Content
Research Area D
Activation of Melanoma Differentiation-Associated Gene 5 Causes Rapid Involution of the Thymus
20.01.2010
The Journal of Immunology,
2009,
182,
6044-50
published on 20.01.2010
The Journal of Immunology, online article
The Journal of Immunology, online article
In the course of infection, the detection of pathogen-associated molecular patterns by specialized pattern recognition receptors in
the host leads to activation of the innate immune system. Whereas the subsequent induction of adaptive immune responses in secondary lymphoid organs is well described, little is known about the effects of pathogen-associated molecular pattern-induced activation on primary lymphoid organs. Here we show that activation of innate immunity through the virus-sensing melanoma differentiation-associated gene 5 (MDA-5) receptor causes a rapid involution of the thymus. We observed a strong decrease in thymic cellularity associated with characteristic alterations in thymic subpopulations and microanatomy. In contrast, immune stimulation with potent TLR agonists did not lead to thymic involution or induce changes in thymic subpopulations, demonstrating that thymic pathology is not a general consequence of innate immune activation. We determined that suppression of thymocyte proliferation and enhanced apoptosis are the essential cellular mechanisms involved in the decrease in thymic size upon MDA-5 activation. Further, thymic involution critically depended on type I IFN. Strikingly however, no direct action of type I IFN on thymocytes was required, given that the decrease in thymic size was still observed in mice with a selective deletion of the type I
IFN receptor on T cells. All changes observed were self-limiting, given that cessation of MDA-5 activation led to a rapid recovery
of thymic size. We show for the first time that the in vivo activation of the virus-sensing MDA-5 receptor leads to a rapid and
reversible involution of the thymus.
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Defects in 18S or 28S rRNA processing activate the p53 pathway
07.01.2010
The Journal of Biological Chemistry,
2010,
doi: 10.1074/jbc.M109.054734,
published on 07.01.2010
The Journal of Biological Chemistry, online article
The Journal of Biological Chemistry, online article
The p53 tumor suppressor pathway is activated by defective ribosome synthesis. Ribosomal proteins are released from the nucleolus and block Hdm2 that targets p53 for degradation. However, it remained elusive how abrogation of individual rRNA processing pathways contributes to p53 stabilization. Here we show that selective inhibition of 18S rRNA processing provokes accumulation of p53 as efficiently as abrogated 28S rRNA maturation. We describe hUTP18 as a novel mammalian rRNA processing factor that is specifically involved in 18S rRNA production. hUTP18 was essential for the cleavage of the 5'ETS leader sequence from the primary Pol I transcript, but dispensable for rRNA transcription. As maturation of the 28S rRNA was unaffected in hUTP18 depleted cells, our results suggest that the integrity of both, the 18S and 28S rRNA synthesis pathways, can be independently monitored by the p53 pathway. Interestingly, accumulation of p53 after hUTP18 knockdown required the ribosomal protein L11. Therefore, cells survey the maturation of the small and large ribosomal subunits by separate molecular routes, which may merge in an L11-dependent signalling pathway for p53 stabilization.
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The multi-domain protein Np95 connects DNA methylation and histone modification
21.12.2009
DNA methylation and histone modifications play
a central role in the epigenetic regulation of gene
expression and cell differentiation. Recently, Np95
(also known as UHRF1 or ICBP90) has been found
to interact with Dnmt1 and to bind hemimethylated
DNA, indicating together with genetic studies a
central role in the maintenance of DNA methylation.
Using in vitro binding assays we observed a
weak preference of Np95 and its SRA (SET- and
Ring-associated) domain for hemimethylated CpG
sites. However, the binding kinetics of Np95 in
living cells was not affected by the complete loss
of genomic methylation. Investigating further links
with heterochromatin, we could show that Np95
preferentially binds histone H3 N-terminal tails
with trimethylated (H3K9me3) but not acetylated
lysine 9 via a tandem Tudor domain. This domain
contains three highly conserved aromatic amino
acids that form an aromatic cage similar to the
one binding H3K9me3 in the chromodomain of
HP1ß. Mutations targeting the aromatic cage of
the Np95 tandem Tudor domain (Y188A and Y191A)
abolished specific H3 histone tail binding. These
multiple interactions of the multi-domain protein
Np95 with hemimethylated DNA and repressive
histone marks as well as with DNA and histone
methyltransferases integrate the two major
epigenetic silencing pathways.
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Modulation of protein properties in living cells using nanobodies
13.12.2009
Nature Structural & Molecular Biology,
2009,
doi:10.1038/nsmb.1727
published on 13.12.2009
Nature Structural & Molecular Biology, online article
Nature Structural & Molecular Biology, online article
Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP–nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.
Interaction of HP1 and Brg1/Brm with the Globular Domain of Histone H3 Is Required for HP1-Mediated Repression
11.12.2009
The heterochromatin-enriched HP1 proteins play a critical role in regulation of transcription. These proteins contain two
related domains known as the chromo- and the chromoshadow-domain. The chromo-domain binds histone H3 tails
methylated on lysine 9. However, in vivo and in vitro experiments have shown that the affinity of HP1 proteins to native
methylated chromatin is relatively poor and that the opening of chromatin occurring during DNA replication facilitates their
binding to nucleosomes. These observations prompted us to investigate whether HP1 proteins have additional histone
binding activities, envisioning also affinity for regions potentially occluded by the nucleosome structure. We find that the
chromoshadow-domain interacts with histone H3 in a region located partially inside the nucleosomal barrel at the entry/exit
point of the nucleosome. Interestingly, this region is also contacted by the catalytic subunits of the human SWI/SNF
complex. In vitro, efficient SWI/SNF remodeling requires this contact and is inhibited in the presence of HP1 proteins. The
antagonism between SWI/SNF and HP1 proteins is also observed in vivo on a series of interferon-regulated genes. Finally,
we show that SWI/SNF activity favors loading of HP1 proteins to chromatin both in vivo and in vitro. Altogether, our data
suggest that HP1 chromoshadow-domains can benefit from the opening of nucleosomal structures to bind chromatin and
that HP1 proteins use this property to detect and arrest unwanted chromatin remodeling.
