Content
2008
The Chromosomal High-Affinity Binding Sites for the Drosophila Dosage Compensation Complex
12.12.2008
PLOS Genetics,
2008,
doi:10.1371/journal.pgen.1000302,
published on 12.12.2008
PLOS genetics, online article
PLOS genetics, online article
Dosage compensation in male Drosophila relies on the X chromosome–specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are
unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called ‘‘high-affinity sites’’ (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing highresolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various
means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage
compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on noncoding
parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.
The Universal Stress Protein UspC Scaffolds the KdpD/KdpE Signaling Cascade of Escherichia coli under Salt Stress
11.12.2008
Journal of Molecular Biology,
2008,
doi:10.1016/j.jmb.2008.12.007,
published on 11.12.2008
Journal of Molecular Biology , online article
Journal of Molecular Biology , online article
The sensor kinase KdpD and the response regulator KdpE control induction of the kdpFABC operon encoding the high-affinity K+-transport system KdpFABC in response to K+ limitation or salt stress. Under K+ limiting conditions the Kdp system restores the intracellular K+ concentration, while in response to salt stress K+ is accumulated far above the normal content. The kinase activity of KdpD is inhibited at high concentrations of K+, so it has been puzzling how the sensor can be activated in response to salt stress. Here, we demonstrate that the universal stress protein UspC acts as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with a Usp domain in KdpD of the UspA subfamily under salt stress. Escherichia coli encodes three single domain proteins of this subfamily, UspA, UspC, and UspD, whose expression is up-regulated under various stress conditions. Among these proteins only UspC stimulated the in vitro
reconstructed signaling cascade (KdpD→KdpE→DNA) resulting in phosphorylation of KdpE at a K+ concentration that would otherwise almost prevent phosphorylation. In agreement, in a ΔuspC mutant KdpFABC production was down-regulated significantly when cells were exposed to salt stress, but unchanged under K+ limitation. Biochemical studies revealed
that UspC interacts specifically with the Usp domain in the stimulus perceiving N-terminal domain of KdpD. Furthermore, UspC stabilized the KdpD/KdpE∼P/DNA complex and is therefore believed to act as a scaffolding protein. This study describes the stimulation of a bacterial twocomponent system under distinct stress conditions by a scaffolding protein, and highlights a new role of the universal stress proteins.
Recruitment of RNA polymerase II cofactor PC4
01.12.2008
The multifunctional nuclear protein positive cofactor 4 (PC4) is involved in various cellular processes including transcription, replication, and chromatin organization. Recently, PC4 has been identifi ed as a suppressor of oxidative mutagenesis in Escherichia coli and Saccharomyces cerevisiae . To investigate a potential role of PC4 in mammalian DNA repair, we used a combination of live cell microscopy, microirradiation, and fl uorescence recovery after photobleaching analysis. We found a clear accumulation of endogenous PC4 at DNA damage sites introduced by either chemical agents or laser microirradiation. Using fl uorescent fusion proteins and specific mutants, we demonstrated that the rapid recruitment of PC4 to laser-induced DNA damage sites is
independent of poly(ADP-ribosyl)ation and y-H2AX but depends on its single strand binding capacity. Furthermore, PC4 showed a high turnover at DNA damages sites compared with the repair factors replication protein A and proliferating cell nuclear antigen. We propose that PC4 plays a role in the early response to DNA damage by recognizing single-stranded DNA and may thus initiate or
facilitate the subsequent steps of DNA repair.
