2012

CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo

28.12.2012

Bo Gu, Dirk Eick, Olivier Bensaude

Nucleic Acids Research, 2012, doi:10.1093/nar/gks1327, 1–13 published on 28.12.2012
Nucleic Acids Research, online article
Co-transcriptional pre-mRNA processing relies on reversible phosphorylation of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAP II). In this study, we replaced in live cells the endogenous Rpb1 by S2A Rpb1, where the second serines (Ser2) in the CTD heptapeptide repeats were switched to alanines, to prevent phosphorylation. Although slower, S2A RNAP II was able to transcribe. However, it failed to recruit splicing components such as U2AF65 and U2 snRNA to transcription sites, although the recruitment of U1 snRNA was not affected. As a consequence, co-transcriptional splicing was impaired. Interestingly, the magnitude of the S2A RNAP II splicing defect was promoter dependent. In addition, S2A RNAP II showed an impaired recruitment of the cleavage factor PCF11 to pre-mRNA and a defect in 3′-end RNA cleavage. These results suggest that CTD Ser2 plays critical roles in co-transcriptional pre-mRNA maturation in vivo: It likely recruits U2AF65 to ensure an efficient co-transcriptional splicing and facilitates the recruitment of pre-mRNA 3′-end processing factors to enhance 3′-end cleavage.

Eick_Gu2012_NAR_500



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Global DNA Hypomethylation Prevents Consolidation of Differentiation Programs and Allows Reversion to the Embryonic Stem Cell State

27.12.2012

Christine S. Schmidt, Sebastian Bultmann, Daniela Meilinger, Benedikt Zacher, Achim Tresch, Kerstin C. Maier, Christian Peter, Dietmar E. Martin, Heinrich Leonhardt, Fabio Spada

PLOS ONE, 2012, doi:10.1371/journal.pone.0052629, Volume 7, Issue 12, e52629 published on 27.12.2012
PLOS ONE, online article
DNA methylation patterns change dynamically during mammalian development and lineage specification, yet scarce information is available about how DNA methylation affects gene expression profiles upon differentiation. Here we determine genome-wide transcription profiles during undirected differentiation of severely hypomethylated (Dnmt1−/−) embryonic stem cells (ESCs) as well as ESCs completely devoid of DNA methylation (Dnmt1−/−;Dnmt3a−/−;Dnmt3b−/− or TKO) and assay their potential to transit in and out of the ESC state. We find that the expression of only few genes mainly associated with germ line function and the X chromosome is affected in undifferentiated TKO ESCs. Upon initial differentiation as embryoid bodies (EBs) wild type, Dnmt1−/− and TKO cells downregulate pluripotency associated genes and upregulate lineage specific genes, but their transcription profiles progressively diverge upon prolonged EB culture. While Oct4 protein levels are completely and homogeneously suppressed, transcription of Oct4 and Nanog is not completely silenced even at late stages in both Dnmt1−/− and TKO EBs. Despite late wild type and Dnmt1−/− EBs showing a much higher degree of concordant expression, after EB dissociation and replating under pluripotency promoting conditions both Dnmt1−/− and TKO cells, but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation seems not to be critical for initial activation of differentiation programs, it is crucial for permanent restriction of developmental fate during differentiation.

 

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Controlling The Mobility Of Oligonucleotides In The Nanochannels Of Mesoporous Silica

27.12.2012

Timo Lebold, Axel Schlossbauer, Katrin Schneider, Lothar Schermelleh, Heinrich Leonhardt, Thomas Bein, Christoph Bräuchle

Advanced Functional Materials, 2012, DOI: 10.1002/adfm.201101365, 22, 106–112 published on 27.12.2012
Advanced Functional Materials, online article
Oligonucleotides used in gene therapy and silencing are fragile compounds that degrade easily in biological environments. Porous biocompatible carrier particles may provide a useful strategy to deliver these therapeutics to their target sites. Development of appropriate delivery vehicles, however, requires a better understanding of the oligonucleotide-host interactions and the oligonucleotide dynamics inside carrier particles. We investigated template-free SBA-15 type mesoporous silica particles and report their loading characteristics with siRNA depending on the surface functionalization of their porous network. We show that the siRNA uptake capability of the particles can be controlled by the composition of the functional groups. Fluorescence recovery after photobleaching measurements revealed size-dependent mobility of siRNA and double-stranded DNA oligonucleotides within the functionalized silica particles and provided evidence for the stability of the oligonucleotides inside the pores. Hence, our study demonstrates the potential of mesoporous silica particles as a means for alternative gene delivery in nanomedicine.

Leonhardt_Brauchle_2012_Lebold_AdvFunctMater_500



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Towards a structural understanding of PARP1 activation and related signalling ADP-ribosyl-transferases

15.12.2012

Markus Hassler, Andreas G Ladurner

Structural Biology, 2012, http://dx.doi.org/10.1016/j.sbi.2012.08.005, 22:721–729 published on 15.12.2012
Structural Biology, online article
ADP-ribosylation is a post-translational modification of proteins that occurs mostly in response to cellular stress and is catalysed by members of the diverse poly-ADP-ribose (PAR) polymerase (PARP/ARTD) family. The founding member of the family, PARP1, is best recognized for its function as a sensor of DNA strand lesions, but ADP-ribosylation has been implicated in transcriptional regulation, chromatin dynamics, telomere maintenance, apoptosis and neuronal signalling. Here we summarize a number of exciting recent breakthroughs in our understanding of the structural and mechanistic aspects of how PARP1 recognizes DNA, how PARPs are regulated, how ADP-ribose modifications are set onto specific targets and how the cellular machinery recognizes this elusive post-translational modification.

Hassler und Ladurner 2012_500



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Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches

13.12.2012

Susanne Ude, Jürgen Lassak, Agata L. Starosta, Tobias Kraxenberger, Daniel N. Wilson, Kirsten Jung

Science, 2012, DOI: 10.1126/science.1228985, Vol. 339 no. 6115 pp. 82-85 published on 13.12.2012
Science, online article
Translation elongation factor P (EF-P) is critical for virulence in bacteria. EF-P is present in all bacteria and orthologous to archaeal and eukaryotic initiation factor 5A, yet the biological function has so far remained enigmatic. Here, we demonstrate that EF-P is an elongation factor that enhances translation of polyproline-containing proteins: In the absence of EF-P, ribosomes stall at polyproline stretches, whereas the presence of EF-P alleviates the translational stalling. Moreover, we demonstrate the physiological relevance of EF-P to fine-tune the expression of the polyproline-containing pH receptor CadC to levels necessary for an appropriate stress response. Bacterial, archaeal, and eukaryotic cells have hundreds to thousands of polyproline-containing proteins of diverse function, suggesting that EF-P and a/eIF-5A are critical for copy-number adjustment of multiple pathways across all kingdoms of life.

