The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of ...methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters.
Display omitted
► The H3K4me3/2 demethylase Kdm5c interacts with gene-specific transcription factors ► Genomic profiles of Kdm5c in ESCs reveal promoter-proximal and -distal binding ► Kdm5c represses promoter activity but stimulates enhancer function ► Dynamic H3K4me3/2 thus modulates promoter and enhancer function
Regulatory regions of eukaryotic genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine 4 (H3K4). Using embryonic stem cells, Timmers and colleagues report that the Kdm5c/Jarid1c/SMCX demethylase controls H3K4 mono- and trimethylation with different transcriptional outcomes. At promoters, Kdm5c dampens transcription, but at enhancers, Kdm5c stimulates their activity. Impairment of enhancer function activates an intrinsic promoter activity of Kdm5c sites. This demonstrates that Kdm5c plays a dynamic role in the functional discrimination between genomic elements.
Malaria kills >1 million people each year, in particular in sub-Saharan Africa. Although asexual forms are directly responsible for disease and death, sexual stages account for the transmission of ...Plasmodium parasites from human to the mosquito vector and therefore the spread of the parasite in the population. Development of a malaria vaccine is urgently needed to reduce morbidity and mortality. Vaccines against sexual stages of Plasmodium falciparum are meant to decrease the force of transmission and consequently reduce malaria burden. Pfs48/45 is specifically expressed in sexual stages and is a well established transmission-blocking (TB) vaccine candidate. However, production of correctly folded recombinant Pfs48/45 protein with display of its TB epitopes has been a major challenge. Here, we show the production of a properly folded Pfs48/45 C-terminal fragment by simultaneous coexpression with four periplasmic folding catalysts in Escherichia coli. This C-terminal fragment fused to maltose binding protein was produced at medium scale with >90% purity and a stability over at least a 9-month period. It induces uniform and high antibody titers in mice and elicits functional TB antibodies in standard membrane feeding assays in 90% of the immunized mice. Our data provide a clear perspective on the clinical development of a Pfs48/45-based TB malaria vaccine.
Coloration of plant organs such as fruit, leaves and flowers through anthocyanin production is governed by a combination of MYB and bHLH type transcription factors (TFs). In this study we introduced ...Rosea1 (ROS1, a MYB type) and Delila (DEL, a bHLH type), into Nicotiana benthamiana leaves by agroinfiltration. ROS1 and DEL form a pair of well-characterized TFs from Snapdragon (Antirrhinum majus), which specifically induce anthocyanin accumulation when expressed in tomato fruit. In N. benthamiana, robust induction of a single anthocyanin, delphinidin-3-rutinoside (D3R) was observed after expression of both ROS1 and DEL. Surprisingly in addition to D3R, a range of additional metabolites were also strongly and specifically up-regulated upon expression of ROS1 and DEL. Except for the D3R, these induced compounds were not derived from the flavonoid pathway. Most notable among these are nornicotine conjugates with butanoyl, hexanoyl, and octanoyl hydrophobic moieties, and phenylpropanoid-polyamine conjugates such as caffeoyl putrescine. The defensive properties of the induced molecules were addressed in bioassays using the tobacco specialist lepidopteran insect Manduca sexta. Our study showed that the effect of ROS1 and DEL expression in N. benthamiana leaves extends beyond the flavonoid pathway. Apparently the same transcription factor may regulate different secondary metabolite pathways in different plant species.
Plants are increasingly used as production platforms of various heterologous proteins, but rapid protein turnover can seriously limit the steady-state expression level. Little is known about specific ...plant proteases involved in this process. In an attempt to obtain potato (Solanum tuberosum cv Desirée) plants resistant to Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, the protease inhibitor equistatin was expressed under the control of strong, light-inducible and constitutive promoters and was targeted to the secretory pathway with and without endoplasmic reticulum retention signal. All constructs yielded similar stepwise protein degradation patterns, which considerably reduced the amount of active inhibitor in planta and resulted in insufficient levels for resistance against Colorado potato beetle larvae. Affinity purification of the degradation products and N-terminal sequencing allowed the identification of the amino acid P1-positions (asparagine Asn-13, lysine-56, Asn-82, and arginine-151) that were cleaved in planta. The proteases involved in the equistatin degradation were characterized with synthetic substrates and inhibitors. Kininogen domain 3 completely inhibited equistatin degradation in vitro. The results indicate that arginine/lysine-specific and legumain-type Asn-specific cysteine proteases seriously impede the functional accumulation of recombinant equistatin in planta. General strategies to improve the resistance to proteases of heterologous proteins in plants are proposed.
