Direct imaging of human SWI/SNF-remodeled mono- and polynucleosomes by atomic force microscopy employing carbon nanotube tips.

Chromatin-remodeling complexes alter chromatin structure to facilitate, or in some cases repress, gene expression. Recent studies have suggested two potential pathways by which such regulation might occur. In the first, the remodeling complex repositions nucleosomes along DNA to open or occlude regulatory sites. In the second, the remodeling complex creates an altered dimeric form of the nucleosome that has altered accessibility to transcription factors. The extent of translational repositioning, the structure of the remodeled dimer, and the presence of dimers on remodeled polynucleosomes have been difficult to gauge by biochemical assays. To address these questions, ultrahigh-resolution carbon nanotube tip atomic force microscopy was used to examine the products of remodeling reactions carried out by the human SWI/SNF (hSWI/SNF) complex. We found that mononucleosome remodeling by hSWI/SNF resulted in a dimer of mononucleosomes in which approximately 60 bp of DNA is more weakly bound than in control nucleosomes. Arrays of evenly spaced nucleosomes that were positioned by 5S rRNA gene sequences were disorganized by hSWI/SNF, and this resulted in long stretches of bare DNA, as well as clusters of nucleosomes. The formation of structurally altered nucleosomes on the array is suggested by a significant increase in the fraction of closely abutting nucleosome pairs and by a general destabilization of nucleosomes on the array. These results suggest that both the repositioning and structural alteration of nucleosomes are important aspects of hSWI/SNF action on polynucleosomes.

Isolation of histones and nucleosome cores from mammalian cells.

In vitro analysis of DNA in chromatin is often important for understanding mechanisms of regulation of transcription and other processes that occur on DNA. The basic unit of chromatin is the nucleosome core, containing two copies each of the core histones H2A, H2B, H3, and H4 to form a histone octamer that wraps 145 base pairs of DNA in a left-handed superhelix. In vivo, chromatin is associated with linker histones (such as H1), which facilitate the ordered packing of nucleosomes. This linker histone-containing particle is properly termed the nucleosome (or chromatosome), while the linker histone-free particle is the nucleosome core. Pure polynucleosome cores and histones can be readily isolated from mammalian tissue culture cells. This unit describes procedures for isolation and purification of nuclei, isolation of polynucleosomes lacking linker histones from these nuclei, isolation of pure populations of mono- and dinucleosome cores from oligonucleosome fractions, and isolation of core histones from purified nuclei. The methods presented here do not denature the histones, and may yield histones that are more active for in vitro assemblies.

ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor.

The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

Octamer transfer and creation of stably remodeled nucleosomes by human SWI-SNF and its isolated ATPases.

Chromatin remodeling complexes help regulate the structure of chromatin to facilitate transcription. The multisubunit human (h) SWI-SNF complex has been shown to remodel mono- and polynucleosome templates in an ATP-dependent manner. The isolated hSWI-SNF ATPase subunits BRG1 and hBRM also have these activities. The intact complex has been shown to produce a stable remodeled dimer of mononucleosomes as a product. Here we show that the hSWI-SNF ATPases alone can also produce this product. In addition, we show that hSWI-SNF and its ATPases have the ability to transfer histone octamers from donor nucleosomes to acceptor DNA. These two reactions are characterized and compared. Our results are consistent with both products of SWI-SNF action being formed as alternative outcomes of a single remodeling mechanism. The ability of the isolated ATPase subunits to catalyze these reactions suggests that these subunits play a key role in determining the mechanistic capabilities of the SWI-SNF family of remodeling complexes.

Chromatin deacetylation by an ATP-dependent nucleosome remodelling complex.

