Identification of two transcription factors that bind to specific elements in the promoter of the adenovirus early-region 4

1988 ◽  
Vol 8 (3) ◽  
pp. 1290-1300
Author(s):  
H Watanabe ◽  
T Imai ◽  
P A Sharp ◽  
H Handa
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Base Pairs ◽  
Early Region ◽  
Dnase I Footprinting ◽  
Nuclear Extracts ◽  
Early Region 4

Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.

scholarly journals Identification of two transcription factors that bind to specific elements in the promoter of the adenovirus early-region 4.

1988 ◽  
Vol 8 (3) ◽  
pp. 1290-1300 ◽  
Author(s):  
H Watanabe ◽  
T Imai ◽  
P A Sharp ◽  
H Handa
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Base Pairs ◽  
Early Region ◽  
Dnase I Footprinting ◽  
Nuclear Extracts ◽  
Early Region 4

Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.

Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene

1991 ◽  
Vol 11 (5) ◽  
pp. 2832-2841
Author(s):  
N Mechti ◽  
M Piechaczyk ◽  
J M Blanchard ◽  
P Jeanteur ◽  
B Lebleu
Keyword(s):  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Rna Transcripts ◽  
First Intron ◽  
Nuclear Extracts ◽  
Intron 1 ◽  
A Cell ◽  
Sequence Requirements

A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.

A downstream-element-binding factor facilitates assembly of a functional preinitiation complex at the simian virus 40 major late promoter

1990 ◽  
Vol 10 (7) ◽  
pp. 3635-3645
Author(s):  
D E Ayer ◽  
W S Dynan
Keyword(s):  
Rna Polymerase Ii ◽  
Simian Virus 40 ◽  
Initiation Site ◽  
Simian Virus ◽  
Recognition Sequence ◽  
Base Pairs ◽  
Transcriptional Initiation ◽  
Nuclear Extracts ◽  
Major Late Promoter ◽  
Time Required

Recent work has shown that many promoters recognized by eucaryotic RNA polymerase II contain essential sequences located downstream of the transcriptional initiation site. We show here that the activity of a promoter element centered 28 base pairs downstream of the simian virus 40 major late initiation site appears to be mediated by a DNA-binding protein, which was isolated by affinity chromatography from HeLa cell nuclear extracts. In the absence of the other components of the transcriptional machinery, the protein bound specifically but weakly to its recognition sequence, with a Kd of approximately 10(-8) M. Analysis of kinetic data showed that mutation of the downstream element decreased the number of functional preinitiation complexes assembled at the promoter without significantly altering the time required for half the complexes to assemble. This suggests that in the absence of the downstream activating protein, preinitiation complexes are at least partially assembled but are not transcriptionally competent.

Effect of DNA supercoiling on in vitro transcription from the adenovirus early region 4

FEBS Letters ◽  
1989 ◽  
Vol 249 (1) ◽  
pp. 17-20 ◽  
Author(s):  
Hiroshi Handa ◽  
Hajime Watanabe ◽  
Yoshiaki Suzuki ◽  
Susumu Hirose
Keyword(s):  
Dna Supercoiling ◽  
In Vitro Transcription ◽  
Early Region ◽  
Early Region 4

New Approaches for Mapping Epigenome Dynamics

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-21-SCI-21
Author(s):  
Steven Henikoff
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Conflicts Of Interest ◽  
Regulation Of Transcription ◽  
Base Pairs ◽  
Simple Method ◽  
Genome Wide ◽  
Resolution Mapping

