Content
2010
The histone methyltransferase Dot1 is required for DNA damage repair and proper development in Dictyostelium
25.12.2010
Posttranslational histone modifications play an important role in modulating gene expression and chro- matin structure. Here we report the identification of histone H3K79 dimethylation in the simple eukary- ote Dictyostelium discoideum. We have deleted the D. discoideum Dot1/KMT4 homologue and demonstrate that it is the sole enzyme responsible for histone H3K79me2. Cells lacking Dot1 are reduced in growth and delayed in development, but do not show apparent changes in cell cycle regulation. Furthermore, our results indicate that Dot1 contributes to UV damage resistance and DNA repair in D. discoideum. In summary, the data support the view that the machinery controlling the setting of histone marks is evo- lutionary highly conserved and provide evidence that D. discoideum is a suitable model system to analyze these modifications and their functions during development and differentiation.
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Fast signals and slow marks: the dynamics of histone modifications
01.11.2010
Trends in Biochemical Sciences,
01.11.2010 13:00,
doi:10.1016/j.tibs.2010.05.006,
published on 25.12.2010
TIBS, online article
TIBS, online article
Most multi-cellular organisms adopt a specific gene expression pattern during cellular differentiation. Once established, this pattern is frequently maintained over several cell divisions despite the fact that the initiating signal is no longer present. Differential packaging into chromatin is one such mechanism that allows fixation of transcriptional activity. Recent genome-wide studies demonstrate that actively transcribed regions are characterized by a specific modification pattern of histones, the main protein component of chromatin. These findings support the hypothesis that a histone code uses histone post-translational modifications to stably inscribe particular chromatin structures into the genome. Experiments on the dynamics of histone modifications reveal a striking kinetic difference between methylation, phosphorylation and acetylation, suggesting different roles of these modifications in epigenetically fixing specific gene expression patterns.
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Delivery of Immunostimulatory RNA Oligonucleotides by Gelatin Nanoparticles Triggers an Efficient Antitumoral Response
01.12.2010
Journal of Immunotherapy,
2010,
doi: 10.1097/CJI.0b013e3181f5dfa7,
Volume 33 - Issue 9 - pp 935-944
published on 01.12.2010
Journal of Immunotherapy, online article
Journal of Immunotherapy, online article
RNA oligonucleotides have emerged as a new class of biologicals that can silence gene expression but also stimulate immune responses through specific pattern-recognition receptors. The development of effective delivery systems remains a major challenge for the therapeutic application of the RNA oligonucleotides. In this study, we have established a novel biodegradable carrier system that is highly effective for the delivery of immunostimulatory RNA oligonucleotides. Formulation of RNA oligonucleotides with cationized gelatin nanoparticles potentiates immune activation through the Toll-like receptor 7 (TLR7) in both myeloid and plasmacytoid dendritic cells. Further, nanoparticle-delivered RNA oligonucleotides trigger production of the antitumoral cytokines IL-12 and IFN-α. Binding to gelatin nanoparticles protects RNA oligonucleotides from degradation by nucleases, facilitates their uptake by dendritic cells, and targets these nucleic acids to the endosomal compartment in which they are recognized by TLR7. In these effects, the nanoparticles are superior to the conventional transfection reagents lipofectamine, polyethylenimine, and DOTAP. In vivo, the delivery of TLR7-activating RNA oligonucleotides by gelatin nanoparticles triggers antigen-specific CD8+ T-cell and antibody responses. Indeed, immunization with RNA-loaded nanoparticles leads to an efficient antitumoral immune response in two different mouse tumor models. Thus, gelatin-based nanoparticles represent a novel delivery system for immunostimulatory RNA oligonucleotides that is both effective and nontoxic.
