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.

An Arabidopsis mutant missing one acid phosphatase isoform.

Plant response to phosphorus starvation includes the increased production and secretion of acid phosphatase. We have isolated a mutant of Arabidopsis thaliana (L.) Heynh., phosphatase-underproducer 1 (pup1), that has reduced histochemical staining for acid phosphatase activity in roots of plants grown under phosphorus-starvation conditions. Although pup1 is defective in the production of one inducible acid phosphatase isoform, the most abundant inducible isoform is present. The pup1 mutants are able to respond to phosphorus-deficient conditions by an increase in overall levels of acid phosphatase activity, accumulation of anthocyanins, an increase of the root-to-shoot ratio, and changes in the partitioning of phosphorus between roots and shoots. The gross morphology of the mutants appears normal, except that a small difference in the root to shoot ratio was observed in plants grown under nonstressed conditions. The pup1 gene is incompletely dominant and it is located between 40.2 (+/- 6.2) and 44.9 (+/- 9.9) cM on chromosome 2. This mutant will be useful for determining the role of this acid phosphatase isoform in plant response to phosphorus starvation.

Separation of cis elements responsive to ethylene, fruit development, and ripening in the 5'-flanking region of the ripening-related E8 gene.

The E8 gene is expressed at a high level during fruit ripening, and is transcriptionally activated by ethylene. We have identified a 428 bp fragment of the E8 5'-flanking region, from -1528 to -1100, that makes a minimal 35S promoter responsive to ethylene. This fragment confers ethylene-responsiveness only in the 5'-to-3' orientation; in the reverse orientation it results in increased expression in unripe fruit. Interestingly, this ethylene-responsive construct does not have high levels of expression during fruit ripening, indicating that sequences required for high level expression during fruit ripening are separate from sequences required for ethylene response. The ethylene-responsive sequences of the E8 5'-flanking region interact with the same DNA-binding protein that interacts with sequences required for ethylene responsiveness of the coordinately regulated E4 gene. We also conducted experiments to test the function of a second DNA-binding protein that interacts with both E4 and E8 5'-flanking sequences, the E4/E8-binding protein (E4/E8BP). We examined the effect of an internal deletion from -1088 to -863, which includes the binding site for E4/E8BP, on gene expression. This deletion did not affect expression in ripening fruit, and did not impair ethylene responsiveness. The deletion had a negative effect on expression in unripe fruit, but resulted in increased expression in leaves. These results suggest that the E4/E8BP is not critical for high levels of expression during fruit ripening or for ethylene response, but may play a role in organ-specific gene transcription.

Characterization of a DNA-binding protein that interacts with 5' flanking regions of two fruit-ripening genes.

The E4/E8 binding protein (E4/E8BP) interacts with sequences in the 5' flanking regions of two genes, E4 and E8, that are coordinately regulated by ethylene during tomato fruit ripening. The DNA-binding activity of this protein increases during fruit ripening, and it may play a role in regulation of these genes. To begin to understand the function of this protein, a cDNA has been isolated that encodes a protein, E4/E8BP-1, with DNA-binding specificity similar to that of E4/E8BP. This DNA-binding protein is closely related to a DNA binding protein from tobacco, 3AF1, that interacts with the promoter of the pea rbcS-3A gene. A repeated domain was identified within the predicted 3AF1 amino acid sequence, which includes a series of histidines and cysteines, suggestive of zinc binding, and this repeat is conserved in E4/E8BP-1. Interaction of both E4/E8BP-1 and nuclear extracts from ripening fruit with the E8 recognition sequence is sensitive to 1,10-phenanthroline, indicating that a metal is required for binding of both the native and recombinant proteins. The mRNA for E4/E8BP-1 is moderately abundant in fruit, and increases slightly during fruit ripening, consistent with a role in fruit ripening. A truncated version of E4/E8BP-1 was able to transactivate the E4 promoter in transient assay, demonstrating that this DNA-binding protein can interact with the E4 promoter in vivo to enhance gene transcription.

The Tomato E8 Gene Influences Ethylene Biosynthesis in Fruit but Not in Flowers.