Abstract The protein complexes that package our genomes must be mobilized for active processes to occur, including replication and transcription, but until recently we have only had a static, low resolution view of the "epigenome". Genomes are packaged into nucleosomes, octamers of four core histones wrapped by 147 base pairs of DNA. Nucleosomes present obstacles to transcription, which over genes is the RNA Polymerase II (RNAPII) complex, and one current challenge is to understand what happens to a nucleosome when RNAPII transcribes through the DNA that it occupies. We study this process by developing methods for following nucleosomes as they are evicted and replaced. Among the factors that we have implicated in the process is torsional stress, which we can now measure genome-wide. RNAPII movement can unwrap nucleosomes and thus destabilize them, causing them to be occasionally evicted and replaced. Interestingly, we find that destabilization of nucleosomes during transcription is enhanced by anthracycline compounds, widely used chemotherapeutic drugs that intercalate between DNA base pairs, thus suggesting a new mechanism for cell killing during chemotherapy. We are also interested in what happens to RNAPII during its encounter with a nucleosomes. In vitro, RNAPII cannot transcribe completely through a nucleosome, but rather stalls as it tries to unwrap the DNA from around the core. We have been studying this process in vivo, and have developed a simple method for precisely mapping RNAPII genome-wide. We have used this method to show exactly where RNAPII stalls as it unwraps a nucleosome in vivo, surprisingly in a different place in vivo from where it stalls in vitro. We also have discovered that a variant histone, H2A.Z, which is found in essentially all eukaryotes, helps to reduce the nucleosome barrier to transcription, and in this way may modulate transcription. Other protein components of the epigenome involved in dynamic processes are nucleosome remodelers, which use the energy of ATP to slide or even evict nucleosomes from DNA. Some remodelers help RNAPII get started and others help it overcome the nucleosome barrier to transcription, and by mapping them at base-pair resolution, we can gain insight into how they act. We have also applied our high-resolution mapping tools to transcription factors, which bind DNA at specific sites to regulate transcription and other processes. Our ability to achieve high spatial and temporal resolution mapping of the binding and action of nucleosomes, transcription factors, remodelers and RNAPII provides us with a detailed picture of epigenome dynamics. By using these tools we are beginning to understand how DNA sequence and conformation are recognized for regulation of transcription and other epigenomic processes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro.

1987 ◽  
Vol 7 (10) ◽  
pp. 3402-3408 ◽  
Author(s):  
Z D Cao ◽  
E A Barron ◽  
A J Carillo ◽  
Z D Sharp
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Expression System ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
Nuclear Extracts ◽  
Prolactin Gene ◽  
Flanking Region ◽  
Imperfect Palindrome

We present evidence for the existence of prolactin upstream factor 1 (PUF-1) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived GH3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. GH3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28-bp region (-63 to -36)which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed.

scholarly journals Multiple sequence elements are required for maximal in vitro transcription of a human histone H2B gene.

1986 ◽  
Vol 6 (10) ◽  
pp. 3329-3340 ◽  
Author(s):  
H L Sive ◽  
N Heintz ◽  
R G Roeder
Keyword(s):  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Multiple Sequence ◽  
Histone H2b ◽  
Cis Acting ◽  
Promoter Sequences ◽  
Nuclear Extracts ◽  
Sequence Elements

As part of our studies on the cell cycle regulation of human histone gene expression, we examined the elements governing transcription of a human histone H2B gene in nuclear extracts derived from human HeLa cells. Circular templates were transcribed at 5- to 10-fold higher levels than were linear templates. A series of deletion, linker-substitution, and point mutants defined cis-acting promoter sequences that were recognized in nuclear extracts. These sequences extended from 118 to 21 base pairs 5' to the transcription initiation site. Elements recognized included (from 5' to 3') a series of direct repeats, a CCAAT homology, a human histone-specific hexamer, an H2B consensus element, and a TATA box. Sequence elements 5' to the hexamer were required for its function. In contrast, the H2B consensus element could function independently of more-5' promoter elements and in turn was essential for the function of upstream elements. An interesting feature of this consensus is that its core octanucleotide (ATTTGCAT) is found in several nonhistone genes. By comparison with functional elements in an H4 promoter, we infer that a combinatorial interaction of general and gene-specific factors may contribute to the S-phase elevation of H2B transcription.

A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes

1988 ◽  
Vol 8 (10) ◽  
pp. 4441-4449
Author(s):  
A K Lofquist ◽  
A D Garcia ◽  
S J Sharp
Keyword(s):  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Trna Genes ◽  
Selection Mechanism ◽  
Base Pairs ◽  
Discrete Region ◽  
Flanking Sequences ◽  
Actual Sequence

We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5Asn genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters.

Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3402-3408
Author(s):  
Z D Cao ◽  
E A Barron ◽  
A J Carillo ◽  
Z D Sharp
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Expression System ◽  
In Vitro Transcription ◽  
Gh3 Cells ◽  
Nuclear Extracts ◽  
Prolactin Gene ◽  
Flanking Region ◽  
Imperfect Palindrome

We present evidence for the existence of prolactin upstream factor 1 (PUF-1) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived GH3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. GH3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28-bp region (-63 to -36)which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed.

Sign in / Sign up

Export Citation Format

Share Document