Musso ◽  
G. Karsenty ◽  
W.H. Klein
Keyword(s):  
Binding Site ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Mobility Shift ◽  
Specific Expression ◽  
Lytechinus Pictus ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Electrophoretic Mobility Shift Assays

The LpS1 alpha and beta genes of Lytechinus pictus are activated at the late cleavage stage of embryogenesis, with LpS1 mRNAs accumulating only in lineages contributing to aboral ectoderm. We had shown previously that 762 bp of 5' flanking DNA from the LpS1 beta gene was sufficient for proper temporal and aboral ectoderm specific expression. In the present study, we identified a strong positive cis-regulatory element at −70 bp to −75 bp in the LpS1 beta promoter with the sequence (G)6 and a similar, more distal cis-element at −721 bp to −726 bp. The proximal ‘G-string’ element interacted with two nuclear factors, one specific to ectoderm and one to endoderm/mesoderm nuclear extracts, whereas the distal G-string element interacted only with the ectoderm factor. The ectoderm and endoderm/mesoderm G-string factors were distinct based on their migratory behavior in electrophoretic mobility shift assays, binding site specificities, salt optima and EDTA sensitivity. The proximal G-string element shared homology with a binding site for the mammalian transcription factor IF1, a protein that binds to negative cis-regulatory elements in the mouse alpha 1(I) and alpha 2(I) collagen gene promoters. Competition experiments using wild-type and mutant oligonucleotides indicated that the ectoderm G-string factor and IF1 have similar recognition sites. Partially purified IF1 specifically bound to an oligonucleotide containing the proximal G-string of LpS1 beta. From our results, we suggest that the ectoderm G-string factor, a member of the G-rich DNA-binding protein family, activates the LpS1 gene in aboral ectoderm cells by binding to the LpS1 promoter at the proximal G-string site.

Transcriptional regulation of the rat tissue type plasminogen activator gene: localization of DNA elements and nuclear factors mediating constitutive and cyclic AMP-induced expression

1993 ◽  
Vol 13 (1) ◽  
pp. 266-275
Author(s):  
M Ohlsson ◽  
G Leonardsson ◽  
X C Jia ◽  
P Feng ◽  
T Ny
Keyword(s):  
Cyclic Amp ◽  
Plasminogen Activator ◽  
Granulosa Cells ◽  
Constitutive Expression ◽  
Cell Types ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Type Plasminogen Activator

We have characterized tissue type plasminogen activator (tPA) promoter elements and nuclear factors required for follicle-stimulating hormone (FSH)-induced transcription of the rat tPA gene in granulosa cells and constitutive expression of the gene in the rat neuroblastoma cell line B103. Run-on transcription analysis of isolated nuclei revealed that B103 cells transcribe the tPA gene at a high and constitutive level, while FSH was found to induce tPA gene transcription in a rapid and transient manner in granulosa cells. The maximal FSH-induced transcription rate was obtained after 20 min and was similar in the absence or presence of the protein synthesis inhibitor cycloheximide. However, in the presence of cycloheximide, tPA transcription was not turned off but continued at a high rate for several hours. This phenomenon may at least partly explain the earlier finding that tPA mRNA is superinduced by FSH in the presence of cycloheximide. DNase I footprinting analysis of the first 621 bp of the tPA promoter revealed a total of six regions that interact with nuclear factors from B103 and granulosa cells. Deletion of the promoter region from positions -269 to -621, a region that includes the two most-upstream footprints, had no effect on constitutive or FSH-induced transcription in transient expression experiments. Nuclear extracts from both granulosa cells and B103 cells showed strong binding to a consensus cyclic AMP-responsive element (CRE) at positions -178 to -185 and a neighboring binding site for nuclear factor 1 (NF1) at positions -145 to -158. The factors binding to these two regions were identified as members of the CRE-binding protein and NF1 families of transcription factors, respectively. Footprints were also obtained over two GC boxes at positions -64 to -71 and -41 to -49. These footprints were more pronounced with nuclear extracts from B103 cells than with extracts from untreated or FSH-treated granulosa cells, but gel shift assays indicate that similar amounts of two distinct factors bind to the two GC boxes in both cell types. Transfection experiments using promoter constructs with inactivated promoter elements indicate that both the CRE and NF1 sites contribute to the FSH responsiveness of the rat tPA gene in granulosa cells, while only the NF1 site is important for constitutive expression in B103 cells. The two GC boxes were found to be necessary both for constitutive expression in B103 cells and for FSH-induced expression in granulosa cells, and inactivation of both GC boxes essentially eliminated the tPA promoter activity in both cell types.

scholarly journals Interaction of distinct nuclear proteins with sequences controlling the expression of polyomavirus early genes.

