Content
2011
Histidine kinases and response regulators in networks
13.12.2011
Current Opinion in Microbiology,
2011,
DOI 10.1016/j.mib.2011.11.009,
15, 1–7
published on 13.12.2011
Current Opinion in Microbiology, online article
Current Opinion in Microbiology, online article
Two-component systems, composed of a histidine kinase (HK) and a response regulator (RR), are the major signal transduction devices in bacteria. Originally it was thought that these two components function as linear, phosphorylationdriven stimulus–response system. Here, we will review how accessory proteins are employed by HKs and RRs to mediate signal integration, scaffolding, interconnection and allosteric regulation, and how these two components are embedded in regulatory networks.
Full View
Full View
Probing the conformation of the ISWI ATPase domain with genetically encoded photoreactive crosslinkers and mass spectrometry
13.12.2011
Molecular & Cellular Proteomics,
2011,
doi: 10.1074/mcp.M111.012088,
published on 13.12.2011
Molecular & Cellular Proteomics, online article
Molecular & Cellular Proteomics, online article
We present a strategy for rapidly gaining structural information about a protein from crosslinks formed by genetically encoded unnatural amino acids. We applied it to ISWI, a chromatin remodeling enzyme involved in chromatin assembly, DNA replication and transcription. ISWI is part of the vast Snf2 family of helicase-related proteins, many of which constitute the catalytic cores of chromatin remodeling complexes. Structural information about this family is scarce, hampering our mechanistic understanding of chromatin remodeling. Making use of cells that harbour a special tRNA/aminoacyl-tRNA synthetase pair, several residues within the ATPase domain of ISWI were individually substituted with the UV-reactive unnatural amino acid p-benzoyl-p-phenylalanine. Intramolecular crosslinks could be mapped with amino acid precision by high resolution tandem mass spectrometry and the novel bioinformatic tool "Crossfinder". Most crosslinks were fully consistent with published crystal structures of ISWI-related ATPases. A subset of crosslinks, however, disagreed with the conformations previously captured in crystal structures. We built a structural model using the distance information obtained from the crosslinks and the structure of the closest crystallized relative, Chd1. The model shows the ATPase lobes strongly rotated against each other, a movement postulated earlier to be necessary to achieve a catalytically competent state. The minimal requirements for solubility and protein amounts make our approach ideal for studying structures and conformations of proteins that are not amenable to conventional structural techniques.
Full View
Full View
Engineering antibodies and proteins for molecular in vivo imaging
01.12.2011
Science Direct,
2011,
doi:10.1016/j.copbio.2011.06.007,
Volume 22, Issue 6, Pages 882-887
published on 01.12.2011
Science Direct, online article
Science Direct, online article
The rapid and ongoing discovery of new disease related biomarkers leads to a dramatic paradigm change in human healthcare and constitutes the basis for a truly personalized medicine. Molecular imaging enables early detection and classification of human diseases and provides valuable data for optimized, target-oriented therapies. By now, the biochemical and physiological properties of antibody derivatives or alternative protein scaffolds can be engineered for the detection of a wide range of target structures. The
successful application of these reagents in animals, xenograft models and cells in preclinical research clearly demonstrate their utility for molecular imaging. Despite these promising perspectives, only a few antibodies and recombinant proteins are used yet for molecular imaging in human medicine. Especially the high safety demands and the need to eliminate off target effects in humans require extensive research and development efforts.
Full View
Full View
In breast cancer, a high ratio of tumour-infiltrating intraepithelial CD8+ to FoxP3+ cells is characteristic for the medullary subtype
17.11.2011
Histopathology,
2011,
DOI: 10.1111/j.1365-2559.2011.04040.x,
Volume 59, Issue 5, pages 965–974,
published on 17.11.2011
Histopathology, online article
Histopathology, online article
In breast cancer, a high ratio of tumour-infiltrating intraepithelial CD8+ to FoxP3+ cells is characteristic for the medullary subtype.
Full View
Full View
CD103 is a hallmark of tumor-infiltrating regulatory T cells
15.11.2011
International Journal of Cancer,
2011,
DOI: 10.1002/ijc.25902,
Volume 129, Issue 10, pages 2417–2426
published on 15.11.2011
International Journal of Cancer, online article
International Journal of Cancer, online article
Regulatory T cells (Treg) mediate tolerance towards self-antigens by suppression of innate and adaptive immunity. In cancer patients, tumor-infiltrating FoxP3+ Treg suppress local anti-tumor immune responses and are often associated with poor prognosis. Markers that are selectively expressed on tumor-infiltrating Treg may serve as targets for immunotherapy of cancer. Here we show that CD103, an integrin mediating lymphocyte retention in epithelial tissues, is expressed at high levels on tumor-infiltrating FoxP3+ Treg in several types of murine cancer. In the CT26 model of colon cancer up to 90% of the intratumoral FoxP3+ cells expressed CD103 compared to less than 20% in lymphoid organs. CD103+ Treg suppressed T effector cell activation more strongly than CD103neg Treg. Expression of CD103 on Treg closely correlated with intratumoral levels of transforming growth factor β (TGF-β) and could be induced in a TGF-β-dependent manner by tumor cell lines. In vivo, gene silencing of TGF-β reduced the frequency of CD103+ Treg, demonstrating that CD103 expression on tumor-infiltrating Treg is driven by intratumoral TGF-β. Functional blockade of CD103 using a monoclonal antibody did however not reduce the number of intratumoral Treg, indicating that CD103 is not involved in homing or retention of FoxP3+ cells in the tumor tissue. In conclusion, expression of CD103 is a hallmark of Treg that infiltrate TGF-β-secreting tumors. CD103 thus represents an interesting target for selective depletion of tumor-infiltrating Treg, a strategy that may help to improve anti-cancer therapy.
Full View
Full View
Developmental regulation of N-terminal H2B methylation in Drosophila melanogaster
03.11.2011
Nucleic Acids Research,
2011,
doi: 10.1093/nar/gkr935,
1-14,
published on 03.11.2011
Nucl. Acids Res., online article
Nucl. Acids Res., online article
Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo. Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. Here, we show that a major modification of H2B in Drosophila melanogaster is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless
of their cell cycle distribution. We identified dNTMT (CG1675) as the enzyme responsible for H2B methylation. We also found that the level of N-terminal methylation is regulated by dART8, an arginine
methyltransferase that physically interacts with dNTMT and asymmetrically methylates H3R2. Our results demonstrate the existence of a complex containing two methyltransferases enzymes, which negatively influence each other’s activity.