The dynamic assembly and remodelling of eukaryotic chromosomes facilitate fundamental cellular processes such as DNA replication and gene transcription. The repeating unit of eukaryotic chromosomes is the nucleosome core, consisting of DNA wound about a defined octamer of histone proteins. Two enzymatic processes that regulate transcription by targeting elements of the nucleosome include ATP-dependent nucleosome remodelling and reversible histone acetylation. The histone deacetylases, however, are unable to deacetylate oligonucleosomal histones in vitro. The protein complexes that mediate ATP-dependent nucleosome remodelling and histone acetylation/deacetylation in the regulation of transcription were considered to be different, although it has recently been suggested that these activities might be coupled. We report here the identification and functional characterization of a novel ATP-dependent nucleosome remodelling activity that is part of an endogenous human histone deacetylase complex. This activity is derived from the CHD3 and CHD4 proteins which contain helicase/ATPase domains found in SWI2-related chromatin remodelling factors, and facilitates the deacetylation of oligonucleosomal histones in vitro. We refer to this complex as the nucleosome remodelling and deacetylating (NRD) complex. Our results establish a physical and functional link between the distinct chromatin-modifying activities of histone deacetylases and nucleosome remodelling proteins.

Human SWI/SNF interconverts a nucleosome between its base state and a stable remodeled state.

The human SWI/SNF complex remodels nucleosome structure in an ATP-dependent manner, although the nature of this change has not been determined. Here we show that hSWI/SNF and ATP generate an altered nucleosomal structure that is stable in the absence of SWI/SNF. This product has an altered sensitivity to digestion by DNAse, restriction enzymes, and micrococcal nuclease, and an increased affinity for GAL4. It has the same protein composition but is approximately twice the size of a normal nucleosome. Incubation of the altered nucleosome with hSWI/SNF converts this structure back to a standard nucleosome in an ATP-dependent process. These results suggest that hSWI/ SNF acts by facilitating an exchange between normal and altered, more accessible, nucleosome conformations.

Nucleosome disruption by human SWI/SNF is maintained in the absence of continued ATP hydrolysis.

We have examined the requirement for ATP in human (h) SWI/SNF-mediated alteration of nucleosome structure and facilitation of transcription factor binding to nucleosomal DNA. hSWI/SNF-mediated nucleosome alteration requires hydrolysis of ATP or dATP. The alteration is stable upon removal of ATP from the reaction or upon inhibition of activity by excess ATPgammaS, indicating that continued ATP hydrolysis is not required to maintain the altered nucleosome structure. This stable alteration is sufficient to facilitate binding of a transcriptional activator protein; concurrent ATP hydrolysis was not required to facilitate binding. These data suggest sequential steps that can occur in the process by which transcription factors gain access to nucleosomal DNA.

RNA polymerase II holoenzyme contains SWI/SNF regulators involved in chromatin remodeling.

The RNA polymerase II holoenzyme contains RNA polymerase II, a subset of general transcription factors and SRB regulatory proteins. We report here that SWI and SNF gene products, previously identified as global gene regulators whose functions include remodeling chromatin, are also integral components of the yeast RNA polymerase II holoenzyme. The SWI/SNF proteins are components of the SRB complex, also known as the mediator, which is tightly associated with the RNA polymerase II C-terminal repeat domain. The SWI/SNF components provide the holoenzyme with the capacity to disrupt nucleosomal DNA and thus facilitate stable binding of various components of the transcription initiation complex at promoters.

Serpentine cAMP receptors may act through a G protein-independent pathway to induce postaggregative development in Dictyostelium.

The transcription factor G box-binding factor (GBF) is required for the developmental switch between aggregative and postaggregative gene expression, cell-type differentiation, and morphogenesis. We show that constitutive expression of GBF allows ectopic expression of postaggregative genes, but only in response to exogenous cAMP. GBF activation requires the serpentine cAMP receptors required for aggregation, but not the coupled G alpha 2 or the G beta subunit, suggesting a novel signaling pathway. In response to high cAMP, g alpha 2-null cells can bypass the aggregation stage, expressing cell type-specific genes and forming fruiting bodies. Our results demonstrate that the same receptors regulate aggregation and cell-type differentiation, but via distinct pathways depending upon whether the receptor perceives a pulsatile or sustained signal.