A New Mechanism of Phosphoregulation in Signal Transduction Pathways
10.11.2009
Histidine protein kinases and serine, threonine, or tyrosine protein kinases play essential roles in signal transduction in prokaryotes and eukaryotes. A third type of protein kinase, an arginine protein kinase, has been identified. McsB of Bacillus subtilis hosphorylates the heat shock transcriptional regulator CtsR and can be regarded as the founding member of arginine protein kinases.
RNAPII CTD SER-7 phosphorylation is established in a mediator-dependent fashion
09.11.2009
The largest subunit of RNA polymerase II (RNAPII) carboxy-terminal heptarepeat domain (CTD) is subject to phosphorylation during initiation and elongation of transcription by RNA polymerase II. Here we study the molecular mechanisms leading to phosphorylation of serine-7 (ser-7) in the human enzyme. Ser-7 becomes phosphorylated before initiation of transcription at promoter regions. We identify CDK7 as one responsible kinase. Phosphorylation of both ser-5 and ser-7 is fully dependent on the cofactor complex Mediator. A subform of Mediator associated with an active RNAPII is critical for preinitiation complex formation and CTD phosphorylation. The Mediator-RNAPII complex independently recruits TFIIB and CDK7 to core promoter regions. CDK7 phosphorylates ser-7 selectively in the context of an intact preinitiation complex. CDK7 is not the only kinase that can modify ser-7 of the CTD. ChIP experiments with chemical inhibitors provide evidence that other yet to be identified kinases further phosphorylate ser-7 in coding regions.
Measurement of replication structures at the nanometer scale using super-resolution light microscopy
28.10.2009
Nucleic Acids Research,
2009,
doi:10.1093/nar/gkp901,
1-11
published on 28.10.2009
Nucleic Acids Research, online article
Nucleic Acids Research, online article
DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish
and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary
in size from 210nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3Dstructured illumination microscopy (3D-SIM) both showed an average size of 125nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved
only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.
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CIPSM researcher Prof. Dr. Thomas Cremer is honored by the German Academy of Sciences Leopoldina with the Schleiden Medal
05.10.2009
2009,
published on 05.10.2009
German Academy of Sciences Leopoldina
German Academy of Sciences Leopoldina
CIPSM is very proud Thomas Cremer was honored by the prestigious Schleiden Medal of the German Academy of Sciences Leopoldina for his outstanding scientific work on the topograhy of chromosomes in the nucleus of Eucaryota.
German press release:
Die Deutsche Akademie der Naturforscher Leopoldina - Nationale Akademie der Wissenschaften würdigt die wissenschaftliche Arbeit ihres Mitglieds Prof. Dr. Thomas Cremer, München, mit der Vergabe der Schleiden-Medaille im Rahmen der Feierlichen Eröffnung der Leopoldina- Jahresversammlung am 2. Oktober 2009 in Halle (Saale). Thomas Cremer erhält die Auszeichnung für seine prägenden Arbeiten auf dem Gebiet der Zellkernarchitektur.
Prof. Dr. Thomas Cremer (Jg. 1945), Professor für Anthropologie und Humangenetik an der Universität
München, hat sich bereits nach dem Studium der Medizin der Methodenentwicklung für experimentelle Zellforschung zugewandt. Sein zentrales Thema war stets die Lage der Chromosomen im Zellkern bei Eukaryonten. Cremer vermutete, dass die Chromosomen im Zellkern - auch in der Interphase - nicht zufällig verteilt sind, sondern eine bestimmte, möglicherweise zelltyp-spezifische Topographie haben. Diese Vermutung hat er durch eine Fülle von Daten belegt, die heute zum allgemein akzeptierten Wissen gehören. In seinen Untersuchungen gelang es ihm, die Richtigkeit der zuerst von Carl Rabl (1885) und Theodor
Boveri (1909) vorgeschlagene Hypothese der "Chromosomen-Territorien" im Zellkern zu beweisen und in
ein Modell der funktionalen Zellkernarchitektur zu integrieren. Lage und Struktur der Chromosomenterritorien haben Bedeutung für die Regulierung der Genexpression und für das neue Gebiet der Epigenetik. Diese wissenschaftlichen Einsichten wären ohne neue methodische Wege aber nicht möglich gewesen. So entwickelte Thomas Cremer zusammen mit seinem Bruder, dem Physiker Christoph Cremer (Universität Heidelberg), ein Verfahren zur Laser-UV-Mikrobestrahlung des Zellkerns in lebenden Zellen, die erste Beweise für die Existenz der Chromosomenterritorien und ihre Anordnung lieferte. Später hat er mit Peter Lichter (Deutsches Krebsforschungszentrum Heidelberg) entscheidend zur Entwicklung der heute weltweit genutzten Methode der "Chromosomalen-in-situ-Suppressions-Hybridisierung" ("Chromosome Painting") beigetragen. Thomas Cremer hat mit seinen Arbeiten das heutige Wissen über die Zellkernarchitektur maßgeblich mitgeprägt. Er ist seit 2006 Mitglied der Leopoldina-Sektion Humangenetik und Molekulare Medizin.
Zur Schleiden-Medaille: Die Schleiden-Medaille, benannt nach dem Akademie-Mitglied Matthias Jacob
Schleiden (1804-1881), Botaniker und Mitbegründer der Zelltheorie, wird seit 1955 von der Leopoldina für
hervorragende Erkenntnisse auf dem Gebiet der Zellbiologie vergeben.
Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells
02.10.2009
EMBO reports,
2009,
doi: 10.1038/embor.2009.201,
published on 02.10.2009
EMBO reports, online article
EMBO reports, online article
Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi-methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.
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Bundesministerium supports CIPSM cooperation of CIPSM Investigators Leonhardt and Schotta
01.10.2009
The cooperation "Experimental and theoretical methods for dissecting the dynamics of epigenetic gene silencing in living cells" between Prof. Heinrich Leonhardt's and Prof. Gunnar Schotta's CIPSM groups and scientists from the University of Heidelberg will be supported for the next three years by the "New methods in systembiology" program of the BMBF financially. The cooperation examplifies nicely how successful sicentists can work together. CIPSM gratulates and wishes all the best!
