Christian Hoischen

Publications of Christian Hoischen

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

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

  Nucleus (Austin, Tex.). 01/2012; 3(1).

  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 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.

• Dynamics of CENP-N kinetochore binding during the cell cycle.

  Authors: Daniela Hellwig, Stephan Emmerth, Tobias Ulbricht, Volker Döring, Christian Hoischen, Ronny Martin, Catarina P Samora, Andrew D McAinsh, Christopher W Carroll, Aaron F Straight, Patrick Meraldi, Stephan Diekmann

  Journal of cell science. 11/2011; 124(Pt 22):3871-83.

  Accurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding ofAccurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.

• Premitotic assembly of human CENPs -T and -W switches centromeric chromatin to a mitotic state.

  Authors: Lisa Prendergast, Chelly van Vuuren, Agnieszka Kaczmarczyk, Volker Doering, Daniela Hellwig, Nadine Quinn, Christian Hoischen, Stephan Diekmann, Kevin F Sullivan

  PLoS biology. 06/2011; 9(6):e1001082.

  Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They areCentromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.

• Segrosome assembly at the pliable parH centromere.

  Authors: Meiyi Wu, Massimiliano Zampini, Malte Bussiek, Christian Hoischen, Stephan Diekmann, Finbarr Hayes

  Nucleic acids research. 03/2011; 39(12):5082-97.

  The segrosome of multiresistance plasmid TP228 comprises ParF, which is a member of the ParA ATPase superfamily, and the ParG ribbon-helix-helix factor that assemble jointly on the parH centromere.The segrosome of multiresistance plasmid TP228 comprises ParF, which is a member of the ParA ATPase superfamily, and the ParG ribbon-helix-helix factor that assemble jointly on the parH centromere. Here we demonstrate that the distinctive parH site (∼100-bp) consists of an array of degenerate tetramer boxes interspersed by AT-rich spacers. Although numerous consecutive AT-steps are suggestive of inherent curvature, parH lacks an intrinsic bend. Sequential deletion of parH tetramers progressively reduced centromere function. Nevertheless, the variant subsites could be rearranged in different geometries that accommodated centromere activity effectively revealing that the site is highly elastic in vivo. ParG cooperatively coated parH: proper centromere binding necessitated the protein's N-terminal flexible tails which modulate the centromere binding affinity of ParG. Interaction of the ParG ribbon-helix-helix domain with major groove bases in the tetramer boxes likely provides direct readout of the centromere. In contrast, the AT-rich spacers may be implicated in indirect readout that mediates cooperativity between ParG dimers assembled on adjacent boxes. ParF alone does not bind parH but instead loads into the segrosome interactively with ParG, thereby subtly altering centromere conformation. Assembly of ParF into the complex requires the N-terminal flexible tails in ParG that are contacted by ParF.

• Theoretical study of lipid biosynthesis in wild-type Escherichia coli and in a protoplast-type L-form using elementary flux mode analysis.

  Authors: Dimitar Kenanov, Christoph Kaleta, Andreas Petzold, Christian Hoischen, Stephan Diekmann, Roman A Siddiqui, Stefan Schuster

  The FEBS journal. 02/2010; 277(4):1023-34.

  In the present study, we investigated lipid biosynthesis in the bacterium Escherichia coli by mathematical modeling. In particular, we studied the interaction between the subsystems producingIn the present study, we investigated lipid biosynthesis in the bacterium Escherichia coli by mathematical modeling. In particular, we studied the interaction between the subsystems producing unsaturated and saturated fatty acids, phospholipids, lipid A, and cardiolipin. The present analysis was carried out both for the wild-type and for several in silico knockout mutants, using the concept of elementary flux modes. Our results confirm that, in the wild type, there are four main products: L1-phosphatidylethanolamine, lipid A, lipid A (cold-adapted), and cardiolipin. We found that each of these compounds is produced on several different routes, indicating a high redundancy of the system under study. By analysis of the elementary flux modes remaining after the knockout of genes of lipid biosynthesis, and comparison with publicly available data on single-gene knockouts in vivo, we were able to determine the metabolites essential for the survival of the cell. Furthermore, we analyzed a set of mutations that occur in a cell wall-free mutant of Escherichia coli W1655F+. We postulate that the mutant is not capable of producing both forms of lipid A, when the combination of mutations is considered to make a nonfunctional pathway. This is in contrast to gene essentiality data showing that lipid A synthesis is indispensable for the survival of the cell. The loss of the outer membrane in the cell wall-free mutant, however, shows that lipid A is dispensable as the main component of the outer surface structure in this particular E. coli strain.

• Sequence-specific physical properties of African green monkey alpha-satellite DNA contribute to centromeric heterochromatin formation.