We investigated the function of the tomato (Lycopersicon esculentum) E8 gene. Previous experiments in which antisense suppression of E8 was used suggested that the E8 protein has a negative effect on ethylene evolution in fruit. E8 is expressed in flowers as well as in fruit, and its expression is high in anthers. We introduced a cauliflower mosaic virus 35S-E8 gene into tomato plants and obtained plants with overexpression of E8 and plants in which E8 expression was suppressed due to co-suppression. Overexpression of E8 in unripe fruit did not affect the level of ethylene evolution during fruit ripening; however, reduction of E8 protein by cosuppression did lead to elevated levels during ripening. Levels for ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ACC oxidase mRNA were increased approximately 7-fold in fruit of plants with reduced E8 protein. Levels of ACC synthase 2 mRNA were increased 2.5-fold, and ACC synthase 4 mRNA was not affected. Reduction of E8 protein in anthers did not affect the accumulation of ACC or of mRNAs encoding enzymes involved in ethylene biosynthesis. Our results suggest that the product of the E8 reaction participates in feedback regulation of ethylene biosynthesis during fruit ripening.

Ethylene control of E4 transcription during tomato fruit ripening involves two cooperative cis elements.

E4 gene transcription is controlled by ethylene during tomato fruit ripening. To define the ethylene-responsive promoter elements, we have tested the activity of mutations of the E4 promoter, and of chimeric genes in transient assay. Using a set of linker scan mutations of the region from -160 to -91, we determined that sequences located between -150 and -121 bp from the transcription start site are required for normal levels of ethylene-regulated transcription. However, E4 sequences from -193 to -40 were not able to confer ethylene-responsiveness to the minimal (-46) 35S promoter. The E4/E8 binding protein (E4/E8 BP) interacts with sequences in the 5'-flanking regions of both E4 and the coordinately regulated E8 gene, and its role in regulation of E4 transcription was investigated. The E4 binding site spans the E4 TATA box, and so mutations of this site were limited to those that did not disrupt the E4 TATA box. Mutations of this site which reduced affinity for the E4/E8 BP also resulted in reduced activity in transient assay, supporting a role for this element in normal regulation of the gene. Fusion of the 35S enhancer to E4 sequences from -85 to +65 did not result in an ethylene-responsive promoter, indicating that the E4/E8 BP-binding site is not sufficient for ethylene response. We conclude that at least two cis elements are required for ethylene-responsive transcription of the E4 gene during fruit ripening, one between -150 and -121 and the other between -40 and +65.

Induction of Anthocyanin Accumulation by Cytokinins in Arabidopsis thaliana.

Arabidopsis thaliana plants treated with exogenous cytokinins accumulate anthocyanin pigments. We have characterized this response because it is potentially useful as a genetic marker for cytokinin responsiveness. Levels of mRNAs for four genes of the anthocyanin biosynthesis pathway, phenylalanine ammonia lyase 1 (PAL1), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) were shown to increase coordinately in response to benzyladenine (BA). However, nuclear run-on transcription experiments suggested that although CHS and DFR are controlled by BA at the transcriptional level, PAL1 and CHI are controlled by BA posttranscriptionally. CHS mRNA levels increased within 2 h of BA spray application, and peaked by 3 h. Levels of PAL1 mRNA did not increase within 6 h of BA spray. We also showed that PAL1, CHS, CHI, and DFR mRNA levels fluctuate during a 24-h period and appear to be controlled by a circadian clock. The relation between cytokinin regulation and light regulation of CHS gene transcription is discussed.

A novel cytokinin-resistant mutant of Arabidopsis with abbreviated shoot development.

Cytokinins influence several fundamental processes of plant growth and development, including cell division and organogenesis. To identify genes involved in cytokinin response, a screen was carried out for mutants of Arabidopsis thaliana (L.) Heynh. that are resistant to elevated levels of exogenous cytokinins. One such mutant was isolated and named cyr1. A cross to another Arabidopsis cytokinin-resistant mutant, ckr1 (Su and Howell 1992), indicated that the two mutants are not allelic. The recessive cyr1 mutation causes tenfold decreased sensitivity to benzyladenine in a root-elongation assay but does not confer resistance of roots to indole-3-acetic acid, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, or abscisic acid. The mutant has increased sensitivity to abscisic acid. This mutation is apparently pleiotropic, giving rise to shoot abnormalities. The phenotype of the cyr1 shoot includes abbreviated development with reduction in cotyledon and leaf expansion, limited leaf production, reduced chlorophyll accumulation, failure to accumulate anthocyanins in response to cytokinins, and the formation of a single infertile flower. These traits, as well as root resistance to cytokinin, are all consistent with a defect in cytokinin action, and are probably due to mutation of a single gene. The cyr1 gene is located on chromosome 5, between 76.2 and 77.6 cM.

