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R Treisman

Publications and source records attributed to R Treisman.

At least 55 records · Page 3Linked to original sources
PubMed1992

Spatial flexibility in ternary complexes between SRF and its accessory proteins.

We investigated the sequence requirements for ternary complex formation by the transcription factor SRF and its Ets domain accessory factors Elk-1 and SAP-1. Ternary complex formation is specified by an SRF consensus site CC(A/T)6GG and a neighbouring Ets motif (C/A)(C/A)GGA(A/T), which is contacted by Elk-1/SAP-1. Both the spacing of these sequences and their relative orientation can be substantially altered with little effect on the efficiency of ternary complex formation. Efficient ternary complex formation by Elk-1 is mediated by the B box, a conserved 21 amino acid region located 50 residues C-terminal to the Ets domain, which also acts to inhibit autonomous DNA binding. Binding studies with the isolated Ets domains indicate that ternary complex formation compensates for low affinity Ets domain-DNA interactions. Several naturally occurring SREs containing Ets motifs at different locations to that in the human c-fos SRE allow SAP-1 and Elk-1 recruitment in vitro. We discuss the mechanism of ternary complex formation.

Base Sequence↗
PubMed1992

The serum response element.

The promoters of many genes whose transcription is rapidly and transiently induced following growth factor or mitogen stimulation of susceptible cells contain a common regulatory element, the serum response element (SRE). As the transcription factors that bind the SRE and the signalling molecules that affect its activity are characterized in more detail, the major challenge is to elucidate the signalling pathways that link cell-surface receptors to the SRE, and to determine the mechanism by which signalling events modulate transcription factor activity.

Animals↗
PubMed1992

The SRF and MCM1 transcription factors.

The mammalian transcription factor SRF (serum-response factor) and the related Saccharomyces cerevisiae transcription factor MCM1 are the prototypes of a new class of dimeric DNA-binding proteins. Their function is regulated in part by the interactions of their DNA-binding domains with accessory proteins. Recent work has advanced the functional characterization of the contributions of SRF and MCM1, and their accessory proteins to transcriptional activation.

Amino Acid Sequence↗
PubMed1991

Human SRF-related proteins: DNA-binding properties and potential regulatory targets.

Serum response factor (SRF) is a transcription factor that binds the sequence CC(A/T)6GG found in a number of growth factor-inducible and muscle-specific promoters. We describe the isolation and characterization of cDNA clones encoding a family of three human SRF-related DNA-binding proteins. Each of these RSRF (related to SRF) proteins contains an 86-amino-acid amino-terminal region related to the SRF DNA-binding domain: In RSRFC4 and RSRFC9, this region is identical, whereas that present in RSRFR2 differs by seven conservative amino acid substitutions. The DNA-binding specificity of the RSRF proteins, which recognize the consensus sequence CTA(A/T)4TAG, is distinct from that of SRF. The entire RSRF common region is required for DNA binding, and the differential sequence specificity of the RSRFs and SRF is the result of differences in the basic amino-terminal part of this domain. The RSRF proteins bind DNA as dimers and can dimerize with one another but not with SRF. Although the RSRF mRNAs are expressed in many cell types, RSRFR2 mRNA is expressed at elevated levels in several B-cell lines. Consistent with this, extracts from many cell types form CTA(A/T)4TAG-binding complexes that contain RSRF proteins, and oligonucleotides containing RSRF-binding sites function as promoter elements in transfection assays. Like SRF-binding sites, RSRF-binding sites are found in the regulatory sequences of a number of growth factor-inducible and muscle-specific genes, and we show that RSRF polypeptides are components of previously characterized binding activities that interact with these elements. We discuss the potential role of RSRF proteins in the regulation of these genes.

3T3 Cells↗
PubMed1990

A sensitive method for the determination of protein-DNA binding specificities.

