Biomedical subjects

Karsten Rippe

Publications and source records attributed to Karsten Rippe.

15 recordsLinked to original sources

PubMed2026

HP1α binding creates a local barrier against transcription activation and persists during chromatin decondensation.

Mouse pericentric heterochromatin forms compacted, transcriptionally silent domains, termed chromocenters, that are enriched in heterochromatin protein 1 (HP1). Whether HP1α represses chromocenters by binding locally or by maintaining a phase-separated compartment is unresolved. We investigated this question by recruiting transcriptional activators to mouse fibroblast chromocenters and quantifying repression at a transcription reporter. HP1α established a promoter-proximal barrier that suppressed weaker activators (VP16) but was overcome by stronger ones (VP64-p65-Rta [VPR] and p65). Activator-induced decondensation and transcription occurred without displacing HP1α or H3K9 trimethylation, and HP1α retained its granular distribution and stoichiometric binding dynamics. Multi-color super-resolution imaging revealed spatial segregation of transcribed and HP1α-bound regions at the nanodomain scale. A nanodomain model captured how repeat clusters independently transition between silenced and activated states. These data establish that HP1α and H3K9me3 act through independently switchable nanodomains, accounting for chromocenter regulation without invoking phase separation.

Animals↗

PubMed2006

Structure of a bacterial pyridoxal 5'-phosphate synthase complex.

Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a glutamine amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5'-phosphate (PLP), directly from a triose and a pentose saccharide and glutamine. Here we report the 3D structure of the PLP synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined glutamine amidotransferases, macromolecular assembly is directed by an N-terminal alpha-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel.

Bacillus subtilis↗

PubMed2006

RNA: Networks & Imaging.

The past few years have brought about a fundamental change in our understanding and definition of the RNA world and its role in the functional and regulatory architecture of the cell. The discovery of small RNAs that regulate many aspects of differentiation and development have joined the already known non-coding RNAs that are involved in chromosome dosage compensation, imprinting, and other functions to become key players in regulating the flow of genetic information. It is also evident that there are tens or even hundreds of thousands of other non-coding RNAs that are transcribed from the mammalian genome, as well as many other yet-to-be-discovered small regulatory RNAs. In the recent symposium RNA: Networks & Imaging held in Heidelberg, the dual roles of RNA as a messenger and a regulator in the flow of genetic information were discussed and new molecular genetic and imaging methods to study RNA presented.

Animals↗

PubMed2006

On the mechanism of nucleosome assembly by histone chaperone NAP1.

The process of mononucleosome assembly mediated by histone chaperone NAP1 was investigated using DNA fragments 146 and 207 bp in length containing the Lytechinus variegatus 5 S rDNA nucleosome positioning sequence. A quantitative description was derived using gel electrophoresis and fluorescent anisotropy data. First, NAP1-bound H3.H4 was released forming a DNA-histone tetramer complex with a time constant of k(1) = (2.5 +/- 0.7) . 10(4) m(-1) s(-1). The tetrasome was converted quickly (k(2) = (4.1 +/- 3.5) . 10(5) m(-1) s(-1)), by the addition of a single H2A.H2B dimer, into a "hexasome," i.e. a nucleosome lacking one H2A.H2B dimer. From this intermediate a nucleosome was formed by the addition of a second H2A.H2B dimer with an average rate constant k(3) = (6.6 +/- 1.4) . 10(3) m(-1) s(-1). For the back-reaction, significant differences were observed between the 146- and 207-bp DNA upon substitution of the canonical H2A histone with H2A.Z. The distinct nucleosome/hexasome ratios were reflected in the corresponding equilibrium dissociation constants and revealed some differences in nucleosome stability. In a fourth reaction, NAP1 mediated the binding of linker histone H1 to the nucleosome, completing the chromatosome structure with k(4) = (7.7 +/- 3.7) . 10(3) m(-1) s(-1). The activity of the chromatin remodeling complex ACF did not increase the kinetics of the mononucleosome assembly process.

Anisotropy↗

PubMed2005

Histone acetylation increases chromatin accessibility.