5´-triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma
02.11.2008
Genetic and epigenetic plasticity allows tumors to evade single-targeted treatments. Here we direct Bcl2-specific short interfering
RNA (siRNA) with 5¢-triphosphate ends (3p-siRNA) against melanoma. Recognition of 5¢-triphosphate by the cytosolic antiviral
helicase retinoic acid–induced protein I (Rig-I, encoded by Ddx58) activated innate immune cells such as dendritic cells and
directly induced expression of interferons (IFNs) and apoptosis in tumor cells. These Rig-I–mediated activities synergized with
siRNA-mediated Bcl2 silencing to provoke massive apoptosis of tumor cells in lung metastases in vivo. The therapeutic activity
required natural killer cells and IFN, as well as silencing of Bcl2, as evidenced by rescue with a mutated Bcl2 target, by sitespecific
cleavage of Bcl2 messenger RNA in lung metastases and downregulation of Bcl-2 protein in tumor cells in vivo. Together,
3p-siRNA represents a single molecule–based approach in which Rig-I activation on both the immune- and tumor cell level
corrects immune ignorance and in which gene silencing corrects key molecular events that govern tumor cell survival.
Short-term activation induces multifunctional dendritic cells that generate potent antitumor T-cell responses in vivo
25.10.2008
Cancer Immunol Immunother,
2008,
10.1007/s00262-008-0606-2,
published on 25.10.2008
Cancer Immunol Immunother, online article
Cancer Immunol Immunother, online article
Dendritic cell (DC) vaccines have emerged as a promising strategy to induce antitumoral cytotoxic T cells for the immunotherapy of cancer. The maturation state of DC is of critical importance for the success of vaccination, but the most effective mode of maturation is still a matter of debate. Whereas immature DC carry the risk of inducing tolerance, extensive stimulation of DC may lead to DC unresponsiveness and exhaustion. In this study, we investigated how short-term versus long-term DC activation with a Toll-like receptor 9 agonist influences DC phenotype and function. Murine DC were generated in the presence of the hematopoietic factor Flt3L (FL-DC) to obtain both myeloid and plasmacytoid DC subsets. Short activation of FL-DC for as little as 4 h induced fully functional DC that rapidly secreted IL-12p70 and IFN-alpha, expressed high levels of costimulatory and MHC molecules and efficiently presented antigen to CD4 and CD8 T cells. Furthermore, short-term activated FL-DC overcame immune suppression by regulatory T cells and acquired high migratory potential toward the chemokine CCL21 necessary for DC recruitment to lymph nodes. In addition, vaccination with short-term activated DC induced a strong cytotoxic T-cell response in vivo and led to the eradication of tumors. Thus, short-term activation of DC generates fully functional DC for tumor immunotherapy. These results may guide the design of new protocols for DC generation in order to develop more efficient DC-based tumor vaccines.
Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin
24.10.2008
Messenger ribonucleoprotein particles (mRNPs) move randomly
within nucleoplasm before they exit from the nucleus. To further
understand mRNP trafficking, we have studied the intranuclear
movement of a specific mRNP, the BR2 mRNP, in salivary gland cells in Chironomus tentans. Their polytene nuclei harbor giant chromosomes separated by vast regions of nucleoplasm, which allows us to study mRNP mobility without interference of chromatin. The particles were fluorescently labeled with microinjected oligonucleotides (DNA or RNA) complementary to BR2 mRNA or with the RNA-binding protein hrp36, the C. tentans homologue of hnRNP A1. Using high-speed laser microscopy, we followed the intranuclear trajectories of single mRNPs and characterized their motion within the nucleoplasm. The Balbiani ring (BR) mRNPs moved randomly, but unexpectedly, in a discontinuous manner. When mobile, they diffused with a diffusion coefficient corresponding to their size. Between mobile phases, the mRNPs were slowed down 10-to 250-fold but were never completely immobile. Earlier electron microscopy work has indicated that BR particles can attach to thin nonchromatin fibers, which are sometimes connected to discrete fibrogranular clusters. We propose that the observed discontinuous movement reflects transient interactions between freely diffusing BR particles and these submicroscopic structures.
Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes
20.10.2008
The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affi nity purifi cation strategy to identify specifi c interactors that combines quantitative SILAC-based mass spectrometry with characterization
of common contaminants binding to affi nity matrices (bead proteomes). This strategy can be applied to affi nity purifi cation of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the
integration of biochemical protein interaction data with in vivo measurements using fl uorescence microscopy. Proteins binding nonspecifi cally to the most commonly used affi nity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and and nonquantitative pull-down and
immunoprecipitation experiments.
Camelid immunoglobulins and nanobody technology
17.10.2008
Veterinary Immunology and Immunopathology,
2008,
doi:10.1016/j.vetimm.2008.10.299
published on 17.10.2008
Veterinary Immunology and Immunopathology, online article
Veterinary Immunology and Immunopathology, online article
It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies fromother species, these special antibodies are devoid of light chains and are composed of a heavy-chainhomodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V–D–J rearrangement and require dedicated constant g-genes. An
immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigenbinding
fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in
bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.
Combined Use of RNAi and Quantitative Proteomics to Study Gene Function in Drosophila
05.09.2008
RNA interference is a powerful way to study gene function and is frequently combined with microarray analysis. Here we introduce a similar technology at the protein level by simultaneously applying Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and RNA interference (RNAi) to Drosophila SL2 cells. After knockdown of ISWI, an ATP-hydrolyzing motor of different chromatin remodeling complexes, we obtained a quantitative proteome of more than 4,000 proteins. ISWI itself was reduced 10-fold as quantified by SILAC. Several hundred proteins were significantly regulated and clustered into distinct functional categories. Acf-1, a direct interaction partner of ISWI, is severely depleted at the protein, but not the transcript, level; this most likely results from reduced protein stability. We found little overall correlation between changes in the transcriptome and proteome with many protein changes unaccompanied by message changes. However, correlation was high for those mRNAs that changed significantly by microarray.
PR-SET7 and SUV4-20H regulate H4 lysine-20 methylation at imprinting control regions in the mouse
22.08.2008
Imprinted genes are important in development and their allelic
expression is mediated by imprinting control regions (ICRs). On
their DNA-methylated allele, ICRs are marked by trimethylation at H3 Lys 9 (H3K9me3) and H4 Lys 20 (H4K20me3), similar to pericentric heterochromatin. Here, we investigate which histone methyltransferases control this methylation of histone at ICRs. We found that inactivation of SUV4-20H leads to the loss of H4K20me3 and increased levels of its substrate, H4K20me1. H4K20me1 is controlled by PR-SET7 and is detected on both parental alleles. The disruption of SUV4-20H or PR-SET7 does not affect methylation of DNA at ICRs but influences precipitation of H3K9me3, which is suggestive of a trans-histone change. Unlike at pericentric heterochromatin, however, H3K9me3 at ICRs does not depend on SUV39H. Our data show not only new similarities but also differences between ICRs and heterochromatin, both of which show constitutive maintenance of methylation of DNA in somatic cells.
Function of Transmembrane Domain IX in the Na+/Proline Transporter PutP
30.07.2008
Selected residues of transmembrane domain (TM) IX were previously shown to play key roles in ligand binding and transport in members of the Na+/solute symporter family. Using the Na+/proline transporter PutP as a model, a complete Cys scanning mutagenesis of TM IX (positions 324 to 351) was performed here to further investigate the functional significance of
the domain. G328, S332, Q345, and L346 were newly identified as important for Na+-coupled proline uptake. Placement of Cys at one of these positions altered Km(pro) (S332C and L346C, 3- and 21-fold decreased, respectively; Q345C, 38-fold increased), K0.5(Na+) (S332C, 13-fold decreased; Q345C, 19-
fold increased), and/or Vmax [G328C, S332C, Q345C, and L346C, 3-, 22-, 2-, and 8-fold decreased compared to PutP(wild type), respectively]. Membrane- permeant N-ethylmaleimide inhibited proline uptake into cells containing PutP with Cys at distinct positions in the middle (T341C) and cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) and had little
or no effect on all other single Cys PutP variants. The inhibition pattern was in agreement with the pattern of labeling with fluorescein-5-maleimide. In addition, Cys placed into the cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) was protected from fluorescein-5-maleimide labeling by proline while Na+ alone had no effect. Membrane-impermeant
methanethiosulfonate ethyltrimethylammonium modified Cys in the middle (A337C and T341C) and periplasmic half (L331C) but not in the cytoplasmic half of TM IX in intact cells. Furthermore, Cys at the latter positions was partially protected by Na+ but not by proline. Based on these results, a model is discussed according to which residues of TM IX participate in the formation of ligand-sensitive, hydrophilic cavities in the protein that may reconstitute part of the Na+ and/or proline translocation
pathway of PutP.