Ketoconazole and 25-hydroxycholesterol produce reciprocal changes in the rate of transcription of the human LDL receptor gene.

Sterol-dependent regulation of low-density lipoprotein (LDL) receptor gene expression was studied in the human hepatoma HepG2 cell line. Incubation of HepG2 cells with 20 microM ketoconazole increased the level of LDL receptor mRNA. After a lag of approx. 1.0 h the level rose 6.5-fold within 8.0 h and remained elevated for up to 24 h. Incubation with 10 micrograms 25-hydroxycholesterol/ml for 24 h produced a 40-50% reduction in the level of LDL receptor mRNA. Ketoconazole- and 25-hydroxycholesterol-induced changes in LDL receptor mRNA accumulation were due to alterations in the relative rate of LDL receptor gene transcription as measured by nuclear run-on transcription. Incubation with 20 microM ketoconazole for 4 h or 10 micrograms 25-hydroxycholesterol/ml for 24 h produced a 3.6-fold increase and a 40% reduction, respectively, in the transcription rate of LDL receptor gene. Removal of the Alu-like sequence elements within the LDL receptor cDNA was required to consistently measure changes in LDL receptor gene transcription. No significant changes were noted in the half-life of LDL receptor mRNA in ketoconazole or 25-hydroxycholesterol-treated cells. These data demonstrate that sterol-dependent changes in the level of LDL receptor mRNA can be completely accounted for by changes in the rate of LDL receptor gene transcription.

Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp.

The tomato fruit consists of a thick, fleshy pericarp composed predominantly of highly vacuolated parenchymatous cells, which surrounds the seeds. During ripening, the activation of gene expression results in dramatic biochemical and physiological changes in the pericarp. The polygalacturonase (PG) gene, unlike many fruit ripening-induced genes, is not activated by the increase in ethylene hormone concentration associated with the onset of ripening. To investigate ethylene concentration-independent gene transcription in ripe tomato fruit, we analyzed the expression of chimeric PG promoter-beta-glucuronidase (GUS) reporter gene fusions in transgenic tomato plants. We determined that a 1.4-kb PG promoter directs ripening-regulated transcription in outer pericarp but not in inner pericarp cells, with a sharp boundary of PG promoter activity located midway through the pericarp. Promoter deletion analysis indicated that a minimum of three promoter regions influence the spatial regulation of PG transcription. A positive regulatory region from -231 to -134 promotes gene transcription in the outer pericarp of ripe fruit. A second positive regulatory region from -806 to -443 extends gene activity to the inner pericarp. However, a negative regulatory region from -1411 to -1150 inhibits gene transcription in the inner pericarp. DNase I footprint analysis showed that nuclear proteins in unripe and ripe fruit interact with DNA sequences within each of these three regulatory regions. Thus, temporal and spatial control of PG transcription is mediated by the interaction of negative and positive regulatory promoter elements, resulting in gene activity in the outer pericarp but not the inner pericarp of ripe tomato fruit. The expression pattern of PG suggests that, although they are morphologically similar, there is a fundamental difference between the parenchymatous cells within the inner and outer pericarp.

Identification of an ethylene-responsive region in the promoter of a fruit ripening gene.

Transcription of the E4 gene is controlled by an increase in ethylene concentration during tomato fruit ripening. To investigate the molecular basis for ethylene regulation, we have examined the E4 promoter to identify cis elements and trans-acting factors that are involved in E4 gene expression. In transgenic tomato plants a chimeric gene construct containing a 1.4-kilobase E4 promoter fused to a beta-glucuronidase reporter gene is rapidly induced by ethylene in ripening fruit. Deletion of E4 promoter sequences to 193 base pairs reduces the level of GUS activity but does not affect ethylene induction. Transient expression of E4 promoter-luciferase chimeric gene constructs containing various deletions, introduced into tomato fruit pericarp by particle bombardment, indicates that a positive ethylene-responsive region is localized between nucleotides -161 and -85 relative to the transcription start site. DNase I footprint analysis shows that a nuclear factor in unripe fruit interacts specifically with sequences in this element, from -142 to -110, which are required for the ethylene response. The DNase I footprint of this factor is reduced in ethylene-treated unripe fruit and undetectable in ripe fruit. Based on the correlation of a nuclear factor binding site with promoter sequences required for ethylene induction, we propose that this in vitro DNA-binding activity may represent a factor that is involved in ethylene-regulated E4 gene expression.

