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R E Kingston

Publications and source records attributed to R E Kingston.

At least 19 recordsLinked to original sources
PubMed1992

Nucleosome core displacement in vitro via a metastable transcription factor-nucleosome complex.

In order to function, transcription factors must compete for DNA binding with structural components of chromatin, including nucleosomes. Mechanisms that could be used in this competition have been characterized with the use of the DNA binding domain of the yeast GAL4 protein. The binding of GAL4 to a nucleosome core resulted in a ternary complex containing GAL4, the core histone proteins, and DNA. This ternary complex was unstable; upon the addition of nonspecific competitor DNA, it dissociated into either the original nucleosome core particle or GAL4 bound to naked DNA. Nucleosome core destabilization by GAL4 did not require a transcriptional activation domain. These data demonstrate the displacement of nucleosome cores as a direct result of binding by a regulatory factor. Similar mechanisms might affect the establishment of factor occupancy of promoters and enhancers in vivo.

Base Sequence↗
PubMed1992

Mammalian promoter element function in the fission yeast Schizosaccharomyces pombe.

We have analyzed the function of several mammalian promoter elements in the fission yeast, Schizosaccharomyces pombe. Mutants of the human HSP70 promoter were introduced into S. pombe as single copy integrants at a specific location. Transcription initiation sites utilized in S. pombe with the HSP70 TATA element were similar to those used in mammalian cells. Of three mammalian TATA elements tested, only the HSP70 TATA element functioned in S. pombe. The adenovirus Ella TATA element had little or no activity in S. pombe, indicating that S. pombe is deficient in the factor(s) necessary for recognition of this element. Of upstream promoter elements tested, the CCAAT, Sp1 binding, ATF binding and heat shock elements were functional in S. pombe. Strains containing mutant promoters fused to the ble gene were used to demonstrate that phleomycin can be used as a graded selection in S. pombe. These data demonstrate that S. pombe should provide a useful system in which to characterize and isolate mammalian factors involved in initiation site determination and transcriptional regulation.

3T3 Cells↗
PubMed1992

DNA binding activities of c-Myc purified from eukaryotic cells.

c-Myc is a nuclear phosphoprotein which contains both a leucine zipper and a helix-loop-helix dimerization motif. These are adjacent to a basic region believed to make specific contacts with DNA upon dimerization. We report the purification of full-length c-Myc to near homogeneity from two independent eukaryotic systems: the baculovirus overexpression system using an insect cell host, and Chinese hamster ovary cells containing heat-inducible c-myc genes. The DNA binding capabilities of these preparations were characterized. Both preparations contain two distinct activities that bind specifically to sequences with a core of CACGTG. The Myc protein is solely responsible for one of these binding activities. Specific sequences that bound to c-Myc were selected from a large pool of random DNA sequence. Sequencing of individual binding sites selected by this procedure yielded a 12-base consensus, PuACCACGTGCTC, for c-Myc binding. Both protein preparations additionally demonstrated a distinct complex, containing both c-Myc and a copurifying 26-29-kDa protein, that bound to DNA with higher affinity than Myc alone. Selection of specific DNA sequences by this complex revealed a consensus binding site similar to the 12-base consensus described above. These data demonstrate that c-Myc isolated from eukaryotic cells is capable of sequence-specific DNA binding and further refine the optimal sequence for c-Myc binding. These protein preparations should prove useful in further characterizing the biochemical properties of c-Myc.

Amino Acid Sequence↗
PubMed1992

Regulation of DNA replication in vitro by the transcriptional activation domain of GAL4-VP16.

Studies of DNA viruses have provided evidence that eukaryotic transcriptional activator proteins can enhance the efficiency of DNA replication as well as transcription. The mechanism of this effect was studied in vitro using the chimeric transcription factor GAL4-VP16 and a DNA template containing GAL4 binding sites adjacent to the simian virus 40 origin of DNA replication. The binding of GAL4-VP16 prevented the repression of DNA replication which otherwise occurred when the template was assembled into chromatin. Relief of repression by GAL4-VP16 required both its DNA-binding and transcriptional activation domains but did not require RNA synthesis. The results are consistent with a general model in which transcriptional activators stimulate eukaryotic DNA replication by modifying the outcome of the competition between initiation factors and histones for occupancy of the origin.

DNA Replication↗
PubMed1992

HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors.