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Every methyl counts – Epigenetic calculus
23.11.2010
Histone modifications play an important role in the formation of an epigenetic memory system that maintains cellular identity. Their complex patterns have been suggested to constitute a histone code, which encodes for specific forms of chromatin. According to the histone code hypothesis these specific patterns are passed on from one cell generation to the next. This enables cells to keep a specific gene expression pattern even in absence of the specific transcription factors that initiated the expression of lineage determining genes. The methylation of specific lysine residues within the histone tails plays a particularly important role in defining the histone modification pattern as mutations of the enzymes that catalyze the formation or the removal of methyl groups have severe effects on cellular physiology. Lysines can get mono-, di- or trimethylated, but the molecular function of the different modification states is still not fully understood. In the following review we will highlight recent data that try to tackle this question and discuss their potential impact for our understanding of the role of histone methylation in epigenetic inheritance.
CIPSM sponsors Kirsten Jung's project at the International Genetically Engineered Machine competition (iGEM)
15.10.2010
2010,
published on 15.10.2010
iGEM 2010 CIPSM LMU team
iGEM 2010 CIPSM LMU team
We thank the CIPSM iGEM team members and Kirsten Jung and wish them good luck at this years competition!
The International Genetically Engineered Machine competition (iGEM) is the premiere undergraduate Synthetic Biology competition.
The iGEM team LMU-Munich works on the project: "Production of azobenzene derivates in Escherichia coli and selection of successful transformants by apoptosis.
The topic “Pathway” involves the creation of an artificial metabolic pathway for the synthesis of azobenzene derivates in E. coli. This will be accomplished by expressing the required enzymes enclosed in a proteinaceous bacterial microcompartment. This microcompartment is necessary to shield the cytoplasm from toxic intermediates which would otherwise make this biosynthesis impossible. Azobenzene derivates are interesting in the field of biochemistry because of their properties as synthetic molecular switches.
The topic “ApoControl” is divided into three subprojects on controllable cell-death. The goal is to develop a system to improve the efficiency and specificity of gene expression in eukaryotic cell-lines and more specifically, to select cells expressing the target gene against cells that do not. Here, proapoptotic genes instead of antibiotic resistance are used as selection marker to induce clean cell-death at different stimuli.
Nucleosome dynamics and epigenetic stability
20.09.2010
Essays Biochem.,
2010,
48,
63-74
published on 20.09.2010
Nucleosome remodelling is an essential principle to assure that the packaging of eukaryotic genomes in chromatin remains flexible and adaptable to regulatory needs. Nucleosome remodelling enzymes spend the energy of ATP to alter histone–DNA interactions, to catalyse nucleosome displacement and reassembly, on histone exchange and on the relocation of histone octamers on DNA. Despite these dynamics, chromatin structures encode ‘epigenetic’ information that governs the expression of the underlying genes. These information-bearing structures must be maintained over extended periods of
time in resting cells and may be suffi ciently stable to resist the turmoil of the cell cycle to be passed on to the next cell generation. Intuitively, nucleosome remodelling should antagonize the maintenance of stable structures. However,
upon closer inspection it becomes evident that nucleosome remodelling is intimately involved in the assembly of stable chromatin structures that correspond to functional states. Remodellers may even contribute structural information themselves. Their involvement can be seen at several structural
levels: at the levels of positioning individual nucleosomes, homoeostasis of linker histones, histone variants and non-histone proteins, as well as the differential folding of the nucleosome fibre. All of them may contribute to the
assembly of heritable epigenetic structures.
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Developmental role for ACF1-containing nucleosome remodellers in chromatin organisation
15.09.2010
The nucleosome remodelling complexes CHRAC and ACF of Drosophila are thought to play global roles in chromatin assembly and nucleosome dynamics. Disruption of the gene encoding the common ACF1 subunit compromises fly viability. Survivors show defects in chromatin assembly and chromatin-mediated gene repression at all developmental stages. We now show that ACF1 expression is under strict developmental control. The expression is strongly diminished during embryonic development and persists at high levels only in undifferentiated cells, including the germ cell precursors and larval neuroblasts. Constitutive expression of ACF1 is lethal. Cell-specific ectopic expression perturbs chromatin organisation and nuclear programmes. By monitoring heterochromatin formation during development, we have found that ACF1-containing factors are involved in the initial establishment of diversified chromatin structures, such as heterochromatin. Altering the levels of ACF1 leads to global and variegated deviations from normal chromatin organisation with pleiotropic defects.
