Content
2009
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.
Superior Protective Immunity against Murine Listeriosis by Combined Vaccination with CpG DNA and Recombinant Salmonella enterica Serovar Typhimurium
01.12.2009
Infection and Immunity,
2009,
77, 12,
5501 - 08
published on 01.12.2009
Infection and Immunity, online article
Infection and Immunity, online article
Preexisting antivector immunity can severely compromise the ability of Salmonella enterica serovar Typhimurium live vaccines to induce protective CD8 T-cell frequencies after type III secretion system-mediated heterologous protein translocation in orally immunized mice. To circumvent this problem, we injected CpG DNA admixed to the immunodominant p60217-225 peptide from Listeria monocytogenes subcutaneously into BALB/c mice and coadministered a p60-translocating Salmonella strain by the orogastric route. The distribution of tetramer-positive p60217-225-specific effector and memory CD8 T cells was analyzed by costaining of lymphocytes with CD62L and CD127. In contrast to the single oral application of recombinant Salmonella or single immunization with CpG and p60, in the spleens from mice immunized with a combination of both vaccine types a significantly higher level of p60-specific CD8 T cells with a predominance of the effector memory T-cell subset was detected. In vivo protection studies revealed that this CD8 T-cell population conferred sterile protective immunity against a lethal infection with L. monocytogenes. However, p60-specific central memory CD8 T cells induced by single vaccination with CpG and p60 were not able confer effective protection against rapidly replicating intracellular Listeria. In conclusion, we provide compelling evidence that the combination of Salmonella type III-mediated antigen delivery and CpG immunization is an attractive novel vaccination strategy to modulate CD8 differentiation patterns toward distinct antigen-specific T-cell subsets with favorable protective capacities.
Immunostimulatory RNA Oligonucleotides Induce an Effective Antitumoral NK Cell Response through the TLR71
15.11.2009
The Journal of Immunology,
2009,
doi: 10.4049/jimmunol.0901594,
vol. 183 no. 10 6078-6086
published on 15.11.2009
The Journal of Immunology, online article
The Journal of Immunology, online article
RNA oligonucleotides containing immune-activating sequences promote the development of cytotoxic T cell and B cell responses to Ag. In this study, we show for the first time that immunostimulatory RNA oligonucleotides induce a NK cell response that prevents growth of NK-sensitive tumors. Treatment of mice with immunostimulatory RNA oligonucleotides activates NK cells in a sequence-dependent manner, leading to enhanced IFN-γ production and increased cytotoxicity. Use of gene-deficient mice showed that NK activation is entirely TLR7-dependent. We further demonstrate that NK activation is indirectly induced through IL-12 and type I IFN production by dendritic cells. Reconstitution of TLR7-deficient mice with wild-type dendritic cells restores NK activation upon treatment with immunostimulatory RNA oligonucleotides. Thus, by activating both NK cells and CTLs, RNA oligonucleotides stimulate two major cellular effectors of antitumor immunity. This dual activation may enhance the efficacy of immunotherapeutic strategies against cancer by preventing the development of tumor immune escape variants.
Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1beta production
15.11.2009
Nature Immunology,
2009,
doi:10.1038/ni.1824,
11, 63–69 (2010)
published on 15.11.2009
Nature Immunology, online article
Nature Immunology, online article
Interleukin 1beta (IL-1beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-κB and subsequent processing of pro-IL-1betaby an inflammasome. However, the sensors and mechanisms that facilitate RNA virus–induced production of IL-1beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus–induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-KB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9–Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.
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!
Revealing the high-resolution three-dimensional network of chromatin and interchromatin space: A novel electron-microscopic approach to reconstructing nuclear architecture
17.09.2009
Chromosome Research,
2009,
17, 6,
801 - 10
published on 17.09.2009
Chomosome Research, online article
Chomosome Research, online article
The nuclear architecture is considered an important contributor to genome function. Although the fine structural features of the cell nucleus have been investigated extensively by means of ultrastructural cytochemistry, mainly on ultrathin sections in two dimensions (2D), there was a of lack routine methods for a rapid reconstruction of three-dimensional (3D) distribution of different structural constituents throughout the nuclear volume. We have now filled this gap by the application of a novel approach associating a pre-embedding selective visualization of nuclear components with a method making use of ultramicrotomy combined with scanning electron microscopy (microtome serial block face scanning electron microscopy—‘3View’). We have been able to apply this method to the study of DNA distribution within the nuclear volume and reconstruction of 3D chromatin arrangement in nuclei of rat hepatocytes and endothelial cells. Our observations demonstrate that while chromatin appears to occupy the interior of nuclei rather sparsely on 2D images, once reconstructed in 3D from a series of sequential 2D images it gives the impression of considerably filling the nuclear volume. However, quantitative evaluation of the nuclear volume occupied by DNA in the above two types of nuclei leaves a significant part to the interchromatin space (66.2% for hepatic cells and 41.7% for endothelial cells, including nuclear space occupied by nucleoli). Detailed analysis of the reconstructed nuclei reveals a high degree of superposition of chromatin domains, giving rise to a false impression that they fill a much larger part of the nuclear volume than they really do. Our results show the importance of the contribution of such reconstruction techniques to our understanding of the nuclear architecture.