Proteins containing the basic-helix-loop-helix (B-HLH) domain have been shown to be important in regulating cellular differentiation. We have isolated a cDNA for a human B-HLH factor, denoted HEB, that shares nearly complete identity in the B-HLH domain with the immunoglobulin enhancer binding proteins encoded by the E2A and ITF2 genes (E proteins). Functional characterization of the protein expressed from this cDNA indicates that HEB is a third member of the E-protein class of B-HLH factors. HEB mRNA was found to be expressed in several tissues and cell types, including skeletal muscle, thymus, and a B-cell line. HEB, ITF2, and the E12 product of the E2A gene all bound to a similar spectrum of E-box sequences as homo-oligomers. All three factors also formed hetero-oligomers with myogenin, and the DNA-binding specificity and binding off-rates (dissociation rates) were modulated after hetero-oligomerization. Both homo- and hetero-oligomers of these proteins were able to distinguish between very closely related E-box sequences. In addition, HEB was shown to form hetero-oligomers with the E12 and ITF2 proteins. Finally, HEB was able to activate gene expression. These data demonstrate that HEB shares characteristics with other E proteins and show that HEB can interact with members of both the myogenic regulatory class and the E-protein class of B-HLH factors. HEB is therefore likely to play an important role in regulating lineage-specific gene expression.

Amino Acid Sequence↗
PubMed1991

Isolation of a cDNA for HSF2: evidence for two heat shock factor genes in humans.

The heat shock response is transcriptionally regulated by an evolutionarily conserved protein termed heat shock factor (HSF). We report the purification to homogeneity and the partial peptide sequence of HSF from HeLa cells. The peptide sequence was used to isolate a human cDNA with a predicted open reading frame that has homology to the DNA binding domains of both Saccharomyces cerevisiae and Drosophila HSFs. The cDNA directs the synthesis of a protein that binds to the heat shock element with specificity identical to HeLa HSF and stimulates transcription from a heat shock promoter. The expressed protein cross-reacts with anti-HSF antibodies. Surprisingly, however, this cDNA does not encode all of the peptides obtained from purified HeLa HSF. These peptides are encoded by a distinct human cDNA, HSF1, described by Rabindran et al. [Rabindran, S. K., Giorgi, G., Clos, J. & Wu, C. (1991) Proc. Natl. Acad. Sci. USA 88, 6906-6910.] It therefore appears that there is a human heat shock factor gene family and that at least two separate but related HSF proteins regulate the stress response in humans.

Amino Acid Sequence↗
PubMed1991

Activation domains of stably bound GAL4 derivatives alleviate repression of promoters by nucleosomes.

GAL4 derivatives containing an activation domain alleviated repression of a promoter during nucleosome assembly. A GAL4 derivative lacking an activation domain stably bound the promoter during nucleosome assembly but was not sufficient to preserve promoter function. The activation domain of GAL4 derivatives was essential for preserving promoter function, and thus the transcriptional stimulatory activity attributable to these activation domains increased dramatically during nucleosome assembly. Furthermore, promoter-bound activation domains allowed the formation of preinitiation complexes after nucleosome assembly. Finally, GAL4 derivatives containing activation domains significantly stimulated transcription through bacterially produced yeast TFIID only from nucleosome-assembled templates. These data indicate that acidic activation domains stimulate transcription by enhancing the ability of basal transcription factors to compete with nucleosomes for occupancy of the promoter.

Animals↗
PubMed1991

RNA polymerase II carboxy-terminal domain contributes to the response to multiple acidic activators in vitro.

The largest subunit of RNA polymerase II contains a unique carboxy-terminal domain (CTD) that consists of repeats of the heptapeptide YSPTSPS. RNA polymerase II CTD truncation mutations affect the ability to induce transcription of a subset of yeast genes in vivo, and the lack of response to induction maps to the upstream activating sequences of these genes. Here, we report that progressive truncation of the yeast RNA polymerase II CTD causes progressive loss of trans-activator-dependent transcription in nuclear extracts but has little effect on elongation or termination. Specific transcription, which is reduced by up to 50-fold in these assays, can be restored in the defective nuclear extracts by adding purified wild-type RNA polymerase II. The defects in factor-dependent transcription are observed with templates that are assembled into nucleosomes as well as with templates that are not so assembled. Defects in factor-independent transcription are also observed, but these are not as profound as those observed in the presence of trans-activators. These results indicate that the RNA polymerase II CTD functions during transcription initiation and is required for normal levels of activated transcription in vitro.

Amino Acid Sequence↗
PubMed1991

Facilitated binding of GAL4 and heat shock factor to nucleosomal templates: differential function of DNA-binding domains.