Jung_Wilson_Sience_ 2013_500



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Identification of a Target Gene and Activating Stimulus for the YpdA/YpdB Histidine Kinase/Response Regulator System in Escherichia coli

07.12.2012

Luitpold Fried, Stefan Behr, Kirsten Jung

Journal of Bacteriology, 2012, doi: 10.1128/​JB.02051-12, vol. 195 no. 4 807-815 published on 07.12.2012
Journal of Bacteriology, online article
Escherichia coli contains 30 two-component systems (TCSs), each consisting of a histidine kinase and a response regulator. Whereas most TCSs are well characterized in this model organism, little is known about the YpdA/YpdB system. To identify YpdB-regulated genes, we compared the transcriptomes of E. coli cells overproducing either YpdB or a control protein. Expression levels of 15 genes differed by more than 1.9-fold between the two strains. A comprehensive evaluation of these genes identified yhjX as the sole target of YpdB. Electrophoretic mobility shift assays with purified YpdB confirmed its interaction with the yhjX promoter. Specifically, YpdB binds to two direct repeats of the motif GGCATTTCAT separated by an 11-bp spacer in the yhjX promoter. yhjX encodes a cytoplasmic membrane protein of unknown function that belongs to the major facilitator superfamily of transporters. Finally, we characterized the pattern of yhjX expression and identified extracellular pyruvate as a stimulus for the YpdA/YpdB system. It is suggested that YpdA/YpdB contributes to nutrient scavenging before entry into stationary phase.

Jung_Fried_etal_2013_500



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The ATPase domain of ISWI is an autonomous nucleosome remodeling machine

02.12.2012

Felix Mueller-Planitz,  Henrike Klinker, Johanna Ludwigsen, Peter B Becker

Nature Structural & Molecular Biology, 2012, doi:10.1038/nsmb.2457, 20, 82–89 (2013), published on 02.12.2012
Nature Structural & Molecular Biology, online article
ISWI slides nucleosomes along DNA, enabling the structural changes of chromatin required for the regulated use of eukaryotic genomes. Prominent mechanistic models imply cooperation of the ISWI ATPase domain with a C-terminal DNA-binding function residing in the HAND-SANT-SLIDE (HSS) domain. Contrary to these models, we show by quantitative biochemical means that all fundamental aspects of nucleosome remodeling are contained within the compact ATPase module of Drosophila ISWI. This domain can independently associate with DNA and nucleosomes, which in turn activate ATP turnover by inducing a conformational change in the enzyme, and it can autonomously reposition nucleosomes. The role of the HSS domain is to increase the affinity and specificity for nucleosomes. Nucleosome-remodeling enzymes may thus have evolved directly from ancestral helicase-type motors, and peripheral domains have furnished regulatory capabilities that bias the remodeling reaction toward different structural outcomes.

 

Becker_2012 Müller_Planitz_NSMB_500



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Direct and Dynamic Detection of HIV-1 in Living Cell

28.11.2012

Jonas Helma, Katrin Schmidthals, Vanda Lux, Stefan Nüske, Armin M. Scholz,  Hans- Georg Kräusslich, Ulrich Rothbauer, Heinrich Leonhardt

PLOS ONE, 2012, doi:10.1371/journal.pone.0050026, Volume 7, Issue 11, e50026 published on 28.11.2012
PLOS ONE, online article
In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

Leonhardt_Nov2012_Helma_PloSOne_500



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Impact of Histone H4 Lysine 20 Methylation on 53BP1 Responses to Chromosomal Double Strand Breaks

28.11.2012

Andrea J. Hartlerode, Yinghua Guan., Anbazhagan Rajendran, Kiyoe Ura, Gunnar Schotta, Anyong Xie, Jagesh V. Shah, Ralph Scully

Plos one, 2012, doi:10.1371/journal.pone.0049211, 7(11): e49211 published on 28.11.2012
Plos one, online article
Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.

 

Schotta_journalpone2012_500



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Autoinducers Act as Biological Timers in Vibrio harveyi

26.10.2012

Claudia Anetzberger, Matthias Reiger, Agnes Fekete, Ursula Schell, Nina Stambrau Laure Plener, Joachim Kopka, Phillippe Schmitt-Kopplin, Hubert Hilbi, Kirsten Jung

Plos, 2012, doi:10.1371/journal.pone.0048310, 7(10): e48310 published on 26.10.2012
Plos, online article
Quorum sensing regulates cell density-dependent phenotypes and involves the synthesis, excretion and detection of so-called autoinducers. Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbellii), one of the best-characterized model organisms for the study of quorum sensing, produces and responds to three autoinducers. HAI-1, AI-2 and CAI-1 are recognized by different receptors, but all information is channeled into the same signaling cascade, which controls a specific set of genes. Here we examine temporal variations of availability and concentration of the three autoinducers in V. harveyi, and monitor the phenotypes they regulate, from the early exponential to the stationary growth phase in liquid culture. Specifically, the exponential growth phase is characterized by an increase in AI-2 and the induction of bioluminescence, while HAI-1 and CAI-1 are undetectable prior to the late exponential growth phase. CAI-1 activity reaches its maximum upon entry into stationary phase, while molar concentrations of AI-2 and HAI-1 become approximately equal. Similarly, autoinducer-dependent exoproteolytic activity increases at the transition into stationary phase. These findings are reflected in temporal alterations in expression of the luxR gene that encodes the master regulator LuxR, and of four autoinducer-regulated genes during growth. Moreover, in vitro phosphorylation assays reveal a tight correlation between the HAI-1/AI-2 ratio as input and levels of receptor-mediated phosphorylation of LuxU as output. Our study supports a model in which the combinations of autoinducers available, rather than cell density per se, determine the timing of various processes in V. harveyi populations.

