Unfractionated Nuclear Extracts Research Articles

TRAP/SMCC/mediator-dependent transcriptional activation from DNA and chromatin templates by orphan nuclear receptor hepatocyte nuclear factor 4.

The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liver-specific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regulatory module of the transcription apparatus. Using a system that has been reconstituted from purified transcription factors, as well as one consisting of unfractionated nuclear extract from which TRAP/SMCC/Mediator has been depleted by specific antibodies, we demonstrate a strong dependence of HNF-4 function on this coactivator. Importantly, we further show a TRAP/SMCC/Mediator-dependence for HNF-4 transcriptional activation from chromatin templates. The latter involves cooperation with the histone acetyltransferase-containing coactivator p300, in accord with a synergistic mode of action of the two divergent coactivators. We also show that HNF-4 and TRAP/SMCC/Mediator can interact physically. This interaction likely involves primary HNF-4 activation function 2 (AF-2)-dependent interactions with the TRAP220 subunit of TRAP/SMCC/Mediator and secondary (AF-2-independent) interactions with TRAP170/RGR1. Finally, recruitment experiments using immobilized templates strongly suggest that the functional consequences of the physical interaction probably are manifested at a postrecruitment step in the activation pathway.

The TRAP/Mediator coactivator complex interacts directly with estrogen receptors alpha and beta through the TRAP220 subunit and directly enhances estrogen receptor function in vitro.

Target gene activation by nuclear hormone receptors, including estrogen receptors (ERs), is thought to be mediated by a variety of interacting cofactors. Here we identify a number of nuclear extract-derived proteins that interact with immobilized ER ligand binding domains in a 17beta-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)/Mediator coactivator complex, which interacts with ERalpha and ERbeta in both unfractionated nuclear extracts and purified form. Studies with extracts from TRAP220(-/-) fibroblasts reveal that these interactions depend on TRAP220, a TRAP/Mediator subunit previously shown to interact with ER and other nuclear receptors in a ligand-dependent manner. The physiological relevance of the in vitro interaction is documented further by the isolation of an ERalpha-TRAP/Mediator complex from cultured cells expressing an epitope-tagged ERalpha. Finally, the complete TRAP/Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP/Mediator, through TRAP220, in ER function.

Coactivation by OCA-B: definition of critical regions and synergism with general cofactors.

Molecular dissection of the B-cell-specific transcription coactivator OCA-B has revealed distinct regions important, respectively, for recruitment to immunoglobulin promoters through interaction with octamer-bound Oct-1 and for subsequent coactivator function. Further analysis of general coactivator requirements showed that selective removal of PC4 from the essential USA fraction severely impairs Oct-1 and OCA-B function in a cell-free system reconstituted with partially purified factors. Full activity can be restored by the combined action of recombinant PC4 and the PC4-depleted USA fraction, thus suggesting a joint requirement for PC4 and another, USA-derived component(s) for optimal function of Oct-1/OCA-B in the reconstituted system. Indeed, USA-derived PC2 was found to act synergistically with PC4 in reproducing the function of intact USA in the assay system. Consistent with the requirement for PC4 in the reconstituted system, OCA-B was found to interact directly with PC4. Surprisingly, however, removal of PC4 from the unfractionated nuclear extract has no detrimental effect on OCA-B/Oct-1-dependent transcription. These results lead to a general model for the synergistic function of activation domains in Oct-1 and OCA-B (mediated by the combined action of the multiple USA components) and, further, suggest a functional redundancy in general coactivators.

One-step affinity purification protocol for human telomerase.

Human telomerase is a ribonucleoprotein (RNP) enzyme, comprising protein components and an RNA template that catalyses telomere elongation through the addition of TTAGGG repeats. Telomerase function has been implicated in aging and cancer cell immortalization. We report a rapid and efficient one-step purification protocol to obtain highly active telomerase from human cells. The purification is based on affinity chromatography of nuclear extracts with antisense oligonucleotides complementary to the template region of the human telomerase RNA component. Bound telomerase is eluted with a displacement oligonucleotide under mild conditions. The resulting affinity-purified telomerase is active in PCR-amplified telomerase assays. The purified telomerase complex has a molecular mass of approximately 550 kDa compared to the approximately 1000 kDa determined for the telomerase RNP in unfractionated nuclear extracts. The purification protocol provides a rapid and efficient tool for functional and structural studies of human telomerase.