Regulatory factors must contend with chromatin structure to function. Although nucleosome structure and position on promoters can be important in determining factor access, the intrinsic ability of factors to bind to nucleosomal DNA might also play an essential regulatory role. We have used templates where nucleosomes were either randomly positioned or rotationally phased to demonstrate that two transcription factors, heat shock factor (HSF) and GAL4, differ significantly in their ability to bind to nucleosomes. GAL4 was able to bind to nucleosomal templates. Surprisingly, in contrast to its behavior on naked DNA, GAL4 bound better to multiple GAL4 sites than to a single GAL4 site on these templates. HSF alone was not able to bind to nucleosomal templates. HSF was able to bind to nucleosomal templates, however, when the TATA-binding factor TFIID was present. Consequently, binding to nucleosomal templates could be facilitated by adjacent binding of the same protein in the case of GAL4 but required binding of a second protein in the case of HSF. Taken together, these data demonstrate that regulatory factors differ in their inherent ability to bind to nucleosomal templates. These differences are likely to be important to the function of these factors in vivo.

Animals↗
PubMed1991

Regulation of heat shock factor in Schizosaccharomyces pombe more closely resembles regulation in mammals than in Saccharomyces cerevisiae.

The heat shock response appears to be universal. All eucaryotes studied encode a protein, heat shock factor (HSF), that is believed to regulate transcription of heat shock genes. This protein binds to a regulatory sequence, the heat shock element, that is absolutely conserved among eucaryotes. We report here the identification of HSF in the fission yeast Schizosaccharomyces pombe. HSF binding was not observed in extracts from normally growing S. pombe (28 degrees C) but was detected in increasing amounts as the temperature of heat shock increased between 39 and 45 degrees C. This regulation is in contrast to that observed in Saccharomyces cerevisiae, in which HSF binding is detectable at both normal and heat shock temperatures. The S. pombe factor bound specifically to the heat shock element, as judged by methylation interference and DNase I protection analysis. The induction of S. pombe HSF was not inhibited by cycloheximide, suggesting that induction occurs posttranslationally, and the induced factor was shown to be phosphorylated. S. pombe HSF was purified to near homogeneity and was shown to have an apparent mobility of approximately 108 kDa. Since heat-induced DNA binding by HSF had previously been demonstrated only in metazoans, the conservation of heat-induced DNA binding by HSF among S. pombe and metazoans suggests that this mode of regulation is evolutionarily ancient.

Base Sequence↗
PubMed1990

An upstream transcription factor, USF (MLTF), facilitates the formation of preinitiation complexes during in vitro chromatin assembly.

During in vitro chromatin assembly the formation of transcription complexes is in direct competition with the assembly of promoter sequences into nucleosomes. Under these conditions the fold stimulation of transcription by an upstream transcription factor (USF) was greater than that observed in the absence of nucleosome assembly. Function of USF during nucleosome assembly required the simultaneous presence of the TATA box binding protein TFIID. Unlike TFIID, USF alone was unable to prevent repression of the promoter during nucleosome assembly. Furthermore, USF displayed reduced or no transcriptional stimulatory activity when added to previously assembled minichromosomes. Under conditions of nucleosome assembly, USF increased the number of assembled minichromosomes which contained stable preinitiation complexes. Subsequent to assembly, the rate at which preformed complexes initiated transcription appeared to be independent of the presence of USF. Thus USF potentiated the subsequent transcriptional activity of the promoter indirectly, apparently by increasing the rate or stability of TFIID binding. This activity resulted in the promoter becoming resistant to nucleosome mediated repression. These observations suggest that some ubiquitous upstream factors, e.g. USF, may play an important role in establishing the transcriptional potential of cellular genes during chromatin assembly.

Animals↗
PubMed1990

Minimal deviation adenocarcinoma (adenoma malignum) of the endocervix: a histochemical and immunohistochemical study of two cases.

The histopathological diagnosis of minimal deviation adenocarcinoma (adenoma malignum) of the endocervix may be difficult. Two cases of minimal deviation adenocarcinoma (MDA) were examined using mucin histochemistry and immunocytochemistry with antibodies to epithelial membrane antigens (HMFG1, Ep1), low-molecular-weight cytokeratins (CAM 5.2), carcinoembryonic antigen (CEA), and alpha-amylase. The results were compared with those for normal endocervical glands. Reactivity for CEA in MDA was focal and would be unreliable for biopsy diagnosis. Both cases of MDA contained abundant neutral mucins and sialomucins, whereas sulfomucins were rarely detected; this pattern contrasted with that of normal endocervix. Neoplastic glandular epithelial cells in MDA consistently showed both luminal and cytoplasmic reactivity with Ep1 and HMFG1, whereas normal cervix showed luminal labeling only. Thus, mucin histochemistry and immunohistochemical detection of epithelial membrane antigens may distinguish between extremely well differentiated neoplastic glands in MDA and normal endocervical glands, and hence may aid diagnosis in biopsy specimens.