We describe a sensitive and rapid method for determination of the sequence specificity of DNA binding proteins. The method allows recovery of specific sites using the small amounts of protein present in crude cell extracts or produced by cell-free translation reactions. Extract proteins are incubated with a pool of random sequence oligonucleotides, complexes purified by immunoprecipitation, and bound DNA amplified by the Polymerase Chain Reaction (PCR). This DNA is then used in further rounds of binding, immunoprecipitation, and amplification, until specific binding is detectable. With the transcription factor SRF as a model system, we demonstrate that authentic high affinity binding sites are recovered, and show that epitope tagging can be used to allow recovery of sites when specific antibodies are unavailable. We also show that specific sites bound by the Fos protein, which binds DNA with high affinity only when complexed with other polypeptides, are easily recovered by this technique.

Animals↗
PubMed1990

The SRE: a growth factor responsive transcriptional regulator.

Cellular immediate-early genes are rapidly and transiently activated when cells are stimulated with many different growth factors. This review examines the function of a short immediate-early gene regulatory sequence, the Serum Response Element (SRE), that is sufficient for this transient transcriptional activation. The structures of SREs and SRE-containing promoters are presented, followed by a summary of SRE regulatory properties. The roles of other SRE-like regulatory sequences in mammalian and yeast cells are then considered. The properties of SRE binding proteins are reviewed, followed by a discussion of the effects of mutations on both SRE function and protein binding. Finally, possible models for SRE function are discussed.

Base Sequence↗
PubMed1989

Muscle-specific (CArG) and serum-responsive (SRE) promoter elements are functionally interchangeable in Xenopus embryos and mouse fibroblasts.

The Xenopus cardiac actin gene contains four copies of a promoter element, the CArG box, which is conserved amongst striated muscle actin genes and is essential for tissue-specific expression in the developing Xenopus embryo. Our aim is to identify embryo and muscle proteins that interact with the CArG box as a step towards understanding the molecular basis of this developmentally regulated gene expression. The CArG box shares some sequence similarity with the Serum Response Element (SRE), which mediates the transcriptional activation by serum of genes such as c-fos and cytoskeletal actin. We show here that the most proximal cardiac actin CArG box is recognized by the same binding activity as the cytoskeletal actin SRE in nuclear extracts from both Xenopus embryos and mammalian muscle cells. This activity is indistinguishable from the previously characterized HeLa cell SRE-binding activity, Serum Response Factor (SRF). Importantly, we extend these in vitro studies to demonstrate that the CArG box and SRE are functionally interchangeable, both in Xenopus embryos and mouse fibroblasts. This implies that the CArG box and SRE can bind the same protein in vivo, as well as in vitro. Our results identify an SRF-like protein as a CArG box-binding factor and we discuss the implication that a common mechanism may be utilized in both muscle-specific gene expression and serum-responsive transcription.

Actins↗
PubMed1988

Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element.

The serum response element (SRE) is a sequence required for transient transcriptional activation of genes in response to growth factors. We have isolated cDNA clones encoding serum response factor (SRF), a ubiquitous nuclear protein that binds to the SRE. The SRF gene is highly conserved through evolution, and in cultured cells its transcription is itself transiently increased following serum stimulation. A cDNA clone of SRF expressed in vitro generates protein that forms complexes indistinguishable from those formed with HeLa cell SRF, as judged by DNA binding specificity and the ability to promote SRE-dependent in vitro transcription. SRF binds DNA as a dimer, and the DNA binding/dimerization domain of the protein exhibits striking homology to two yeast regulatory proteins.

Base Sequence↗
PubMed1988

Fos C-terminal mutations block down-regulation of c-fos transcription following serum stimulation.