Jung_Anetzberger_2012_500



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MSL2 Combines Sensor and Effector Functions in Homeostatic Control of the Drosophila Dosage Compensation Machinery

18.10.2012

Raffaella Villa, Ignasi Forné, Marisa Müller, Axel Imhof, Tobias Straub, Peter B. Becker

Molecular Cell,, 2012, 10.1016/j.molcel.2012.09.012, 48, 1–8, published on 18.10.2012
Molecular Cell, online article
The process of dosage compensation (DC) in Drosophila counterbalances the monosomy of the X chromosome in male flies by increasing the transcription from this unique chromosome in the two-fold range. Upon exclusive expression of male-specific lethal 2 (MSL2) in males, the dosage compensation machinery assembles on active X-chromosomal genes. Overexpression of MSL proteins leads to aberrant binding of complex components to autosomes. Accordingly, MSL levels have to be carefully regulated. Here we describe a new mechanism through which MSL2 can fulfill its role as the central regulator of the faithful biogenesis and functionality of the DC machinery. MSL2 is an E3 ligase that ubiquitylates itself and the other associated components when their stoichiometry is unbalanced, uncovering proteasome-dependent degradation as an additional layer of homeostatic control of MSL levels. Furthermore, systematic mapping of modification sites by mass spectrometry and chromatin interaction studies on the target protein MSL1 suggest that the role of MSL2-mediated ubiquitylation goes beyond proteolysis.

Imhof_Becker_12 Villa Mol Cell_500



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Protective and Aggravating Effects of Nlrp3 Inflammasome Activation in IBD Models: Influence of Genetic and Environmental Factors

11.10.2012

Christian Bauer, Peter Duewell, Hans-Anton Lehr, Stefan Endres, Max Schnurr

Digestive Diseases, 2012, DOI: 10.1159/000341681, 30 (Suppl. 1):82-90 published on 11.10.2012
Digestive Diseases, online article
Background: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation due to dysregulation of the mucosal immune system. The cytokines IL-1β and IL-18 appear early in intestinal inflammation and their pro-forms are processed via the caspase-1-activating multiprotein complex, the Nlrp3 inflammasome. Previously, we reported that the uptake of dextran sodium sulfate (DSS) by macrophages activates the Nlrp3 inflammasome and that Nlrp3–/– mice are protected in the acute DSS colitis model. Of note, other groups have reported opposing effects in regards to DSS susceptibility in Nlrp3–/– mice. Recently, mice lacking inflammasomes were found to develop a distinct intestinal microflora. Methods: To reconcile the contradicting observations, we investigated the role of Nlrp3 deficiency in two different IBD models: acute DSS colitis and TNBS (2,4,6-trinitrobenzene sulfonic acid)-induced colitis. In addition, we investigated the impact of the intestinal flora on disease severity by performing cohousing experiments of wild-type and Nlrp3–/– mice, as well as by antibiotic treatment. Results: Nlrp3–/– mice treated with either DSS or TNBS exhibited attenuated colitis and lower mortality. This protective effect correlated with an increased frequency of CD103+ lamina propria dendritic cells expressing a tolerogenic phenotype in Nlrp3–/– mice in steady state conditions. Interestingly, after cohousing, Nlrp3–/– mice were as susceptible as wild-type mice, indicating that transmission of endogenous bacterial flora between the two mouse strains might increase susceptibility of Nlrp3–/– mice towards DSS-induced colitis. Accordingly, treatment with antibiotics almost completely prevented colitis in the DSS model. Conclusions: The composition of the intestinal microflora significantly influences disease severity in IBD models comparing wild-type and Nlrp3–/– mice. This observation may – at least in part – explain contradictory results concerning the role of the inflammasome in different labs. Further studies are required to define the role of the Nlrp3 inflammasome in noninflamed mucosa under steady state conditions and in IBD.

Bauer_etal_Endres2012_500



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Histone H2A variants in nucleosomes and chromatin: more or less stable?

21.09.2012

Clemens Bönisch, Sandra B. Hake,

Nucl. Acids Res., 2012, doi: 10.1093/nar/gks865, published on 21.09.2012
Nucl. Acids Res., online article
In eukaryotes, DNA is organized together with histones and non-histone proteins into a highly complex nucleoprotein structure called chromatin, with the nucleosome as its monomeric subunit. Various interconnected mechanisms regulate DNA accessibility, including replacement of canonical histones with specialized histone variants. Histone variant incorporation can lead to profound chromatin structure alterations thereby influencing a multitude of biological processes ranging from transcriptional regulation to genome stability. Among core histones, the H2A family exhibits highest sequence divergence, resulting in the largest number of variants known. Strikingly, H2A variants differ mostly in their C-terminus, including the docking domain, strategically placed at the DNA entry/exit site and implicated in interactions with the (H3–H4)2-tetramer within the nucleosome and in the L1 loop, the interaction interface of H2A–H2B dimers. Moreover, the acidic patch, important for internucleosomal contacts and higher-order chromatin structure, is altered between different H2A variants. Consequently, H2A variant incorporation has the potential to strongly regulate DNA organization on several levels resulting in meaningful biological output. Here, we review experimental evidence pinpointing towards outstanding roles of these highly variable regions of H2A family members, docking domain, L1 loop and acidic patch, and close by discussing their influence on nucleosome and higher-order chromatin structure and stability.

 

Hake_NAR 2012_500



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Step-Wise Assembly, Maturation and Dynamic Behavior of the Human CENP-P/O/R/Q/U Kinetochore Sub- Complex

18.09.2012

Anja Eskat, Wen Deng, Antje Hofmeister, Sven Rudolphi, Stephan Emmerth, Daniela Hellwig, Tobias Ulbricht,Volker Döring, James M. Bancroft, Andrew D. McAinsh, M. Cristina Cardoso, Patrick Meraldi, Christian Hoischen, Heinrich Leonhardt, Stephan Diekmann

PLOS ONE, 2012, doi:10.1371/journal.pone.0044717.g001, Volume 7, Issue 9, e44717 published on 18.09.2012
PLOS ONE, online article
Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment.