Full View
Full View
De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation
01.11.2011
Journal of Cell Science,
2011,
doi: 10.1242/jcs.084681,
124, 3603–3618,
published on 01.11.2011
Journal of Cell Science, online article
Journal of Cell Science, online article
Telomerase-negative tumor cells use an alternative lengthening of telomeres (ALT) pathway that involves DNA recombination and repair to maintain their proliferative potential. The cytological hallmark of this process is the accumulation of promyelocytic leukemia (PML) nuclear1 protein at telomeric DNA to form ALT-associated PML bodies (APBs). Here, the de novo formation of a telomeric PML nuclear subcompartment was investigated by recruiting APB protein components.We show that functionally distinct proteins were able to initiate the formation of bona fide APBs with high efficiency in a self-organizing and self-propagating manner. These included: PML and Sp100 as the constituting components of PML nuclear bodies, telomere repeat binding factors 1 and 2 (TRF1 and TRF2, respectively), the DNA repair protein NBS1 and the SUMO E3 ligase MMS21, as well as the isolated SUMO1 domain, through an interacting domain of another protein factor. By contrast, the repair factors Rad9, Rad17 and Rad51 were less efficient in APB nucleation but were recruited to preassembled APBs. The artificially created APBs induced telomeric extension through a DNA repair mechanism, as inferred from their colocalization with sites of
non-replicative DNA synthesis and histone H2A.X phosphorylation, and an increase of the telomere repeat length. These activities were absent after recruitment of the APB factors to a pericentric locus and establish APBs as functional intermediates of the ALT pathway.
Full View
Full View
The MOF-containing NSL complex associates globally with housekeeping genes, but activates only a defined subset
27.10.2011
Nucl. Acids Res.,
2011,
doi: 10.1093/nar/gkr869,
published on 27.10.2011
Nucleic Acids Research, online article
Nucleic Acids Research, online article
The MOF (males absent on the first)-containing NSL (non-specific lethal) complex binds to a subset of active promoters in Drosophila melanogaster and is thought to contribute to proper gene expression. The determinants that target NSL to specific pro- moters and the circumstances in which the complex engages in regulating transcription are currently unknown. Here, we show that the NSL complex pri- marily targets active promoters and in particular housekeeping genes, at which it colocalizes with the chromatin remodeler NURF (nucleosome remodeling factor) and the histone methyltransferase Trithorax. However, only a subset of housekeeping genes associated with NSL are actually activated by it. Our analyses reveal that these NSL-activated pro- moters are depleted of certain insulator binding pro- teins and are enriched for the core promoter motif ‘Ohler 5’. Based on these results, it is possible to predict whether the NSL complex is likely to regulate a particular promoter. We conclude that the regula- tory capacity of the NSL complex is highly context- dependent. Activation by the NSL complex requires a particular promoter architecture defined by com- binations of chromatin regulators and core promoter motifs.
Full View
Full View
Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription
21.10.2011
Nature Cell Biology,
2011,
doi:10.1038/ncb2365,
published on 21.10.2011
Nature Cell Biology, online article
Nature Cell Biology, online article
Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit2,3 and for segregation of repetitive regions4. Cdc14 is also a subunit of the silencing complex RENT (refs 5,6), but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y′ repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants4 correlate with the presence of subtelomeric Y′ elements and can be rescued by transcriptional inhibition of RNA polymerase II.
Full View
Full View
Suv4-20h Abrogation Enhances Telomere Elongation during Reprogramming and Confers a Higher Tumorigenic Potential to iPS Cells
12.10.2011
Plos,
2011,
doi:10.1371/journal.pone.0025680,
6(10): e25680
published on 12.10.2011
Plos, online article
Plos, online article
Reprogramming of adult differentiated cells to induced pluripotent stem cells (iPS) cells has been achieved by over-expression of specific transcription factors. Nuclear reprogramming induces a series of profound changes at the telomeres of the parental differentiated cells, including a telomerase-dependent telomere elongation and the remodeling of telomeric chromatin. In particular, iPS cells show a decreased density of H4K20me3 heterochromatic mark at telomeres compared to the parental cells. Suv4-20h1 and Suv4-20h2 histone methytransferases (HMTases) are responsible for the trimethylation of H4K20 at telomeres, as cells deficient for both HMTases show decreased levels of H4K20me3 at telomeric chromatin. Here, we set to address the role of the Suv4-20h enzymes in telomere reprogramming by generating bona-fide iPS cells from mouse embryonic fibroblasts (MEFs) double null for both HMTases (Suv4-20dn MEFs). We found that Suv4-20h deficiency enhances telomere elongation during reprogramming without altering their ability to protect the chromosome ends or the efficiency of reprogramming. Moreover, teratomas generated from Suv4-20dn iPS cells also have elongated telomeres and an increased growth rate when compared to wild-type controls. These results indicate that abrogation of Suv4-20h enzymes and loss of heterochromatic mark H4K20me3 at telomeric heterochromatin facilitates telomere reprogramming and provides an increased tumorigenic potential to the resulting iPS cells.
Full View
Full View
Regulation of DNA methyltransferase 1 by interactions and modifcations
30.09.2011
Nucleus,
2011,
http://dx.doi.org/10.4161/nucl.2.5.17928,
Volume 2, Issue 5, Pages 392 - 402
published on 30.09.2011
Nucleus, online article
Nucleus, online article
DNA methylation plays a central role in the epigenetic regulation of gene expression during development and disease. Remarkably, the complex and changing patterns of genomic DNA methylation are established and maintained by only three DNA methyltransferases. Here we focus on DNMT1, the major and ubiquitously expressed DNA methyltransferase in vertebrates, to outline possible regulatory mechanisms. A list of all protein interactions and post-translational
modi!cations reported for DNMT1 clearly shows that DNMT1, and by extension also DNA methylation in general, are functionally linked with several other epigenetic pathways and cellular processes. General themes of these interactions and modi!cations include the activation, stabilization and recruitment of DNMT1 at speci!c sites and heterochromatin regions. For a comprehensive understanding of the regulation of DNA methylation it is now necessary to systematically quantify the interactions and modi!cations of DNMT1, to elucidate their function at the molecular level and to integrate
these data at the cellular level.