Organization of Ripening and Ethylene Regulatory Regions in a Fruit-Specific Promoter from Tomato (Lycopersicon esculentum).

Tomato (Lycopersicon esculentum) fruit ripening is initiated by an increase in ethylene hormone concentration. E8 gene transcription is fruit-specific and is activated at the onset of ripening and in unripe fruit treated with exogenous ethylene. To understand how E8 gene transcription is controlled during ripening, we analyzed the effect of deletions of flanking DNA sequences on E8 gene expression in transgenic tomato fruit. We found that a minimum of three 5' and one 3' regions influence E8 gene expression during fruit ripening. DNA sequences that confer responsiveness to exogenous ethylene in unripe fruit are distinct from DNA sequences that are sufficient for expression during fruit ripening.

Interaction of a developmentally regulated DNA-binding factor with sites flanking two different fruit-ripening genes from tomato.

To investigate mechanisms that control fruit development, we have begun experiments to identify proteins that control gene expression during tomato fruit ripening. We focused on the regulation of two different genes, E4 and E8, whose transcription is coordinately activated at the onset of fruit ripening. We report here that a DNA-binding protein specifically reacts with similar sequences flanking the E4 and E8 genes. The E4 binding site is at position -34 to -18 and, therefore, overlaps the region (TATA box) that in many eukaryotic genes serves to determine the efficiency and initiation site of transcription. In contrast, the E8 binding site is distal, located at -936 to -920 relative to the start of E8 gene transcription. Gel electrophoresis mobility retardation experiments indicate that the DNA binding activity that interacts with these two sites increases at the onset of fruit ripening. Taken together, these results suggest that this DNA-binding protein may function to coordinate E4 and E8 gene expression during fruit ripening.

Interaction of a DNA binding factor with the 5'-flanking region of an ethylene-responsive fruit ripening gene from tomato.

To understand how fruit development is controlled, we have begun experiments to identify DNA sequences and proteins that regulate gene expression during tomato (Lycopersicon esculentum) fruit ripening. We have focused on the E8 gene because its transcription is responsive to ethylene hormone and is activated at the onset of fruit ripening. We report here that sequences required for ethylene-responsive and developmentally regulated E8 gene expression in transgenic tomato plants are contained on a 4.4 kb restriction fragment which includes sequences 2 kb 5' and 0.5 kb 3' to the gene. In addition, we have identified a DNA-binding factor that specifically interacts with DNA sequences that flank the E8 gene. This DNA-binding activity is low in unripe fruit and increases during fruit ripening. This factor also binds to the 5'-flanking region of another ethylene-responsive gene which is coordinately expressed during tomato fruit ripening. These data suggest that the DNA binding-factor may be involved in the regulation of gene expression during fruit ripening.

Regulation of the accumulation of mRNA for alpha-amylase in barley aleurone.

The effect of gibberellic acid and Ca(2+) on the accumulation of alpha-amylase mRNAs in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) was studied using cDNA clones containing sequences of mRNAs for the high and low isoelectric point (pI) alpha-amylases. There is no significant hybridization between the two alpha-amylase cDNA clones under the hybridization and washing conditions employed. These clones were therefore used to monitor levels of mRNAs for high and low pI alpha-amylases. It is shown that although the synthesis of the high pI alpha-amylase proteins depends on the presence of Ca(2+) in the incubation medium, the accumulation of mRNA for this group occurs to the same degree in the presence or the absence of Ca(2+). The accumulation of low pI alpha-amylase mRNA is also not affected by the presence or absence of Ca(2+) in the incubation medium. These results establish gibberellic acid, not Ca(2+), as the principal regulator of alpha-amylase mRNA accumulation in barley aleurone, while Ca(2+) controls high pI alpha-amylase synthesis at a later step in the biosynthetic pathway.

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers.

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA(3)) with or without 5 millimolar CaCl(2) shows that alpha-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca(2+). A cDNA clone for alpha-amylase was isolated and used to measure alpha-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca(2+). No difference was observed in alpha-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA(3) with 5 millimolar CaCl(2) and layers incubated in GA(3) alone. RNA isolated from layers incubated for 12 hours in GA(3) with and without Ca(2+) was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca(2+) in the incubation medium. Immunoprecipitation of translation products showed that the RNA for alpha-amylase synthesized in Ca(2+)-deprived aleurone layers was translatable. Ca(2+) is required for the synthesis of alpha-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.