A fluorescent two-hybrid (F2H) assay for direct visualization of protein interactions in living cells
12.07.2008
Molecular & Cellular Proteomics,
2008,
published on 12.07.2008
Molecular & Cellular Proteomics , online article
Molecular & Cellular Proteomics , online article
Genetic high-throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac
repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid (F2H) assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the F2H assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real-time.
Uncoupling the replication machinery
01.07.2008
Cell Cycle,
2008,
7, issue 13,
1983-90
published on 01.07.2008
www.landesbioscience.com, online article
www.landesbioscience.com, online article
The precise coordination of the different steps of DNA replication
is critical for the maintenance of genome stability. We have
probed the mechanisms coupling various components of the
replication machinery and their response to polymerase stalling
by inhibition of the DNA polymerases in living mammalian cells
with aphidicolin. We observed little change in the behaviour of
proteins involved in the initiation of DNA replication. In contrast,
we detected a marked accumulation of the single stranded DNA
binding factor RPA34 at sites of DNA replication. Finally, we
demonstrate that proteins involved in the elongation step of
DNA synthesis dissociate from replication foci in the presence
of aphidicolin. Taken together, these data indicate that inhibition
of processive DNA polymerases uncouples the initiation of
DNA replication from subsequent elongation steps. We, therefore,
propose that the replication machinery is made up of distinct functional sub-modules that allow a flexible and dynamic response to challenges during DNA replication.
Targeting CpG Oligonucleotides to the Lymph Node by Nanoparticles Elicits Efficient Antitumoral Immunity
21.06.2008
Viral nucleic acids are recognized by specific pattern-recognition receptors of the Toll-like and RIG-I-like receptor families.
Synthetic DNA and RNA oligonucleotides can activate the immune system through these receptors and potentiate Ab and
CD8 cytotoxic responses to Ags. Systemic application of immunostimulatory oligonucleotides however also results in a
generalized, non-Ag-specific stimulation of the immune system. In this study, we have dissociated the induction of an Ag-specific response from the systemic immune activation generally associated with immunostimulatory oligonucleotides. Delivery of CpG oligodeoxynucleotides that bind TLR9 by cationized gelatin-based nanoparticles potentiates the in vivo generation of an Ag-specific cytotoxic T cell and Ab response. Furthermore, immunization with CpG-loaded nanoparticles induces a protective antitumoral response in a murine model of melanoma. The systemic release of proinflammatory cytokines and widespread immunostimulation associated with free CpG is however completely abolished. In addition, we show that gelatin nanoparticle formulation prevents the destruction of lymphoid follicles mediated by CpG. Nanoparticle-delivered CpG, in contrast to free CpG, are selectively targeted to APCs in the lymph nodes where they mediate local immune stimulation. We describe a novel strategy to target immunostimulatory oligonucleotides to the initiation site of the immune
response while at the same time protecting from an indiscriminate and generalized activation of the immune system.