Full View
Full View
Proteomic Interrogation of Human Chromatin
14.09.2011
PLoS ONE,
2011,
doi:10.1371/ journal.pone.0024747,
6(9): e24747.
published on 14.09.2011
PLoS ONE, online article
PLoS ONE, online article
Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the ‘‘Chromatome’’) is still a continuing
process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by
identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone
proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance
Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a
glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel
post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed
methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a
compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt
to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational
modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that
may be involved in directing gene expression and other chromatin-related processes.
Full View
Full View
Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin
08.09.2011
Nucleic Acids Research,
2011,
doi: 10.1093/nar/gkr723,
published on 08.09.2011
Nucleic Acids Research, online article
Nucleic Acids Research, online article
The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1−/− cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.
Full View
Full View
Estrogen-Dependent Gene Transcription in Human Breast Cancer Cells Relies upon Proteasome-Dependent Monoubiquitination of Histone H2B
23.08.2011
Cancer Research,
2011,
doi: 10.1158/0008-5472.CAN-11-1896,
published on 23.08.2011
Cancer Research, online article
Cancer Research, online article
The estrogen receptor-alpha (ERa) determines the phenotype of breast cancers where it serves as a positive
prognostic indicator. ERa is a well-established target for breast cancer therapy, but strategies to target its
function remain of interest to address therapeutic resistance and further improve treatment. Recent findings
indicate that proteasome inhibition can regulate estrogen-induced transcription, but how ERa function might
be regulated was uncertain. In this study, we investigated the transcriptome-wide effects of the proteasome
inhibitor bortezomib on estrogen-regulated transcription in MCF7 human breast cancer cells and showed that
bortezomib caused a specific global decrease in estrogen-induced gene expression. This effect was specific
because gene expression induced by the glucocorticoid receptor was unaffected by bortezomib. Surprisingly, we
observed no changes in ERa recruitment or assembly of its transcriptional activation complex on ERa target
genes. Instead, we found that proteasome inhibition caused a global decrease in histone H2B monoubiquitination
(H2Bub1), leading to transcriptional elongation defects on estrogen target genes and to decreased
chromatin dynamics overall. In confirming the functional significance of this link, we showed that RNA
interference–mediated knockdown of the H2B ubiquitin ligase RNF40 decreased ERa-induced gene transcription.
Surprisingly, RNF40 knockdown also supported estrogen-independent cell proliferation and activation of
cell survival signaling pathways. Most importantly, we found that H2Bub1 levels decrease during tumor
progression. H2Bub1 was abundant in normal mammary epithelium and benign breast tumors but absent
in most malignant and metastatic breast cancers. Taken together, our findings show how ERa activity is blunted
by bortezomib treatment as a result of reducing the downstream ubiquitin-dependent function of H2Bub1. In
supporting a tumor suppressor role for H2Bub1 in breast cancer, our findings offer a rational basis to pursue
H2Bub1-based therapies for future management of breast cancer.
Full View
Full View
Cooperative DNA and Histone Binding by Uhrf2 Links the Two Major Repressive Epigenetic Pathways
18.08.2011
Journal of Cellular Biochemistry,
2011,
DOI: 10.1002/jcb.23185,
Volume 112, Issue 9, pages 2585–2593
published on 18.08.2011
Journal of Cellular Biochemistry, online article
Journal of Cellular Biochemistry, online article
Gene expression is regulated by DNA as well as histone modifications but the crosstalk and mechanistic link between these epigenetic signals are still poorly understood. Here we investigate the multi-domain protein Uhrf2 that is similar to Uhrf1, an essential cofactor of maintenance DNA methylation. Binding assays demonstrate a cooperative interplay of Uhrf2 domains that induces preference for hemimethylated DNA, the substrate of maintenance methylation, and enhances binding to H3K9me3 heterochromatin marks. FRAP analyses revealed that localization and binding dynamics of Uhrf2 in vivo require an intact tandem Tudor domain and depend on H3K9 trimethylation but not on DNA methylation. Besides the cooperative DNA and histone binding that is characteristic for Uhrf2, we also found an opposite expression pattern of uhrf1 and uhrf2 during differentiation. While uhrf1 is mainly expressed in pluripotent stem cells, uhrf2 is upregulated during differentiation and highly expressed in differentiated mouse tissues. Ectopic expression of Uhrf2 in uhrf1 -/- embryonic stem cells did not restore DNA methylation at major satellites indicating functional differences. We propose that the cooperative interplay of Uhrf2 domains may contribute to a tighter epigenetic control of gene expression in differentiated cells.
Full View
Full View
Splicing enhances recruitment of methyltransferase HYPB/Setd2 and methylation of histone H3 Lys36
24.07.2011
Nature Structural & Molecular Biology,
2011,
18,
977 - 83
published on 24.07.2011
Nature Structual & Molecular Biology, online article
Nature Structual & Molecular Biology, online article
Several lines of recent evidence support a role for chromatin in splicing regulation. Here, we show that splicing can also contribute to histone modification, which implies bidirectional communication between epigenetic mechanisms and RNA processing. Genome-wide analysis of histone methylation in human cell lines and mouse primary T cells reveals that intron-containing genes are preferentially marked with histone H3 Lys36 trimethylation (H3K36me3) relative to intronless genes. In intron-containing genes, H3K36me3 marking is proportional to transcriptional activity, whereas in intronless genes, H3K36me3 is always detected at much lower levels. Furthermore, splicing inhibition impairs recruitment of H3K36 methyltransferase HYPB (also known as Setd2) and reduces H3K36me3, whereas splicing activation has the opposite effect. Moreover, the increase of H3K36me3 correlates with the length of the first intron, consistent with the view that splicing enhances H3 methylation. We propose that splicing is mechanistically coupled to recruitment of HYPB/Setd2 to elongating RNA polymerase II.