CIPSM football team rules the cup!
11.09.2009
Yeah, the CIPSM football team won the 2009 ScieKickIn football tournament. We thank its members around Prof. Axel Imhof for their excellent performance and passion.
The regulatory interplay between membrane-integrated sensors and transport proteins in bacteria
27.08.2009
Molecular Microbiology,
2009,
73(6),
982–991, doi:10.1111/j.1365-2958.2009.06847.x
published on 27.08.2009
Molecular Microbiology, online article
Molecular Microbiology, online article
Bacteria sense environmental stimuli and transduce this information to cytoplasmic components of the signal transduction machinery to cope with and to adapt to ever changing conditions. Hence, bacteria are equipped with numerous membrane-integrated proteins responsible for sensing such as histidine kinases, chemoreceptors and ToxR-like proteins. There is increasing evidence that sensors employ transport proteins as co-sensors. Transport proteins are well-suited information carriers as they bind lowmolecular-weight molecules in the external medium and transport them into the cytoplasm, allowing them to provide dynamic information on the metabolic flux.
This review explores the sensing capabilities of secondary permeases, primary ABC-transporters, and soluble substrate-binding proteins. Employing transporters as co-sensors seems to be a sophisticated and probably widely distributed mechanism.
The dosage compensation complex shapes the conformation of the X chromosome in Drosophila
26.08.2009
The dosage compensation complex (DCC) in Drosophila
globally increases transcription from the X chromosome
in males to compensate for its monosomy.We discovered
a male-specific conformation of the X chromosome that
depends on the associations of high-affinity binding sites
(HAS) of the DCC. The core DCC subunits MSL1–MSL2
are responsible for this male-specific organization. Contrary
to emerging concepts, we found that neither DCC assembly nor the conformation of the male X chromosome are influenced by nuclear pore components. We propose that nuclear organization of HAS is central to the faithful distribution of the DCC along the X chromosome.
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Induction Kinetics of a Conditional pH Stress Response System in Escherichia coli
21.08.2009
J. Mol. Biol.,
2009,
393,
272–286, doi:10.1016/j.jmb.2009.08.037
published on 21.08.2009
J. Mol. Biol., online article
J. Mol. Biol., online article
The analysis of stress response systems in microorganisms can reveal molecular strategies for regulatory control and adaptation. In this study, we focused on the Cad module, a subsystem of Escherichia coli’s response to acidic stress that is conditionally activated at low pH only when lysine is available. When expressed, the Cad system counteracts the elevated H+ concentration by converting lysine to cadaverine under the consumption of H+ and exporting cadaverine in exchange for external lysine. Surprisingly, the cad operon displays a transient response, even when the conditions for its induction persist. To quantitatively characterize the regulation of the Cad module, we experimentally recorded and theoretically modeled the dynamics of important system variables. We established a quantitative model that adequately describes and predicts the transient expression behavior for various initial conditions. Our quantitative analysis of the Cad system supports negative feedback by external cadaverine as the origin of the transient response. Furthermore, the analysis puts causal constraints on the precise mechanism of signal transduction via the regulatory protein CadC.
Proteome-wide prediction of acetylation substrates
18.08.2009
Acetylation is a well-studied posttranslational modification that has been associated with a broad spectrum of biological processes, notably gene regulation. Many studies have contributed to our
knowledge of the enzymology underlying acetylation, including efforts to understand the molecular mechanism of substrate recognition by several acetyltransferases, but traditional experiments to
determine intrinsic features of substrate site specificity have proven challenging. Here,wecombine experimental methods with clustering analysis of protein sequences to predict protein acetylation based on the sequence characteristics of acetylated lysines within histones with our unique prediction tool PredMod. We define a local amino acid
sequence composition that represents potential acetylation sites by implementing a clustering analysis of histone and nonhistone sequences. We show that this sequence composition has predictive power on 2 independent experimental datasets of acetylation marks. Finally, we detect acetylation for selected putative substrates using mass spectrometry, and report several nonhistone acetylated substrates
in budding yeast. Our approach, combined with more traditional experimental methods, may be useful for identifying acetylated substrates proteome-wide.
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Heterogeneity in quorum sensing-regulated bioluminescence of Vibrio harveyi
23.07.2009
Mol Microbiol,
2009,
73 Issue 2,
267-77
published on 23.07.2009
Molecular Microbiology, online article
Molecular Microbiology, online article
Quorum sensing (QS) refers to the ability of bacterial populations to read out the local environment for cell density and to collectively activate gene expression. Vibrio harveyi, one of the best characterized model organisms in QS, was used to address the question how single cells behave within a QS-activated community in a homogeneous environment. Analysis of the QS-regulated bioluminescence of a wild type strain revealed that even at high cell densities only 69% of the cells of the population produced bioluminescence, 25% remained dark and 6% were dead. Moreover, light intensities greatly varied from cell to cell at high population density. Addition of autoinducer to a bright liquid culture of V. harveyi increased the percentage of luminescent cells up to 98%, suggesting that V. harveyi produces and/or keeps the autoinducers at non-saturating concentrations. In contrast, all living cells of a constitutive QS-active mutant (DeltaluxO) produced light. We also found that QS affects biofilm formation in V. harveyi. Our data provide first evidence that a heterogeneous population produces more biofilm than a homogeneous one. It is suggested that even a QS-committed population of V. harveyi takes advantage of heterogeneity, which extends the current view of QS-regulated uniformity.
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Domain swapping reveals that the N-terminal domain of the sensor kinase KdpD in Escherichia coli is important for signaling
09.07.2009
BMC Microbiology,
2009,
9(133),
doi:10.1186/1471-2180-9-133
published on 09.07.2009
BMC Microbiology, online article
BMC Microbiology, online article
The KdpD/KdpE two-component system of Escherichia coli regulates expression of the kdpFABC operon encoding the high affinity K+ transport system KdpFABC. The input domain of KdpD comprises a domain that belongs to the family of universal stress proteins (Usp). It has been previously demonstrated that UspC binds to this domain, resulting in KdpD/KdpE scaffolding under salt stress. However the mechanistic significance of this domain for signaling remains unclear. Here, we employed a "domain swapping" approach to replace the KdpD-Usp domain with four homologous domains or with the six soluble Usp proteins of E. coli.