Functional significance of the TATA element major groove in transcription initiation by RNA polymerase II.

The binding of TFIID to the TATA element initiates assembly of a preinitiation complex and thus represents one of the most important steps for transcriptional regulation. The fact that the TATA binding protein (TBP), a subunit of TFIID, exclusively contacts the minor groove of the TATA element led us to ask whether the major groove of the TATA element plays any role in transcription initiation or its regulation. Our results show that modifications of the major groove of the TATA element in the adenovirus major late promoter have no effect on TFIID binding affinity or on transcription in a cell-free system reconstituted with purified factors. However, major groove modifications do decrease the levels of both basal and activator-mediated transcription in unfractionated nuclear extracts, indicating that the intact structure of the major groove of the TATA element is functionally important for transcription initiation in a more physiological context.

A Novel Mn++-Dependent Ribonuclease That Functions in U16 SnoRNA Processing inX.Laevis.

The intron-encoded U16 small nucleolar RNA (snoRNA) is a component of a new family of molecules which originate by processing of pre-mRNA in which they are contained. The mechanism of U16 snoRNA biosynthesis involves an initial step of endonucleolytic cleavage of the pre-mRNA with the release of a pre-ssnoRNA molecule; the subsequent step consists of exonucleolytic trimming that produces mature U16 molecules. In order to identify the molecular components involved in this peculiar biosynthetic pathway, we have undertaken the characterization of the endonucleolytic activity by biochemical fractionation ofXenopus laevisoocyte nuclear extract. In this paper we show the production of a protein fraction (BSF) which is highly enriched for a specific endonucleolytic activity that exactly reproduces the cleavage pattern of the U16-containing pre-mRNA identifiedin vivoinX.laevisoocytes and in unfractionated nuclear extract.

RNA polymerase II/III transcription specificity determined by TATA box orientation.

The TATA box sequence in eukaryotes is located about 25 bp upstream of many genes transcribed by RNA polymerase II (Pol II) and some genes transcribed by RNA polymerase III (Pol III). The TATA box is recognized in a sequence-specific manner by the TATA box-binding protein (TBP), an essential factor involved in the initiation of transcription by all three eukaryotic RNA polymerases. We have investigated the recognition of the TATA box by the Pol II and Pol III basal transcription machinery and its role in establishing the RNA polymerase specificity of the promoter. Artificial templates were constructed that contained a canonical TATA box as the sole promoter element but differed in the orientation of the 8-bp TATA box sequence. As expected, Pol II initiated transcription in unfractionated nuclear extracts downstream of the "forward" TATA box. In distinct contrast, transcription that initiated downstream of the "reverse" TATA box was carried out specifically by Pol III. Importantly, this effect was observed regardless of the source of the DNA either upstream or downstream of the TATA sequence. These findings suggest that TBP may bind in opposite orientations on Pol II and Pol III promoters and that opposite, yet homologous, surfaces of TBP may be utilized by the Pol II and Pol III basal machinery for the initiation of transcription.

Transcriptional trans activation by human immunodeficiency virus type 1 Tat requires specific coactivators that are not basal factors

Expression of human immunodeficiency virus type 1 (HIV-1) genes is regulated by the trans activator Tat. Tat exerts its effects by increasing the rate of transcription, but the mechanism by which it does so is still unknown. To study the cellular factors required for Tat trans activation, we have expressed functional Gst-Tat fusion protein and used it to construct affinity columns. Our findings are as follows. (i) A Gst-Tat affinity matrix depleted HeLa nuclear extracts of a factor(s) required for Tat function. A Tat mutant bearing the missense mutation lysine to alanine at position 41 was incapable of this depletion. (ii) Tat trans activation was recovered by addition of unfractionated nuclear extract, the 0.5 M KCl elution fraction from the Tat affinity column, or sedimentation gradient fractions of HeLa extracts. The activity from the gradients sedimented with an apparent molecular mass of 200 kDa. (iii) Tat trans activation could not be recovered by use of recombinant human TATA-binding protein or partially purified TFIID. (iv) trans activation by Tat was blocked by heating of the nuclear extract under conditions in which basal transcription was not decreased. Our data demonstrate for the first time the existence of unique Tat coactivators distinct from factors required for general basal transcription.

