Разработка устройства сканирования пучков используемых для производства изотопов

Expression of the C(4)-specific phosphoenolpyruvate carboxylase (C(4)-PEPC) gene in maize (Zea mays) is regulated in a tissue-specific manner, but affected by light and nutrient availability. We manipulated these stimuli in a combinatorial manner and analyzed concomitant changes in histone acetylation of the nucleosomes associated with the C(4)-PEPC gene in relation to transcriptional activity and steady-state mRNA levels. Whereas the transition from the lowest activity to an intermediate activity was observed in the absence of histone acetylation, the light-induced boost to full activity was associated with strong enhancement of the acetylation of both histones H3 and H4 limited to the gene region. Once activated by light, prolonged darkness was necessary to reduce both transcription and, in parallel, histone acetylation. Unexpectedly, histone acetylation was also induced in bundle sheath cells, although the transcriptional activity did not respond to illumination in this tissue. Furthermore, we were able to down-regulate the promoter by nitrogen depletion in the light without any decrease in the hyperacetylation of histone H4. When plants kept in prolonged darkness were nitrogen depleted and then exposed to light, transcription was not induced, but the promoter chromatin became hyperacetylated. We suggest a model where inhibition of a histone deacetylase in the light triggers H4 hyperacetylation at the C(4)-PEPC gene promoter regardless of the transcriptional activity of the gene. Our data indicate that an understanding of the interplay between histone modification and transcription requires analysis of signal integration on promoters in vivo

Epigenetische Mechanismen zur Regulation der Transkription C 4-typischer Gene in Mais (Zea mays L.)

Nuclear genes are organised in chromatin. The transcriptional activation of chromatin is associated with various epigenetic mechanisms and covalent histone modifications play a central role in this process. Among these modifications, methylation and acetylation of the N-terminal regions of histones H3 and H4 are most important. Acetylation of histones normally correlates with transcriptional activation whereas histone methylation can show either activating or repressing effects. Methylation of DNA can also act as a signal that is often associated with gene repression. An appropriate model system for the analysis of epigenetic effects on transcription are genes involved in the C4-metabolism. As these genes show a light-induced and tissue-specific gene regulation, different transcriptional levels can be easily induced and analysed. In this study, the transcriptional activation of C4-PEPC, C4-ME, C4-PPDK and the small subunit of RubisCO RbcS2 was determined by Real-Time-PCR. Histone methylation was analysed via ChIP-analysis and DNA methylation patterns were measured with restriction enzymes sensitive or insensitive to cytosine methylation. Upon light induction, all genes studied are expressed either in the mesophyll or in bundle sheath cells. An impact of DNA methylation on light-dependent or tissue-specific regulation of transcription could not be detected, but a strong correlation of certain histone modifications with the potential to activate genes in specific tissues was observed. Both coding and promotor regions of the tested genes showed trimethylation of lysine 4 on histone H3 (=H3K4) in the tissues where the genes could be activated by light. In contrast, tissues where transcription could not be activated display increased values of mono- and dimethylation of the lysine residue. These modifications were independent of transcription itself, in particular light was not necessary to establish the modification pattern. The strongest effects were monitored at the genes encoding C4-PEPC and C4-ME, but the same tendencies were observed at the genes for C4-PPDK and RbcS2. Unexpectedly, dimethylation of lysine 9 on histone H3, a modification normally associated with repressed chromatin, was also found on the C4-PEPC-gene. Signals were as strong as measured at a transcriptionally totally inactive retrotransposon sequence. Comparison of the histone methylation patterns in leaf and root tissues indicates a strong reduction of H3K4-trimethylation in the root. Final results could not be obtained for the other modifications. Data are discussed against the background of the histone code hypothesis and a model is suggested where different environmental stimuli are integrated into a promoter response through the induction of specific histone modifications

Epigenetische Mechanismen zur Regulation der Transkription C 4-typischer Gene in Mais (Zea mays L.)

Nuclear genes are organised in chromatin. The transcriptional activation of chromatin is associated with various epigenetic mechanisms and covalent histone modifications play a central role in this process. Among these modifications, methylation and acetylation of the N-terminal regions of histones H3 and H4 are most important. Acetylation of histones normally correlates with transcriptional activation whereas histone methylation can show either activating or repressing effects. Methylation of DNA can also act as a signal that is often associated with gene repression. An appropriate model system for the analysis of epigenetic effects on transcription are genes involved in the C4-metabolism. As these genes show a light-induced and tissue-specific gene regulation, different transcriptional levels can be easily induced and analysed. In this study, the transcriptional activation of C4-PEPC, C4-ME, C4-PPDK and the small subunit of RubisCO RbcS2 was determined by Real-Time-PCR. Histone methylation was analysed via ChIP-analysis and DNA methylation patterns were measured with restriction enzymes sensitive or insensitive to cytosine methylation. Upon light induction, all genes studied are expressed either in the mesophyll or in bundle sheath cells. An impact of DNA methylation on light-dependent or tissue-specific regulation of transcription could not be detected, but a strong correlation of certain histone modifications with the potential to activate genes in specific tissues was observed. Both coding and promotor regions of the tested genes showed trimethylation of lysine 4 on histone H3 (=H3K4) in the tissues where the genes could be activated by light. In contrast, tissues where transcription could not be activated display increased values of mono- and dimethylation of the lysine residue. These modifications were independent of transcription itself, in particular light was not necessary to establish the modification pattern. The strongest effects were monitored at the genes encoding C4-PEPC and C4-ME, but the same tendencies were observed at the genes for C4-PPDK and RbcS2. Unexpectedly, dimethylation of lysine 9 on histone H3, a modification normally associated with repressed chromatin, was also found on the C4-PEPC-gene. Signals were as strong as measured at a transcriptionally totally inactive retrotransposon sequence. Comparison of the histone methylation patterns in leaf and root tissues indicates a strong reduction of H3K4-trimethylation in the root. Final results could not be obtained for the other modifications. Data are discussed against the background of the histone code hypothesis and a model is suggested where different environmental stimuli are integrated into a promoter response through the induction of specific histone modifications

Developmental and Environmental Signals Induce Distinct Histone Acetylation Profiles on Distal and Proximal Promoter Elements of the C4-Pepc Gene in Maize

The maize C4-Pepc gene is expressed in an organ- and cell-type-specific manner, inducible by light and modulated by nutrient availability and the metabolic state of the cell. We studied the contribution of histone acetylation at five lysine residues to the integration of these signals into a graduated promoter response. In roots and coleoptiles, where the gene is constitutively inactive, three of the five lysines were acetylated and the modifications showed unique patterns with respect to their distribution on the gene. A similar pattern was observed in etiolated leaves, where the gene is poised for activation by light. Here, illumination selectively induced the acetylation of histone H4 lysine 5 and histone H3 lysine 9 in both the promoter and the transcribed region, again with unique distribution patterns. Induction was independent of transcription and fully reversible in the dark. Nitrate and hexose availability modulated acetylation of all five lysines restricted to a distal promoter region, whereas proximal promoter acetylation was highly resistant to these stimuli. Our data suggest that light induction of acetylation is controlled by regulating HDAC activity, whereas metabolic signals regulate HAT activity. Acetylation turnover rates were high in the distal promoter and the transcribed regions, but low on the proximal promoter. On the basis of these results, we propose a model with three levels of stimulus-induced histone modifications that collectively adjust promoter activity. The results support a charge neutralization model for the distal promoter and a stimulus-mediated, but transcription-independent, histone acetylation pattern on the core promoter, which might be part of a more complex histone code