Full response to salt stress was only achieved with a chimera that contains UspC, probably due to unaffected scaffolding of the KdpD/KdpE signaling cascade by soluble UspC. Unexpectedly, chimeras containing either UspF or UspG not only prevented kdpFABC expression under salt stress but also under K+ limiting conditions, although these hybrid proteins exhibited kinase and phosphotransferase activities in vitro. These are the first KdpD derivatives that do not respond to K+ limitation due to alterations in the N-terminal domain. Analysis of the KdpD-Usp tertiary structure revealed that this domain has a net positively charged surface, while UspF and UspG are characterized by net negative surface charges.
The Usp domain within KdpD not only functions as a binding surface for the scaffold UspC, but it is also important for KdpD signaling. We propose that KdpD sensing/signaling involves alterations of electrostatic interactions between the large N- and C-terminal cytoplasmic domains.
CDK9 directs H2B monoubiquitination and controls replication-dependent histone mRNA 3'-end processing
03.07.2009
EMBO reports,
2009,
doi:10.1038/embor.2009.108,
published on 03.07.2009
EMBO reports, online article
EMBO reports, online article
Post-translational histone modifications have essential roles in
controlling nuclear processes; however, the specific mechanisms
regulating these modifications and their combinatorial activities
remain elusive. Cyclin-dependent kinase 9 (CDK9) regulates
gene expression by phosphorylating transcriptional regulatory
proteins, including the RNA polymerase II carboxy-terminal
domain. Here, we show that CDK9 activity is essential for maintaining global and gene-associated levels of histone H2B
monoubiquitination (H2Bub1). Furthermore, CDK9 activity and
H2Bub1 help to maintain correct replication-dependent histone
messenger RNA (mRNA) 30-end processing. CDK9 knockdown
consistently resulted in inefficient recognition of the correct
mRNA 30-end cleavage site and led to increased read-through of
RNA polymerase II to an alternative downstream polyadenylation
signal. Thus, CDK9 acts to integrate phosphorylation
during transcription with chromatin modifications to control
co-transcriptional histone mRNA processing.
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5'-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I
02.07.2009
The ATPase retinoid acid-inducible gene (RIG)-I senses viral RNA in the cytoplasm of infected cells and subsequently activates cellular antiviral defense mechanisms. RIG-I recognizes molecular structures that discriminate viral from host RNA. Here, we show that RIG-I ligands require base-paired structures in conjunction with a free 5′-triphosphate to trigger antiviral signaling. Hitherto unavailable chemically synthesized 5′-triphosphate RNA ligands do not trigger RIG-I-dependent IFN production in cells, and they are unable to trigger the ATPase activity of RIG-I without a base-paired stretch. Consistently, immunostimulatory RNA from cells infected with a virus recognized by RIG-I is sensitive to double-strand, but not single-strand, specific RNases. In vitro, base-paired stretches and the 5′-triphosphate bind to distinct sites of RIG-I and synergize to trigger the induction of signaling competent RIG-I multimers. Strengthening our model of a bipartite molecular pattern for RIG-I activation, we show that the activity of supposedly “single-stranded” 5′-triphosphate RNAs generated by in vitro transcription depends on extended and base-paired by-products inadvertently, but commonly, produced by this method. Together, our findings accurately define a minimal molecular pattern sufficient to activate RIG-I that can be found in viral genomes or transcripts.
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DNA Methylation-Mediated Epigenetic Control
29.06.2009
During differentiation and development cells undergo dramatic morphological, and functional changes without any change in the DNA sequence. The underlying changes of gene expression patterns are established and maintained by epigenetic processes. Early mechanistic insights came from the observation that gene activity and repression states correlate with the DNA methylation level of their promoter region. DNA methylation is a postreplicative modification that occurs exclusively at the C5 position of cytosine residues (5mC) and predominantly in the context of CpG dinucleotides in vertebrate cells. Here, three major DNA methyltransferases (Dnmt1, 3a, and 3b) establish specific DNA methylation patterns during differentiation and maintain them over many cell division cycles. CpG methylation is recognized by at least three protein families that in turn recruit histone modifying and chromatin remodeling enzymes and thus translate DNA methylation
into repressive chromatin structures. By now a multitude of histone modifications have been linked in various ways with DNA methylation. We will discuss some of the basic connections and the emerging complexity of these regulatory networks.
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Chromosome shattering: a mitotic catastrophe due to chromosome condensation failure
19.06.2009
European Biophysics Journal,
2009,
38, 6,
729 - 47
published on 19.06.2009
European Biophysics Journal, online article
European Biophysics Journal, online article
Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a “shattered” chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted.
Establishment of Histone Modifications after Chromatin Assembly
18.06.2009
ScienceDirect,
2009,
doi:10.1093/nar/gkp518,
published on 18.06.2009
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Every cell has to duplicate its entire genome during S-phase of the cell cycle. After replication, the newly synthesized DNA is rapidly assembled into chromatin. The newly assembled chromatin ‘matures’ and adopts a variety of different conformations. This differential packaging of DNA plays an important role
for the maintenance of gene expression patterns and has to be reliably copied in each cell division. Posttranslational histone modifications are prime candidates for the regulation of the chromatin structure. In order to understand the maintenance of
chromatin structures, it is crucial to understand the replication of histone modification patterns. To study the kinetics of histone modifications in vivo, we have pulse-labeled synchronized cells with an isotopically labeled arginine (15N4) that is 4 Da
heavier than the naturally occurring 14N4 isoform. As most of the histone synthesis is coupled with replication, the cells were arrested at the G1/S boundary, released into S-phase and simultaneously incubated in the medium containing heavy arginine, thus labeling all newly synthesized proteins.
This method allows a comparison of modification
patterns on parental versus newly deposited histones.