A GBF-binding site and a novel AT element define the minimal sequences sufficient to direct prespore-specific expression in Dictyostelium discoideum.

In order to better understand the molecular mechanisms of cellular differentiation in Dictyostelium discoideum, we have identified the minimum regulatory sequences of the prespore-specific gene SP60/cotC that are sufficient to confer cell-type-specific expression on a heterologous promoter. This region includes at least two essential cis-acting elements: a novel AT-rich element (or elements) and CAE3. The essential function of the AT element is confirmed through point mutations that decrease expression below the level of detection. CAE3 is one of three CA-rich elements (CAEs) required for the induction of SP60/cotC during development or in response to extracellular cyclic AMP. The CAEs have differential affinities for a specific developmentally induced nuclear activity (CAE1 > CAE2 >> CAE3). Here, we identify this activity as G-box-binding factor (GBF) and show that in vitro-transcribed and -translated GBF binds all three SP60/cotC CAEs in a sequence-specific manner. Previous studies have suggested that GBF mediates the induction of some prestalk genes, and these results demonstrate that it also has a specific role in prespore gene activation.

Enormous hemangiosarcoma of the heart.

This report describes a 26-year-old patient with hemangiosarcoma of the heart and summarizes the clinicopathological features in previous reports of patients with cardiac angiosarcoma. The patient was admitted to our hospital because of a syncope and one episode of nocturnal dyspnea and hemoptysis. In his history he complained of progressive weakness and loss of weight over the past 2 months. Echocardiography and computed tomography of the chest showed inhomogeneous masses in the pericardial cavity completely surrounding the heart and involving the ascending aorta and the superior vena cava. Histological examination of the tissue obtained from the mass by fine needle technique revealed a poorly differentiated malignant tumor of mesenchymal origin. Exploratory thoracotomy followed by tumor biopsies showed an inoperable cardiac hemangiosarcoma of enormous size with multiple metastases in both lungs. Palliative tumor resection was not performed. During the postoperative course the patient still required controlled ventilation. After 3 days of cytostatic chemotherapy no regression of tumor mass was seen by chest radiography. Cardiorespiratory insufficiency was progressive, and the patient died within 3 weeks after admission.

Cloning and characterization of the G-box binding factor, an essential component of the developmental switch between early and late development in Dictyostelium.

During Dictyostelium development, the cAMP-regulated induction of cell-type-specific late genes marks a developmental switch from the initial formation of the multicellular organism to the differentiation of the various cell types that mediate morphogenesis and eventually give rise to the mature fruting body. The G-box binding factor (GBF) is a developmentally regulated Dictyostelium transcription factor whose affinity for a DNA sequence correlates with the ability of that sequence to confer inducibility to late gene promoters in response to high, continuous levels of extracellular cAMP. We report the purification of GBF and cloning of the gene that encodes it, as confirmed by in vitro production of GBF activity. The predicted protein is highly basic and contains two putative zinc fingers. Disruption of the GBF gene by homologous recombination results in the loss of all GBF DNA-binding activity, developmental arrest at the loose aggregate stage, and the loss of late gene induction during development or in response to extracellular cAMP. Constitutive expression of GBF complements the null phenotype and allows for the rapid activation of a class of late genes in response to cAMP. Our results indicate that GBF acts as an extracellular cAMP-responsive transcriptional activator regulating late gene expression and is an essential component of a developmental switch between aggregation and cellular morphogenesis.

A G-rich sequence element common to Dictyostelium genes which differ radically in their patterns of expression.

Removal of a G-rich element from the DIF-inducible, prestalk-, and stalk-specific ecmB gene reduces expression but cell-type specificity is retained. The ecmB element will functionally substitute for a homologous sequence upstream of CP2, a cAMP-inducible gene and is bound by GBF, the factor which interacts with the CP2 G box. These results suggest that the G box may play a similar stimulatory role in these two independently regulated genes where it presumably interacts with different ancillary promoter elements.

