Browsing by Subject "Transkriptionsfaktor"

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Functional characterization of the COOH-terminal kinase activity of the TBP-associated factor TAF1
(2006) Maile, Tobias; Sauer, Frank
Activation of eukaryotic transcription involves an orchestrated interplay between transcription factors and the general RNA polymerase II (Pol II) transcription machinery (GTM), which consists of Pol II and general transcription factors (GTFs). The GTF TFIID consists of the TATA-box binding protein (TBP) and several TBP-associated factors (TAFs). The binding of TFIID to promoters can nucleate transcription. TAF1 is the largest subunit of TFIID and plays a central role within the nucleating function of TFIID in transcription. TAF1 mediates the binding of TFIID to promoters and interacts with enhancer-bound transcription factors and several GTFs. Additionally, TAF1 contains four enzymatic activities that are essential for viability of eukaryotes and mediate posttranslational modification of GTFs and histones. TAF1 is a bipartite protein kinase and contains an NH2-terminal kinase domain (NTK) and a COOH-terminal kinase domain (CTK). A previous study demonstrated that the CTK phosphorylates serine-residue 33 in histone H2B (H2BS33). However, the role of TAF1-mediated phosphorylation in transcription regulation remained unknown. In this study, the functional importance of H2BS33 phosphorylation (H2BS33P) by TAF1 was investigated by using a combination of biochemical and in vivo assays. In vitro kinase assays uncovered the two essential kinase motifs in TAF1CTK, the ATP-binding motif and the serine/threonine-specific catalytic motif, and indicate that the TAF1 CTK has intrinsic kinase-activity. Western blot analysis using an antibody to H2BS33P revealed that H2BS33 is phosphorylated in Drosophila. RNA-interference (RNAi) assays, designed to attenuate TAF1 expression (TAF1RNAi), revealed that TAF1 is a major kinase for H2BS33 in Drosophila Schneider cells. Flow-cytometry analysis of TAF1RNAi cells indicated that loss of TAF1 expression results in cell cycle arrest in G2/M-phase. Screening the transcription of cell cycle genes in TAF1RNAi cells by using reverse-transcriptase-PCR demonstrated that the transcription of the cell cycle gene string (stg) is reduced in the absence of TAF1. Chromatin immunoprecipitation assays (XChIP) indicate that H2BS33P is detectable at the transcriptionally active stg promoter but not at the silent stg promoter in TAF1RNAi cells. These results demonstrate that phosphorylation of H2BS33 is involved in stg transcription. XChIP-assays using chromatin prepared from Drosophila embryos, which express a mutant TAF1 lacking the CTK, revealed that CTK-mediated phosphorylation of H2BS33 plays an essential role in the activation of transcription of the Drosophila segmentation gene giant. In vitro kinase assays demonstrate that Bdf1 and Bdf2, the yeast homologues of the TAF1CTK, phosphorylate histones suggesting that the kinase activity of the TAF1CTK is phylogenetically conserved. The results of this work demonstrate that TAF1CTK is a major histone kinase of H2BS33 and that TAF1-mediated phosphorylation of H2BS33 plays an essential role in the transcription events during cell cycle progression and development.
Metabolic engineering of flavonoid biosynthesis in hop (Humulus lupulus L.) for enhancing the production of pharmaceutically active secondary metabolites
(2012) Gatica Arias, Andres Mauricio; Weber, Gerd
For a long time, hop (Humulus lupulus L.) has been used in the brewing industry as flavoring and preserving agent. Moreover, the hop plant has been used for medicinal purposes. Recently, xanthohumol and desmethylxanthohumol have received special attention due to their potential cancer chemopreventive properties. Hop breeding programs have been mainly focused on the development of new cultivars with a high content of α- and β-acids in order to satisfy the demand of the brewing industry. However, due to the medical and pharmaceutical importance of hops, new breeding efforts have been done to create new cultivars with a higher content of xanthohumol and desmethylxanthohumol. In order, to complement these efforts, metabolic engineering of flavonoid biosynthesis offers tremendous potential to modify the production of these compounds. The flavonoid biosynthetic pathway has been intensively studied in plants. Often single target genes are regulated by several transcription factors. The R2R3 MYB transcription factor family plays an important role in the regulation of the biosynthesis of phenylpropanoids and flavonoids. Ectopic expression of these transcription factors in transgenic plants stimulated the production and enhanced the quantity of flavonoids. The main objective of the research presented here was to modulate the production of pharmaceutical metabolites in hop through metabolic engineering of the flavonoid biosynthesis pathway. Towards this goal, in a first approach, genetic engineering of hop cv. Tettnanger with the heterologous transcription factor PAP1/AtMYB75 from Arabidopsis thaliana L. was successfully accomplished. It was shown that PAP1/AtMYB75 was stably incorporated and expressed in the hop genome. The transgenic events showed reddish to pink female flowers and cones. Moreover, compared to the wildtype plants, the expression of the structural genes CHS_H1, CHI, and F3´H was elevated in transgenic hop plants. In addition, the production of anthocyanins, rutin, isoquercitin, kaempferol-7-O-glucoside, kaempferol-7-O-glucoside-malonate, desmethylxanthohumol, xanthohumol, α-acids, and β-acids in transgenic hop plants was influenced by the PAP1/AtMYB75 transcription factor. In a second approach, the homologous transcription factor HlMYB3 from H. lupulus L. was genetically introduced and expressed in the hop genome. The effect of the over-expression of the transgene on the expression rate of structural flavonoid and phloroglucinol biosynthetic genes, like PAL, C4H, 4CL, CHS_H1, CHI, F3H, F3´H, FLS, F3´5´H, OMT1, HlPT1, and VPS was examined. Transgenic events with an elevated expression of genes of flavonoid and phloroglucinol biosynthesis were identified. For quite some time successful plant tissue culture and Agrobacterium tumefaciens-mediated transformation procedures are available to genetically modify hop. However, these procedures are characterized by the low regeneration and transformation rates. Moreover, A. tumefaciens-mediated transformation is a laborious and time consuming process. For that reason, in order to evaluate further homologous or heterologous transcription factors with respect to the regulation of flavonoid and phloroglucinol biosynthesis in hop was highly desirable to have a simple and fast transformation system. A. rhizogenes-mediated transformation represents an alternative to express genes in hairy roots. Therefore, hop explants were genetically transformed with A. rhizogenes strains K599 and 15834. Hairy roots were only induced by A. rhizogenes 15834. The transgenity of the obtained hairy roots was confirmed by histochemical GUS assay. The integration of rolC and mgfp5 genes in transgenic hairy roots was confirmed by PCR. Particle bombardment combined with the regeneration of plants in temporary immersion bioreactors could provide another alternative for hop genetic transformation. In this study, a protocol for the micropropagation and shoot induction from organogenic calli of hop cv. Tettnanger using the temporary immersion bioreactors was developed. Furthermore, a procedure for particle bombardment was established using the following parameters: helium pressure of 900 psi, and target distance of 6 cm. The importance of hop relies on the secondary metabolites contain in the lupulin glands of the female cones. The present thesis demonstrated that the production of these secondary metabolites in transgenic hop plants could be influenced and enhanced by the expression of homologous or heterologous transcription factors. Moreover, the new developed transformation methods open the possibility for evaluating further genes that might influence the composition of secondary metabolites in the lupulin glands of hop.