Plants accumulate secondary metabolites to adapt to environmental conditions. These compounds, here exemplified by the purple-colored anthocyanins, are accumulated upon high temperatures, UV-light, ...drought, and nutrient deficiencies, and may contribute to tolerance to these stresses. Producing compounds is often part of a more broad response of the plant to changes in the environment. Here we investigate how a transcription-factor-mediated program for controlling anthocyanin biosynthesis also has effects on formation of specialized cell structures and changes in the plant root architecture. A systems biology approach was developed in tomato (Solanum lycopersicum) for coordinated induction of biosynthesis of anthocyanins, in a tissue- and development-independent manner. A transcription factor couple from Antirrhinum that is known to control anthocyanin biosynthesis was introduced in tomato under control of a dexamethasone-inducible promoter. By application of dexamethasone, anthocyanin formation was induced within 24 h in vegetative tissues and in undifferentiated cells. Profiles of metabolites and gene expression were analyzed in several tomato tissues. Changes in concentration of anthocyanins and other phenolic compounds were observed in all tested tissues, accompanied by induction of the biosynthetic pathways leading from Glc to anthocyanins. A number of pathways that are not known to be involved in anthocyanin biosynthesis were observed to be regulated. Anthocyanin-producing plants displayed profound physiological and architectural changes, depending on the tissue, including root branching, root epithelial cell morphology, seed germination, and leaf conductance. The inducible anthocyanin-production system reveals a range of phenomena that accompanies anthocyanin biosynthesis in tomato, including adaptions of the plants architecture and physiology.
Histone methylation patterns are correlated with eukaryotic gene transcription. High‐affinity binding of the plant homeodomain (PHD) of TFIID subunit TAF3 to trimethylated lysine‐4 of histone H3 ...(H3K4me3) is involved in promoter recruitment of this basal transcription factor. Here, we show that for transcription activation the PHD of TAF3 can be replaced by PHDs of other high‐affinity H3K4me3 binders. Interestingly, H3K4me3 binding of TFIID and the TAF3‐PHD is decreased by phosphorylation of the adjacent threonine residue (H3T3), which coincides with mitotic inhibition of transcription. Ectopic expression of the H3T3 kinase haspin repressed TAF3‐mediated transcription of endogenous and of reporter genes and decreased TFIID association with chromatin. Conversely, immunofluorescence and live‐cell microscopy studies showed an increased association of TFIID with mitotic chromosomes upon haspin knockdown. Based on our observations, we propose that a histone H3 phospho–methyl switch regulates TFIID‐mediated transcription during mitotic progression of the cell cycle.
The role of histone modifications in regulating transcriptional activation remains incompletely understood. Here, the mitotic phosphorylation of H3T3 by haspin kinase results in the release of H3K4me3‐mediated binding of TFIID from chromosomes thereby inhibiting transcription.
To improve the expression of equistatin, a proteinase inhibitor from the sea anemone Actinia equina, in the yeast Pichia pastoris, we prepared gene variants with yeast-preferred codon usage and lower ...repetitive AT and GC content. The full gene optimization approximately doubled the level of steady-state mRNA and protein accumulated in the culture medium. The removal of a short stretch of 12 additional nucleotides from the multiple cloning site (MCS) sequence in the vector pPIC9 had an enhancement effect similar to full gene optimization (factor 1.5) at the mRNA level. However, at the protein level, this increase was 4- to 10-fold. The optimized gene without the MCS sequence yielded 1.66 g/L active protein in a bioreactor and was purified by a new two-step procedure with a recovery of activity that was >95%. This production level constitutes an overall improvement of about 20-fold relative to our previously published results. The characteristics of the MCS sequence element are discussed in the light of its apparent ability to act as negative expression regulator.
Summary
Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), cause very large economic damage on a variety of field and greenhouse crops. In this study, plant ...resistance against thrips was introduced into transgenic potato plants through the expression of novel, custom‐made, multidomain protease inhibitors. Representative classes of inhibitors of cysteine and aspartic proteases kininogen domain 3 (K), stefin A (A), cystatin C (C), potato cystatin (P) and equistatin (EIM) were fused into reading frames consisting of four (K‐A‐C‐P) to five (EIM‐K‐A‐C‐P) proteins, and were shown to fold into functional inhibitors in the yeast Pichia pastoris. The multidomain proteins were expressed in potato and found to be more resistant to degradation by plant proteases than the individual domains. In a time span of 14–16 days, transgenic potato plants expressing EIMKACP and KACP at a similar concentration reduced the number of larvae and adults to less than 20% of the control. Leaf damage on protected plants was minimal. Engineered multidomain cysteine protease inhibitors thus provide a novel way of controlling western flower thrips in greenhouse and field crops, and open up possibilities for novel insect resistance applications in transgenic crops.
Summary
In this study, the effects of the accumulation of cysteine protease inhibitors on the food preferences of adult female western flower thrips, Frankliniella occidentalis (Pergande), were ...investigated. Representative members of the cystatin and thyropin gene families (stefin A, cystatin C, kininogen domain 3 and equistatin) were expressed in potato (Solanum tuberosum) cv. Impala, Kondor and Line V plants. In choice assays, a strong time‐ and concentration‐dependent deterrence from plants expressing stefin A and equistatin was observed. Cystatin C and kininogen domain 3 were not found to be active. All tested inhibitors were equally or more active than stefin A at inhibiting the proteolytic activity of thrips, but, in contrast with stefin A, they were all expressed in potato as partially degraded proteins. The resistance of cysteine protease inhibitors against degradation in planta by endogenous plant proteases may therefore be relevant in explaining the observed differences in the deterrence of thrips. The results demonstrate that, when given a choice, western flower thrips will select plants with low levels of certain cysteine protease inhibitors. The novel implications of the defensive role of plant cysteine protease inhibitors as both deterrents and antimetabolic proteins are discussed.
PDF