Full View
Full View
Stage-Specific Histone Modification Profiles Reveal Global Transitions in the Xenopus Embryonic Epigenome
22.07.2011
PLoS ONE,
2011,
doi:10.1371/journal.pone.0022548,
Volime 6,
published on 22.07.2011
PLoS ONE, online article
PLoS ONE, online article
Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these
precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs) for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development..
Full View
Full View
Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1
21.07.2011
Cell Death and Disease,
2011,
e182,
doi:10.1038/cddis.2011.66
published on 21.07.2011
Cell Death and Disease, online article
Cell Death and Disease, online article
The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser2. This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.
Full View
Full View
Tuning communication fidelity
18.07.2011
Nature Chemical Biology,
2011,
doi:10.1038/nchembio.618,
502–503
published on 18.07.2011
Nature Chemical Biology, online article
Nature Chemical Biology, online article
Bacteria communicate by sending and receiving chemical cues in a process termed ‘quorum sensing’. New research shows how five feedback loops of the Vibrio harveyi quorum sensing cascade ensure signal integration and transmission fidelity, with one loop controlling signal sensitivity by regulating receptor ratios.
Full View
Full View
Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters
17.07.2011
Nature Structural & Molecular Biology,
2011,
18,
956 - 63
published on 17.07.2011
Nature Structual & Molecular Biology, online article
Nature Structual & Molecular Biology, online article
Recent work has shown that RNA polymerase (Pol) II can be recruited to and transcribe distal regulatory regions. Here we analyzed transcription initiation and elongation through genome-wide localization of Pol II, general transcription factors (GTFs) and active chromatin in developing T cells. We show that Pol II and GTFs are recruited to known T cell–specific enhancers. We extend this observation to many new putative enhancers, a majority of which can be transcribed with or without polyadenylation. Importantly, we also identify genomic features called transcriptional initiation platforms (TIPs) that are characterized by large areas of Pol II and GTF recruitment at promoters, intergenic and intragenic regions. TIPs show variable widths (0.4–10 kb) and correlate with high CpG content and increased tissue specificity at promoters. Finally, we also report differential recruitment of TFIID and other GTFs at promoters and enhancers. Overall, we propose that TIPs represent important new regulatory hallmarks of the genome
Full View
Full View
Role for hACF1 in the G2/M damage checkpoint
11.07.2011
Nucleic Acids Research,
2011,
doi: 10.1093/nar/gkr435,
pp. 1-12
published on 11.07.2011
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Active chromatin remodelling is integral to the DNA damage response in eukaryotes, as damage sensors, signalling molecules and repair enzymes gain access to lesions. A variety of nucleosome remodel- ling complexes is known to promote different stages of DNA repair. The nucleosome sliding factors CHRAC/ACF of Drosophila are involved in chromatin organization during development. Involvement of corresponding hACF1-containing mammalian nucleosome sliding factors in replication, transcription and very recently also non-homologous end-joining of DNA breaks have been suggested. We now found that hACF1-containing factors are more generally involved in the DNA damage response. hACF1 depletion increases apoptosis, sensitivity to radiation and compromises the G2/M arrest that is activated in response to UV- and X-rays. In the absence of hACF1, cH2AX and CHK2ph signals are diminished. hACF1 and its ATPase partner SNF2H rapidly accu- mulate at sites of laser-induced DNA damage. hACF1 is also required for a tight checkpoint that is induced upon replication fork collapse. ACF1- depleted cells that are challenged with aphidicolin enter mitosis despite persistence of lesions and accumulate breaks in metaphase chromosomes. hACF1-containing remodellers emerge as global facilitators of the cellular response to a variety of different types of DNA damage.
Full View
Full View
Recognition of 5-Hydroxymethylcytosine by the Uhrf1 SRA Domain
22.06.2011
PLoS ONE,
2011,
doi:10.1371/journal.pone.0021306,
6(6): e21306.
published on 22.06.2011
PLoS ONE, online article
PLoS ONE, online article
Recent discovery of 5-hydroxymethylcytosine (5hmC) in genomic DNA raises the question how this sixth base is recognized by cellular proteins. In contrast to the methyl-CpG binding domain (MBD) of MeCP2, we found that the SRA domain of Uhrf1, an essential factor in DNA maintenance methylation, binds 5hmC and 5-methylcytosine containing substrates with similar affinity. Based on the co-crystal structure, we performed molecular dynamics simulations of the SRA:DNA complex with the flipped cytosine base carrying either of these epigenetic modifications. Our data indicate that the SRA binding pocket can accommodate 5hmC and stabilizes the flipped base by hydrogen bond formation with the hydroxyl group.
Full View
Full View
Systemic Cancer Therapy with a Small Molecule Agonist of Toll-like Receptor 7 Can Be Improved by Circumventing TLR Tolerance
22.06.2011
Cancer Research,
2011,
doi: 10.1158/0008-5472.CAN-10-3903,
published on 22.06.2011
Cancer Research, online article
Cancer Research, online article
Topical application of small molecule Toll-like receptor 7 (TLR7) agonists is highly effective for the treatment of skin tumors, whereas their systemic application has been largely unsuccessful for cancer therapy. One reason may be that repeated systemic application of TLR ligands can induce a state of immune unresponsiveness, termed TLR tolerance. We show here that a single injection of the TLR7 agonist R848 in mice induces a short period of increased response to TLR stimulation followed by a state of hyporesponsiveness lasting several days. This state is characterized by inhibited secretion of the key cytokines IL-12p70 and IL-6 as well as by a block in IFN-α production. We demonstrate for the first time that at the cellular level, TLR7 tolerance occurs in both plasmacytoid and myeloid dendritic cells, two cell populations that play a critical role in the initiation and amplification of antitumor immune responses. We further show that TLR7 tolerance in plasmacytoid dendritic cells is accompanied by downregulation of the adaptor protein interleukin-1 receptor-associated kinase (IRAK-1). Based on these findings, we have designed a novel strategy for the treatment of tumors using cycles of repeated R848 injections separated by treatment-free intervals. We show in CT26 tumor-bearing mice that this protocol circumvents TLR7 tolerance and improves the efficacy of cancer immunotherapy.