  Authors: Malte Bussiek, Christian Hoischen, Stephan Diekmann, Martin L Bennink

  Journal of structural biology. 04/2009;

  Satellite DNA, a major component of eukaryotic centromeric heterochromatin, is potentially associated with the processes ensuring the faithful segregation of the genetic material during cellSatellite DNA, a major component of eukaryotic centromeric heterochromatin, is potentially associated with the processes ensuring the faithful segregation of the genetic material during cell division. Structural properties of alpha-satellite DNA (AS) from African green monkey were studied. Atomic force microscopy imaging showed smaller end-to-end distances of AS fragments than would be expected for the persistence length of random sequence DNA. The apparent persistence length of the AS was determined as 35 nm. Gel-electrophoresis indicated only a weak contribution of intrinsic curvature to the DNA conformations suggesting an additional contribution of an elevated bending flexibility to the reduced end-to-end distances. Next, the force-extension behavior of the naked AS and in complex with nucleosomes was studied using optical tweezers. The naked AS showed a reduced overstretching transition force (-18% the value determined for random DNA) and higher forces required to straighten the DNA. Finally, reconstituted AS nucleosomes disrupted at significantly higher forces as compared with random DNA nucleosomes which is probably due to structural properties of the AS which stabilize the nucleosomes. The data support that the AS plays a role in the formation of centromeric heterochromatin due to specific structural properties and suggest that a relatively higher mechanical stability of nucleosomes is important in AGM-AS chromatin.

• Dynamics of component exchange at PML nuclear bodies.

  Authors: Stefanie Weidtkamp-Peters, Thorsten Lenser, Dmitri Negorev, Norman Gerstner, Thomas G Hofmann, Georg Schwanitz, Christian Hoischen, Gerd Maul, Peter Dittrich, Peter Hemmerich

  Journal of cell science. 09/2008; 121(Pt 16):2731-43.

  PML nuclear bodies (NBs) are involved in the regulation of key nuclear pathways but their biochemical function in nuclear metabolism is unknown. In this study PML NB assembly dynamics were assessedPML nuclear bodies (NBs) are involved in the regulation of key nuclear pathways but their biochemical function in nuclear metabolism is unknown. In this study PML NB assembly dynamics were assessed by live cell imaging and mathematic modeling of its major component parts. We show that all six nuclear PML isoforms exhibit individual exchange rates at NBs and identify PML V as a scaffold subunit. SP100 exchanges at least five times faster at NBs than PML proteins. Turnover dynamics of PML and SP100 at NBs is modulated by SUMOylation. Exchange is not temperature-dependent but depletion of cellular ATP levels induces protein immobilization at NBs. The PML-RARalpha oncogene exhibits a strong NB retention effect on wild-type PML proteins. HIPK2 requires an active kinase for PML NB targeting and elevated levels of PML IV increase its residence time. DAXX and BLM turn over rapidly and completely at PML NBs within seconds. These findings provide a kinetics model for factor exchange at PML NBs and highlight potential mechanisms to regulate intranuclear trafficking of specific factors at these domains.

• Dynamics of inner kinetochore assembly and maintenance in living cells.

  Authors: Peter Hemmerich, Stefanie Weidtkamp-Peters, Christian Hoischen, Lars Schmiedeberg, Indri Erliandri, Stephan Diekmann

  The Journal of cell biology. 04/2008; 180(6):1101-14.

  To investigate the dynamics of centromere organization, we have assessed the exchange rates of inner centromere proteins (CENPs) by quantitative microscopy throughout the cell cycle in human cells.To investigate the dynamics of centromere organization, we have assessed the exchange rates of inner centromere proteins (CENPs) by quantitative microscopy throughout the cell cycle in human cells. CENP-A and CENP-I are stable centromere components that are incorporated into centromeres via a "loading-only" mechanism in G1 and S phase, respectively. A subfraction of CENP-H also stays stably bound to centromeres. In contrast, CENP-B, CENP-C, and some CENP-H and hMis12 exhibit distinct and cell cycle-specific centromere binding stabilities, with residence times ranging from seconds to hours. CENP-C and CENP-H are immobilized at centromeres specifically during replication. In mitosis, all inner CENPs become completely immobilized. CENPs are highly mobile throughout bulk chromatin, which is consistent with a binding-diffusion behavior as the mechanism to scan for vacant high-affinity binding sites at centromeres. Our data reveal a wide range of cell cycle-specific assembly plasticity of the centromere that provides both stability through sustained binding of some components and flexibility through dynamic exchange of other components.

• Centromere anatomy in the multidrug-resistant pathogen Enterococcus faecium.

  Authors: Andrew Derome, Christian Hoischen, Malte Bussiek, Ruth Grady, Malgorzata Adamczyk, Barbara Kedzierska, Stephan Diekmann, Daniela Barillà, Finbarr Hayes

  Proceedings of the National Academy of Sciences of the United States of America. 03/2008; 105(6):2151-6.

  Multidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is aMultidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is a driving force in antibiotic-resistance evolution in enterococci. The segregation locus of a high-level gentamicin-resistance plasmid, pGENT, in Enterococcus faecium was identified and dissected. This locus includes overlapping genes encoding PrgP, a member of the ParA superfamily of segregation proteins, and PrgO, a site-specific DNA binding homodimer that recognizes the cenE centromere upstream of prgPO. The centromere has a distinctive organization comprising three subsites, CESII separates CESI and CESIII, each of which harbors seven TATA boxes spaced by half-helical turns. PrgO independently binds both CESI and CESIII, but with different affinities. The topography of the complex was probed by atomic force microscopy, revealing discrete PrgO foci positioned asymmetrically at the CESI and CESIII subsites. Bending analysis demonstrated that cenE is intrinsically curved. The organization of the cenE site and of certain other plasmid centromeres mirrors that of yeast centromeres, which may reflect a common architectural requirement during assembly of the mitotic apparatus in yeast and bacteria. Moreover, segregation modules homologous to that of pGENT are widely disseminated on vancomycin and other resistance plasmids in enterococci. An improved understanding of segrosome assembly may highlight new interventions geared toward combating antibiotic resistance in these insidious pathogens.

• Assembly of the inner kinetochore proteins CENP-A and CENP-B in living human cells.

  Authors: Sandra Orthaus, Christoph Biskup, Birgit Hoffmann, Christian Hoischen, Sabine Ohndorf, Klaus Benndorf, Stephan Diekmann

  Chembiochem : a European journal of chemical biology. 02/2008; 9(1):77-92.

  DNA segregation in mammalian cells during mitosis is an essential cellular process that is mediated by a specific subchromosomal protein complex, the kinetochore. Malfunction of this complex resultsDNA segregation in mammalian cells during mitosis is an essential cellular process that is mediated by a specific subchromosomal protein complex, the kinetochore. Malfunction of this complex results in aneuploidy and can cause cancer. A subkinetochore complex, the "inner kinetochore", is present at the centromere during the entire cell cycle. Its location seems to be defined by the settlement of CENP-A (CENH3), which replaces histone H3 in centromeric nucleosomes. This suggests that CENP-A can recruit further inner kinetochore proteins by direct binding. Surprisingly, intense in vitro studies could not identify an interaction of CENP-A with any other inner kinetochore protein. Instead, centromere identity seems to be maintained by a unique nucleosome, which might have a modified structure or epigenetic state that serves to distinguish the centromere from the rest of the chromosome. We investigated the association of CENP-A and CENP-B by fluorescence intensity and lifetime-based FRET measurements in living human HEp-2 cells. We observed Förster resonance energy transfer (FRET) between CENP-A and CENP-B at centromere locations; this indicates that these proteins are in the molecular vicinity (<10 nm) of each other. In addition, we analysed protein-protein interactions within the centromeric nucleosome. We could detect energy transfer between CENP-A and histone H4 as well as between CENP-A molecules themselves. On the other hand, no FRET was detected between CENP-A and H2A.1 or H3.1. Our data support the view that two CENP-A molecules are packed with H4, but not with H3, in a single centromeric nucleosome.

• The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli.

  Authors: Roman A Siddiqui, Christian Hoischen, Otto Holst, Ivonne Heinze, Bernhard Schlott, Johannes Gumpert, Stephan Diekmann, Frank Grosse, Matthias Platzer

  FEMS microbiology letters. 06/2006; 258(2):305-11.

  Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divideCell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.

• Centromere parC of plasmid R1 is curved.

  Authors: Christian Hoischen, Alexander Bolshoy, Kenn Gerdes, Stephan Diekmann

  Nucleic acids research. 02/2004; 32(19):5907-15.

  The centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by itsThe centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by its migration anomaly in polyacrylamide gels. The 159 bp long parC is strongly curved with anomaly values (k-factors) close to 2. The properties of the parC curvature agree with those of other curved DNA sequences. parC contains two regions of 5-fold repeated iterons separated by 39 bp. We modified 4 bp within this intermediate sequence so that we could analyse the two 5-fold repeated regions independently. The analysis shows that the two repeat regions are not independently curved parts of parC but that the overall curvature is a property of the whole fragment. Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate that curvature might be a general property of centromeres.

• Novel bacterial membrane surface display system using cell wall-less L-forms of Proteus mirabilis and Escherichia coli.

  Authors: Christian Hoischen, Christine Fritsche, Johannes Gumpert, Martin Westermann, Katleen Gura, Beatrix Fahnert

  Applied and environmental microbiology. 03/2002; 68(2):525-31.

  We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli andWe describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 microg ml(-1), with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.

• Use of cell wall-less bacteria (L-forms) for efficient expression and secretion of heterologous gene products

  Authors: Johannes Gumpert, Christian Hoischen

  Current Opinion in Biotechnology.

  In spite of many efforts and achievements to optimize the prokaryotic expression systems, there are still general and specific problems in obtaining sufficient yields of the functionally active geneIn spite of many efforts and achievements to optimize the prokaryotic expression systems, there are still general and specific problems in obtaining sufficient yields of the functionally active gene products. The main problems concern the formation of inclusion bodies, incorrect folding, toxicity for the producer cells and degradation by proteases. One way to overcome these problems is with expression systems alternative to those of Escherichia coli. For the first time, cell wall-less L-form bacteria were used to establish such an alternative expression system and test its practicability. The results showed that various recombinant proteins can be synthesized in considerable amounts as soluble, functionally active products with these cell wall-less strains.