Nuclear transit of the RNA-binding protein She2 is required for translational control of localized ASH1 mRNA
20.06.2008
EMBO reports,
2008,
doi:10.1038/embor.2008.112,
published on 20.06.2008
EMBO reports, online article
EMBO reports, online article
Cytoplasmic localization and localized translation of messenger
RNAs contribute to asymmetrical protein distribution. Recognition
of localized mRNAs by RNA-binding proteins can occur in
the cytoplasm or, alternatively, co- or post-transcriptionally in
the nucleus. In budding yeast, mRNAs destined for localization
are bound by the She2 protein before their nuclear export. Here,
we show that a specific transcript, known as ASH1 mRNA, and
She2 localize specifically to the nucleolus when their nuclear
export is blocked. Nucleolar She2 localization is enhanced in a
She2 mutant that cannot bind to RNA. A fusion protein of the
amino terminus of She3 and She2 (She3N-She2) fails to enter the
nucleus, but does not impair ASH1 mRNA localization. Instead,
these cells fail to distribute Ash1 protein asymmetrically, which is
caused by a defective translational control of ASH1 mRNA. Our
results indicate that the nucleolar transit of RNA-binding proteins
such as She2 is necessary for the correct assembly of translationally
silenced localizing messenger ribonucleoproteins.
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Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy
06.06.2008
Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities
to analyze subcellular structures beyond the diffraction limit of the emitted light.
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A chromatin-wide transition to H4K20 monomethylation impairs genome integrity and programmed DNA rearrangements in the mouse
30.05.2008
H4K20 methylation is a broad chromatin modification that has been linked with diverse epigenetic functions. Several enzymes target H4K20 methylation, consistent with distinct mono-, di-, and trimethylation states controlling different biological outputs. To analyze the roles of H4K20 methylation states, we generated conditional null alleles for the two Suv4-20h histone methyltransferase (HMTase) genes in the mouse. Suv4-20h-double-null (dn) mice are perinatally lethal and have lost nearly all H4K20me3 and H4K20me2 states. The genome-wide transition to an H4K20me1 state results in increased sensitivity to damaging stress, since Suv4-20h-dn chromatin is less efficient for DNA double-strand break (DSB) repair and prone to chromosomal aberrations. Notably, Suv4-20h-dn B cells are defective in immunoglobulin class-switch recombination, and Suv4-20h-dn deficiency impairs the stem cell pool of lymphoid progenitors. Thus,
conversion to an H4K20me1 state results in compromised chromatin that is insufficient to protect genome integrity and to process a DNA-rearranging differentiation program in the mouse.
The PHD Domain of Np95 (mUHRF1) Is Involved in Large-Scale Reorganization of Pericentromeric Heterochromatin
28.05.2008
Molecular Biology of the Cell,
2008,
19,
3554-63
published on 28.05.2008
www.molbiolcell.org , online article
www.molbiolcell.org , online article
Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD
domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH.
Photorhabdus luminescens genes induced upon insect infection
19.05.2008
BMC Genomics,
2008,
9:229,
doi:10.1186/1471-2164-9-229
published on 19.05.2008
BMC Genomics, online article
BMC Genomics, online article
Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is
simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a pathogenic stage, characterized by release from the nematode into the hemocoel of insect larvae, resulting in rapid insect death caused by bacterial toxins. P. luminescens appears to sense and adapt to the novel host environment upon changing hosts, which facilitates the production of factors involved in survival within the host, host-killing, and -exploitation. Results: A differential fluorescence induction (DFI) approach was applied to identify genes that are up-regulated in the bacterium after infection of the insect host Galleria mellonella. For this purpose, a P. luminescens promoter-trap library utilizing the mCherry fluorophore as a reporter was constructed, and approximately 13,000 clones were screened for fluorescence induction in the
presence of a G. mellonella larvae homogenate. Since P. luminescens has a variety of regulators that potentially sense chemical molecules, like hormones, the screen for up-regulated genes or operons was performed in vitro, excluding physicochemical signals like oxygen, temperature or osmolarity
as variables. Clones (18) were obtained exhibiting at least 2.5-fold induced fluorescence and regarded as specific responders to insect homogenate. In combination with a bioinformatics approach, sequence motifs were identified in these DNA-fragments that are similar to 29 different promoters within the P. luminescens genome. By cloning each of the predicted promoters upstream of the reporter gene, induction was verified for 27 promoters in vitro, and for 24 promoters in
viable G. mellonella larvae. Among the validated promoters are some known to regulate the expression of toxin genes, including tccC1 (encoding an insecticidal toxin complex), and others
encoding putative toxins. A comparably high number of metabolic genes or operons were observed to be induced upon infection; among these were eutABC, hutUH, and agaZSVCD, which encode proteins involved in ethanolamine, histidine and tagatose degradation, respectively. The results reflect rearrangements in metabolism and the use of other metabolites available from the insect. Furthermore, enhanced activity of promoters controlling the expression of genes encoding enzymes linked to antibiotic production and/or resistance was observed. Antibiotic production and resistance may influence competition with other bacteria, and thus might be important for a successful infection. Lastly, several genes of unknown function were identified that may represent novel pathogenicity factors.
