Jacek Lichota

Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus

Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4–wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin

In mammals and plants, formation of heterochromatin is associated with hypermethylation of DNA at CpG sites and histone H3 methylation at lysine 9. Previous studies have revealed that maintenance of DNA methylation in Neurospora and Arabidopsis requires histone H3 methylation. A feedback loop from DNA methylation to histone methylation, however, is less understood. Its recent examination in Arabidopsis with a partial loss of function in DNA methyltransferase 1 (responsible for maintenance of CpG methylation) yielded conflicting results. Here we report that complete removal of CpG methylation in an Arabidopsis mutant null for DNA maintenance methyltransferase results in a clear loss of histone H3 methylation at lysine 9 in heterochromatin and also at heterochromatic loci that remain transcriptionally silent. Surprisingly, these dramatic alterations are not reflected in heterochromatin relaxation.

Linker Histones Play a Role in Male Meiosis and the Development of Pollen Grains in Tobacco

To examine the function of linker histone variants, we produced transgenic tobacco plants in which major somatic histone variants H1A and H1B were present at ∼25% of their usual amounts in tobacco chromatin. The decrease in these major variants was accompanied by a compensatory increase in the four minor variants, namely, H1C to H1F. These minor variants are smaller and less highly charged than the major variants. This change offered a unique opportunity to examine the consequences to a plant of major remodeling of its chromatin set of linker histones. Plants with markedly altered proportions of H1 variants retained normal nucleosome spacing, but their chromosomes were less tightly packed than those of control plants. The transgenic plants grew normally but showed characteristic aberrations in flower development and were almost completely male sterile. These features correlated with changes in the temporal but not the spatial pattern of expression of developmental genes that could be linked to the abnormal flower phenotypes. Preceding these changes in flower morphology were strong aberrations in male gametogenesis. The earliest symptoms may have resulted from disturbances in correct pairing or segregation of homologous chromosomes during meiosis. No aberrations were observed during mitosis. We conclude that in plants, the physiological stoichiometry and distribution of linker histone variants are crucial for directing male meiosis and the subsequent development of functional pollen grains.

Macromolecular drug transport into the brain using targeted therapy

J. Neurochem. (2010) 10.1111/j.1471‐4159.2009.06544.x

Gene delivery by pullulan derivatives in brain capillary endothelial cells for protein secretion.

The blood-brain barrier (BBB) formed by brain capillary endothelial cells protects the brain against potentially harmful substances present in the circulation, but also restricts exogenous substances such as pharmacologically acting drugs or proteins from entering the brain. A novel and rather unchallenged approach to allow proteins to enter the brain is gene therapy based on delivery of genetic material into brain capillary endothelial cells. In theory in vivo transfection will allow protein expression and secretion from brain capillary endothelial cells and further into the brain. This would denote a new paradigm for therapy to transport proteins across the BBB. The aim of this study was to investigate the possibility to use brain capillary endothelial cells as factories for recombinant protein production. Non-viral gene carriers were prepared from pullulan, a polysaccharide, and spermine, a naturally occurring polyamine that were additionally conjugated with plasmid DNA. We were able to transfect rat brain endothelial cells (RBE4s) and human brain microvascular endothelial cells (HBMECs). Transfection of HBMECs with pullulan-spermine conjugated with plasmid DNA bearing cDNA encoding human growth hormone 1 (hGH1), led to secretion of hGH1 protein into the growth medium. Hence, the pullulan-spermine delivery system is a very promising method for delivering DNA to brain endothelial cells with potential for using these cells as factories for secretion of proteins.

Uptake and transport of superparamagnetic iron oxide nanoparticles through human brain capillary endothelial cells.

The blood-brain barrier (BBB) formed by brain capillary endothelial cells (BCECs) constitutes a firm physical, chemical, and immunological barrier, making the brain accessible to only a few percent of potential drugs intended for treatment inside the central nervous system. With the purpose of overcoming the restraints of the BBB by allowing the transport of drugs, siRNA, or DNA into the brain, a novel approach is to use superparamagnetic iron oxide nanoparticles (SPIONs) as drug carriers. The aim of this study was to investigate the ability of fluorescent SPIONs to pass through human brain microvascular endothelial cells facilitated by an external magnet. The ability of SPIONs to penetrate the barrier was shown to be significantly stronger in the presence of an external magnetic force in an in vitro BBB model. Hence, particles added to the luminal side of the in vitro BBB model were found in astrocytes cocultured at a remote distance on the abluminal side, indicating that particles were transported through the barrier and taken up by astrocytes. Addition of the SPIONs to the culture medium did not negatively affect the viability of the endothelial cells. The magnetic force-mediated dragging of SPIONs through BCECs may denote a novel mechanism for the delivery of drugs to the brain.