1986 ◽  
Vol 6 (5) ◽  
pp. 1401-1411 ◽  
Author(s):  
E Böhnlein ◽  
P Gruss
Keyword(s):  
Dna Sequences ◽  
Common Factor ◽  
Simian Virus 40 ◽  
Dna Interaction ◽  
Simian Virus ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Murine Sarcoma Virus ◽  
Lymphotropic Papovavirus

The interaction between cellular factors and polyoma virus (Py) DNA was investigated by using a gel retention assay. Nuclear extracts from various cell lines (NIH 3T3, NIH 3T6, LTK-, F9) contained proteins that formed specific and distinct complexes with Py B enhancer fragments of either wild-type or F9-1 mutant origin. The presence of an excess amount of other well-characterized DNA sequences, including the Py A enhancer, the murine sarcoma virus enhancer, and the simian virus 40 enhancer-promoter region, did not interfere with this protein-DNA interaction. However, a fragment previously defined as containing the lymphotropic papovavirus enhancer shares the binding of some common factor. This observation, in combination with the results of retention gel assays at different Mg2+ concentrations, indicates the interaction of several nuclear factors and Py DNA. The assay systems that were used allowed a distinction between some factors on the basis of their different biochemical and sequence requirements. The contact sites of these complexes were mapped to the B enhancer region of Py with Bal 31-derived mutant restriction fragments and ExoIII nuclease and are compatible with the functional domains determined in vivo.

scholarly journals Transcriptional regulation of the rat tissue type plasminogen activator gene: localization of DNA elements and nuclear factors mediating constitutive and cyclic AMP-induced expression.

1993 ◽  
Vol 13 (1) ◽  
pp. 266-275 ◽  
Author(s):  
M Ohlsson ◽  
G Leonardsson ◽  
X C Jia ◽  
P Feng ◽  
T Ny
Keyword(s):  
Cyclic Amp ◽  
Plasminogen Activator ◽  
Granulosa Cells ◽  
Constitutive Expression ◽  
Cell Types ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Type Plasminogen Activator

We have characterized tissue type plasminogen activator (tPA) promoter elements and nuclear factors required for follicle-stimulating hormone (FSH)-induced transcription of the rat tPA gene in granulosa cells and constitutive expression of the gene in the rat neuroblastoma cell line B103. Run-on transcription analysis of isolated nuclei revealed that B103 cells transcribe the tPA gene at a high and constitutive level, while FSH was found to induce tPA gene transcription in a rapid and transient manner in granulosa cells. The maximal FSH-induced transcription rate was obtained after 20 min and was similar in the absence or presence of the protein synthesis inhibitor cycloheximide. However, in the presence of cycloheximide, tPA transcription was not turned off but continued at a high rate for several hours. This phenomenon may at least partly explain the earlier finding that tPA mRNA is superinduced by FSH in the presence of cycloheximide. DNase I footprinting analysis of the first 621 bp of the tPA promoter revealed a total of six regions that interact with nuclear factors from B103 and granulosa cells. Deletion of the promoter region from positions -269 to -621, a region that includes the two most-upstream footprints, had no effect on constitutive or FSH-induced transcription in transient expression experiments. Nuclear extracts from both granulosa cells and B103 cells showed strong binding to a consensus cyclic AMP-responsive element (CRE) at positions -178 to -185 and a neighboring binding site for nuclear factor 1 (NF1) at positions -145 to -158. The factors binding to these two regions were identified as members of the CRE-binding protein and NF1 families of transcription factors, respectively. Footprints were also obtained over two GC boxes at positions -64 to -71 and -41 to -49. These footprints were more pronounced with nuclear extracts from B103 cells than with extracts from untreated or FSH-treated granulosa cells, but gel shift assays indicate that similar amounts of two distinct factors bind to the two GC boxes in both cell types. Transfection experiments using promoter constructs with inactivated promoter elements indicate that both the CRE and NF1 sites contribute to the FSH responsiveness of the rat tPA gene in granulosa cells, while only the NF1 site is important for constitutive expression in B103 cells. The two GC boxes were found to be necessary both for constitutive expression in B103 cells and for FSH-induced expression in granulosa cells, and inactivation of both GC boxes essentially eliminated the tPA promoter activity in both cell types.

scholarly journals Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene.

1988 ◽  
Vol 8 (10) ◽  
pp. 4362-4369 ◽  
Author(s):  
Y Li ◽  
R F Shen ◽  
S Y Tsai ◽  
S L Woo
Keyword(s):  
Rat Liver ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Flanking Sequence ◽  
Tissue Specific ◽  
Hepatic Expression ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Alpha 1 Antitrypsin

The human alpha-1-antitrypsin (AAT) gene is expressed in the liver, and its deficiency causes pulmonary emphysema. We have demonstrated that its 5'-flanking region contains cis-acting elements capable of directing proper transcription in the presence of rat liver nuclear extract. The in vitro transcription system is tissue-specific in that the AAT promoter is functional in nuclear extracts prepared from the liver but not from HeLa cells. Experiments in which rat liver and HeLa nuclear extracts were mixed suggested the presence of a specific activator(s) in hepatocytes rather than a repressor(s) in nonproducing cells. Two protected regions were detected in the promoter by DNase I footprinting analysis with rat liver nuclear extracts. Region one spanned -78 to -52 and region two spanned -125 to -100 in the 5'-flanking sequence of the gene. By gel retardation assays with synthetic oligonucleotides, at least two distinct liver nuclear factors were identified, HNF-1 and HNF-2 (hepatocyte nuclear factors), which bound specifically to the first and second region, respectively. We present evidence that HNF-1 and HNF-2 are positively acting, tissue-specific transcription factors that regulate hepatic expression of the human AAT gene.

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