Full View
Full View
The RNA Helicase Rm62 Cooperates with SU(VAR)3-9 to Re-Silence Active Transcription in Drosophila melanogaster
02.06.2011
PLoS ONE,
2011,
doi:10.1371/journal.pone.0020761,
6(6)
published on 02.06.2011
PLoS ONE, online article
PLoS ONE, online article
Gene expression is highly dynamic and many genes show a wide range in expression over several orders of magnitude. This
regulation is often mediated by sequence specific transcription factors. In addition, the tight packaging of DNA into chromatin can provide an additional layer of control resulting in a dynamic range of gene expression covering several orders of magnitude. During transcriptional activation, chromatin barriers have to be eliminated to allow an efficient progression of the RNA polymerase. This repressive chromatin structure has to be re-established quickly after it has been activated in order to tightly regulate gene activity. We show that the DExD/H box containing RNA helicase Rm62 is targeted to a site of rapid induction of transcription where it is responsible for an increased degree of methylation at H3K9 at the heat shock locus after removal of the heat shock stimulus. The RNA helicase interacts with the well-characterized histone methyltransferase SU(VAR)3-9 via its N-terminus, which provides a potential mechanism for the targeting of H3K9 methylation to highly regulated genes. The recruitment of SU(VAR)3-9 through interaction with a RNA helicase to a site of active transcription might be a general mechanism that allows an efficient silencing of highly regulated genes thereby enabling a cell to fine tune its gene activity over a wide range.
Full View
Full View
Twists and turns of DNA methylation
31.05.2011
DNA methylation, the postreplicative transfer of a methyl group to the C5 position of cytosine bases, was the first epigenetic modification identified and has been intensively studied for more than half a century. By now it is clear that Dnmt1, the major eukaryotic DNA methyltransferase, faithfully maintains genome-wide methylation patterns and plays an essential role in the epigenetic network controlling gene expression and genome stability during development. However, the molecular mechanisms that ultimately control DNA methylation still remain elusive. This is, in part, attributable to the remarkable complexity of the DNA methylation reaction, the apparent involvement of several inter- and intramolecular protein interactions, and the limited structural information. The crystal structure of Dnmt1 presented in PNAS (1) now provides detailed insights into the inner workings and possible regulation of one of the most intriguing enzymes.
Full View
Full View
Kirsten Jung's program of emphasis on individuality of bacteria gets DFG funding
20.04.2011
2011,
published on 20.04.2011
Click here to read the german press article
Click here to read the german press article
The DFG will fund the program of emphasis on inidividuality of bacteria which is coordinated by CIPSM's Kirsten Jung.
In this program scientists from a range of disciplines are trying to show whether or not bacteria of a population which are genetically identical and have the same phenotype show the same properties and behave in the same manner or not.
The DFG funds this program with 5,8 Mio. Euro over a period of three years.
Full View
Full View
Detection and function of an intramolecular disulfide bond in the pH-responsive CadC of Escherichia coli
12.04.2011
BMC Microbiology,
2011,
doi:10.1186/1471-2180-11-74,
11:74
published on 12.04.2011
BMC Microbiology, online article
BMC Microbiology, online article
Background:
In an acidic and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, the lysine decarboxylase, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family. Activation of CadC requires two stimuli, lysine and low pH. Whereas lysine is detected by an interplay between CadC and the lysine-specific transporter LysP, pH alterations are sensed by CadC directly. Crystal structural analyses revealed a close proximity between two periplasmic cysteines, Cys208 and Cys272.
Results:
Substitution of Cys208 and/or Cys272 by alanine resulted in CadC derivatives that were active in response to only one stimulus, either lysine or pH 5.8. Differential in vivo thiol trapping revealed a disulfide bond between these two residues at pH 7.6, but not at pH 5.8. When Cys208 and Cys272 were replaced by aspartate and lysine, respectively, virtually wild-type behavior was restored indicating that the disulfide bond could be mimicked by a salt bridge.
Conclusion:
A disulfide bond was found in the periplasmic domain of CadC that supports an inactive state of CadC at pH 7.6. At pH 5.8 disulfide bond formation is prevented which transforms CadC into a semi-active state. These results provide new insights into the function of a pH sensor.
Full View
Full View
Functional Nuclear Organization of Transcription and DNA Replication
05.04.2011
Cold Spring Harbor Symposia on Quantitative Biology,
2011,
doi: 10.1101/sqb.2010.75.042,
published on 05.04.2011
Cold Spring Harbor Symposia on Quantitative Biology, online article
Cold Spring Harbor Symposia on Quantitative Biology, online article
We studied the nuclear topography of RNA transcription and DNA replication in mammalian cell types with super-resolution fluorescence microscopy, which offers a resolution beyond the classical Abbe/Raleigh limit. Three-dimensional structured illumination microscopy (3D-SIM) demonstrated a network of channels and wider lacunas, called the interchromatin compartment (IC). The IC starts at nuclear pores and expands throughout the nuclear space. It is demarcated from the compact interior of higher-order chromatin domains (CDs) by a 100–200-nm thick layer of decondensed chromatin, termed the perichromatin region (PR). Nascent DNA, nascent RNA, RNA polymerase II (RNA Pol II), as well as histone modifications for transcriptionally competent/active chromatin, are highly enriched in the PR, whereas splicing speckles are observed in the interior of the IC. In line with previous electron microscopic evidence, spectral precision distance/position determination microscopy (SPDM) confirmed the presence of RNA Pol II clusters indicative of transcription factories. Still, a substantial part of transcription apparently takes place outside of such factories. Previous electron microscopic evidence has suggested that the functional nuclear organization of DNA replication depends on Brownian movements of chromatin between the CD interior and the PR. As an incentive for future studies, we hypothesize that such movements also take place during transcription, i.e., only the actually transcribed part of a gene may be located within the PR, whereas its major part, including previously or later transcribed sequences, is embedded in a higher-order chromatin configuration in the interior of the CD.