Leonhardt_2012_Eskat_PLoSOne_500



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Deactivation of the E. coli pH Stress Sensor CadC by Cadaverine

17.09.2012

Ina Haneburger, Georg Fritz, Nicole Jurkschat, Larissa Tetsch, Andreas Eichinger, Arne Skerra, Ulrich Gerland, Kirsten Jung

Journal of Molecular Biology, 2012, http://dx.doi.org/10.1016/j.jmb.2012.08.023,, Volume 424, Issues 1–2, Pages 15–27 published on 17.09.2012
Journal of Molecular Biology, online article
At acidic pH and in the presence of lysine, the pH sensor CadC activates transcription of the cadBA operon encoding the lysine/cadaverine antiporter CadB and the lysine decarboxylase CadA. In effect, these proteins contribute to acid stress adaptation in Escherichia coli. cadBA expression is feedback inhibited by cadaverine, and a cadaverine binding site is predicted within the central cavity of the periplasmic domain of CadC on the basis of its crystallographic analysis. Our present study demonstrates that this site only partially accounts for the cadaverine response in vivo. Instead, evidence for a second, pivotal binding site was collected, which overlaps with the pH-responsive patch of amino acids located at the dimer interface of the periplasmic domain. The temporal response of the E. coli Cad module upon acid shock was measured and modeled for two CadC variants with mutated cadaverine binding sites. These studies supported a cascade-like binding and deactivation model for the CadC dimer: binding of cadaverine within the pair of central cavities triggers a conformational transition that exposes two further binding sites at the dimer interface, and the occupation of those stabilizes the inactive conformation. Altogether, these data represent a striking example for the deactivation of a pH sensor.

Jung_Haneburger et al 2012_500



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Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells

11.09.2012

Silvia Stingele, Gabriele Stoehr, Karolina Peplowska, Jürgen Cox, Matthias Mann, Zuzana Storchova

Molecular Systems Biology, 2012, doi:10.1038/msb.2012.40, published on 11.09.2012
Molecular Systems Biology, online article
Extra chromosome copies markedly alter the physiology of eukaryotic cells, but the underlying reasons are not well understood. We created human trisomic and tetrasomic cell lines and determined the quantitative changes in their transcriptome and proteome in comparison with their diploid counterparts. We found that whereas transcription levels reflect the chromosome copy number changes, the abundance of some proteins, such as subunits of protein complexes and protein kinases, is reduced toward diploid levels. Furthermore, using the quantitative data we investigated the changes of cellular pathways in response to aneuploidy. This analysis revealed specific and uniform alterations in pathway regulation in cells with extra chromosomes. For example, the DNA and RNA metabolism pathways were downregulated, whereas several pathways such as energy metabolism, membrane metabolism and lysosomal pathways were upregulated. In particular, we found that the p62-dependent selective autophagy is activated in the human trisomic and tetrasomic cells. Our data present the first broad proteomic analysis of human cells with abnormal karyotypes and suggest a uniform cellular response to the presence of an extra chromosome.

Mann_Storchova_Stingele2012_msb201240_500



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Tyrosine-1 and threonine-4 phosphorylation marks complete the RNA polymerase II CTD phospho-code

09.09.2012

Martin Heidemann, Dirk Eick

RNA Biology, 2012, http://dx.doi.org/10.4161/rna.21726, Volume 9, Issue 9, Pages 1144 - 1146 published on 09.09.2012
RNA Biology, online article
Eukaryotic RNA polymerase II (RNAP II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation of Ser2, Ser5 and Ser7 residues orchestrates the binding of transcription and RNA processing factors to the transcription machinery. Recent studies show that the two remaining potential phosphorylation sites, tyrosine-1 and threonine-4, are phosphorylated as well and contribute to the previously proposed “CTD code“. With the impairment of binding of CTD interacting factors, these novel phosphorylation marks add an accessory layer of regulation to the RNAP II transcription cycle.

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Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription

06.09.2012

Martin Heidemann, Corinna Hintermair, Kirsten Voß, Dirk Eick

Biochimica et Biophysica Acta, 2012, http://dx.doi.org/10.1016/j.bbagrm.2012.08.013, published on 06.09.2012
Biochimica et Biophysica Acta, online article
The eukaryotic RNA polymerase II (RNAPII) catalyzes the transcription of all protein encoding genes and is also responsible for the generation of small regulatory RNAs. RNAPII has evolved a unique domain composed of heptapeptide repeats with the consensus sequence Tyr1–Ser2–Pro3–Thr4–Ser5–Pro6–Ser7 at the C-terminus (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation patterns of serine residues in CTD during gene transcription coordinate the recruitment of factors to the elongating RNAPII and to the nascent transcript. Recent studies identified threonine 4 and tyrosine 1 as new CTD modifications and thereby expanded the “CTD code”. In this review, we focus on CTD phosphorylation and its function in the RNAPII transcription cycle. We also discuss in detail the limitations of the phosphospecific CTD antibodies, which are used in all studies. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

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Myb-binding Protein 1a (Mybbp1a) Regulates Levels and Processing of Pre-ribosomal RNA

13.07.2012

Julia Hochstatter1, Michael Hölzel, Michaela Rohrmoser, Lothar Schermelleh, Heinrich Leonhardt, Rebecca Keough, Thomas J. Gonda, Axel Imhof, Dirk Eick, Gernot Längst, Attila Németh

The Journal of Biological Chemistry, 2012, doi: 10.1074/jbc.M111.303719, 287, 24365-24377. published on 13.07.2012
The Journal of Biological Chemistry, online article
Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes.

Eick_Imhof_Hochstatter 2012, JBC_500



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Nucleosome Remodeler SNF2L Suppresses Cell Proliferation and Migration and Attenuates Wnt Signaling

06.07.2012

Maren Eckey, Silke Kuphal, Tobias Straub, Petra Rümmele, Elisabeth Kremmer, Anja K. Bosserhoff, Peter B. Becker

Molecular and Cellular Biology, 2012, doi: 10.1128/​MCB.06619-11, vol. 32 no. 13 2359-2371 published on 06.07.2012
Molecular and Cellular Biology, online article
ISWI is an evolutionarily conserved ATPase that catalyzes nucleosome remodeling in different macromolecular complexes. Two mammalian ISWI orthologs, SNF2H and SNF2L, are thought to have specialized functions despite their high sequence similarity. To date, the function of SNF2L in human cells has not been a focus of research. Newly established specific monoclonal antibodies and selective RNA interference protocols have now enabled a comprehensive characterization of loss-of-function phenotypes in human cells. In contrast to earlier results, we found SNF2L to be broadly expressed in primary human tissues. Depletion of SNF2L in HeLa cells led to enhanced proliferation and increased migration. These phenomena were explained by transcriptome profiling, which identified SNF2L as a modulator of the Wnt signaling network. The cumulative effects of SNF2L depletion on gene expression portray the cell in a state of activated Wnt signaling characterized by increased proliferation and chemotactic locomotion. Accordingly, high levels of SNF2L expression in normal melanocytes contrast with undetectable expression in malignant melanoma. In summary, our data document an inverse relationship between SNF2L expression and features characteristic of malignant cells.