Transient accumulation of c-fos RNA following serum stimulation requires both a conserved 5' regulatory element and sequences at the 3' end of the gene. Here we show that mutations at the C terminus of Fos protein, of the type found in a virally-transduced actively transforming Fos variant, prevent the rapid down-regulation of c-fos transcription that occurs following serum-induced activation. Fos mutants that prevent down-regulation are dominant, acting in trans to prevent down-regulation of a co-transfected c-fos gene. Co-transfection experiments suggest that this effect is mediated by multiple sequence elements in the 5'-flanking region. Analysis of different Fos mutants showed that replacement of Fos sequences C-terminal to amino acid 337 with heterologous polypeptide, rather than simple truncation of the protein, is required to produce mutants defective in down-regulation. The results are discussed with reference to transformation by Fos.

Amino Acid Sequence↗
PubMed1988

Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3' AU-rich sequences.

The c-fos proto-oncogene provides a good system to study the processes underlying messenger RNA degradation. After growth factor stimulation of susceptible cells, the c-fos transcription rate transiently increases from a low basal level by as much as 50-fold, producing a large amount of exceedingly unstable c-fos mRNA that is rapidly degraded. Here, we investigate the c-fos mRNA degradation process, and find that: (1) ongoing translation of the c-fos mRNA itself is required for its degradation; (2) after synthesis, the mRNA poly(A) tail is rapidly removed, in a translation-dependent manner, leading to accumulation of apparently deadenylated RNA; (3) deletion or replacement of an AU-rich sequence at the mRNA 3' end significantly stabilizes the mRNA; (4) deletion of the 3' AU-rich sequences dramatically slows the poly(A) shortening rate. These results suggest that the 3' AU-rich sequences act to destabilize the mRNA by directing rapid removal of the mRNA poly(A) tract.

Base Sequence↗
PubMed1987

Identification and purification of a polypeptide that binds to the c-fos serum response element.

A short DNA sequence element, the serum response element (SRE), which binds a nuclear protein, serum response factor (SRF), mediates transient transcriptional activation of c-fos and cytoskeletal actin genes in response to serum factors. Variant SRE sequences with different affinities for HeLa cell SRF were synthesised. Binding of SRF to these sites in vitro correlates with the transcriptional properties of these elements in vivo, suggesting that SRF is a positively acting transcription factor. A 67-kd polypeptide was identified as the DNA-binding component of SRF by photoactivated DNA-protein cross-linking in vitro. The high affinity SRF-binding site was used to purify this polypeptide to virtual homogeneity in a single DNA affinity chromatography step.

Animals↗
PubMed1987

Xenopus cytoskeletal actin and human c-fos gene promoters share a conserved protein-binding site.

Xenopus laevis cytoskeletal actin gene promoters contain a 20-bp sequence homologous to the serum response element (SRE) required for transient human c-fos gene transcription in response to serum factors. Both sequences bind the same factor in HeLa cell extracts, as shown by binding competition, DNase I and dimethylsulphate (DMS) protection and DMS interference assays. A similar protein is present in Xenopus laevis oocytes. Sequences containing the SRE homology are essential for constitutive activity of the actin promoter in both Xenopus and mouse cells, and a synthetic SRE functions as a promoter element in these cells. In mouse cells, transcription of both transfected Xenopus actin and actin/c-fos fusion genes is activated following serum stimulation. These data suggest that the SRE and its cognate protein form part of a regulatory pathway that has been highly conserved during evolution.

Actins↗
PubMed1986

Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors.

Transient transcriptional activation of the c-fos gene following serum stimulation of susceptible cells requires a conserved DNA element located 300 bp 5' to the mRNA cap site. A DNA-binding gel electrophoresis assay was used to detect a protein(s) in HeLa cell nuclear extracts that specifically binds to the 5' activating element. The protein recognizes a region of dyad symmetry within the 5' activating element, defined by binding competition, dimethylsulphate (DMS) interference and DNAase I and DMS protection studies. A single 22 bp synthetic copy of the dyad symmetry element will both compete efficiently for protein binding and restore serum regulation to c-fosH genes that lack the 5' activating element.

Base Sequence↗
PubMed1986

Beta-thalassemia: analysis of mRNA precursors of a mutant human globin gene with defective splicing using peripheral blood nucleated red blood cells.