Full View
Full View
The C-Terminal Domain of RNA Polymerase II Is Modified by Site-Specific Methylation
01.04.2011
Science,
2011,
DOI: 10.1126/science.1202663,
Vol. 332 no. 6025 pp. 99-103
published on 01.04.2011
Science, online article
Science, online article
The carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) in mammals undergoes extensive posttranslational modification, which is essential for transcriptional initiation and elongation. Here, we show that the CTD of RNAPII is methylated at a single arginine (R1810) by the coactivator-associated arginine methyltransferase 1 (CARM1). Although methylation at R1810 is present on the hyperphosphorylated form of RNAPII in vivo, Ser2 or Ser5 phosphorylation inhibits CARM1 activity toward this site in vitro, suggesting that methylation occurs before transcription initiation. Mutation of R1810 results in the misexpression of a variety of small nuclear RNAs and small nucleolar RNAs, an effect that is also observed in Carm1−/− mouse embryo fibroblasts. These results demonstrate that CTD methylation facilitates the expression of select RNAs, perhaps serving to discriminate the RNAPII-associated machinery recruited to distinct gene types.
Full View
Full View
H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation
31.03.2011
PLoS Genetics,
2011,
7,3,
doi:10.1371/journal.pgen.1001354
published on 31.03.2011
PLOS Genetics, online article
PLOS Genetics, online article
Methylation of histone H3 lysine 4 (H3K4me) is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac) has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs) Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC) Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP), we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3), a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS), which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1Δ mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me).
Full View
Full View
Sequential Establishment of Marks on Soluble Histones H3 and H4
29.03.2011
Much progress has been made concerning histone function in
the nucleus; however, following their synthesis, how their marking andsubcellular trafficking are regulated remains tobeexplored.To gainaninsight into these issues, wefocusedonsoluble histonesand analyzed endogenous and tagged H3 histones in parallel. We distinguished six complexes that we could place to account for maturation events occurringonhistonesH3andH4from their synthesis
onward. In each complex, a different set of chaperones is involved, and we found specific post-translational modifications. Interestingly, we revealed that histones H3 and H4 are transiently poly- (ADP-ribosylated). The impact of these marks in histone metabolism proved to be important as we found that acetylation of lysines 5 and 12 on histone H4 stimulated its nuclear translocation. Furthermore, we showed that, depending on particular histone H3 modifications, the balance in the presence of the different translocation complexes changes. Therefore, our results enabled us to propose a regulatory means of these marks for controlling cytoplasmic/ nuclear shuttling and the establishment of early modification patterns.
Full View
Full View
Identification of ArgP and Lrp as Transcriptional Regulators of lysP, the Gene Encoding the Specific Lysine Permease of Escherichia coli
14.03.2011
The Journal of Bacteriology,
2011,
doi:10.1128/JB.00815-10,
2536-2548, Vol. 193,
published on 14.03.2011
The Journal of Bacteriology, online article
The Journal of Bacteriology, online article
Expression of lysP, which encodes the lysine-specific transporter LysP in Escherichia coli, is regulated by the concentration of exogenous available lysine. In this study, the LysR-type transcriptional regulator ArgP was identified as the activator of lysP expression. At lysine concentrations higher than 25 µM, lysP expression was shut off and phenocopied an argP deletion mutant. Purified ArgP-His6 bound to the lysP promoter/control region at a sequence containing a conserved T-N11-A motif. Its affinity increased in the presence of lysine but not in the presence of the other known coeffector, arginine. In vivo data suggest that lysine-loaded ArgP and arginine-loaded ArgP compete at the lysP promoter. We propose that lysine-loaded ArgP prevents lysP transcription at the promoter clearance step, as described for the lysine-dependent regulation of argO (R. S. Laishram and J. Gowrishankar, Genes Dev. 21:1258-1272, 2007). The global regulator Lrp also bound to the lysP promoter/control region. An lrp mutant exhibited reduced lysP expression in the absence of external lysine. These results indicate that ArgP is a major regulator of lysP expression but that Lrp modulates lysP transcription under lysine-limiting conditions.
Full View
Full View
PARG is recruited to DNA damage sites through poly(ADP-ribose)- and PCNA-dependent mechanisms
11.03.2011
Nucl. Acids Res.,
2011,
39 no. 12,
5045-56
published on 11.03.2011
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Post-translational poly(ADP-ribosyl)ation has diverse essential functions in the cellular response to DNA damage as it contributes to avid DNA damage detection and assembly of the cellular repair machinery but extensive modification eventually also induces cell death. While there are 17 human poly(ADP-ribose) polymerase (PARP) genes, there is only one poly(ADP-ribose) glycohydrolase (PARG) gene encoding several PARG isoforms located in different subcellular compartments. To investigate the recruitment of PARG isoforms to DNA repair sites we locally introduced DNA damage by laser microirradiation. All PARG isoforms were recruited to DNA damage sites except for a mitochondrial localized PARG fragment. Using PARP knock out cells and PARP inhibitors, we showed that PARG recruitment was only partially dependent on PARP-1 and PAR synthesis, indicating a second, PAR-independent recruitment mechanism. We found that PARG interacts with PCNA, mapped a PCNA binding site and showed that binding to PCNA contributes to PARG recruitment to DNA damage sites. This dual recruitment mode of the only nuclear PARG via the versatile loading platform PCNA and by a PAR dependent mechanism likely contributes to the dynamic regulation of this posttranslational modification and ensures the tight control of the switch between efficient DNA repair and cell death.
Full View
Full View
Global Analysis of the Relationship between JIL-1 Kinase and Transcription
10.03.2011
PLOSGenetics,
2011,
doi:10.1371/journal.pgen.1001327,
published on 10.03.2011
PLOSGenetics, online article
PLOSGenetics, online article
The ubiquitous tandem kinase JIL-1 is essential for Drosophila development. Its role in defining decondensed domains of larval
polytene chromosomes is well established, but its involvement in transcription regulation has remained controversial. For a first comprehensive molecular characterisation of JIL-1, we generated a high-resolution, chromosome-wide interaction profile of the kinase in Drosophila cells and determined its role in transcription. JIL-1 binds active genes along their entire length. The presence of the kinase is not proportional to average transcription levels or polymerase density. Comparison of JIL-1 association with elongating RNA polymerase and a variety of histone modifications suggests two distinct targeting principles. A basal level of JIL-1 binding can be defined that correlates best with the methylation of histone H3 at lysine 36, a mark that is placed co-transcriptionally. The additional acetylation of H4K16 defines a second state characterised by approximately twofold
elevated JIL-1 levels, which is particularly prominent on the dosage-compensated male X chromosome. Phosphorylation of the histone H3 N-terminus by JIL-1 in vitro is compatible with other tail modifications. In vivo, phosphorylation of H3 at serine 10, together with acetylation at lysine 14, creates a composite histone mark that is enriched at JIL-1 binding regions. Its depletion by RNA interference leads to a modest, but significant, decrease of transcription from the male X chromosome. Collectively, the results suggest that JIL-1 participates in a complex histone modification network that characterises active, decondensed chromatin. We hypothesise that one specific role of JIL-1 may be to reinforce, rather than to establish, the status of active chromatin through the phosphorylation of histone H3 at serine 10.