Experiments using various pulse/chase times show that particular modifications have considerably different kinetics until they have acquired a modification pattern indistinguishable from the parental histones.
MBD4 and MLH1 are required for apoptotic induction in xDNMT1-depleted embryos
05.06.2009
Loss of the of the maintenance methyltransferase xDNMT1 during Xenopus development results in premature transcription and
activation of a p53-dependent apoptotic program that accounts for embryo lethality. Here, we show that activation of the
apoptotic response is signalled through the methyl-CpG binding protein xMBD4 and the mismatch repair pathway protein xMLH1.
Depletion of xMBD4 or xMLH1 increases the survival rate of xDNMT1-depleted embryos, whereas overexpression of these proteins in embryos induces programmed cell death at the onset of gastrulation. MBD4 interacts directly with both DNMT1 and MLH1,
leading to recruitment of the latter to heterochromatic sites that are coincident with DNMT1 localisation. Time-lapse microscopy of
micro-irradiated mammalian cells shows that MLH1/MBD4 (like DNMT1) can accumulate at DNA damage sites. We propose that
xMBD4/xMLH1 participates in a novel G2 checkpoint that is responsive to xDNMT1p levels in developing embryos and cells.
TFIIH Kinase Places Bivalent Marks on the Carboxy-Terminal Domain of RNA Polymerase II
15.05.2009
Posttranslational modifications of the carboxyterminal
domain (CTD) of the largest subunit of RNA polymerase II (Pol II) specify a molecular recognition code that is deciphered by proteins involved in RNA biogenesis. The CTD is comprised of a repeating heptapeptide (Y1S2P3T4S5P6S7). Recently, phosphorylation
of serine 7 was shown to be important for cotranscriptional processing of two snRNAs in mammalian cells. Here we report that Kin28/Cdk7, a subunit of the evolutionarily conserved TFIIH
complex, is a Ser7 kinase. The ability of Kin28/Cdk7 to phosphorylate Ser7 is particularly surprising because this kinase functions at promoters of protein-coding genes, rather than being restricted to promoter-distal regions of snRNA genes. Kin28/
Cdk7 is also known to phosphorylate Ser5 residues
of the CTD at gene promoters. Taken together, our results implicate the TFIIH kinase in placing bivalent Ser5 and Ser7 marks early in gene transcription. These bivalent CTD marks, in concert with cues
within nascent transcripts, specify the cotranscriptional
engagement of the relevant RNA processing machinery.
Stimulation of the potassium sensor KdpD kinase activity by interaction with the phosphotransferase protein IIANtr in Escherichia coli
29.04.2009
Molecular Microbiology,
2009,
72 Issue 4,
978-94
published on 29.04.2009
Molecular Microbiology, online article
Molecular Microbiology, online article
Proteins EINtr, NPr and IIANtr form a phosphoryl group transfer chain (Ntr-PTS) working in parallel to the phosphoenolpyruvate:carbohydrate phosphotransferase
system (transport-PTS) in Escherichia coli. Recently, it was shown that dephosphorylated IIANtr binds and inhibits TrkA, a low-affinity potassium transporter. Here we report that the Ntr-PTS also regulates expression of the high-affinity K+ transporter KdpFABC, which rescues K+ uptake at limiting
K+ concentrations. Transcription initiation at the kdpFABC promoter is positively controlled by the two-component system KdpD/KdpE in response to K+ availability. We found that kdp promoter activity is stimulated by the dephosphorylated form of IIANtr. Two-hybrid data and biochemical analysis revealed
that IIANtr interacts with sensor kinase KdpD and stimulates kinase activity, resulting in increased levels of phosphorylated response regulator KdpE. The data suggest that exclusively dephosphorylated IIANtr binds and activates KdpD. As there is cross-talk between the Ntr-PTS and the transport-PTS, carbon
source utilization affects kdpFABC expression. Expression is enhanced, when cells utilize preferred carbohydrates like glucose, which results in preferential dephosphorylation of the transport-PTS and also of IIANtr. Taken together, the data show that the Ntr-PTS has an important role in maintaining K+
homeostasis and links K+ uptake to carbohydrate
metabolism.
Drosophila ISWI Regulates the Association of Histone H1 With Interphase Chromosomes in Vivo
20.04.2009
Although tremendous progress has been made toward identifying factors that regulate nucleosome structure and positioning, the mechanisms that regulate higher-order chromatin structure remain poorly understood. Recent studies suggest that the ISWI chromatin-remodeling factor plays a key role in this process by promoting the assembly of chromatin containing histone H1. To test this hypothesis, we investigated the function of H1 in Drosophila. The association of H1 with salivary gland polytene chromosomes is regulated by a dynamic, ATP-dependent process. Reducing cellular ATP levels triggers the dissociation of H1 from polytene chromosomes and causes chromosome defects similar to those
resulting from the loss of ISWI function. H1 knockdown causes even more severe defects in chromosome structure and a reduction in nucleosome repeat length, presumably due to the failure to incorporate H1 during replication-dependent chromatin assembly. Our findings suggest that ISWI regulates higher-order chromatin structure by modulating the interaction of H1 with interphase chromosomes.
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Nuclear Architecture of Rod Photoreceptor Cells Adapts to Vision in Mammalian Evolution
17.04.2009
We show that the nuclear architecture of rod photoreceptor cells differs fundamentally in nocturnal and diurnal mammals. The rods of diurnal retinas possess the conventional architecture found in nearly all eukaryotic cells, with most heterochromatin situated
at the nuclear periphery and euchromatin residing toward the nuclear interior. The rods of nocturnal retinas have a unique inverted pattern, where heterochromatin localizes in the nuclear center, whereas euchromatin, as well as nascent transcripts and
splicing machinery, line the nuclear border. The inverted pattern forms by remodeling of the conventional one during terminal differentiation of rods. The inverted rod nuclei act as collecting lenses, and computer simulations indicate that columns of such
nuclei channel light efficiently toward the lightsensing
rod outer segments. Comparison of the two patterns suggests that the conventional architecture prevails in eukaryotic nuclei because it results in more flexible chromosome arrangements, facilitating positional regulation of nuclear functions.