Both LyF-1 and an Ets Protein Interact With a Critical Promoter Element in the Murine Terminal Transferase Gene

Terminal deoxynucleotidyltransferase (TdT) is a template-independent DNA polymerase that is expressed transiently during the earliest stages of B- and T-cell ontogeny. Previously, we characterized the promoter for the murine TdT gene and identified a novel DNA-binding protein, called LyF-1, that interacts with a DNA sequence element found to be critical for transcriptional activity in lymphoid cell lines. Here, we present a more detailed analysis of this 30-bp control element, called the TdT D' element, which is centered approximately 60 bp upstream of the transcription start site. We found that both the murine and human D' elements are recognized by multiple proteins, including LyF-1 and at least two Ets family proteins, Ets-1 and Fli-1. Additional protein-DNA interactions were identified through studies using unfractionated nuclear extracts, in which the D' element was apparently incorporated into a multiprotein complex, possibly containing an Ets protein as a core component. By analyzing a series of substitution mutations, two adjacent binding sites for LyF-1 were identified in the murine D' element, with the Ets protein binding site closely coinciding with the proximal, lower-affinity LyF-1 site. Transient transfection analysis with these mutations revealed that only a 10-bp region, containing precisely the Ets and proximal LyF-1 binding sites, was needed for D' activity. These results suggest an important role for an Ets family protein in the expression of the TdT gene. The role of LyF-1 is less clear; it might act in conjunction with the Ets protein bound at the D' element or it might be unnecessary for D' activity.

Both LyF-1 and an Ets protein interact with a critical promoter element in the murine terminal transferase gene.

Terminal deoxynucleotidyltransferase (TdT) is a template-independent DNA polymerase that is expressed transiently during the earliest stages of B- and T-cell ontogeny. Previously, we characterized the promoter for the murine TdT gene and identified a novel DNA-binding protein, called LyF-1, that interacts with a DNA sequence element found to be critical for transcriptional activity in lymphoid cell lines. Here, we present a more detailed analysis of this 30-bp control element, called the TdT D' element, which is centered approximately 60 bp upstream of the transcription start site. We found that both the murine and human D' elements are recognized by multiple proteins, including LyF-1 and at least two Ets family proteins, Ets-1 and Fli-1. Additional protein-DNA interactions were identified through studies using unfractionated nuclear extracts, in which the D' element was apparently incorporated into a multiprotein complex, possibly containing an Ets protein as a core component. By analyzing a series of substitution mutations, two adjacent binding sites for LyF-1 were identified in the murine D' element, with the Ets protein binding site closely coinciding with the proximal, lower-affinity LyF-1 site. Transient transfection analysis with these mutations revealed that only a 10-bp region, containing precisely the Ets and proximal LyF-1 binding sites, was needed for D' activity. These results suggest an important role for an Ets family protein in the expression of the TdT gene. The role of LyF-1 is less clear; it might act in conjunction with the Ets protein bound at the D' element or it might be unnecessary for D' activity.

The CACC box upstream of human embryonic epsilon globin gene binds Sp1 and is a functional promoter element in vitro and in vivo

DNA sequences 179 base pairs upstream and 23 base pairs downstream of the cap site of human embryonic epsilon globin gene exhibit promoter activity in transfected cell cultures. The nuclear factor binding in vitro of this epsilon promoter region was studied by DNase I foot-printing, methylation interference, and gel mobility shift assay. Four major nuclear factor-binding sites are detected in complexes formed with unfractionated nuclear extracts: NF-E1 at -163, epsilon F1 at -143, CACC box at -111, and CBF at -81, respectively. Of these, NF-E1 is an erythroid-specific factor. epsilon F1 is probably a ubiquitous factor because it is present in both erythroid K562 cells and nonerythroid HeLa cells. The epsilon F1-binding site exhibits sequence similarity to that of the cAMP response element-binding protein family of transcription factors. Finally, the CCAAT box-binding protein (CBF)-binding site centers around the CCAAT promoter box. Comparative binding studies with unfractionated nuclear extracts and affinity purified HeLa Sp1 demonstrated that the epsilon-globin CACC box at -111 is a binding site of Sp1. The spatial arrangements of the NF-E1-binding site, the CACC box, and CCAAT box, with respect to their mutual separations by approximately integral numbers of helical turns, is well conserved in all mammalian embryonic epsilon globin promoters. Transient expression assay, using human growth hormone gene as the receptor, demonstrated that similar to the other two human beta-like globin genes (beta and gamma), the CACC box of epsilon globin gene is an essential promoter element for epsilon globin gene expression in vivo in K562 cells. This CACC promoter box also functions in vitro in nuclear extracts prepared from K562 cells. These data, together with Sp1-binding studies of the human beta and gamma globin CACC boxes, suggest that the general transcription factor Sp1, through its differential interactions with different forms of CACC promoter boxes, is an essential component of the machinery that controls the developmental program of mammalian globin gene regulation.