Full View
Full View
Kirsten Jung Interview about CIPSMs Gender Equality Program
08.03.2011
2011,
published on 08.03.2011
Click here to read the interview on Laborjournal.de
Click here to read the interview on Laborjournal.de
Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5–hydroxymethylcytosine
04.03.2011
Nucl. Acids Res.,
2011,
doi: 10.1093/nar/gkr118,
published on 04.03.2011
Nucl. Acids Res., online article
Nucl. Acids Res., online article
In mammalian genomes a sixth base, 5-hydroxymethylcytosine (hmC), is generated by enzymatic oxidation of 5-methylcytosine (mC). This discovery has raised fundamental questions about the functional relevance of hmC in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare hmC against the far more abundant mC is technically challenging and to date no methods for direct sequencing of hmC have been reported. Here, we report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves hmC-containing sequences. We determined the consensus cleavage site of PvuRts1I as hmCN11–12/N9–10G and show first data on its potential to interrogate hmC patterns in mammalian genomes.
Full View
Full View
Histone acetylation controls the inactive X chromosome replication dynamics
01.03.2011
Nature Communications,
2011,
DOI: 10.1038/ncomms1218,
published on 01.03.2011
Nature Communications, online article
Nature Communications, online article
In mammals, dosage compensation between male and female cells is achieved by inactivating one female X chromosome (Xi). Late replication of Xi was proposed to be involved in the maintenance of its silenced state. Here, we show a highly synchronous replication of the Xi within 1 to 2 h during early-mid S-phase by following DNA replication in living mammalian cells with green fluorescent protein-tagged replication proteins. The Xi was replicated before or concomitant with perinuclear or perinucleolar facultative heterochromatin and before constitutive heterochromatin. Ectopic expression of the X-inactive-specific transcript (Xist) gene from an autosome imposed the same synchronous replication pattern. We used mutations and chemical inhibition affecting different epigenetic marks as well as inducible Xist expression and we demonstrate that histone hypoacetylation has a key role in controlling Xi replication. The epigenetically controlled, highly coordinated replication of the Xi is reminiscent of embryonic genome replication in flies and frogs before genome activation and might be a common feature of transcriptionally silent chromatin.
Full View
Full View
Transcription modulation chromosome-wide: universal features and principles of dosage compensation in worms and flies
11.02.2011
Science Direct,,
2011,
doi:10.1016/j.gde.2011.01.012,
published on 11.02.2011
Science Direct, online article
Science Direct, online article
Dosage compensation processes in flies and worms provide a
unique opportunity to study common regulatory principles of
thousands of genes.Technological advancement in the recent
years has allowed for the comprehensive description of key
aspects such as the targeting of the regulatory factors,the
emerging chromatin structure changes and the ensuing subtle
transcriptional alterations.With plenty of data at hand the
challenge remains to integrate the findings into coherent models that appreciatetheglobalnatureoftheunderlying principles leaving the experimental anecdotes behind while avoiding the numerical burlesque.
Full View
Full View
H4K20 monomethylation faces the WNT
11.02.2011
Growth factor signaling pathways regulate a broad spectrum of
cellular processes ranging from proliferation to differentiation
and tissue homeostasis. Activation of a signaling pathway ultimately leads to transcriptional changes in specific target genes. Although the molecular identities of many signaling pathway components have been revealed over the last years,
there is still very little knowledge of how these components induce changes in the chromatin structure of target genes, a requirement
for activation or repression of these genes. A new link is provided by an interesting paper in PNAS that demonstrates that in the context of Wnt signaling the histone methyltransferase SETD8
(PR-SET7, KMT5a, SET8) is recruited to enhancer regions of Wnt-regulated genes. SETD8 establishes H4K20 monomethylation (H4K20me1) at these regulatory regions, which is crucial for full activation of these target genes.
Full View
Full View
Cortical Constriction During Abscission Involves Helices of ESCRT-III–Dependent Filaments
10.02.2011
Science Express,
2011,
DOI: 10.1126/science.1201847,
published on 10.02.2011
Science, online article
Science, online article
Following partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells, using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nm-diameter filaments, which narrowed the cortex of the intercellular bridge to a single stalk. The Endosomal Sorting Complex Required for Transport (ESCRT)-III colocalized with constriction zones and was required for assembly of 17-nm-diameter filaments. Simultaneous Spastin-mediated removal of underlying microtubules enabled full constriction at the abscission site. The identification of contractile filament helices at the intercellular bridge has broad implications for the understanding of cell division and of ESCRT-III–mediated fission of large membrane structures.
Full View
Full View
Interferon Antagonist NSs of La Crosse Virus Triggers a DNA Damage Response-like Degradation of Transcribing RNA Polymerase II
04.02.2011
La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.
Full View
Full View
Different Binding Properties and Function of CXXC Zinc Finger Domains in Dnmt1 and Tet1
02.02.2011
Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1−/− embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1−/− ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.