Drosophila HP1c Is Regulated by an Auto-Regulatory Feedback Loop through Its Binding Partner Woc
07.04.2009
HP1 is a major component of chromatin and regulates gene expression through its binding to methylated histone H3. Most
eukaryotes express at least three isoforms of HP1 with similar domain architecture. However, despite the common
specificity for methylated histone H3, the three HP1 isoforms bind to different regions of the genome. Most of the studies so
far focused on the HP1a isoform and its role in transcriptional regulation. As HP1a requires additional factors to bind
methylated chromatin in vitro, we wondered whether another isoform might also require additional targeting factors.
Indeed, we found that HP1c interacts with the DNA binding factors Woc and Row and requires Woc to become targeted to
chromatin in vivo. Moreover, we show that the interaction between HP1c and Woc constitutes a transcriptional feedback
loop that operates to balance the concentration of HP1c within the cell. This regulation may prevent HP1c from binding to
methylated heterochromatin.
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Synergy between CD26/DPP-IV Inhibition and G-CSF Improves Cardiac Function after Acute Myocardial Infarction
03.04.2009
Ischemic cardiomyopathy is one of the main causes
of death, which may be prevented by stem cell-based
therapies. SDF-1a is the major chemokine attracting
stem cells to the heart. Since SDF-1a is cleaved
and inactivated by CD26/dipeptidylpeptidase IV
(DPP-IV), we established a therapeutic concept—
applicable to ischemic disorders in general—by
combining genetic and pharmacologic inhibition of
DPP-IV with G-CSF-mediated stem cell mobilization
after myocardial infarction in mice. This approach
leads to (1) decreased myocardial DPP-IV activity,
(2) increased myocardial homing of circulating
CXCR-4+ stem cells, (3) reduced cardiac remodeling,
and (4) improved heart function and survival. Indeed,
CD26 depletion promoted posttranslational stabilization
of active SDF-1a in heart lysates and preserved
the cardiac SDF-1-CXCR4 homing axis. Therefore,
we propose pharmacological DPP-IV inhibition and
G-CSF-based stem cell mobilization as a therapeutic
concept for future stem cell trials after myocardial
infarction.
Active promoters and insulators are marked by the centrosomal protein 190
19.02.2009
For the compact Drosophila genome, several factors mediating insulator function, such as su(Hw) and dCTCF, have been identified. Recent analyses showed that both these insulator-binding factors are functionally dependent on the same cofactor, CP190. Here we analysed genome-wide binding of CP190 and dCTCF. CP190 binding was detected at CTCF, su(Hw) and GAF sites and unexpectedly at the transcriptional start sites of actively transcribed genes. Both insulator and transcription start site CP190-binding elements are strictly marked by a depletion of histone H3 and, therefore, a loss of nucleosome occupancy. In addition, CP190/dCTCF double occupancy was seen at the borders of many H3K27me3 'islands'. As before, these sites were also depleted of H3. Loss of either dCTCF or CP190 causes an increase of H3 and H3K27 trimethylation at these sites. Thus, for both types of cis-regulatory elements, domain borders and promoters, the chromatin structure is dependent on CP190.
How are signals transduced across the cytoplasmic membrane? Transport proteins as transmitter of information
08.02.2009
In order to adapt to ever changing environmental conditions, bacteria sense environmental stimuli, and convert them into signals that are transduced intracellularly. Several mechanisms have evolved by which receptors transmit signals across the cytoplasmic membrane. Stimulus perception may trigger receptor dimerization and/or conformational changes. Another mechanism involves the proteolytic procession of a receptor whereby a diffusible cytoplasmic protein is generated. Finally, there is increasing evidence that transport proteins play an important role in transducing signals across the membrane. Transport proteins either directly translocate signaling molecules into the cytoplasm, or transmit information via conformational changes to their interacting partners such as membrane-integrated or soluble components of signal transduction cascades. Employing transport proteins as sensors and regulators of signal transduction represents a sophisticated way of interconnecting metabolic flux and transcriptional regulation in cells.
Spatiotemporal Dynamics of Regulatory Protein Recruitment at DNA Damage Sites
02.02.2009
Journal of Cellular Biochemistry,
2009,
104,
1562–1569
published on 02.02.2009
Journal of Cellular Biochemistry, online article
Journal of Cellular Biochemistry, online article
Mammalian cells are constantly threatened by multiple types ofDNAlesions arising from various sources like irradiation, environmental agents, replication errors or by-products of the normal cellular metabolism. If not readily detected and repaired these lesions can lead to cell death or to the transformation of cells giving rise to life-threatening diseases like cancer. Multiple specialized repair pathways have evolved to preserve the genetic integrity of a cell. The
increasing number of DNA damage sensors, checkpoint regulators, and repair factors identified in the numerous interconnected repair pathways raises the question of howDNArepair is coordinated. In the last decade, various methods have been developed that allow the induction of DNA lesions and subsequent real-time analysis of repair factor assembly at DNA repair sites in living cells. This combination of biophysical and molecular cell biology methods has yielded
interesting newinsights into the order and kinetics of protein recruitment and identified regulatory sequences and selective
loading platforms for the efficient restoration of the genetic and epigenetic integrity of mammalian cells.
Dimerization of DNA Methyltransferase 1 Is Mediated by Its Regulatory Domain
27.01.2009
Journal of Cellular Biochemistry,
2009,
105,
521-28
published on 27.01.2009
Journal of Cellular Biochemistry, online article
Journal of Cellular Biochemistry, online article
DNA methylation is a major epigenetic modification and plays a crucial role in the regulation of gene expression. Within the family of DNA methyltransferases (Dnmts), Dnmt3a and 3b establish methylation marks during early development, while Dnmt1 maintains methylation patterns after DNA replication. The maintenance function of Dnmt1 is regulated by its large regulatory N-terminal domain that interacts with other chromatin factors and is essential for the recognition of hemi-methylated DNA. Gelfiltration analysis showed that purified Dnmt1 elutes at an apparent molecular weight corresponding to the size of a dimer. With protein interaction assays we could show that Dnmt1 interacts with itself through its N-terminal regulatory domain. By deletion analysis and co-immunoprecipitations we mapped the dimerization domain to the targeting sequence TS that is located in the center of the N-terminal domain (amino acids 310–629) and was previously shown to mediate replication independent association with heterochromatin at chromocenters. Further mutational analyses suggested that the dimeric complex
has a bipartite interaction interface and is formed in a head-to-head orientation. Dnmt1 dimer formation could facilitate the discrimination of hemi-methylated target sites as has been found for other palindromic DNA sequence recognizing enzymes. These results assign an additional function to the TS domain and raise the interesting question how these functions are spatially and temporarily co-ordinated.