We show that a DFI screen is useful for identifying genes or operons induced by chemical stimuli, such as diluted insect homogenate. A bioinformatics comparison of motifs similar
to known promoters is a powerful tool for identifying regulated genes or operons. We conclude that signals for the regulation of those genes or operons induced in P. luminescens upon insect
infection may represent a wide variety of compounds that make up the insect host. Our results provide insight into the complex response to the host that occurs in a bacterial pathogen,
particularly reflecting the potential for metabolic shifts and other specific changes associated with virulence.
Simultaneous Transport of Different Localized mRNA Species Revealed by Live-Cell Imaging
09.05.2008
Traffic,
2008,
doi:10.1111/j.1600-0854.2008.00763.x,
published on 09.05.2008
Traffic, online article
Traffic, online article
Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, differentmRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 differentmRNAs to the bud with the help of theMyo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the
RNAtag sequences.Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the
transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles andcotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.
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Molecular evolution of the RNA polymerase II CTD
08.05.2008
In higher eukaryotes, an unusual C-terminal domain (CTD) is crucial to the function of RNA polymerase II in transcription. The CTD consists of multiple heptapeptide repeats; differences in the number of repeats between organisms and their degree of conservation have intrigued researchers for two decades. Here, we review the evolution of the CTD at the molecular level. Several primitive motifs have been integrated into compound heptads that can be readily amplified. The selection of phosphorylatable residues in the heptad repeat provided
the opportunity for advanced gene regulation in eukaryotes. Current findings suggest that the CTD should be considered as a collection of continuous overlapping motifs as opposed to a specific functional unit defined by a heptad.
Generation and Characterization of a Rat Monoclonal Antibody Specific for Multiple Red Fluorescent Proteins
27.04.2008
Fluorescent proteins (FP) are widely used as in vivo reporter molecules and are available in multiple colors
spanning almost the entire visible light spectrum. Genetically fused to any protein target, FPs offer a powerful tool to study protein localization and dynamics. After the isolation of the prototypical green fluorescent protein (GFP) from the jellyfish Aequorea victoria, a red fluorescent protein (DsRed) was discovered in the coral Discosoma sp. that provided a better spectral separation from cellular autofluorescence and allowed multicolor tracking of fusion proteins. However, the obligate tetramerization of DsRed caused serious problems for its use in live-cell imaging. Subsequent mutageneses of the red progenitor have resulted in several monomeric red FPs (mRFP1, mCherry, mOrange, mPlum, etc). These improved red FPs are characterized by higher brightness and photostability, complete chromophore maturation, and promise a wide variety of features for biological imaging and multicolor labeling. Here we report the generation and characterization of the first rat monoclonal antibody
(MAb) against multiple red FPs, designated as multi-red 5F8. We demonstrate that multi-red 5F8 is a
MAb with high affinity and specificity against the DsRed derivatives and corresponding fusion proteins, and
that it is suitable for ELISA, immunoblotting, immunoprecipitation, and immunofluorescence assays. Applying our versatile antibody, one and the same red fluorescent protein tag can be used to perform not only microscopic studies, but also multiple biochemical assays.
Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination
24.04.2008
Microbial Cell Factories,
2008,
7:14,
doi:10.1186/1475-2859-7-14
published on 24.04.2008
Microb Cell Fact, online article
Microb Cell Fact, online article
Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems.
Here we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red®/ET® Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed.
Chromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before.
DNA vaccination efficiently induces antibodies to Nogo-A and does not exacerbate experimental autoimmune encephalomyelitis
16.04.2008
Eur J Pharmacol.,
2008,
588(1),
99-105
published on 16.04.2008
European Journal of Pharmacology, online article
European Journal of Pharmacology, online article
Antibodies against the neurite outgrowth inhibitor Nogo-A enhance axonal regeneration following spinal cord injury. However, antibodies directed against myelin components can also enhance CNS inflammation. The present study was designed to assess the efficacy of DNA vaccination for generating antibodies against Nogo-A and to study their pathogenic potential in a mouse model for multiple sclerosis. Mice were immunized by a single i.m. injection of a plasmid expression vector encoding either full length membraneintegral Nogo-A equipped with a signal peptide or two versions of its large N-terminal extramembrane region. The presence of serum antibodies to Nogo-A was measured 4 weeks after injection by ELISA, Western
blotting and immunohistochemistry. DNA vaccination efficiently induced production of Nogo-A-specific antibodies that recognized recombinant, intracellular Nogo-A in cell culture but also stained native Nogo-A on the oligodendrocyte surface. Experimental autoimmune encephalomyelitis was induced in DNAvaccinated
mice by immunization with proteolipid peptide (a.a. 139–154). In contrast to vaccination with DNA encoding myelin oligodendrocyte glycoprotein that exacerbates this disease, Nogo-A DNA vaccination did not enhance clinical severity of disease. In summary, DNA vaccination is a simple and efficient method for
generating an antibody response to Nogo-A. No pathogenicity was observed even during a full-blown inflammatory response of the central nervous system.
ATP-dependent chromatosome remodeling
01.04.2008
Chromatin serves to package, protect and organize the complex eukaryotic genomes to assure their stable inheritance over many cell generations. At the same time, chromatin must be dynamic to allow continued use of DNA during a cell’s lifetime. One important principle
that endows chromatin with flexibility involves ATPdependent
‘remodeling’ factors, which alter DNA-histone interactions to form, disrupt or move nucleosomes. Remodeling is well documented at the nucleosomal level, but little is known about the action of remodeling factors in a more physiological chromatin environment. Recent findings suggest that some remodeling machines can
reorganize even folded chromatin fibers containing the linker histone H1, extending the potential scope of remodeling reactions to the bulk of euchromatin.
CIPSM-Researcher Heinrich Leonhardt wins Binder-Innovation Award 2008
19.03.2008
2008,
published on 19.03.2008
Heinrich Leonhardt
Heinrich Leonhardt
CIPSM-Researchers Heinrich Leonhardt and Ulrich Rothbauer win the Binder-Innovation-Award for their work on the usage of fluorescent nano-antibodies to detect biological structures and processes in living cells.