Full View
Full View
Usp7 and Uhrf1 control ubiquitination and stability of the maintenance DNA methyltransferase Dnmt1
25.01.2011
Journal of Cellular Biochemistry,
2011,
DOI: 10.1002/jcb.22998,
published on 25.01.2011
Journal of Cellular Biochemistry, online article
Journal of Cellular Biochemistry, online article
In mammals Dnmt1 is the DNA methyltransferase chiefly responsible for maintaining genomic methylation patterns through DNA replication cycles, but how its maintenance activity is controlled is still not well understood. Interestingly, Uhrf1, a crucial cofactor for maintenance of DNA methylation by Dnmt1, is endowed with E3 ubiquitin ligase activity. Here, we show that both Dnmt1 and Uhrf1 coprecipitate with ubiquitin specific peptidase 7 (Usp7), a de-ubiquitinating enzyme. Overexpression of Uhrf1 and Usp7 resulted in opposite changes in the ubiquitination status and stability of Dnmt1. Our findings suggest that, by balancing Dnmt1 ubiquitination, Usp7 and Uhrf1 fine tune Dnmt1 stability.
Full View
Full View
Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR-like protein family
24.01.2011
Protein Science,
2011,
DOI: 10.1002/pro.594,
656-669
published on 24.01.2011
Protein Science, online article
Protein Science, online article
The membrane-integral transcriptional activator CadC comprises sensory and transcriptional regulatory functions within one polypeptide chain. Its C-terminal periplasmic
domain, CadCpd, is responsible for sensing of environmental pH as well as for binding of the feedback inhibitor cadaverine. Here we describe the crystal structure of CadCpd (residues 188–512) solved at a resolution of 1.8 A ° via multiple wavelength anomalous dispersion (MAD) using a ReCl6 22 derivative. CadCpd reveals a novel fold comprising two subdomains: an N-terminal subdomain dominated by a b-sheet in contact with three a-helices and a C-terminal subdomain formed by an eleven-membered a-helical bundle, which is oriented almost perpendicular to the helices in the first subdomain. Further to the native protein, crystal structures were also solved for its variants D471N and D471E, which show functionally different behavior in pH sensing. Interestingly, in
the heavy metal derivative of CadCpd used for MAD phasing a ReCl6 22 ion was found in a cavity located between the two subdomains. Amino acid side chains that coordinate this complex ion are conserved in CadC homologues from various bacterial species, suggesting a function of the cavity in the binding of cadaverine, which was supported by docking studies. Notably, CadCpd forms a homodimer in solution, which can be explained by an extended, albeit rather polar interface between two symmetry-related monomers in the crystal structure. The occurrence of several acidic residues in this region suggests protonation-dependent changes in the mode of dimerization, which could eventually trigger transcriptional activation by CadC in the bacterial cytoplasm.
Full View
Full View
Nuclei of chicken neurons in tissues and three-dimensional cell cultures are organized into distinct radial zones
20.01.2011
Chromosome Research,
2011,
DOI: 10.1007/s10577-010-9182-3,
published on 20.01.2011
Chromosome Research, online article
Chromosome Research, online article
We used chicken retinospheroids (RS) to study the nuclear architecture of vertebrate cells in a three-dimensional (3D) cell culture system. The results showed that the different neuronal cell types of RS displayed an extreme form of radial nuclear organization. Chromatin was arranged into distinct radial zones which became already visible after DAPI staining. The distinct zones were enriched in different chromatin modifications and in different types of chromosomes. Active isoforms of RNA polymerase II were depleted in the outermost zone. Also chromocenters and nucleoli were radially aligned in the nuclear interior. The splicing factor SC35 was enriched at the central zone and did not show the typical speckled pattern of distribution. Evaluation of neuronal and non-neuronal chicken tissues showed that the highly ordered form of radial nuclear organization was also present in neuronal chicken tissues. Furthermore, the data revealed that the neuron-specific nuclear organization was remodeled when cells spread on a flat substrate. Monolayer cultures of a chicken cell line did not show this extreme form of radial organization. Rather, such monolayer cultures displayed features of nuclear organization which have been described before for many different types of monolayer cells. The finding that an extreme form radial nuclear organization, which has not been described before, is present in RS and tissues, but not in cells spread on a flat substrate, suggests that it would be important to complement studies on nuclear architecture performed with monolayer cells by studies on 3D cell culture systems and tissues.
Full View
Full View
CpG Blocks Immunosuppression by Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice
13.01.2011
Clinical Cancer Research,
2011,
doi: 10.1158/1078-0432.CCR-10-2672,
published on 13.01.2011
Clinical Cancer Research, online article
Clinical Cancer Research, online article
Purpose: The Toll-like receptor (TLR) 9 ligand CpG has been used successfully for the immunotherapy of cancer. Chronic CpG application in tumor-free hosts leads, however, to the expansion of myeloid-derived suppressor cells (MDSC), which can cause T-cell suppression and may thus hamper the development of an effective immune response. Here, we investigated the effect of TLR9 activation on the function of MDSC in tumor-bearing mice.
Full View
Full View
New Insights into the Signaling Mechanism of the pH-responsive, Membrane-integrated Transcriptional Activator CadC of Escherichia coli
07.01.2011
The membrane-integrated transcriptional regulator CadC of Escherichia coli activates expression of the cadBA operon at low external pH with concomitantly available lysine, providing adaptation to mild acidic stress. CadC is a representative of the ToxR-like proteins that combine sensory, signal transduction, and DNA-binding activities within a single polypeptide. Although several ToxR-like regulators such as CadC, as well as the main regulator of Vibrio cholerae virulence, ToxR itself, which activate gene expression at acidic pH, have been intensively investigated, their molecular activation mechanism is still unclear. In this study, a structure-guided mutational analysis was performed to elucidate the mechanism by which CadC detects acidification of the external milieu. Thus, a cluster of negatively charged amino acids (Asp-198, Asp-200, Glu-461, Glu-468, and Asp-471) was found to be crucial for pH detection. These amino acids form a negatively charged patch on the surface of the periplasmic domain of CadC that stretches across its two subdomains. The results of different combinations of amino acid replacements within this patch indicated that the N-terminal subdomain integrates and transduces the signals coming from both subdomains to the transmembrane domain. Alterations in the phospholipid composition did not influence pH-dependent cadBA expression, and therefore, interplay of the acidic surface patch with the negatively charged headgroups is unlikely. Models are discussed according to which protonation of these acidic amino acid side chains reduces repulsive forces between the two subdomains and/or between two monomers within a CadC dimer and thereby enables receptor activation upon lowering of the environmental pH.
Full View
Full View
