Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.

Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently.

Transcriptional and posttranscriptional gene silencing are mechanistically related.

Two distinct gene-silencing phenomena are observed in plants: transcriptional gene silencing (TGS), which involves decreased RNA synthesis because of promoter methylation, and posttranscriptional gene silencing (PTGS), which involves sequence-specific RNA degradation. PTGS is induced by deliberate [1-4] or fortuitous production (R.v.B., unpublished data) of double-stranded RNA (dsRNA). TGS could be the result of DNA pairing [5], but could also be the result of dsRNA, as was shown by the dsRNA-induced inactivation of a transgenic promoter [6]. Here, we show that when targeting flower pigmentation genes in Petunia, transgenes expressing dsRNA can induce PTGS when coding sequences are used and TGS when promoter sequences are taken. For both types of silencing, small RNA species are found, which are thought to be dsRNA decay products [7] and determine the sequence specificity of the silencing process [8, 9]. Furthermore, silencing is accompanied by the methylation of DNA sequences that are homologous to dsRNA. DNA methylation is assumed to be essential for regulating TGS and important for reinforcing PTGS [10]. Therefore, we conclude that TGS and PTGS are mechanistically related. In addition, we show that dsRNA-induced TGS provides an efficient tool to generate gene knockouts, because not only does the TGS of a PTGS-inducing transgene fully revert the PTGS phenotype, but also an endogenous gene can be transcriptionally silenced by dsRNA corresponding to its promoter.

Distinct features of post-transcriptional gene silencing by antisense transgenes in single copy and inverted T-DNA repeat loci.

The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as-RNA) are expected to result in strong silencing but often there is no clear correlation between as-RNA levels and the degree of silencing. To obtain insight into these puzzling observations, we have analyzed several petunia transformants of which the pigmentation gene chalcone synthase (Chs) is post-transcriptionally silenced in corollas by antisense (as) Chs transgenes. The transformants were examined with respect to the steady-state as-RNA level, transcription level of the as-transgenes, the repetitiveness and structure of the integrated T-DNAs, and the methylation status of the transgenes. This revealed that the transformants can be divided in two classes: the first class contains a single copy (S) T-DNA of which the as-Chs gene is transcribed, although several-fold lower than the endogenous Chs genes. As there are not sufficient as-RNAs to degrade every mRNA, we speculate that silencing is induced by double-stranded RNA. The second class contains two T-DNAs which are arranged as inverted repeats (IRs). These IR loci are severely methylated and the as-Chs transgenes transcriptionally barely active. The strongest silencing was observed with IR loci in which the as-Chs transgenes were proximal to the centre of the IR. Similar features have been described for co-suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisense IRs also occurs by co-suppression, either via ectopic DNA pairing or via dsRNA.

Developmental abnormalities associated with deoxyadenosine methylation in transgenic tobacco.

As in other higher eukaryotes, DNA methylation in plants is predominantly found at deoxycytosine residues, while deoxyadenosine residues are not methylated at significant levels. 6mdA methylation has been successfully introduced into yeast and Drosophila via expression of a heterologous methyltransferase, but similar attempts in tobacco had, up until now, proved unsuccessful despite the correct expression of a methyltransferase construct. It was unclear whether this result reflected the failure of heterologous methyltransferases to enter the nucleus, or whether 6mdA methylation, which has been shown to interfere with promoter activity, was toxic for plants. Here we show that 6mdA methylation can be successfully introduced into transgenic tobacco plants via expression of the bacterial dam enzyme. The efficiency of 6mdA methylation was directly proportional to expression levels of the dam construct, and methylation of all GATC sites was observed in a highly expressing line. Increasing expression levels of the enzyme in different plants correlated with increasingly abnormal phenotypes affecting leaf pigmentation, apical dominance, and leaf and floral structure. Whilst introduction of dam-specific methylation does not cause any developmental abnormalities in yeast or Drosophila, our data suggest that methylation of deoxyadenine residues in plants interferes with the expression of genes involved in leaf and floral development.

Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing.

The chromatin structures of two epigenetic alleles of a transgene were investigated by measuring the local accessibility of transgene chromatin to endonucleases. The two epialleles represented the active, hypomethylated state of a transgene in line 17-I of Petunia hybrida, and a transcriptionally inactive, hypermethylated derivative of the same transgene in line 17-IV. In nuclear preparations the inactive epiallele was significantly less sensitive to DNasel digestion and nuclease S7 digestion than the transcriptionally active epiallele, whereas no significant differences in accessibility were observed between naked DNA samples of the two epialleles. Our data suggest that a condensed chromatin structure is specifically imposed on transcribed regions of the construct in line 17-IV. In contrast, in both epialleles the plasmid region of the transgene, which is not transcriptionally active in plants, retains the same accessibility to endonucleases as the chromosomal integration site. These data suggest that transcriptional inactivation is linked to the process of transcription, and imply that control of transgene expression via the use of inducible or tissue-specific promoters might prevent transgene silencing and conserve the active state of transgenes during sexual propagation.

The relationship between cardiovascular and catecholamine reactions to laboratory and real-life stress.

Studies on the predictive value of stress testing in the laboratory for the reaction to real-life stress have shown equivocal results. The variety of laboratory tasks differ for unknown reasons in their predictive power, and the results vary unsystematically among the physiological parameters measured. Most studies have focused on the prediction of ambulatory levels of blood pressure. Many other influences, besides stress, however, influence ambulatory levels. Therefore, a better operationalization of real-life stress is to measure a person in a resting position during a period of well defined real-life stress. The present study investigates whether the difference in ability of laboratory stress tasks to predict real-life stress values is due to the different type of physiological response they induce. Hence, in the present study a more detailed measurement of the stress response was performed. A second question was whether stress testing would add to the prediction of the real-life stress reactions above the prediction based on resting levels. This question was answered for both cardiovascular and catecholamine reactions to laboratory tasks. Two active coping tasks, one inducing a mainly cardiac-sympathetic reaction and the other a relatively more vascular response, and a cold pressor test were administered to 33 healthy young males. Real-life stress consisted of the anticipation of the public defence of the PhD thesis. Tasks indeed differed in predictive power, but this was not a function of the type of response they induced.(ABSTRACT TRUNCATED AT 250 WORDS)

Gibberellic Acid Regulates Chalcone Synthase Gene Transcription in the Corolla of Petunia hybrida.

The pigmentation of Petunia hybrida corollas is regulated by gibberellic acid (GA(3)). It controls the increase of flavonoid enzyme levels and their corresponding mRNAs. We have used an in vitro culture system for corollas to study the regulatory role of GA(3) in the expression of flavonoid genes. By determining steady-state mRNA levels, we show that the accumulation of chalcone synthase (chs) mRNA in young corollas is dependent on the presence of both sucrose and GA(3) in the culture medium. Whereas sucrose had a general metabolic effect on gene expression, the stimulatory role of GA(3) was specific. Analysis of nascent transcripts in isolated corolla nuclei showed that changes in steady-state chs mRNA levels correlated very well with changes in the transcription rate. We therefore conclude that GA(3) controls the expression of chs at the transcriptional level. Preculturing the corollas in sucrose medium without GA(3) resulted in a lower chs mRNA level. The expression could be reinduced by the addition of GA(3). The hormone is thus required for the induction but also for the maintenance of chs transcription. The delayed reinduction of chs expression, the lag time in the kinetics of chs mRNA accumulation, and the inhibitory effect of cycloheximide on the action of GA(3) suggest that GA(3) controls chs transcription in an indirect manner. Our data support a model in which GA(3) induces the production of a regulatory protein such as a receptor or a trans-acting factor that is directly involved in chs transcription.

Regulation of plant gene expression by antisense RNA.