Epigenetic regulation of Arc and c-Fos in the hippocampus after acute electroconvulsive stimulation in the rat

Electroconvulsive stimulation (ECS) remains one of the most effective treatments of major depression. However, the underlying molecular changes still remain to be elucidated. Since ECS causes rapid and significant changes in gene expression we have looked at epigenetic regulation of two important immediate early genes that are both induced after ECS: c-Fos and Arc. We examined Arc and c-Fos protein expression and found Arc present over 4 h, in contrast to c-Fos presence lasting only 1 h. Both genes had returned to baseline expression at 24 h post-ECS. Histone H4 acetylation (H4Ac) is one of the important epigenetic marks associated with gene activation. We show increased H4Ac at the c-Fos promoter at 1 h post-ECS. Surprisingly, we also observed a significant increase in DNA methylation of the Arc gene promoter at 24 h post-ECS. DNA methylation, which is responsible for gene silencing, is a rather stable covalent modification. This suggests that Arc expression has been repressed and may consequently remain inhibited for a prolonged period post-ECS. Arc plays a critical role in the maintenance phase of long-term potentiation (LTP) and consolidation of memory in the rat brain. Thus, this study is one of the first to demonstrate DNA methylation as a regulator of ECS-induced gene expression and it provides a molecular link to the memory deficits observed after ECS.

Heterogenous distribution of ferroportin-containing neurons in mouse brain.

Iron is crucial for a variety of cellular functions in neuronal cells. Neuronal iron uptake is reflected in a robust and consistent expression of transferrin receptors and divalent metal transporter 1 (DMT 1). Conversely, the mechanisms by which neurons neutralize and possibly excrete iron are less clear. Studies indicate that neurons express ferroportin which could reflect a mechanism for iron export. We mapped the distribution of ferroportin in the adult mouse brain using an antibody prepared from a peptide representing amino acid sequences 223–303 of mouse ferroportin. The antibody specifically detected ferroportin in brain homogenates, whereas homogenates of cultured endothelial cells were devoid of immunoreactivity. In brain sections, ferroportin was confined to neuronal cell bodies and peripheral processes of cerebral cortex, hippocampus, thalamus, brain stem, and cerebellum. In brain stem ferroportin-labeling was particularly high in neurons of cranial nerve nuclei and reticular formation. Ferroportin was hardly detectable in striatum, pallidum, or hypothalamus. Among non-neuronal cells, ferroportin was detected in oligodendrocytes and choroid plexus epithelial cells. A comparison with previous studies on the distribution of transferrin receptors in neurons shows that many neuronal pools coincide with those expressing ferroportin. The data therefore indicate that neuronal iron homeostasis consists of a delicate balance between transferrin receptor-mediated uptake of iron-transferrin and ferroportin-related iron excretion. The findings also suggest a particular high turnover of iron in neuronal regions, such as habenula, hippocampus, reticular formation and cerebellum, as several neurons in these regions exhibit a prominent co-expression of transferrin receptors and ferroportin.

Functionality of the beta/six site-specific recombination system in tobacco and Arabidopsis: a novel tool for genetic engineering of plant genomes.

The β recombinase is a member of the prokaryotic site-specific serine recombinases (invertase/resolvase family), which in the presence of a DNA bending cofactor can catalyse DNA deletions between two directly oriented 90-bp six recombination sites. We have examined here whether the β recombinase can be expressed in plants and whether it displays in planta its specific catalytic activity excising DNA sequences that are flanked by six sites. In plant protoplasts, the enzyme could be expressed as a GFP-β recombinase fusion which can localise to the cell nucleus. β recombinase stably expressed in tobacco plants can catalyse deletion of a spacer region that is flanked by directly oriented six sites and has been placed between promoter and a GUS reporter gene (preventing GUS expression). In transient transformation experiments, β recombinase-mediated elimination of the spacer results in transcriptional induction of the GUS gene. Similarly, β recombinase in stably double-transformed Arabidopsis plants deletes specifically the spacer region of a reporter construct that has been incorporated into the genome. In the segregating T1 generation, plants were identified that contain exclusively the recombined reporter construct. In summary, our results demonstrate that functional β recombinase can be expressed in plants and that the enzyme is suitable to precisely eliminate undesired sequences from plant genomes. Therefore, the β/six recombination system (and presumably related recombinases) may become an attractive tool for plant genetic engineering.

Temporal gene expression profile after acute electroconvulsive stimulation in the rat

Electroconvulsive therapy (ECT) remains one of the most effective treatments of major depression. It has been suggested that the mechanisms of action involve gene expression. In recent decades there have been several investigations of gene expression following both acute and chronic electroconvulsive stimulation (ECS). These studies have focused on several distinct gene targets but have generally included only few time points after ECS for measuring gene expression. Here we measured gene expression of three types of genes: Immediate early genes, synaptic proteins, and neuropeptides at six time points following an acute ECS. We find significant increases for c-Fos, Egr1, Neuritin 1 (Nrn 1), Bdnf, Snap29, Synaptotagmin III (Syt 3), Synapsin I (Syn 1), and Psd95 at differing time points after ECS. For some genes these changes are prolonged whereas for others they are transient. Npy expression significantly increases whereas the gene expression of its receptors Npy1r, Npy2r, and Npy5r initially decreases. These decreases are followed by a significant increase for Npy2r, suggesting anticonvulsive adaptations following seizures. In summary, we find distinct changes in mRNA quantities that are characteristic for each gene. Considering the observed transitory and inverse changes in expression patterns, these data underline the importance of conducting measurements at several time points post-ECS.