A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli.

We present a collection of 182 isogenic strains containing genetically linked antibiotic resistance elements located at approximately 1-min intervals around the Escherichia coli chromosome. At most positions both Tn10 (Tetr) and TN10kan (Kanr) elements are available, so that the collection contains a linked set of alternating antibiotic resistance markers. The map position of each insertion has been aligned to the E. coli genetic map as well as to the Kohara ordered clone bank. These strains are designed to be used in a rapid two-step mapping system in E. coli. In the first step, the mutation is localized to a 5- to 15-min region of the chromosome by Hfr mapping with a set of Hfr strains containing either Tn10 or Tn10kan elements located 20 min from their respective origins of transfer. In the second step, the mutation is localized to a 1-min region by P1 transduction, with a collection of isogenic insertion strains as donors. We discuss the uses of this collection of strains to map and eventually to clone a variety of mutations in E. coli.

Phase III study of 5-FU and carmustine versus 5-FU, carmustine, and doxorubicin in advanced gastric cancer.

Seventy-seven patients with advanced gastric carcinoma were prospectively randomized to receive 5-FU and carmustine (FB) with or without doxorubicin (FAB). Thirty-five patients were evaluable for response. Neither the response rates (partial remission, 11% for FB and 24% for FAB) nor survival times (median, 4.0 months for FB and 5.5 months for FAB) were statistically different. The median survival of patients with partial remission (7.8 months) and those with no change (7.4 months) was significantly prolonged compared to patients with progressive disease (4.5 months). The side effects of both regimens after the first treatment cycle (except alopecia in FAB) were low. After the second cycle more pronounced myelosuppression in the FAB arm was observed.

[Prospective randomized study in advanced stomach cancer. Comparison between combinations of 5-fluorouracil and carmustine without and with adriamycin]. / Prospektive randomisierte Studie beim fortgeschrittenen Magenkarzinom. Vergleich der Kombination von 5-Fluorouracil und Carmustin ohne und mit Adriamycin.

In a multi-center study of 77 patients with gastric carcinoma and metastases, the effects of combined 5-fluorouracil and carmustine with or without adriamycin (FB vs FAB) were compared. There was no significant difference between the two treatment groups as to rate of response or survival time, response to treatment was 20% (FB) and 24% (FAB), including "no change" 52% and 56%, respectively. But median survival time among the responders (partial remission and clinical improvement) and of the patients with unchanged findings was significantly longer (8.4 or 7.4 months, respectively) than that among patients with progression (4.5 months). Analysis of prognostic factors in terms of survival curves revealed no difference between patients with or without measurable tumor parameters, with or without local recurrences or liver metastases. However, there was a statistically significant worsening of prognosis for patients whose initial general state was reduced, who had lung metastases or increased WBC counts. Side effects were relatively mild with both therapy regimens, except for alopecia with FAB. After the second treatment cycle myelosuppression was more marked with FAB than FB.

Comparison of ftorafur with 5-fluorouracil in combination chemotherapy of advanced gastrointestinal carcinoma.

The aim of the study was to compare the combination 5-FU-carmustine with ftorafur-carmustine in the treatment of advanced gastrointestinal cancer. To this end, a prospective, multicenter, randomized trial was initiated. Part I of this trial showed that similar response rates can be obtained with 5-FU-carmustine and ftorafur-carmustine in 109 patients (32.7% versus 26.3%). However, median survival was better in patients treated with 5-FU-carmustine (307 days versus 163 days). Part II of the trial revealed that neither a higher dosage of ftorafur (2 g/m2/day X 5 days) nor the addition of vincristine to both regimens changed the previously obtained results significantly. Again, median survival was found to be better in patients treated with 5-FU combination chemotherapy (304 days versus 144 days). Both the 5-FU and the ftorafur combination were tolerated reasonably well. The results suggest that combination chemotherapy including 5-FU is superior to ftorafur at the applied dosages in terms of survival.