From left to right: Prof. Vestweber (President of the German Association of Cellbiology), Prof. Baumeister (MPI Martinsried), Dr. Ankerhold (Carl Zeiss), Dr. Rothbauer and Prof. Leonhardt
From left to right: Heinrich Leonhardt, Annabel, Ulrich Rothbauer
DNA sequence and the organization of chromosomal domains
04.03.2008
Current Opinion in Genetics & Development,
2008,
18,
175-80
published on 04.03.2008
Current Opinion in Genetics & Development, online article
Current Opinion in Genetics & Development, online article
The combination of chromatin structure and the organization of chromosomes in eukaryotic nuclei affects many genome functions. Distinct functional states of genes ranging from ‘highly active’ to ‘silenced’ correlate with particular nucleosome arrangements, histone variants, histone modifications, and interactions of non-histone regulators. Transcription factors that recognize and bind specific DNA sequences recruit chromatin modulators to specific genes via protein interactions. However, little is known about how
chromosomal domains or entire chromosomes are targeted to implement particular chromatin structures and activity states. Here we discuss emerging concepts of how DNA sequence can contribute to chromatin organization at the domain level. Inspiration and motivation for this discourse comes from the unresolved question of how X chromosomes are identified for dosage compensation.
The nucleolar SUMO-specific protease SENP3 reverses SUMO modification of nucleophosmin and is required for rRNA processing
08.02.2008
The ubiquitin-like SUMO system functions by a cyclic process of
modification and demodification, and recent data suggest that the nucleolus is a site of sumoylation–desumoylation cycles.For example, the tumour suppressor ARF stimulates sumoylation of nucleolar proteins. Here, we show that the nucleolar SUMOspecific
protease SENP3 is associated with nucleophosmin (NPM1), a crucial factor in ribosome biogenesis. SENP3 catalyses desumoylation of NPM1–SUMO2 conjugates in vitro and counteracts ARF-induced modification of NPM1 by SUMO2 in vivo. Intriguingly, depletion of SENP3 by short interfering RNA interferes with nucleolar ribosomal RNA processing and inhibits the conversion of the 32S rRNA species to the 28S form, thus phenocopying the processing defect observed on depletion of NPM1. Moreover, mimicking constitutive modification of NPM1 by SUMO2 interferes with 28S rRNA maturation. These results define SENP3 as an essential factor for ribosome biogenesis and suggest that deconjugation of SUMO2 from NPM1 by SENP3 is critically involved in 28S rRNA maturation.
Fine Mapping of Posttranslational Modifications of the Linker Histone H1 from Drosophila melanogaster
06.02.2008
The linker histone H1 binds to the DNA in between adjacent nucleosomes and contributes to chromatin organization and transcriptional control. It is known that H1 carries diverse posttranslational modifications (PTMs), including phosphorylation, lysine methylation and ADP-ribosylation. Their biological functions, however, remain largely unclear. This is in part due to the fact that most of the studies have been performed in organisms that have several H1 variants, which complicates the analyses. We have chosen Drosophila melanogaster, a model organism, which has a single H1 variant, to approach the study of the role of H1 PTMs during embryonic development. Mass spectrometry mapping of the entire sequence of the protein showed phosphorylation only in the ten N-terminal amino acids, mostly at S10. For the first time, changes in the PTMs of a linker H1 during the development of a multicellular organism are reported. The abundance of H1 monophosphorylated at S10 decreases as the embryos age, which suggests that this PTM is related to cell cycle progression and/or cell differentiation. Additionally, we have found a polymorphism in the protein sequence that can be mistaken with lysine methylation if the analysis is not rigorous.
Chromatin proteomics and epigenetic regulatory circuits
01.02.2008
Expert Review of Proteomics,
2008,
5 No. 1,
105-19
published on 01.02.2008
Expert Reviews, online article
Expert Reviews, online article
Many phenotypic changes of eukaryotic cells due to changes in gene expression depend on alterations in chromatin structure. Processes involved in the alteration of chromatin are diverse and include post-translational modifications of histone proteins, incorporation of specific histone variants, methylation of DNA and ATP-dependent chromatin remodeling. Interconnected with these processes are the localization of chromatin domains within the nuclear architecture and the appearance of various classes of noncoding regulatory RNAs. Recent experiments underscore the role of these processes in influencing diverse biological functions. However, the evidence to date implies the importance of an interplay of all these chromatin-changing functions, generating an epigenetic regulatory circuit that is still not well understood.