Studies on the effects of thalassemic mutations on gene function in vivo have clinical as well as scientific implications. Usually these studies have been performed on nucleated red blood cell (RBC) precursors normally present in bone marrow. Many patients with beta-thalassemia are splenectomized and may have high levels of nucleated RBC, orthochromatic normoblasts, in their peripheral blood (1-5% of total RBC). The possibility of exploiting these cells instead of bone marrow as a source for nuclear and cytoplasmic RNA for expression studies was investigated. A simple procedure was developed for enrichment for normoblasts in blood samples withdrawn from patients prior to transfusion. Globin transcripts were analyzed in RNA purified from 12 patients. Unspliced precursor beta-mRNA molecules were observed in a patient with beta o-thalassemia, homozygous for a mutation at the 5' IVS2 splice site of the beta-globin gene. Detailed analyses showed that his mature beta-mRNA was larger than normal, and that a cryptic 5' splice site, approximately 50 nucleotides downstream from the normal one, was utilized. We conclude that peripheral blood can be used as a reliable source of RNA for the analysis of the effects of beta-thalassemia mutations on gene expression and the relationship to the clinical condition. Moreover, this procedure facilitates the comparison of in vivo gene expression with the results obtained from DNA transfection experiments with cloned beta-thalassemia genes.

Adolescent↗
PubMed1985

Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5' element and c-fos 3' sequences.

Transcription of the c-fos gene is transiently activated to generate large amounts of unstable c-fos RNA when quiescent fibroblasts are stimulated by polypeptide mitogens or whole serum. A cloned human c-fos gene (c-fosH) transfected into mouse fibroblasts is regulated in a similar manner. An element essential for transcription activation is located between nucleotides -332 and -276 relative to the mRNA cap site. This element has properties similar to those of previously characterized transcription enhancer elements. However, replacement of the 5' activating element by enhancers from SV40 or Moloney murine leukemia virus does not allow regulated c-fosH expression. The study of fusion genes showed that in addition to the 5' activating element, transient accumulation of c-fosH RNA following serum stimulation requires sequences at the 3' end of the c-fosH gene.

Animals↗
PubMed1984

Human alpha- and beta-globin gene transcription in mouse erythroleukaemia cells.

Human beta-globin genes introduced into mouse erythroleukaemia (MEL) cells by DNA co-transformation are correctly regulated when erythroid cell differentiation is induced by dimethylsulphoxide (DMSO). In contrast, cloned human alpha-globin genes are efficiently transcribed in MEL cells before induction, and no increase in the level alpha-globin mRNA is observed when the cells differentiate. These observations suggest that the mechanisms by which alpha- and beta-globin genes are activated during erythroid cell differentiation are fundamentally different. Analysis of the transcription of hybrid human alpha-beta-globin genes in MEL cells revealed that the sequences responsible for differences in transcription of the intact alpha- and beta-globin genes are located on the 3' side of the mRNA capping site of the two genes, suggesting that cis-acting regulatory sequences are located within the structural genes.

Animals↗
PubMed1984

Differences in human alpha- and beta-globin gene expression in mouse erythroleukemia cells: the role of intragenic sequences.

Human beta-globin genes introduced into mouse erythroleukemia (MEL) cells by DNA cotransformation are correctly regulated when erythroid cell differentiation is induced by dimethylsulfoxide (DMSO). In contrast, cloned human alpha-globin genes are efficiently transcribed in MEL cells prior to induction, and no increase in the level of alpha-globin mRNA is observed when the cells differentiate. These observations suggest that the mechanisms by which alpha- and beta-globin genes are activated during erythroid cell differentiation are fundamentally different. Analysis of the transcription of hybrid human alpha/beta-globin genes in MEL cells revealed that the sequences responsible for differences in transcription of the intact alpha- and beta-globin genes are located on the 3' side of the mRNA capping site of the two genes, suggesting that cis-acting regulatory sequences are located within the structural genes.

Animals↗