Adenocarcinoma↗
PubMed1990

Factor substitution in a human HSP70 gene promoter: TATA-dependent and TATA-independent interactions.

To investigate interactions between transcription factors on mammalian promoters, we constructed a set of 24 variations of the human HSP70 gene promoter in which six upstream sequence motifs are paired in every possible combination with four TATA motifs. These promoters were analyzed for in vivo expression, and selected constructs were examined by in vitro template commitment studies. Activation transcription factor (ATF) and CP1 showed dramatically different interactions with the factor(s) bound to the TATA region. CP1 functioned in vivo regardless of the TATA motif that it was paired with and was not capable of sequestering the core promoter complex in a template commitment assay. ATF activity was dramatically altered by changing the TATA motif, and ATF was able to sequester the core promoter complex. These data suggest that CP1 and ATF function by distinct mechanisms that differ with respect to interaction with the factor(s) at the TATA box. Factor Sp1 also appeared to function by a TATA-independent mechanism. These data imply that the ability of a factor to function is determined not only by the intrinsic properties of the factor but also by promoter context.

Base Sequence↗
PubMed1990

E1a transactivation of human HSP70 gene promoter substitution mutants is independent of the composition of upstream and TATA elements.

We have analyzed 41 deletion, linker scan, and substitution mutants of the human HSP70 gene promoter for activation by the adenovirus E1a region. No natural element of the HSP70 gene promoter was required for activation. To investigate specific interactions between E1a and transcription factors, a set of 24 promoters containing all possible combinations of eight different upstream or TATA motifs was investigated for E1a stimulation. E1a transactivated the promoter regardless of the particular TATA motif present. Furthermore, there was no dramatic correlation between any upstream motif and activation by E1a. These data suggest that E1a does not stimulate transcription via an interaction with any specific transcription factor but instead suggest that E1a interacts via the general transcription machinery.

Adenovirus Early Proteins↗
PubMed1990

TATA-dependent and TATA-independent function of the basal and heat shock elements of a human hsp70 promoter.

We have characterized the interactions between the TATA element and other sequence elements of a human heat shock protein 70 (hsp70) promoter by a mutational approach. Expression of a distal element of this promoter requires an intact TATA element in human cell lines. The hsp70 TATA element can be functionally replaced for this interaction by TATA elements from the simian virus 40 early and adenovirus EIIa promoters. The TATA element in this promoter therefore both determines the appropriate start site and determines strength by allowing function of the distal element. In contrast, three proximal upstream elements necessary for basal and heat-regulated transcription have no requirement either for a TATA element or for any other proximal element. The behavior of promoters multiply mutant in these proximal elements implies that these elements function independently. We examined the interaction between the heat shock element (HSE) and the TATA element as the distance between the two factor-binding sites was increased. It was necessary to create a mutant HSE with an extended consensus sequence in order for the HSE to function at a distance. Moving this extended HSE 500 bases upstream did not increase its dependence on the TATA element, suggesting that the TATA independence of this element is intrinsic to its function and is not determined by distance from the promoter.

Animals↗
PubMed1990

DNA binding of heat shock factor to the heat shock element is insufficient for transcriptional activation in murine erythroleukemia cells.

The heat shock response is among the most highly conserved examples of regulated gene expression, being present in all cellular organisms. Transcriptional activation of heat shock genes by increased temperature or other cellular stresses is mediated by the binding of a heat shock factor (HSF) to a conserved nucleotide sequence (the heat shock element) present in the promoter of heat-inducible genes. Despite the high degree of conservation of this response, embryonic stages of development are characterized by the absence of a heat shock response. Murine erythroleukemia (MEL) cells also lack this response, and we report here a detailed characterization of this defect for one of the most highly conserved of these genes, hsp70. Surprisingly, heat-induced transcriptional activation of this gene does not occur, despite the induction of a protein with the binding specificity of murine HSF. However, the MEL HSF differs slightly in apparent size from the HSF in 3T3 cells, which exhibit a normal heat shock response. These data suggest that activation of mammalian HSF by heat requires at least two separate steps: an alteration of binding activity followed by further modification that activates transcription. MEL cells do not respond to heat shock because they lack the ability to perform this secondary modification. These cells provide a useful system for characterizing heat shock activation in mammals.

Animals↗