Specific down-regulation of an engineered human cyclin D1 promoter by a novel DNA-binding ligand in intact cells.

Cyclin D1 is expressed at abnormally high levels in many cancers and has been specifically implicated in the development of breast cancer. In this report we have extensively analyzed the cyclin D1 promoter in a variety of cancer cell lines that overexpress the protein and identified two critical regulatory elements (CREs), a previously identified CRE at -52 and a novel site at -30. In vivo footprinting experiments demonstrated factors binding at both sites. We have used a novel DNA-binding ligand, GL020924, to target the site at -30 (-30-21) of the cyclin D1 promoter in MCF7 breast cancer cells. A binding site for this novel molecule was constructed by mutating 2 bp of the wild-type cyclin D1 promoter at the -30-21 site. Treatment with GL020924 specifically inhibited expression of the targeted cyclin D1 promoter construct in MCF7 cells in a concentration-dependent manner, thus validating the -30-21 site as a target for minor groove-binding ligands. In addition, this result validates our approach to regulating the expression of genes implicated in disease by targeting small DNA-binding ligands to key regulatory elements in the promoters of those genes.

Nm23/PuF does not directly stimulate transcription through the CT element in vivo.

Decreased levels of the nm23 gene product have been correlated with increased tumor metastatic potential in a variety of malignancies. At least a subset of the regulatory properties of Nm23 has been proposed to be due to transactivation of the human c-myc oncogene through binding to a homopyrimidine tract 140 base pairs upstream of the transcription start site (termed the CT element or the PuF site). Conventional transcription factors possess DNA binding and transactivation domains; Nm23 fusion proteins were used to address two questions. First, if provided with a well characterized DNA binding domain, does Nm23 possess a transactivation domain capable of stimulating transcription of an appropriate reporter? Second, if provided with a potent transactivation domain, is the DNA binding of Nm23 of sufficient specificity and affinity to direct the fusion protein to a CT-dependent reporter? Since reporter gene expression was not stimulated in either case, we conclude that Nm23 does not directly stimulate transcription through binding to the CT element and that its antimetastatic and other reported functions are likely due to other biochemical activities.

Marking of active genes on mitotic chromosomes.

During development and differentiation, cellular phenotypes are stably propagated through numerous cell divisions. This epigenetic 'cell memory' helps to maintain stable patterns of gene expression. DNA methylation and the propagation of specific chromatin structures may both contribute to cell memory. There are two impediments during the cell cycle that can hinder the inheritance of specific chromatin configurations: first, the pertinent structures must endure the passage of DNA-replication forks in S phase; second, the chromatin state must survive mitosis, when chromatin condenses, transcription is turned off, and almost all double-stranded DNA-binding proteins are displaced. After mitosis, the previous pattern of expressed and silent genes must be restored. This restoration might be governed by mass action, determined by the binding affinities and concentrations of individual components. Alternatively, a subset of factors might remain bound to mitotic chromosomes, providing a molecular bookmark to direct proper chromatin reassembly. Here we analyse DNA at transcription start sites during mitosis in vivo and find that it is conformationally distorted in genes scheduled for reactivation but is undistorted in repressed genes. These protein-dependent conformational perturbations could help to re-establish transcription after mitosis by 'marking' genes for re-expression.

Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo.

Transcription activation and repression of eukaryotic genes are associated with conformational and topological changes of the DNA and chromatin, altering the spectrum of proteins associated with an active gene. Segments of the human c-myc gene possessing non-B structure in vivo located with enzymatic and chemical probes. Sites hypertensive to cleavage with single-strand-specific S1 nuclease or the single-strand-selective agent potassium permanganate included the major promoters P1 and P2 as well as the far upstream sequence element (FUSE) and CT elements, which bind, respectively, the single-strand-specific factors FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K in vitro. Active and inactive c-myc genes yielded different patterns of S1 nuclease and permanganate sensitivity, indicating alternative chromatin configurations of active and silent genes. The melting of specific cis elements of active c-myc genes in vivo suggested that transcriptionally associated torsional strain might assist strand separation and facilitate factor binding. Therefore, the interaction of FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K with supercoiled DNA was studied. Remarkably, both proteins recognize their respective elements torsionally strained but not as liner duplexes. Single-strand- or supercoil-dependent gene regulatory proteins may directly link alterations in DNA conformation and topology with changes in gene expression.

Heterogeneous nuclear ribonucleoprotein K is a transcription factor.

The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.

Cellular nucleic acid binding protein regulates the CT element of the human c-myc protooncogene.

The CT element of the c-myc gene is required for promoter P1 usage and can drive expression of a heterologous promoter. Both double strand (Sp1) and single strand (hnRNP K) CT-binding proteins have been implicated as mediators of CT action. Although significant levels of CT activity persisted following Sp1 immunodepletion, EGTA totally abolished transactivation, thus implicating another metal requiring factor in CT element activity. As hnRNP K binds to one strand of the CT element, but has no metal requirement, the opposite (purine-rich strand) was examined as a target for a metal-dependent protein. A zinc-requiring purine strand binding activity was identified as cellular nucleic acid binding protein (CNBP), a protein previously implicated in the regulation of sterol responsive genes. Two forms of CNBP differed in their relative binding to the CT- or sterol-response elements. CNBP was shown to be a bona fide regulator of the CT element by cotransfection of a CNBP expression vector that stimulated expression of a CT-driven but not an AP1-dependent reporter. These data suggest that hnRNP K and CNBP bind to opposite strands and co-regulate the CT element.