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Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y
06.09.2010
JCB,
2010,
190 no. 5,
777-91
published on 06.09.2010
Nucleosomal incorporation of specialized histone variants is an important mechanism to generate different functional chromatin states. Here, we describe the identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y. Their messenger RNAs are found in certain human cell lines, in addition to several normal and malignant human tissues. In keeping with their primate specificity, H3.X and H3.Y are detected in different brain regions. Transgenic H3.X and H3.Y proteins are stably incorporated into chromatin in a similar fashion to the known H3 variants. Importantly, we demonstrate biochemically and by mass spectrometry that endogenous H3.Y protein exists in vivo, and that stress stimuli, such as starvation and cellular density, increase the abundance of H3.Y-expressing cells. Global transcriptome analysis revealed that knockdown of H3.Y affects cell growth and leads to changes in the expression of many genes involved in cell cycle control. Thus, H3.Y is a novel histone variant involved in the regulation of cellular responses to outside stimuli.
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Chemical-genomic dissection of the CTD code
29.08.2010
Nature Structural & Molecular Biology,
2010,
doi:10.1038/nsmb.1900
published on 29.08.2010
Nature Structural & Molecular Biology, online article
Nature Structural & Molecular Biology, online article
Sequential modifications of the RNA polymerase II (Pol II) C-terminal domain (CTD) coordinate the stage-specific association and release of cellular machines during transcription. Here we examine the genome-wide distributions of the 'early' (phospho-Ser5 (Ser5-P)), 'mid' (Ser7-P) and 'late' (Ser2-P) CTD marks. We identify gene class–specific patterns and find widespread co-occurrence of the CTD marks. Contrary to its role in 3′-processing of noncoding RNA, the Ser7-P marks are placed early and retained until transcription termination at all Pol II–dependent genes. Chemical-genomic analysis reveals that the promoter-distal Ser7-P marks are not remnants of early phosphorylation but are placed anew by the CTD kinase Bur1. Consistent with the ability of Bur1 to facilitate transcription elongation and suppress cryptic transcription, high levels of Ser7-P are observed at highly transcribed genes. We propose that Ser7-P could facilitate elongation and suppress cryptic transcription.
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Dosage compensation and the global re-balancing of aneuploid genomes
26.08.2010
Genome Biology,
2010,
8,
doi:10.1186/gb-2010-11-8-216
published on 26.08.2010
Genome Biology, online article
Genome Biology, online article
Diploid genomes are exquisitely balanced systems of gene expression. The dosage-compensation systems that evolved along with monosomic sex chromosomes exemplify the intricacies of compensating for differences in gene copy number by transcriptional regulation.