Backbone Structure of Transmembrane Domain IX of the Naþ/Proline Transporter PutP of Escherichia coli
15.01.2009
The backbone structure is determined by site-directed spin labeling, double electron electron resonance measurements of distances, and modeling in terms of a helix-loop-helix construct for a transmembrane domain that is supposed to line the translocation pathway in the 54.3 kDa Naþ/proline symporter PutP of Escherichia coli. The conformational distribution of the spin labels is accounted for by a rotamer library. An ensemble of backbone models with a root mean-square deviation of less than 2A˚ is obtained. These models exhibit a pronounced kink near residue T341, which is involved in substrate binding. The kink may be associated with a hinge that allows the protein to open and close an inwardly oriented cavity.
A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding
07.01.2009
Nucleic Acids Research Advance Access,
2009,
doi:10.1093/nar/gkn1029,
published on 07.01.2009
Nucleic Acids Research , online article
Nucleic Acids Research , online article
We present a simple, non-radioactive assay for DNA
methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were
subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA
methyltransferases can also be determined by using suicide DNA substrates. These substratescontain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian
DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1C1229W catalytic site mutant showed
DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several
different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity.
Monomethylation of Lysine 20 on Histone H4 Facilitates Chromatin Maturation
22.10.2008
Histone modifications play an important role in shaping chromatin structure. Here, we describe the use of an in vitro chromatin assembly system from Drosophila embryo extracts to investigate the dynamic changes of histone modifications subsequent to histone deposition. In accordance with what has been observed in vivo, we
find a deacetylation of the initially diacetylated isoform of histone H4, which is dependent on chromatin assembly. Immediately after deposition of the histones onto DNA, H4 is monomethylated at K20, which is required for an efficient deacetylation of the H4 molecule. H4K20 methylation-dependent dl(3)MBT association with chromatin and the identification of a dl(3)MBT-dRPD3 complex suggest that a deacetylase is specifically recruited to the monomethylated substrate through interaction with dl(3)MBT. Our data demonstrate that histone modifications are added and removed during chromatin assembly in a highly regulated manner.
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.
Structure-function analysis of the RNA helicase maleless
17.12.2007
Nucleic Acids Research,
2008,
36 No. 3,
950-62
published on 17.12.2007
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster leads to male-specific lethality due to a failure of X chromosome dosage compensation. MLE is presumably involved in incorporating the non-coding roX RNA into the dosage compensation complex (DCC), which is an essential but poorly understood requirement for faithful targeting of the complex to the X chromosome. Sequence comparison predicts several RNA-binding domains in MLE but their properties have not been experimentally verified. We evaluated the RNA-binding characteristics of these conserved motifs and their contributions to RNA-stimulated ATPase activity, to helicase activity, as well as to the targeting of MLE to the nucleus and to the X chromosome territory. We find that RB2 is the dominant, conditional RNA-binding module, which is indispensable for ATPase and helicase activity whereas the N-terminal RB1 motif does not bind RNA, but is involved in targeting MLE to the X chromosome. The C-terminal domain containing a glycine-rich heptad repeat adds potential dimerization and RNA-binding surfaces which are not required for helicase activity.
Seven Ups the Code
14.12.2007
Patters of phosphorylation in a region of RNA polymerase II may constitute a code that controls the recruitment of regulatory factors to control gene expression. Jeffry L. Corden puts CIPSM-researcher Dirk Eick's recent work on transcription in perspective.
The development of higher forms of life would appear to have been influenced by RNA polymerase II. This enzyme transcribes the information coded by genes from DNA into messenger-RNA (mRNA), which in turn is the basis for the production of proteins. RNA polymerase II is highly conserved through evolution, with many of its structural characteristics being conserved between bacteria and humans. Single-cell organisms were already in existence 500 million years ago, with several thousand genes providing different cellular functions. Further developments seemed dependent on producing even more genes. For a highly developed organism like a human, this form of evolution would have resulted in several million genes. Researchers were therefore surprised to learn, following publication of the human genome, that a human only has around 25,000 genes – not many more than a fruit fly or a worm with approximately 15,000 to 20,000 genes. It would appear that, over the last 500 million years, other ways to produce highly complex organisms have evolved. Evolution has simply found more efficient ways to use the genes already there. But what could have made this possible? In the current issue of Science the group of Prof. Dirk Eick at the Institute of Clinical Molecular Biology and Tumor Genetics, GSF – Research Center for Environment and Health, Munich, and the group of Dr. Shona Murphy from Oxford University, England, publish results which represent a piece of the puzzle and shed new light on to the purpose of an unusual structure in RNA polymerase II. They build on earlier observations that gene expression is not just regulated by binding of the enzyme to the gene locus to which it is recruited, but also during the phase of active transcription from DNA into RNA. During this phase, parts of the newly synthesised RNA may be removed and the remaining sequences combined into new RNA message. This ‘splicing’ of RNA occurs during gene transcription, and in extreme cases, can produce RNAs coding for several thousand different proteins from a single gene.
But what was the development that permitted this advance in gene usage? The RNA polymerase II has developed a structure composed of repeats of a 7 amino-acid sequence. In humans this structure – termed “carboxyterminal domain” or CTD – is composed of 52 such repeats. It is placed exactly at the position where RNA emerges from RNA polymerase II. In less complex organisms the CTD is much shorter: a worm has 36 repeats, and yeast as few as 26, but many single-cell organisms and bacteria have never developed an obvious CTD structure.