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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A Technical Note on Quantum Dots for Multi-Color Fluorescence in situ Hybridization
25.08.2009
Cytogenetic and Genome Research,
2009,
124, 3-4,
351 - 59
published on 25.08.2009
Cytogenetic and Genome Research, online article
Cytogenetic and Genome Research, online article
Quantum dots (Qdots) are semiconductor nanocrystals, which are photo-stable, show bright fluorescence with narrow, symmetric emission spectra and are available in multiple resolvable colors. We established a FISH protocol for the simultaneous visualization of up to 6 different DNA probes differentially labeled with Qdots and with conventional organic fluorochromes. Using a Leica SP5 laser scanning confocal microscope for image capture, we tested various combinations of hapten-labeled probes detected with streptavidin-Qdot525, sheep anti-digoxigenin-Qdot605, rat anti-dinitrophenyl-Qdot655 and goat anti-mouse-Qdot655, respectively, together with FITC-dUTP-, Cy3-dUTP- and Texas Red-dUTP-labeled probes. We further demonstrate that Qdots are suitable for imaging of FISH probes using 4Pi microscopy, which promises to push the resolution limits of light microscopy to 100 nanometers or less when applying a deconvolution algorithm, but requires the use of highly photo-stable fluors.
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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Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon–independent apoptosis in human melanoma cells
03.08.2009
JCI,
2009,
119 Issue 8,
2399–411
published on 03.08.2009
The Journal of Clinical Investigation, online article
The Journal of Clinical Investigation, online article
The retinoic acid–inducible gene I (RIG-I) and melanoma differentiation–associated antigen 5 (MDA-5) helicases
sense viral RNA in infected cells and initiate antiviral responses such as the production of type I IFNs. Here we have shown that RIG-I and MDA-5 also initiate a proapoptotic signaling pathway that is independent of type I IFNs. In human melanoma cells, this signaling pathway required the mitochondrial adapter Cardif
(also known as IPS-1) and induced the proapoptotic BH3-only proteins Puma and Noxa. RIG-I– and MDA-5– initiated apoptosis required Noxa but was independent of the tumor suppressor p53. Triggering this pathway led to efficient activation of mitochondrial apoptosis, requiring caspase-9 and Apaf-1. Surprisingly, this
proapoptotic signaling pathway was also active in nonmalignant cells, but these cells were much less sensitive to apoptosis than melanoma cells. Endogenous Bcl-xL rescued nonmalignant, but not melanoma, cells from RIG-I– and MDA-5–mediated apoptosis. In addition, we confirmed the results of the in vitro studies, demonstrating that RIG-I and MDA-5 ligands both reduced human tumor lung metastasis in immunodeficient
NOD/SCID mice. These results identify an IFN-independent antiviral signaling pathway initiated by RIG-I and MDA-5 that activates proapoptotic signaling and, unless blocked by Bcl-xL, results in apoptosis. Due to their immunostimulatory and proapoptotic activity, RIG-I and MDA-5 ligands have therapeutic potential due to their ability to overcome the characteristic resistance of melanoma cells to apoptosis.
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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Changes of higher order chromatin arrangements during major genome activation in bovine preimplantation embryos
15.07.2009
Experimental Cell Research,
2009,
315, 12,
2053 - 63
published on 15.07.2009
Experimental Cell Research, online article
Experimental Cell Research, online article
Gene-dense chromosome territories (CTs) are typically located more interior, gene-poor CTs more peripheral in mammalian cell nuclei. Here, we show that this gene-density correlated CT positioning holds for the most gene-rich and gene-poor bovine chromosomes 19 and 20, respectively, in bovine fibroblast and lymphocyte nuclei. In order to determine the period at which this non-random CT order is established during development, we performed fluorescence in situ hybridization, on three-dimensionally preserved bovine preimplantation embryos generated by in vitro fertilization and investigated the distribution of BTA 19 and 20 CTs. Radial arrangements of CTs 19 and 20 were the same up to the 8-cell stage. At the 10- to 16-cell stage, however, a significant difference became apparent with CTs 19 localized more internally and CTs 20 more peripherally. Since major genome activation in bovine embryos occurs at the 8- to 16-cell stage, our findings demonstrate a temporal correlation between transcriptional activation and a major rearrangement of chromatin topography in blastomere nuclei
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.
The HP1a–CAF1–SetDB1-containing complex provides H3K9me1 for Suv39-mediated K9me3 in pericentric heterochromatin
05.06.2009
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.
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.