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Efficient Eradication of Subcutaneous but Not of Autochthonous Gastric Tumors by Adoptive T Cell Transfer in an SV40 T Antigen Mouse Model
15.08.2010
The Journal of Immunology,
2010,
doi: 10.4049/jimmunol.0903231,
vol. 185 no. 4 2580-2588
published on 15.08.2010
The Journal of Immunology, online article
The Journal of Immunology, online article
In stomach cancer, there is a need for new therapeutic strategies, in particular for the treatment of unresectable tumors and micrometastases. We investigated the efficacy of immunotherapy in an autochthonous model of gastric cancer, the CEA424-SV40 T Ag (TAg) transgenic mice. Treatment efficacy against both the autochthonous tumors and s.c. tumors induced by the derived cell line mGC3 were assessed. In wild-type mice, a dendritic cell vaccine loaded with irradiated tumor cells combined with CpG oligonucleotides induced efficient cytotoxic T cell and memory responses against mGC3 s.c. tumors. In contrast, neither s.c. nor autochthonous tumors responded to vaccination in CEA424-SV40 TAg mice, indicating tolerance to the SV40 TAg. To examine whether tumors in these mice were principally accessible to immunotherapy, splenocytes from immune wild-type mice were adoptively transferred into CEA424-SV40 TAg transgenic mice. Treated mice showed complete regression of the s.c. tumors associated with intratumoral infiltrates of CD8 and CD4 T cells. In contrast, the autochthonous gastric tumors in the same mice were poorly infiltrated and did not regress. Thus, even in the presence of an active anti-tumoral T cell response, autochthonous gastric tumors do not respond to immunotherapy. This is the first comparison of the efficacy of adoptive T cell transfer between transplanted s.c. tumors and autochthonous tumors in the same animals. Our results suggest that in gastric cancer patients, even a strong anti-tumor T cell response will not efficiently penetrate the tumor in the absence of additional therapeutic strategies targeting the tumor microenvironment.
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Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA
21.07.2010
Nucleic Acids Resaerch,
2010,
DOI: 10.1093/nar/gkq684,
published on 21.07.2010
Nucleic Acids Research, online article
Nucleic Acids Research, online article
The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.
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Form and function of dosage-compensated chromosomes – a chicken-and-egg relationship
20.07.2010
BioEssays,
2010,
Volume 32, Issue 8, pages 709–717, August 2010,
DOI: 10.1002/bies.201000029
published on 20.07.2010
BioEssays, online article
BioEssays, online article
Does the three-dimensional (3D) conformation of interphase
chromosomes merely reflect their function or does it actively contribute to gene regulation? The analysis of sex chromosomes that are subject to chromosome-wide dosage compensation processes promises new insight into this question. Chromosome conformations ar dynamic and largely determined by association of distant chromosomal loci in the nuclear space or by their anchoring to the nuclear envelope, effectively generating chromatin loops. The type and extent of such interactions
depend on chromatin-bound transcription regulators and therefore reflects function. Dosage compensation adjusts the overall transcription activity of X chromosomes to assure balanced expression in the two sexes. Initial analyses of mammalian and Drosophila X chromosomes have led to the hypothesis that their conformations may not only reflect their functional state but may in turn contribute to the coordination of chromosome-wide tuning of transcription.
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A guide to super-resolution fluorescence microscopy
19.07.2010
For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.
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Soft skills turned into hard facts: nucleosome remodelling at developmental switches
01.07.2010
Nucleosome remodelling factors are regulators of DNA accessibility in chromatin and lubricators of all major functions of eukaryotic genomes. Their action is transient and reversible, yet can be decisive for irreversible cell-fate decisions during development. In addition to the well-known local actions of nucleosome remodelling factors during transcription initiation, more global and fundamental roles for remodelling complexes in shaping the epigenome during development are emerging.
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The Activation Potential of MOF Is Constrained for Dosage Compensation
25.06.2010
The H4K16 acetyltransferase MOF plays a crucial
role in dosage compensation in Drosophila but has
additional, global functions. We compared the
molecular context and effect of MOF in male and
female flies, combining chromosome-wide mapping
and transcriptome studies with analyses of defined
reporter loci in transgenic flies. MOF distributes
dynamically between two complexes, the dosage
compensation complex and a complex containing
MBD-R2, a global facilitator of transcription. These
different targeting principles define the distribution
of MOF between the X chromosome and autosomes
and at transcription units with 50 or 30 enrichment. The
male X chromosome differs from all other chromosomes
in that H4K16 acetylation levels do not correlate
with transcription output. The reconstitution of
this phenomenon at a model locus revealed that
the activation potential of MOF is constrained in
male cells in the context of the DCC to arrive at the
2-fold activation of transcription characteristic of
dosage compensation.