 

Becker_2012 Eckey MCB_500



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CTD Tyrosine Phosphorylation Impairs Termination Factor Recruitment to RNA Polymerase II

29.06.2012

Andreas Mayer, Martin Heidemann, Michael Lidschreiber, Amelie Schreieck, Mai Sun, Corinna Hintermair, Elisabeth Kremmer, Dirk Eick, Patrick Cramer

Science, 2012, DOI: 10.1126/science.1219651, Vol. 336 no. 6089 pp. 1723-1725 published on 29.06.2012

Science, online article

In different phases of the transcription cycle, RNA polymerase (Pol) II recruits various factors via its C-terminal domain (CTD), which consists of conserved heptapeptide repeats with the sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr1, in addition to Ser2, Thr4, Ser5, and Ser7. Tyr1 phosphorylation stimulates binding of elongation factor Spt6 and impairs recruitment of termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation levels rise downstream of the transcription start site and decrease before the polyadenylation site, largely excluding termination factors from gene bodies. These results show that CTD modifications trigger and block factor recruitment and lead to an extended CTD code that explains transcription cycle coordination on the basis of differential phosphorylation of Tyr1, Ser2, and Ser5.

 

Eick_Cramer_Mayer 2012, Science_500



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A unified phylogeny-based nomenclature for histone variants

21.06.2012

Paul B Talbert, Kami Ahmad, Geneviève Almouzni, Juan Ausió, Frederic Berger, Prem L Bhalla, William M Bonner, W Zacheus Cande, Brian P Chadwick, Simon W L Chan, George A M Cross, Liwang Cui, Stefan I Dimitrov, Detlef Doenecke, José M Eirin-López, Martin A Gorovsky, Sandra B. Hake, Barbara A Hamkalo, Sarah Holec, Steven E Jacobsen, Kinga Kamieniarz, Saadi Khochbin, Andreas G Ladurner, David Landsman, John A Latham, Benjamin Loppin, Harmit S Malik, William F Marzluff, John R Pehrson, Jan Postberg, Robert Schneider, Mohan B Singh, M Mitchell Smith, Eric Thompson, Maria-Elena Torres-Padilla, David John Tremethick, Bryan M Turner, Jakob Harm Waterborg, Heike Wollmann, Ramesh Yelagandula, Bing Zhu, Steven Henikoff

Epigenetics & Chromatin, 2012, doi:10.1186/1756-8935-5-7, 5:7 published on 21.06.2012
Epigenetics & Chromatin, online article
Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

Hake_Ladurner_Epigenetics & Chromatin 2012_500



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First insights into the unexplored two-component system YehU/YehT in Escherichia coli

08.06.2012

Tobias Kraxenberger, Luitpold Fried, Stefan Behr, Kirsten Jung

Journal of Bacteriology, 2012, doi: 10.1128/​JB.00409-12, 4272–4284 published on 08.06.2012
Journal of Bacteriology, online article
Two-component systems (TCSs) consisting of a membrane-anchored histidine kinase (HK) and a response regulator (RR) are major players in signal transduction in prokaryotes. Whereas most TCSs in Escherichia coli are well characterized, almost nothing is known about the LytS-like HK YehU and the corresponding LytTR-like RR YehT. To identify YehT-regulated genes, we compared the transcriptomes of E. coli cells overproducing either YehT or the RR KdpE (control). The expression levels of 32 genes varied by more than 8-fold between the two strains. A comprehensive evaluation of these genes identified yjiY as a target of YehT. Electrophoretic mobility shift assays with purified YehT confirmed that YehT interacts directly with the yjiY promoter. Specifically, YehT binds to two direct repeats of the motif ACC[G/A]CT[C/T]A separated by a 13-bp spacer in the yjiY promoter. The target gene yjiY encodes an inner membrane protein belonging to the CstA superfamily of transporters. In E. coli cells growing in media containing peptides or amino acids as carbon source, yjiY is strongly induced at the onset of the stationary growth phase. Moreover, expression was found to be dependent on cAMP/CRP. It is suggested that YehU/YehT participates in the stationary phase control network.

 

Jung_Kraxenberger et al 2012_500



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Phosphorylation by cyclin-dependent kinase-9 controls ubiquitin-conjugating enzyme-2A function

01.06.2012

Andrei Shchebet, Oleksandra Karpiuk, Elisabeth Kremmer, Dirk Eick, Steven A. Johnsen

Cell Cycle, 2012, http://dx.doi.org/10.4161/cc.20548, Volume 11, Issue 11, 2122 - 2127 published on 01.06.2012
Cell Cycle, online article
Cyclin-dependent kinase-9 (CDK9) plays a central role in transcriptional elongation and controls multiple cotranscriptional histone modifications, including histone H2B monoubiquitination (H2Bub1). Like other CDK9-dependent histone modifications, the role of CDK9 in maintaining H2Bub1 was shown to be partially dependent upon the phosphorylation status of Ser2 of the RNA polymerase II (RNAPII) C-terminal domain (CTD). Since mutation of Ser2 within the RNAPII CTD resulted in a milder effect on H2Bub1 compared with CDK9 knockdown, we explored whether another CDK9 target may also influence H2Bub1. Based on its homology to yeast Bur1, we hypothesized that CDK9 may directly phosphorylate and activate the ubiquitin-conjugating enzyme utilized for H2B monoubiquitination. Indeed, we demonstrate that UBE2A specifically interacts with CDK9, but not CDK2. Furthermore, UBE2A is phosphorylated by CDK9 in vitro and increases UBE2A activity. Interestingly, CDK9 knockdown not only decreases UBE2A phosphorylation and H2Bub1, but also significantly impairs the induction of UBE2A-dependent monoubiquitination of proliferating cell nuclear antigen (PCNA). Thus, we provide the first evidence that CDK9 is required for the activity of UBE2A in humans, and that its activity is not only required for maintaining H2Bub1, but also for the monoubiquitination of PCNA. The common involvement of these two ubiquitinations in distinct DNA repair pathways may provide a mechanistic rationale for further exploring CDK9 as a combinatorial target for increasing the efficacy of existing cancer therapies based on the induction of DNA damage and are repaired by mechanisms which require H2Bub1 and/or PCNA ubiquitination.