In eukaryotes, the interaction of DNA with proteins and supramolecular complexes involved in gene expression is controlled by the dynamic organization of chromatin inasmuch as it defines the DNA accessibility. Here, the nuclear distribution of microinjected fluorescein-labeled dextrans of 42 kDa to 2.5 MDa molecular mass was used to characterize the chromatin accessibility in dependence on histone acetylation. Measurements of the fluorescein-dextran sizes were combined with an image correlation spectroscopy analysis, and three different interphase chromatin condensation states with apparent pore sizes of 16-20 nm, 36-56 nm and 60-100 nm were identified. A reversible change of the chromatin conformation to a uniform 60-100 nm pore size distribution was observed upon increased histone acetylation. This result identifies histone acetylation as a central factor in the dynamic regulation of chromatin accessibility during interphase. In mitotic chromosomes, the chromatin exclusion limit was 10-20 nm and independent of the histone acetylation state.

Acetylation↗

PubMed2005

NAP1 modulates binding of linker histone H1 to chromatin and induces an extended chromatin fiber conformation.

NAP1 (nucleosome assembly protein 1) is a histone chaperone that has been described to bind predominantly to the histone H2A.H2B dimer in the cell during shuttling of histones into the nucleus, nucleosome assembly/remodeling, and transcription. Here it was examined how NAP1 interacts with chromatin fibers isolated from HeLa cells. NAP1 induced a reversible change toward an extended fiber conformation as demonstrated by sedimentation velocity ultracentrifugation experiments. This transition was due to the removal of the linker histone H1. The H2A.H2B dimer remained stably bound to the native fiber fragments and to fibers devoid of linker histone H1. This was in contrast to mononucleosome substrates, which displayed a NAP1-induced removal of a single H2A.H2B dimer from the core particle. The effect of NAP1 on the chromatin fiber structure was examined by scanning/atomic force microscopy. A quantitative image analysis of approximately 36,000 nucleosomes revealed an increase of the average internucleosomal distance from 22.3 +/- 0.4 to 27.6 +/- 0.6 nm, whereas the overall fiber structure was preserved. This change reflects the disintegration of the chromatosome due to binding of H1 to NAP1 as chromatin fibers stripped from H1 showed an average nucleosome distance of 27.4 +/- 0.8 nm. The findings suggest a possible role of NAP1 in chromatin remodeling processes involved in transcription and replication by modulating the local linker histone content.

Chromatin↗

PubMed2005

A 'loop recapture' mechanism for ACF-dependent nucleosome remodeling.

The ATPase ISWI is the molecular motor of several nucleosome remodeling complexes including ACF. We analyzed the ACF-nucleosome interactions and determined the characteristics of ACF-dependent nucleosome remodeling. In contrast to ISWI, ACF interacts symmetrically with DNA entry sites of the nucleosome. Two-color fluorescence cross-correlation spectroscopy measurements show that ACF can bind four DNA duplexes simultaneously in a complex that contains two Acf1 and ISWI molecules. Using bead-bound nucleosomal substrates, nucleosome movement by mechanisms involving DNA twisting was excluded. Furthermore, an ACF-dependent local detachment of DNA from the nucleosome was demonstrated in a novel assay based on the preferred intercalation of ethidium bromide to free DNA. The findings suggest a loop recapture mechanism in which ACF introduces a DNA loop at the nucleosomal entry site that propagates over the histone octamer surface and leads to nucleosome repositioning.

Adenosine Triphosphatases↗

PubMed2005

Mobility of multi-subunit complexes in the nucleus: accessibility and dynamics of chromatin subcompartments.

The cell nucleus contains a number of mobile subnuclear organelles involved in RNA processing, transcriptional regulation and antiviral defence like Cajal and promyelocytic leukaemia (PML) bodies. It remains an open question how these bodies translocate to specific nuclear regions within the nucleus to exert their biological function. The mobility and localisation of macromolecules in the nucleus are closely related to the dynamic organisation and accessibility of chromatin. This relation has been studied with biologically inert fluorescent particles like dextrans, polystyrene nanospheres and inactive protein crystals formed by the Mx1-YFP fusion protein or other ectopically expressed proteins like vimentin. As reviewed here, properties of the chromatin environment can be identified from these experiments that determine the mobility of Cajal and PML bodies and other supramolecular complexes.

Animals↗

PubMed2005

Association states of nucleosome assembly protein 1 and its complexes with histones.

The histone chaperone NAP1 is a carrier of histones during nuclear import, nucleosome assembly, and chromatin remodeling. Analytical ultracentrifugation was used to determine the association states of NAP1 alone and in complexes with core histones. In addition, the concentration dependence of the association was quantified by determining the equilibrium dissociation constant between different NAP1 species. At physiological protein and salt concentrations the prevalent species were the NAP1 dimer and octamer. These were also the association states found to interact with histones in a stoichiometry of one NAP1 monomer per histone. Based on these results a model for a cell cycle-dependent shift of the NAP1 dimer-octamer equilibrium is proposed that reflects different biological functions of NAP1.