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Remodeling of nuclear architecture by the thiodioxoxpiperazine metabolite chaetocin
10.06.2010
Experimental Cell Research,
2010,
doi:10.1016/j.yexcr.2010.03.008,
published on 10.06.2010
Experimental Cell Research, online article
Experimental Cell Research, online article
Extensive changes of higher order chromatin arrangements can be observed during prometaphase, terminal cell differentiation and cellular senescence. Experimental systems where major reorganization of nuclear architecture can be induced under defined conditions, may help to better understand the functional implications of such changes. Here, we report on profound chromatin reorganization in fibroblast nuclei by chaetocin, a thiodioxopiperazine metabolite. Chaetocin induces strong condensation of chromosome territories separated by a wide interchromatin space largely void of DNA. Cell viability is maintained irrespective of this peculiar chromatin phenotype. Cell cycle markers, histone signatures, and tests for cellular senescence and for oxidative stress indicate that chaetocin induced chromatin condensation/clustering (CICC) represents a distinct entity among nuclear phenotypes associated with condensed chromatin. The territorial organization of entire chromosomes is maintained in CICC nuclei; however, the conventional nuclear architecture harboring gene-dense chromatin in the nuclear interior and gene-poor chromatin at the nuclear periphery is lost. Instead gene-dense and transcriptionally active chromatin is shifted to the periphery of individual condensed chromosome territories where nascent RNA becomes highly enriched around their outer surface. This chromatin reorganization makes CICC nuclei an attractive model system to study this border zone as a distinct compartment for transcription. Induction of CICC is fully inhibited by thiol-dependent antioxidants, but is not related to the production of reactive oxygen species. Our results suggest that chaetocin functionally impairs the thioredoxin (Trx) system, which is essential for deoxynucleotide synthesis, but in addition involved in a wide range of cellular functions. The mechanisms involved in CICC formation remain to be fully explored.
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Epigenetic regulation of development by histone lysine methylation
05.05.2010
Hereditary,
2010,
doi: 10.1038/hdy.2010.49,
published on 05.05.2010
Epigenetic mechanisms contribute to the establishment and
maintenance of cell-type-specific gene expression patterns.
In this review, we focus on the functions of histone lysine
methylation in the context of epigenetic gene regulation during
developmental transitions. Over the past few years, analysis of
histone lysine methylation in active and repressive nuclear
compartments and, more recently, genome-wide profiling of
histone lysine methylation in different cell types have revealed
correlations between particular modifications and the transcriptional
status of genes. Identification of histone methyltransferases
(HMTases) and specific binding factors for most methylated
lysine positions has provided a novel insight into the mechanisms
of epigenetic gene regulation. In addition, analyses of HMTase
knockout mice show that histone lysine methylation has
important functions for normal development. In this study, we
review mechanisms of gene activation and repression by histone
lysine methylation and discuss them in the context of the
developmental roles of HMTases.
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CIPSM Excursion to Bayer Schering Pharma with Kirsten Jung
03.05.2010
2010,
published on 03.05.2010
Bayer Schering Pharma
Bayer Schering Pharma
We thank Kirsten Jung for heading the CIPSM Excursion to Bayer Schering Pharma as part of our graduate education series! We heard it was a great time and will enlightingly continue with a follow up excursion.
The tumor suppressor p53 connects ribosome biogenesis to cell cycle control: a double-edged sword
01.05.2010
Since its first description more than 30 years ago p53 has become a paradigm for a protein with versatile functions. P53 sensitizes a large variety of genetic alterations and has been entitled the guardian of the genome. Stabilization of p53 upon DNA damage is accompanied by a complex pattern of modifications, which ascertain the cellular response either in the direction of a reversible or irreversible cell cycle arrest or programmed cell death. More recently it became evident that p53 also responds to non-genotoxic cell stress, in particular if ribosome biogenesis is affected.