 

Eick_Shchebet 2012, Cell Cycle_500



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Versatile Toolbox for High Throughput Biochemical and Functional Studies with Fluorescent Fusion Proteins

11.05.2012

Garwin Pichler, Antonia Jack, Patricia Wolf, Sandra, Sandra B. Hake

PLoS ONE, 2012, doi:10.1371/journal.pone.0036967, 7(5): e36967. published on 11.05.2012
PLoS ONE, online article
Fluorescent fusion proteins are widely used to study protein localization and interaction dynamics in living cells. However, to fully characterize proteins and to understand their function it is crucial to determine biochemical characteristics such as enzymatic activity and binding specificity. Here we demonstrate an easy, reliable and versatile medium/high-throughput method to study biochemical and functional characteristics of fluorescent fusion proteins. Using a new system based on 96- well micro plates comprising an immobilized GFP-binding protein (GFP-mulitTrap), we performed fast and efficient one-step purification of different GFP- and YFP-fusion proteins from crude cell lysate. After immobilization we determined highly reproducible binding ratios of cellular expressed GFP-fusion proteins to histone-tail peptides, DNA or selected RFP-fusion proteins. In particular, we found Cbx1 preferentially binding to di-and trimethylated H3K9 that is abolished by phosphorylation of the adjacent serine. DNA binding assays showed, that the MBD domain of MeCP2 discriminates between fully methylated over unmethylated DNA and protein-protein interactions studies demonstrate, that the PBD domain of Dnmt1 is essential for binding to PCNA. Moreover, using an ELISA-based approach, we detected endogenous PCNA and histone H3 bound at GFP-fusions. In addition, we quantified the level of H3K4me2 on nucleosomes containing different histone variants. In summary, we present an innovative medium/high-throughput approach to analyse binding specificities of fluroescently labeled fusion proteins and to detect endogenous interacting factors in a fast and reliable manner in vitro.

Hake_PloS One 2012_500



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EMBO welcomes 3 CIPSM scientists as new members

09.05.2012

 

EMBO, 2012, published on 09.05.2012

EMBO, online article

The 3 life scientists Ulrike Gaul, Andreas Ladurner and Michael Sattler from CIPSM were today recognised by EMBO for their excellence in research. EMBO elects new members annually on the basis of scientific excellence. The selected researchers will help shape the direction of the life sciences in Europe and beyond by their involvement with the activities of the organization. EMBO Members provide scientific input such as acting on advisory editorial boards of the four scientific journals of the organization, serving on selection committees for EMBO Programmes and giving general advice to the scientific community.

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Cascaded Photoinduced Drug Delivery to Cells from Multifunctional Core–Shell Mesoporous Silica

02.05.2012

Axel Schloßbauer, Anna M. Sauer, Valentina Cauda, Alexandra Schmidt, Hanna Engelke, Ulrich Rothbauer, Kourosh Zolghadr, Heinrich Leonhardt, Christoph Bräuchle, Thomas Bein

Advanced Healthcare Materials, 2012, DOI: 10.1002/adhm.201100033, Volume 1, Issue 3, pages 316–320 published on 02.05.2012
Advanced Healthcare Materials, online article
Different bioactive molecules are released into living cells from lipid-covered mesoporous silica nanoparticles. The release is triggered by light, as the particles feature covalently attached photosensitizers as membrane-opening agents. It is demonstrated that the particles achieve endosomal escape and that they release their cargo into the cytosol.

Brauchle_Schloßbauer Advanced Healthcare Materials 2012_500

 



Threonine-4 of mammalian RNA polymerase II CTD is targeted by Polo-like kinase 3 and required for transcriptional elongation

01.05.2012

Corinna Hintermair, Martin Heidemann, Frederic Koch, Nicolas Descostes, Marta Gut, Ivo Gut, Romain Fenouil, Pierre Ferrier, Andrew Flatley, Elisabeth Kremmer, Rob D Chapman, Jean-Christophe Andrau, Dirk Eick

 

The EMBO Journal, 2012, doi:10.1038/emboj.2012.123, 31, 2784 - 2797 published on 01.05.2012
The EMBO Journal, online article
Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5′ and 3′ regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like kinase 3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3′ region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells.

 

Eick_Hintermair 2012, EMBOJ_500



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Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution

26.04.2012

Lukas Windhager, Thomas Bonfert, Kaspar Burger, Zsolt Ruzsics, Stefan Krebs, Stefanie Kaufmann, Georg Malterer, Anne L'Hernault,  Markus Schilhabel, Stefan Schreiber, Philip Rosenstiel, Ralf Zimmer, Dirk Eick, Caroline C. Friedel, Lars Dölken

Genome Res., 2012, doi: 10.1101/gr.131847.111, published on 26.04.2012
Genome Res., online article
RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression.

Eick_Windhager 2012, Genome Research_500



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H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization

29.03.2012

Clemens Bönisch, Katrin Schneider, Sebastian Pünzeler, Sonja M. Wiedemann, Christina Bielmeier, Marco Bocola, H. Christian Eber, Wolfgang Kuege, Jürgen Neumann, Elisabeth Kremmer, Heinrich Leonhardt, Matthias Mann, Jens Michaelis, Lothar Schermelleh, Sandra B. Hake

Nucleic Acids Research, 2012, doi:10.1093/nar/gks267, 2012, 1–14 published on 29.03.2012
Nucleic Acids Research, online article
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.

Leonhardt_Mann_Michaelis_Hake_NAR_maerz_2012_500



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Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers

02.03.2012

Sebastian Bultmann, Robert Morbitzer, Christine S. Schmidt, Katharina Thanisch, Fabio Spada, Janett Elsaesser, Thomas Lahaye, Heinrich Leonhardt

Nucl. Acids Res., 2012, doi: 10.1093/nar/gks199, 2012, 1–10 published on 02.03.2012
 Nucl. Acids Res., online article
Specific control of gene activity is a valuable tool to study and engineer cellular functions. Recent studies uncovered the potential of transcription activator-like effector (TALE) proteins that can be tailored to activate user-defined target genes. It remains however unclear whether and how epigenetic modifications interfere with TALE-mediated transcriptional activation. We studied the activity of five designer TALEs (dTALEs) targeting the oct4 pluripotency gene. In vitro assays showed that the five dTALEs that target distinct sites in the oct4 promoter had the expected DNA specificity and comparable affinities to their corresponding DNA targets. In contrast to their similar in vitro properties, transcriptional activation of oct4 by these distinct dTALEs varied up to 25-fold. While dTALEs efficiently upregulated transcription of the active oct4 promoter in embryonic stem cells (ESCs) they failed to activate the silenced oct4 promoter in ESC-derived neural stem cells (NSCs), indicating that as for endogenous transcription factors also dTALE activity is limited by repressive epigenetic mechanisms. We therefore targeted the activity of epigenetic modulators and found that chemical inhibition of histone deacetylases by valproic acid or DNA methyltransferases by 5-aza-2′-deoxycytidine facilitated dTALE-mediated activation of the epigenetically silenced oct4 promoter in NSCs. Notably, demethylation of the oct4 promoter occurred only if chemical inhibitors and dTALEs were applied together but not upon treatment with inhibitors or dTALEs only. These results show that dTALEs in combination with chemical manipulation of epigenetic modifiers facilitate targeted transcriptional activation of epigenetically silenced target genes.