A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs.

We have purified and characterized a factor required for accurate polyadenylation of mammalian pre-mRNAs in vitro. This factor, called cleavage-stimulation factor (CstF), is composed of three distinct polypeptide subunits of 77, 64, and 50 kD. Using monoclonal antibodies directed against the 64- and 50-kD subunits, we show that CstF is required for efficient cleavage of polyadenylation substrates. Furthermore, CstF present in unfractionated nuclear extracts interacts with pre-mRNAs containing the signal sequence AAUAAA, but not AAGAAA, in such a manner that the 64-kD subunit can be cross-linked to the RNA by UV light. This polypeptide is thus identical to the previously described 64-kD nuclear protein that binds to AAUAAA-containing RNAs. Finally, indirect immunofluorescence of fixed cells indicates that CstF is distributed diffusely throughout the nucleus in a granular pattern distinct from the "speckled" pattern displayed by factors involved in pre-mRNA splicing, but similar to that of heterogeneous nuclear ribonucleoproteins. A model is presented in which CstF binds specifically to nascent RNA polymerase II transcripts and, by interacting with other factors, results in a rapid initiation of 3'-end processing of pre-mRNAs.

Dioxin-inducible, Ah receptor-dependent transcription in vitro.

We have developed a homologous in vitro transcription system that requires (i) 2,3,7,8-tetrachlorodibenzo-p-dioxin (called TCDD or dioxin), (ii) the Ah receptor, and (iii) a dioxin-responsive enhancer for activity. Unfractionated nuclear extracts from mouse hepatoma cells contain an inhibitor and fail to direct transcription in vitro. However, following phosphocellulose chromatography and reconstitution, the fractionated nuclear extract directs accurate transcription in vitro, using as a template the promoter/enhancer region from the mouse cytochrome P1-450 gene (Cyp1a1) linked to a "G-free cassette" (which generates a transcript with no guanosine residues). Extracts from TCDD-treated cells exhibit higher activity than extracts from untreated cells when transcribing a template containing both the promoter and enhancer but not when transcribing a template containing the promoter alone. Extracts from Ah receptor-defective cells fail to direct in vitro transcription in a TCDD-inducible fashion. A regulatory element that contains two binding sites for the liganded Ah receptor plus a truncated Cyp1a1 promoter suffices to direct TCDD-inducible, Ah receptor-dependent transcription in vitro. The inducible, receptor-dependent, enhancer-dependent properties of this system make it appropriate for analyzing in vitro the mechanism of dioxin action and the function of the Ah receptor.

Promoter-dependent Transcription by RNA Polymerase II Using Immobilized Enzyme Complexes

DNA fragments containing the adenovirus 2 major late or simian virus 40 early promoters were attached to a solid support via a biotin-streptavidin linkage at one end of the fragment, upstream of the RNA start site. Templates immobilized in this manner were incubated with HeLa cell nuclear extracts to form preinitiation complexes containing RNA polymerase II and accessory proteins required for faithful in vitro transcription. These complexes did not require ATP or dATP for assembly, were sensitive to 0.25% Sarkosyl, and were stable to extensive washing. Their incubation with specific combinations of nucleoside triphosphates resulted in the initiation of RNA chain polymerization in situ, while addition of the remaining nucleoside triphosphates was necessary to produce a full length runoff RNA. Transcriptional activity associated with preinitiation complexes was purified approximately 300-fold, relative to the unfractionated nuclear extract. The use of immobilized template permits considerable flexibility in experimental design, as substrates and inhibitors can be added and washed out of the reaction at each step. We exploited this property of the system to dissect the temporal substrate requirements for initiation of RNA synthesis. It is known from prior work that at least one step in the promoter-dependent RNA synthesis reaction requires an adenosine nucleotide that is hydrolyzable at the beta, gamma-position. This requirement is independent of the initiating nucleotide and can be satisfied by dATP, which is not ordinarily incorporated into the RNA product. We show here that the beta, gamma-hydrolyzable adenosine nucleotide must be present simultaneously with the initiating nucleoside triphosphates. No reaction occurred when complexes were incubated with dATP, washed to remove dATP, and incubated subsequently with the two initiating nucleotides.