Although the requirement of CTD for the expression of cellular genes in higher organisms is undisputed, the molecular details for the gene-specific maturation of RNAs is still largely enigmatic. The groups of Dirk Eick and Shona Murphy have now shown a differential requirement for phosphorylation of the amino acid serine at position 7 of CTD in the processing and maturation of specific gene products. These results provide the groundwork for the discovery of further pieces of the CTD puzzle and thus enlarge our knowledge of gene regulation. Given its fundamental importance, understanding the mechanism of gene regulation is essential if we are to understand cancer and other diseases at the molecular level and develop new therapies.
Publications:
-Chapman, R.D., Heidemann, M., Albert, T., Mailhammer, R., Meisterernst, M., Kremmer, E., and Eick, D. (2007) RNA polymerase II CTD is phosphorylated at serine 7 during the transcription cycle. Science, Vol. 318, Issue 5857, December 14, 2007
-Egloff, S., O’Reilly, D., Chapman, R.D., Taylor, A., Tanzhaus, K., Pitts, L., Eick, D., and Murphy, S. (2007) A specific role for serine 7 of the pol II CTD in expression of human snRNA genes. Science, Vol. 318, Issue 5857, December 14, 2007
Transcribing RNA Polymerase II Is Phosphorylated at CTD Residue Serine-7
14.12.2007
RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5′ and 3′ regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.
Serine-7 of the RNA Polymerase II CTD Is Specifically Required for snRNA Gene Expression
14.12.2007
RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene–specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type–specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.
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Probing Intranuclear Environments at the Single-Molecule Level
07.12.2007
Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage.The onlysignificant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds.The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins,when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but
relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentrationof reactants and availability of binding sites.
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NC2 mobilizes TBP on core promoter TATA boxes
11.11.2007
Nature Structural & Molecular Biology,
2007,
doi:10.1038/nsmb1328,
1-6
published on 11.11.2007
www.nature.com/nsmb, online article
www.nature.com/nsmb, online article
The general transcription factors (GTFs) of eukaryotic RNA polymerase II, in a process facilitated by regulatory and accessory factors, target promoters through synergistic interactions with core elements. The specific binding of the TATA box–binding protein (TBP) to the TATA box has led to the assumption that GTFs recognize promoters directly, producing a preinitiation complex at a defined position. Using biochemical analysis as well as biophysical single-pair Fo¨rster resonance energy transfer, we now provide evidence that negative cofactor-2 (NC2) induces dynamic conformational changes in the TBP–DNA complex that allow it to escape and return to TATA-binding mode. This can lead to movement of TBP along the DNA away from TATA.
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A mutagenesis strategy combining systematic alanine scanning with larger mutations to study protein interactions
09.11.2007
Site-directed mutagenesis (SDM) of target DNA is an invaluable tool to study protein structure–function relationships. Alanine-scanning mutagenesis has been successfully applied to systematically map functional binding epitopes. Substitution of target amino acids with alanine removes all side chain atoms past the b-carbon and does not introduce unusual backbone dihedral angle preferences. Therefore, alanine scanning is particularly useful for assessing the contribution of charged residues on the protein surface without disrupting the folding of its core. This approach requires the production of large numbers of point mutants by SDM. Numerous SDM methods have been described (reviewed in Refs.), and commercially available kits offer fast protocols based on oligonucleotide primers harboring the mutation. However, most strategies do not include a selectable marker to distinguish mutant clones from wild-type clones, and when they do so the procedure becomes significantly more laborious and time-consuming because it requires either two cycles of transformation in different bacterial strains or transfer of the target DNA sequence to and from a specialized vector. In addition, most of these procedures are not suited to generate large sequence alterations. Methods based on type IIs restriction enzymes allow precise replacement of individual nucleotides or codons and either selection for mutant clones or generation of larger mutations, but not both possibilities together. Again, when selection for mutant clones is possible, multiple restriction, ligation, fill-in, and transformation reactions are necessary and suitable restriction sites relatively close to the mutagenesis site are required. Other PCR-based approaches allow generation of point and larger mutations and rapid screening for mutant clones but involve amplification of the entire plasmid with consequent risk of introducing additional mutations. Here we describe a simple, fast, and inexpensive strategy to generate alanine substitutions as well as deletions, duplications, insertions, and larger replacements while retaining the ability to screen for mutant clones by restriction analysis.
Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells
03.11.2007
Genome integrity is constantly threatened by DNA lesions arisin from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity.
A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins
21.10.2007
Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein.
Maintenance of imprinting and nuclear architecture in cycling cells
18.09.2007
Dynamic gene repositioning has emerged as an additional level of
epigenetic gene regulation. An early example was the report of a
transient, spatial convergence (<2 micro m) of oppositely imprinted
regions (‘‘kissing’’), including the Angelman syndrome/Prader–
Willi syndrome (AS/PWS) locus and the Beckwith–Wiedemann
syndrome locus in human lymphocytes during late S phase. It was
argued that kissing is required for maintaining opposite imprints in
cycling cells. Employing 3D-FISH with a BAC contig covering the
AS/PWS region, light optical, serial sectioning, and quantitative
3D-image analysis, we observed that both loci always retained a
compact structure and did not form giant loops. Three-dimensional
distances measured among various, homologous AS/PWS segments in 393 human lymphocytes, 132 human fibroblasts, and 129 lymphoblastoid cells from Gorilla gorilla revealed a wide range of distances at any stage of interphase and in G0. At late S phase, 4% of nuclei showed distances <2 micro m, 49% showed distances >6 micro m, and 18% even showed distances >8 micro m. A similar distance variability was found for Homo sapiens (HSA) 15 centromeres in a PWS patient with a deletion of the maternal AS/PWS locus and for the Beckwith–Wiedemann syndrome loci in human lymphocytes. A transient kiss during late S phase between loci widely separated at other stages of the cell cycle seems incompatible with known global constraints of chromatin movements in cycling cells. Further experiments suggest that the previously observed convergence of AS/PWS loci during late S phase was most likely a side effect of the convergence of nucleolus organizer region-bearing acrocentric human chromosomes, including HSA 15.
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