 

Leonhardt_2012_Bultmann_NAR_500



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ATRX-mediated chromatin association of histone variant macroH2A1 regulates a-globin expression

01.03.2012

Kajan Ratnakumar, Luis F. Duarte, Gary LeRoy, Dan Hasson, Daniel Smeets, Chiara Vardabasso, Clemens Bönisch, Tianying Zeng, Bin Xiang, David Y. Zhang, Haitao Li,8 Xiaowo Wang, Sandra B. Hake, Lothar Schermelleh, Benjamin A. Garcia,  Emily Bernstein

Genes & Development, 2012, doi: 10.1101/gad.179416.111, 26: 433-438 published on 01.03.2012
Genes & Development, online article
The histone variant macroH2A generally associates with transcriptionally inert chromatin; however, the factors that regulate its chromatin incorporation remain elusive. Here, we identify the SWI/SNF helicase ATRX (alpha-thalassemia/ MR, X-linked) as a novel macroH2A-interacting protein. Unlike its role in assisting H3.3 chromatin deposition, ATRX acts as a negative regulator ofmacroH2A’s chromatin association. In human erythroleukemic cells deficient for ATRX, macroH2A accumulates at the HBA gene cluster on the subtelomere of chromosome 16, coinciding with the loss of alpha-globin expression. Collectively, our results implicate deregulation of macroH2A’s distribution as a contributing factor to the alpha-thalassemia phenotype of ATRX syndrome.

 

Hake_D_G&D 2012_500



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Structure, function and dynamics of nuclear subcompartments

15.02.2012

M Cristina Cardoso, Katrin Schneider, Robert M Martin, Heinrich Leonhardt

Cell Biology, 2012, DOI 10.1016/j.ceb.2011.12.009, Volume 24, Issue 1, Pages 79–85 published on 15.02.2012
Cell Biology, online article
The nucleus contains a plethora of different dynamic structures involved in the regulation and catalysis of nucleic acid metabolism and function. Over the past decades countless factors, molecular structures, interactions and posttranslational modifications have been described in this context. On the one side of the size scale X-ray crystallography delivers static snapshots of biomolecules at atomic resolution and on the other side light microscopy allows insights into complex structures of living cells and tissues in real time but poor resolution. Recent advances in light and electron microscopy are starting to close the temporal and spatial resolution gap from the atomic up to the cellular level. Old challenges and new insights are illustrated with examples of DNA replication and nuclear protein dynamics.

 

Leonhardt_2012_Cardoso_CurrOpBio_500



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Cytosolic RIG-I–like helicases act as negative regulators of sterile inflammation in the CNS

09.02.2012

Angela Dann, Hendrik Poeck, Andrew Croxford, Stefanie Gaupp, Katrin Kierdorf, Markus Knust, Dietmar Pfeifer, Cornelius Maihoefer, Stefan Endres, Ulrich Kalinke, Sven Meuth, Heinz Wiendl, Klaus-Peter Knobeloch, Shizuo Akira, Ari Waisman, Gunther Hartmann, Marco Prinz

Nature Neuroscience, 2012, 15, doi:10.1038/nn.2964, 98–106, published on 09.02.2012
Nature Neuroscience, online article
The action of cytosolic RIG-I–like helicases (RLHs) in the CNS during autoimmunity is largely unknown. Using a mouse model of multiple sclerosis, we found that mice lacking the RLH adaptor IPS-1 developed exacerbated disease that was accompanied by markedly higher inflammation, increased axonal damage and elevated demyelination with increased encephalitogenic immune responses. Furthermore, activation of RLH ligands such as 5'-triphosphate RNA oligonucleotides decreased CNS inflammation and improved clinical signs of disease. RLH stimulation repressed the maintenance and expansion of committed TH1 and TH17 cells, whereas T-cell differentiation was not altered. Notably, TH1 and TH17 suppression required type I interferon receptor engagement on dendritic cells, but not on macrophages or microglia. These results identify RLHs as negative regulators of TH1 and TH17 responses in the CNS, demonstrate a protective role of the RLH pathway for brain inflammation, and establish oligonucleotide ligands of RLHs as potential therapeutics for the treatment of multiple sclerosis.

 

endres_nature_neurosci_2011_500



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Polycomb Associates Genome-wide with a Specific RNA Polymerase II Variant, and Regulates Metabolic Genes in ESCs

03.02.2012

Emily Brookes,Inês de Santiago,Daniel Hebenstreit, Kelly J. Morris,Tom Carroll,Sheila Q. Xie, Julie K. Stock,Martin Heidemann, Dirk Eick, Naohito Nozaki, Hiroshi Kimura, Jiannis Ragoussis, Sarah A. Teichmann, Ana Pombo

Cell Stem Cell, 2012, DOI 10.1016/j.stem.2011.12.017, Volume 10, Issue 2, 157-170 published on 03.02.2012
Cell Stem Cell, online article
Polycomb repressor complexes (PRCs) are important chromatin modifiers fundamentally implicated in pluripotency and cancer. Polycomb silencing in embryonic stem cells (ESCs) can be accompanied by active chromatin and primed RNA polymerase II (RNAPII), but the relationship between PRCs and RNAPII remains unclear genome-wide. We mapped PRC repression markers and four RNAPII states in ESCs using ChIP-seq, and found that PRC targets exhibit a range of RNAPII variants. First, developmental PRC targets are bound by unproductive RNAPII (S5p+S7p¯S2p¯) genome-wide. Sequential ChIP, Ring1B depletion, and genome-wide correlations show that PRCs and RNAPII-S5p physically bind to the same chromatin and functionally synergize. Second, we identify a cohort of genes marked by PRC and elongating RNAPII (S5p+S7p+S2p+); they produce mRNA and protein, and their expression increases upon PRC1 knockdown. We show that this group of PRC targets switches between active and PRC-repressed states within the ESC population, and that many have roles in metabolism.