Novel antibody derivatives for proteome and high-content analysis
07.04.2010
Analytical and Bioanalytical Chemistry,
2010,
doi: 10.1007/s00216-010-3657-0,
397(8): 3203–3208.
published on 07.04.2010
Analytical and Bioanalytical Chemistry, online article
Analytical and Bioanalytical Chemistry, online article
The understanding of cellular processes and their pathophysiological alterations requires comprehensive data on the abundance, distribution, modification, and interaction of all cellular components. On the one hand, artificially introduced fluorescent fusion proteins provide information about their distribution and dynamics in living cells but not about endogenous factors. On the other hand, antibodies can detect endogenous proteins, posttranslational modifications, and other cellular components but mostly in fixed and permeabilized cells. Here we highlight a new technology based on the antigen-binding domain of heavy-chain
antibodies (VHH) from Camelidae. These extremely stable VHH domains can be produced in bacteria, coupled to matrices, and used for affinity purification and proteome.
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Phosphorylation of SU(VAR)3–9 by the Chromosomal Kinase JIL-1
06.04.2010
PLOSone,
2010,
doi:10.1371/journal.pone.0010042,
1-9
published on 06.04.2010
PLOSone, online article
PLOSone, online article
The histone methyltransferase SU(VAR)3–9 plays an important role in the formation of heterochromatin within the eukaryotic nucleus. Several studies have shown that the formation of condensed chromatin is highly regulated during development, suggesting that SU(VAR)3–9’s activity is regulated as well. However, no mechanism by which this may be achieved has been reported so far. As we and others had shown previously that the N-terminus of SU(VAR)3–9 plays an important role for its activity, we purified interaction partners from Drosophila embryo nuclear extract using as bait a GST fusion protein containing the SU(VAR)3–9 N-terminus. Among several other proteins known to bind Su(VAR)3–9 we isolated the chromosomal kinase JIL-1 as a strong interactor. We show that SU(VAR)3–9 is a substrate for JIL-1 in vitro as well as in vivo and map the site of phosphorylation. These findings may provide a molecular explanation for the observed genetic interaction between SU(VAR)3–9 and JIL-1.
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Heterochromatin dysregulation in human diseases
01.04.2010
J. Appl. Physiol.,
2010,
doi: 10.1152/japplphysiol.00053.2010
published on 01.04.2010
Journal of Applied Physiology, online article
Journal of Applied Physiology, online article
Heterochromatin is a repressive chromatin state which is characterized by densely packed DNA and low transcriptional activity. Heterochromatin-induced gene silencing is important for mediating developmental transitions, and in addition, it has more global functions in ensuring chromosome segregation and genomic integrity. Here we discuss how altered heterochromatic states can impair normal gene expression patterns leading to the development of different diseases. Over the last years, therapeutic strategies which aim toward resetting the epigenetic state of dysregulated genes have been tested. However, due to the complexity of epigenetic gene regulation, the "first generation drugs" that function globally by inhibiting epigenetic machineries might also introduce severe side effects. Thus, detailed understanding of how repressive chromatin states are established and maintained at specific loci will be fundamental for the development of more selective epigenetic treatment strategies in the future.
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Top-Down de Novo Protein Sequencing of a 13.6 kDa Camelid Single Heavy Chain Antibody by Matrix-Associated Laser Desorption Ionization-Time-of-Flight/Time-of-Flight Mass Spectrometry
23.03.2010
Analytical Chemistry,
2010,
doi:10.1021/ac1000515,
published on 23.03.2010
Analytical Chemistry, online article
Analytical Chemistry, online article
The primary structure of a 13.6 kDa single heavy chain camelid antibody (VHH) was determined by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) top-down sequence analysis. The majority of the sequence was obtained by mass spectrometric de novo sequencing, with the N-terminal 14 amino acid residues being determined using T3-sequencing and database interrogation. The determined sequence was confirmed by liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis of a tryptic digest, which also provided high-energy collisionally induced dissociation (CID) data permitting the clear assignment of 3 of the 14 isobaric Leu/Ile residues. Five of the 11 Leu/Ile ambiguities could be resolved by homology comparisons with known VHH sequences. The monoisotopic molecular weight of the VHH was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13610.6066 Da, in excellent agreement with the established sequence. To our knowledge, this is the first time that the entire primary structure of a protein with a molecular weight >13 kDa has been established by mass spectrometric top-down sequencing.