Cell Cycle Proteins↗

PubMed2004

Nuclear body movement is determined by chromatin accessibility and dynamics.

Promyelocytic leukemia (PML) and Cajal bodies are mobile subnuclear organelles, which are involved in activities like RNA processing, transcriptional regulation, and antiviral defense. A key parameter in understanding their biological functions is their mobility. The diffusion properties of PML and Cajal bodies were compared with a biochemically inactive body formed by aggregates of murine Mx1 by using single-particle tracking methods. The artificial Mx1-yellow fluorescent protein body showed a very similar mobility compared with PML and Cajal bodies. The data are described quantitatively by a mechanism of nuclear body movement consisting of two components: diffusion of the body within a chromatin corral and its translocation resulting from chromatin diffusion. This finding suggests that the body mobility reflects the dynamics and accessibility of the chromatin environment, which might target bodies to specific nuclear subcompartments where they exert their biological function.

Adenosine Triphosphate↗

PubMed2004

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin.

The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to >1 microm was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied.

Acetylation↗

PubMed2003

Conformation of reconstituted mononucleosomes and effect of linker histone H1 binding studied by scanning force microscopy.

The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of approximately 1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of DeltaDeltaG approximately 3 kcal mol(-1) as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 +/- 29 degrees and 136 +/- 18 degrees (air-dried samples), and 78 +/- 25 degrees and 137 +/- 25 degrees (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 +/- 34 degrees for complexes in air and 85 +/- 10 degrees in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 +/- 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 +/- 34 basepairs that are wound around the nucleosome.

Air↗

PubMed2002

NUDT9, a member of the Nudix hydrolase family, is an evolutionarily conserved mitochondrial ADP-ribose pyrophosphatase.

We have recently characterized the protein product of the human NUDT9 gene as a highly specific ADP-ribose (ADPR) pyrophosphatase. We now report an analysis of the human NUDT9 gene and its potential alternative transcripts along with detailed studies of the enzymatic properties and cell biological behavior of human NUDT9 protein. Our analysis of the human NUDT9 gene and twenty-two distinct cloned NUDT9 transcripts indicates that the full-length NUDT9 alpha transcript is the dominant form, and suggests that an alternative NUDT9 beta transcript occurs as the result of a potentially aberrant splice from a cryptic donor site within the first exon to the splice acceptor site of exon 2. Computer analysis of the predicted protein of the NUDT9 alpha transcript identified an N-terminal signal peptide or subcellular targeting sequence. Using green fluorescence protein tagging, we demonstrate that the predicted human NUDT9 alpha protein is targeted highly specifically to mitochondria, whereas the predicted protein of the NUDT9 beta transcript, which is missing this sequence, exhibits no clear subcellular localization. Investigation of the physical and enzymatic properties of NUDT9 indicates that it is functional as a monomer, optimally active at near neutral pH, and that it requires divalent metal ions and an intact Nudix motif for enzymatic activity. Furthermore, partial proteolysis of NUDT9 suggests that NUDT9 enzymes consist of two distinct domains: a proteolytically resistant C-terminal domain retaining essentially full specific ADPR pyrophosphatase activity and a proteolytically labile N-terminal portion that functions to enhance the affinity of the C-terminal domain for ADPR.

Amino Acid Sequence↗

PubMed2002

Binding affinity of Escherichia coli RNA polymerase*sigma54 holoenzyme for the glnAp2, nifH and nifL promoters.

Escherichia coli RNA polymerase associated with the sigma54 factor (RNAP*sigma54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase*sigma70 com plex. Promoters for RNAP*sigma54 vary in their overall 'strength' and show differences in their response to the presence of DNA curvature between enhancer and promoter. In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant K(d) for the binding of RNAP*sigma54 to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP*sigma54 to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of K(d) = 0.94 +/- 0.55 nM and K(d) = 0.85 +/- 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with K(d) = 8.5 +/- 1.9 nM. The logarithmic dependence of K(d) on the ionic strength I was -Deltalog(K(d))/Deltalog(I) = 6.1 +/- 0.5 for the glnAp2, 5.2 +/- 1.2 for the nifH and 2.1 +/- 0.1 for the nifL promoter. This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity.

Bacterial Proteins↗

PubMed2002

Capturing chromosome conformation.

We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.

AT Rich Sequence↗