 

Eick_Brookes 2012, Cell Stem Cell_500



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Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development

01.02.2012

Ralph Stadhouders, Supat Thongjuea, Charlotte Andrieu-Soler Robert-Jan Palstra, Jan Christian Bryne Anita van den Heuvel, Mary Stevens Ernie de Boer, Christel Kockx Antoine van der Sloot Mirjam van den Hout, Wilfred van IJcken, Dirk Eick, Boris Lenhard, Frank Grosveld, Eric Soler

The EMBO Journal, 2012, doi:10.1038/emboj.2011.450, 31, 986 - 999 published on 01.02.2012
The EMBO Journal, online article
The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIPSequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized.We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping.

Eick_Stadhouders 2012, EMBOJ_500



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CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin.

31.01.2012

Silvia Dambacher, Wen Deng, Matthias Hahn, Dennis Sadic, Jonathan J. Fröhlich,  Alexander Nuber, Christian Hoischen, Stephan Diekmann, Heinrich Leonhardt, Gunnar Schotta

Nucleus, 2012, http://dx.doi.org/10.4161/nucl.3.1.18955, 101–110 published on 31.01.2012
Nucleus, online article
Centromeres are important structural constituents of chromosomes that ensure proper chromosome segregation during mitosis by providing defined sites for kinetochore attachment. In higher eukaryotes, centromeres have no specific DNA sequence and thus, they are rather determined through epigenetic mechanisms. A fundamental process in centromere establishment is the incorporation of the histone variant CENP-A into centromeric chromatin, which provides a binding platform for the other centromeric proteins. The Mis18 complex, and, in particular, its member M18BP1 was shown to be essential for both incorporation and maintenance of CENP-A. Here we show that M18BP1 displays a cell cycle-regulated association with centromeric chromatin in mouse embryonic stem cells. M18BP1 is highly enriched at centromeric regions from late anaphase through to G1 phase. An interaction screen against 16 core centromeric proteins revealed a novel interaction of M18BP1 with CENP-C. We mapped the interaction domain in M18BP1 to a central region containing a conserved SANT domain and in CENP-C to the C-terminus. Knock-down of CENP-C leads to reduced M18BP1 association and lower CENP-A levels at centromeres, suggesting that CENP-C works as an important factor for centromeric M18BP1 recruitment and thus for maintaining centromeric CENP-A.

 

Leonhardt_Schotta_2012_Dambacher_Nucleus_500



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Connecting Threads: Epigenetics and Metabolism

20.01.2012

Sayako Katada, Axel Imhof, Paolo Sassone-Corsi

Cell, 2012, 148, DOI 10.1016/j.cell.2012.01.001, 24-28, published on 20.01.2012
Cell, online article
Chromatin-modifying enzymes have long been proposed to be the authors of an epigenetic language, but the origin and meaning of the messages they write in chromatin are still mysterious. Recent studies suggesting that the effects of diet can be passed on epigenetically to offspring add weight to the idea that histones act as metabolic sensors, converting changes in metabolism into stable patterns of gene expression. The challenge will now be to understand how localized fluctuations in levels of metabolites control chromatin modifiers in space and time, translating a dynamic metabolic state into a histone map.

 

imhof_cell_2012_500



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Roles of long, non-coding RNA in chromosome-wide transcription regulation: Lessons from two dosage compensation systems

08.01.2012

Sylvain Maenner, Marisa Müller, Peter B. Becker

Biochimie, 2012, doi:10.1016/j.biochi.2011.12.026, Volume 94, Issue 7, Pages 1490–1498 published on 08.01.2012
Biochimie, online article
A large part of higher eukaryotic genomes is transcribed into RNAs lacking any significant open reading frame. This “non-coding part” has been shown to actively contribute to regulating gene expression, but the mechanisms are largely unknown. Particularly instructive examples are provided by the dosage compensation systems, which assure that the single X chromosome in male cells and the two X chromosomes in female cells give rise to similar amounts of gene product. Although this is achieved by very different strategies in mammals and fruit flies, long, non-coding RNAs (lncRNAs) are involved in both cases. Here we summarize recent progress towards unraveling the mechanisms, by which the Xist and roX RNAs mediate the selective association of regulators with individual target chromosomes, to initiate dosage compensation in mammals and fruit flies, respectively.

Becker_12_Maenne_Biochimie_500



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Cytosolic DNA Triggers Mitochondrial Apoptosis via DNA Damage Signaling Proteins Independently of AIM2 and RNA Polymerase III

01.01.2012

Michael Wenzel, Michael Wunderlich, Robert Besch, Hendrik Poeck, Simone Willms, Astrid Schwantes, Melanie Kremer, Gerd Sutter, Stefan Endres, Andreas Schmidt, Simon Rothenfusser

The Journal of Immunology, 2012, doi: 10.4049/​jimmunol.1100523, vol. 188 no. 1 394-403 published on 01.01.2012
The Journal of Immunology, online article
A key host response to limit microbial spread is the induction of cell death when foreign nucleic acids are sensed within infected cells. In mouse macrophages, transfected DNA or infection with modified vaccinia virus Ankara (MVA) can trigger cell death via the absent in melanoma 2 (AIM2) inflammasome. In this article, we show that nonmyeloid human cell types lacking a functional AIM2 inflammasome still die in response to cytosolic delivery of different DNAs or infection with MVA. This cell death induced by foreign DNA is independent of caspase-8 and carries features of mitochondrial apoptosis: dependence on BAX, APAF-1, and caspase-9. Although it does not require the IFN pathway known to be triggered by infection with MVA or transfected DNA via polymerase III and retinoid acid-induced gene I-like helicases, it shows a strong dependence on components of the DNA damage signaling pathway: cytosolic delivery of DNA or infection with MVA leads to phosphorylation of p53 (serines 15 and 46) and autophosphorylation of ataxia telangiectasia mutated (ATM); depleting p53 or ATM with small interfering RNA or inhibiting the ATM/ATM-related kinase family by caffeine strongly reduces apoptosis. Taken together, our findings suggest that a pathway activating DNA damage signaling plays an important independent role in detecting intracellular foreign DNA, thereby complementing the induction of IFN and activation of the AIM2 inflammasome.

 

Endres_Wenzel_JImmunol_2012_500

 



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TU München
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MPI of Neurobiology
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