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Phosphorylation of histone H3T6 by PKCbI controls demethylation at histone H3K4
14.03.2010
Demethylation at distinct lysine residues in histone H3 by lysinespecific demethylase 1 (LSD1) causes either gene repression or activation1,2. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4)1,3. In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9)2. Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbI, also known as PRKCb) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated
at threonine 6 are no longer LSD1 substrates. In vivo,
PKCbI co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression.RNA interference (RNAi)-mediated knockdown of PKCbIabrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of
PKCbI requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1)4. Notably, increased levels of PKCbI and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbI blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent
kinase signalling leads to the writing of the new chromatin
mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.
Replication Stress Interferes with Histone Recycling and Predeposition Marking of New Histones
12.03.2010
To restore chromatin on new DNA during replication, recycling of histones evicted ahead of the fork is combined with new histone deposition. The Asf1 histone chaperone, which buffers excess histones under stress, is a key player in this process. Yet how histones handled by human Asf1 are modified remains unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes induced upon replication stress. In S phase, distinct cytosolic and nuclear Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks, including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest, the predeposition mark H3K9me1 and modifications typical of chromatin accumulate in Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent with evicted histones being trapped with Asf1. During recovery, histones stored with Asf1 are rapidly used as replication resumes. This shows that replication stress interferes with predeposition marking and histone recycling with potential impact on epigenetic stability.
Chemotherapeutic drugs inhibit ribosome biogenesis at various levels
16.02.2010
The Journal of Biological Chemistry,
2010,
doi: 10.1074/jbc.M109.074211
published on 16.02.2010
The Journal of Biological Chemistry, online article
The Journal of Biological Chemistry, online article
Drugs for cancer therapy belong to different categories of chemical substances. The cellular targets for the therapeutic efficacy are often not unambiguously identified. Here, we describe the process of ribosome biogenesis as a target of a large variety of chemotherapeutic drugs. We determined the inhibitory concentration of 36 chemotherapeutic drugs for transcription and processing of ribosomal RNA by in vivo labeling experiments. Inhibitory drug concentrations were correlated to the loss of nucleolar integrity. The synergism of drugs inhibiting ribosomal RNA synthesis at different levels was studied. Drugs inhibited ribosomal RNA synthesis either at the level of (i) rRNA transcription (e.g. Oxaliplatin, Doxorubicin, Mitoxantrone, Methotrexate), (ii) early rRNA processing (e.g. Camptothecin, Flavopiridol, Roscovitine), or (iii) late rRNA processing (e.g. 5-Fluorouracil, MG-132, Homoharringtonine). Blockage of rRNA transcription or early rRNA processing steps caused nucleolar disintegration, while blockage of late rRNA processing steps left the nucleolus intact. Flavopiridol and 5-Fluorouracil showed a strong synergism for inhibition of rRNA processing. We conclude that inhibition of ribosome biogenesis by chemotherapeutic drugs potentially may contribute to the efficacy of therapeutic regimens.
The complexityofthe‘simple’two-component systemKdpD/KdpE in Escherichia coli
20.01.2010
FEMS Microbiology Letters,
2010,
304, 2,
97 - 106
published on 20.01.2010
FEMS Microbiology Letters, online article
FEMS Microbiology Letters, online article
The KdpD/KdpE two-component system of Escherichia coli activates the expression of the kdpFABC operon encoding the high-affinity K+ uptake system KdpFABC in response to K+ limitation or salt stress. Earlier, it was proposed that the histidine kinase KdpD is a turgor sensor; recent studies suggest that KdpD integrates three chemical stimuli from the cytoplasm. The histidine kinase KdpD contains several structural features and subdomains that are important for stimulus perception, modulation of the kinase to phosphatase ratio, and signaling. The response regulator KdpE receives the phosphoryl group from KdpD and induces kdpFABC transcription. The three-dimensional structure of the receiver domain was resolved, providing insights into the activation mechanism of this transcriptional regulator. Two accessory components, the universal stress protein UspC and the phosphotransferase system component IIANtr, are known to interact with KdpD, allowing the modulation of kdpFABC expression under certain physiological conditions. Here, we will discuss the complexity of a 'simple' two-component system and its interconnectivity with metabolism and the general stress response.