Steroid hormone-dependent interaction of human progesterone receptor with its target enhancer element.

We investigated the requirement of steroid hormone for the specific binding of progesterone receptor to its cognate progesterone responsive element (PRE) in cell-free experiments. We prepared unfractionated nuclear extracts from human breast cancer (T47D) cells which are rich in progesterone receptors and used a gel retardation assay to monitor receptor-DNA complex formation. Exposure of receptor to either progesterone, R5020, or the antiprogestin RU38 486 in vivo or in vitro led to the formation of two protein-DNA complexes (1 and 2) which were not detected in nuclear extracts unexposed to hormone. Similar treatment with cortisol or estradiol failed to induce the formation of these complexes. The complexes were specific for PRE, since they could be competed efficiently in binding competition experiments by oligonucleotides containing PRE. A monoclonal antibody which recognizes both A and B forms of human progesterone receptor, interacted with both complexes 1 and 2 and shifted them to slower migrating forms. Another antibody which only recognizes the B form interacted with only complex 1 but not with complex 2, establishing that the complexes 1 and 2 were indeed formed by progesterone receptor forms B and A, respectively. We conclude from the above studies that in vivo or in vitro treatment of nuclear progesterone receptor with either progesterone or R5020 or RU38 486 alone can lead to detection of high affinity complexes formed between the PRE and the receptor present in unpurified nuclear extracts.

Accurate transcription of mouse metallothionein-I gene in a fractionated nuclear extract from a rat hepatoma.

Nuclear extract from Morris hepatoma 3924A was fractionated by DEAE-Sephadex chromatography. The fraction eluting with 300 mM (NH4)2SO4 (DE-C) was used for transcribing cloned mouse metallothionein-I (MT-I) gene in a run-off assay. This fraction contained the majority of RNA polymerase II as well as the transcription factor(s). Accuracy of MT-I DNA transcription was confirmed by S1 nuclease mapping. Low concentrations (1 microgram/ml) of alpha-amanitin inhibited the reaction, indicating that RNA polymerase II directed the transcription. Unfractionated nuclear extracts from the hepatoma or a rat mammary adenocarcinoma as well as whole cell extract obtained from the mammary tumor also transcribed MT-I gene. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-C greater than whole cell extract derived from rat mammary adenocarcinoma cells greater than nuclear extract derived from rat hepatoma or rat mammary adenocarcinoma cells. These studies have demonstrated that a fractionated nuclear extract obtained from a tissue supports efficient and accurate RNA polymerase II-mediated transcription of MT-I DNA.

An exonuclease protection assay reveals heat-shock element and TATA box DNA-binding proteins in crude nuclear extracts.

The ability to identify and purify trans-acting cellular factors that regulate eukaryotic genes is limited by the lack of a practical general assay. Current procedures using crude whole cell or nuclear extracts that restore transcriptional function in vitro or permit reconstruction of native chromatin at control sequences are effective only in select systems. I now present an exonuclease protection assay that is generally applicable for detecting sequence-specific DNA-binding proteins. The assay extends earlier work on the binding to the Drosophila heat-shock gene control element of a protein factor (HAP) present in crude nuclear extracts; the binding was shown by reconstitution of specific exonuclease resistance within a nuclease-hypersensitive site in chromatin. We show here that this same exonuclease resistance can be reconstituted on free linear DNA, despite many nonspecific binding activities present in unfractionated nuclear extracts. We have further applied this assay method to fractionate the protein factor that is bound constitutively to the heat-shock gene TATA box region in native chromatin. Exonuclease protection offers a sensitive, precise and rapid assay for any sequence-specific DNA-binding protein.