Regulation of gene expression by antisense RNA was first discovered as a naturally-occurring phenomenon in bacteria. Recently natural antisense RNAs have been found in a variety of eukaryotic organisms; their in vivo function is, however, obscure. Deliberate expression of antisense RNA in animal and plant systems has lead to successful down-regulation of specific genes. We will review the current status of antisense gene action in plant systems. The recent discovery that 'sense' genes are able to mimic the action of antisense genes indicates that (anti)sense genes must operate by mechanisms other than RNA-RNA interaction.

Chalcone Synthase Promoters in Petunia Are Active in Pigmented and Unpigmented Cell Types.

Chalcone synthase (CHS) catalyzes the first step in the biosynthesis of flavonoids that function in flower pigmentation, protection against stress, and induction of nodulation. The petunia genome contains eight complete chs genes, of which four are differentially expressed in floral tissues and UV-light-induced seedlings. The 5[prime]-flanking regions of these four chs genes were fused to the [beta]-glucuronidase (GUS) reporter gene and introduced into petunia plants by Agrobacterium-mediated transformation. We show that expression of each construct is identical to the expression of the authentic chs gene, implying that the differences in expression pattern between these chs genes are caused at least in part by their promoters. Histochemical analyses of GUS expression show that chs promoters are not only active in pigmented cell types (epidermal cells of the flower corolla and tube and [sub] epidermal cells of the flower stem) but also in a number of unpigmented cell types (mesophylic cells of the corolla, several cell types in the ovary and the seed coat). Comparison of chs-GUS expression and flavonoid accumulation patterns in anthers suggests that intercellular transport of flavonoids and enzymes occurs in this organ. Analysis of the flavonoids accumulated in tissues from mutant lines shows that only a subset of the genes that control flavonoid biosynthesis in the flower operates in the ovary and seed. This implies that (genetic) control of flavonoid biosynthesis is highly tissue specific.

The relation of type A behavior and vital exhaustion with physiological reactions to real life stress.

Type A behavior and a Vital Exhaustion/Depression cluster seem to be the most crucial elements of the psychological 'coronary risk profile'. The question is, what physiological mechanisms intervene between these characteristics and CHD risk. In the present study the relationship was investigated between type A behavior and Vital Exhaustion on the one hand and the reaction of blood pressure, catecholamines and cholesterol to a real life stressor (Ph.D. thesis defence) on the other. Type A was shown to be related to a stronger response of adrenaline and diastolic blood pressure to the stressor. Vital Exhaustion was also positively correlated with the adrenaline reaction, and moreover, with cholesterol base level, stress induced cholesterol change, and noradrenaline and cholesterol stress levels. It was suggested that the relation between Vital Exhaustion and cholesterol parameters may originate in noradrenaline induced lipolysis. Type A and Vital Exhaustion may exert their influence on coronary risk by way of different physiological mechanisms. Type A via exaggerated hemodynamic reactivity, and Vital Exhaustion via lipid metabolism.

Serum-cholesterol: sex specific psychological correlates during rest and stress.

In the present study an attempt was made to assess whether male and female students differ in their cholesterol and catecholamine reaction to examination stress. An answer was also sought to the question of whether cholesterol level and reactivity could be predicted from behavioral traits and mood. In 29 male and 23 female students, cholesterol and urine-catecholamines were measured on an examination day and on a control day. Cholesterol level was found to be higher on the examination day, in both males and females. Although males showed a larger adrenaline reaction than females, their cholesterol reaction did not differ from that of the females. Specifically with regard to males, 62% of the variance in cholesterol base level and 40% of the variance of the stress induced cholesterol rise, were explained by the psychological variables measured. Achievement motivation and depression contributed to both predictions. No significant predictions could be made in the female group. This demonstrates the necessity of taking sex differences into account in psychophysiological studies. It is suggested to pay greater attention in future research to the mediating role that blood lipids play in the relationship between psychological factors and the risk of coronary heart disease.