Trypanosoma brucei mitochondrial ribosomal RNA synthesis, processing and developmentally regulated expression.

The steady-state levels of the mitochondrial ribosomal RNAs of Trypanosoma brucei are repressed in the early bloodstream developmental stage of the parasite and accumulate approximately 30-fold during differentiation to the stage found in the midgut of the insect vector. In order to determine the mechanism regulating this developmental process, we have examined the transcription and processing of the 9S and 12S mitochondrial rRNAs of T. brucei. A short-lived RNA was detected in pulse labeling experiments which contains the mature 12S and 9S rRNAs and at least 1200 nucleotides of RNA transcribed from upstream of the 12S rRNA gene. This putative processing precursor RNA was identified in both intact cells and in run-on experiments using isolated mitochondria. The transcripts containing the upstream sequences are unstable and reach isotopic equilibrium within 15 min. Mature rRNAs in the insect developmental stage are stable and show no detectable turnover during a 36-h chase. Comparison of rRNA synthesis in bloodstream and insect life-stages indicates that mitochondrial rRNA levels are controlled not at the transcriptional level, but rather by a mechanism which likely modulates the stability of the mature rRNAs. These results suggest that a short-lived rRNA precursor is synthesized and processed at comparable rates in both bloodstream and insect stages of the parasite. Thus, it appears that differential stability of the mature 9S and 12S rRNAs plays a major role in modulating mitochondrial gene expression during the developmental cycle of T. brucei.

Organization of minicircle genes for guide RNAs in Trypanosoma brucei.

We have identified four T. brucei minicircle sequences that are complementary to cytochrome oxidase III (COIII) edited mRNA sequence and have shown the existence of transcripts from three of these minicircle sequences. These minicircle transcripts potentially serve as guide RNAs (gRNAs) for RNA editing of the COIII transcript. These gRNAs range in size from 55 to 70 nucleotides, are heterogeneous in sequence, and have a 5' terminal triphosphate. The genes for these gRNAs are flanked by imperfect 18 bp repeats separated by approximately 110 bp. Transcription initiates at the first purine within a conserved sequence, 5'-RYA-YA-3', 31 or 32 bp from the upstream inverted repeat. We propose that these 18 bp inverted repeats are important for minicircle gRNA expression in T. brucei.

Transcription of kinetoplast DNA minicircles.

The mitochondrial DNA of trypanosomes is organized as a network of catenated circular DNA molecules called the kinetoplast. The minicircles of the kinetoplast are 1 kb circular DNA molecules present at 5,000-10,000 copies per network. The maxicircles are 20 kb circular molecules present at 50-100 copies per network. Maxicircles are transcribed and are thus analogous to mitochondrial DNAs. Here we show that, contrary to previous reports, the minicircles of T. brucei are also transcribed. A minicircle transcript of approximately 240 nucleotides is present in bloodstream and insect developmental stages of the parasite, is enriched in purified mitochondrial preparations, and is efficiently synthesized in vitro. The minicircle cDNA overlaps the conserved region of the T. brucei minicircle and is juxtaposed to a 12 base sequence common to all minicircles. These findings indicate that minicircles, in addition to their previously proposed structural role, are transcribed.

Developmental regulation of trypanosome mitochondrial gene expression.

Mitochondrial respiratory activities in the protozoan parasite Trypanosoma brucei are developmentally regulated. The trypanosomes in the mammalian bloodstream derive ATP entirely from glycolysis. The trypanosomes found in the midgut of the insect vector or in culture at 26 degrees C have fully functional mitochondria with cytochrome-mediated respiration. In this paper, we show that the steady state levels of the 9 S and 12 S mitochondrial ribosomal RNAs (rRNAs) are 30-fold lower in an early developmental stage in the mammal, the slender forms, relative to the levels in the stumpy trypanosomes, a later developmental stage in the mammalian infection. Transcripts from three other mitochondrial genes, cytochrome b and subunits I and II of cytochrome oxidase, are undetectable in the slender trypanosomes and increase in the stumpy trypanosomes to levels approaching those in trypanosomes from 26 degrees C cultures. Transcription of other mitochondrial genes, including NADH-dehydrogenase subunit 5, is unregulated during trypanosome development. These results show that the level of some mitochondrial transcripts is developmentally regulated in bloodstream trypanosomes and suggest that the stumpy bloodstream trypanosomes accumulate mitochondrial transcripts prior to development of a functional mitochondrion. These results also show that the developmental activation of mitochondrial activities at 26 degrees C is not controlled at the level of mitochondrial transcription.