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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Immunostimulatory RNA Blocks Suppression by Regulatory T Cells
15.01.2010
The Journal of Immunology,
2010,
doi: 10.4049/jimmunol.0901245,
vol. 184 no. 2 939-946
published on 15.01.2010
The Journal of Immunology, online article
The Journal of Immunology, online article
The role of immune suppression by regulatory T (Treg) cells in the maintenance of immune homeostasis is well established. However, little is known about how Treg cell function is inhibited on viral infection to allow the development of a protective immune response. As viral RNA is a crucial mediator for activation of antiviral immunity, we examined the effects of immunostimulatory RNA and infection with RNA viruses on Treg cell function. We show that synthetic RNA oligonucleotides potently inhibit Treg cell-induced suppression in a sequence-dependent manner. This effect is entirely dependent on TLR7 activation of APCs and subsequent IL-6 production. In addition, stimulation with the RNA viruses encephalomyocarditis virus and Sendai virus that specifically activate the RNA-sensing helicases melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene I (RIG-I) also blocks Treg cell function. Interestingly, this effect is seen even in the absence of APCs. Consistent with this, both Treg and T effector cells express RIG-I and MDA-5. Using MDA-5–deficient mice, we demonstrate that the loss of Treg cell function on infection with encephalomyocarditis virus is strictly dependent on MDA-5 expression by Treg cells. Thus, we show in this study for the first time that activation of a RIG-I–like helicase on Treg cells blocks their suppressive function.
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The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome
12.01.2010
Nucleic Acids Resarch,
2010,
doi: 10.1093/nar/gkq026,
13
published on 12.01.2010
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Dosage compensation in Drosophila melanogaster
involves the selective targeting of the male X chromosome
by the dosage compensation complex (DCC) and the coordinate, 2-fold activation of most genes. The principles that allow the DCC to distinguish the X chromosome from the autosomes are not understood. Targeting presumably involves
DNA sequence elements whose combination or enrichment mark the X chromosome. DNA sequences that characterize ‘chromosomal entry sites’ or ‘high-affinity sites’ may serve such a function. However, to date no DNA binding domain
that could interpret sequence information has been identified within the subunits of the DCC. Early genetic studies suggested that MSL1 and MSL2 serve to recognize high-affinity sites (HAS) in vivo, but a direct interaction of these DCC subunits with DNA has not been studied. We now show that recombinant MSL2, through its CXC domain, directly binds DNA with low nanomolar affinity. The DNA binding of MSL2 or of an MSL2–MSL1 complex does not discriminate between different sequences in vitro, but in a reporter gene assay in vivo, suggesting the existence of an unknown selectivity cofactor. Reporter gene assays and localization of
GFP-fusion proteins confirm the important contribution of the CXC domain for DCC targeting in vivo.
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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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RNA Polymerase II C-terminal Heptarepeat Domain Ser-7 Phosphorylation Is Established in a Mediator-dependent Fashion
01.01.2010
The Jourmal of Biological Chemistry,
2010,
285,
188 - 96
published on 01.01.2010
The Journal of Biological Chemistry, online article
The Journal of Biological Chemistry, online article
The largest subunit of RNA polymerase II (RNAPII) C-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 Ser-7 in the human enzyme. Ser-7 becomes phosphorylated before initiation of transcription at promoter regions. We identify cyclin-dependent kinase 7 (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.
