Biomedical subjects

K van Holde

Publications and source records attributed to K van Holde.

At least 19 recordsLinked to original sources

PubMed2003

Models for chromatin remodeling: a critical comparison.

Nucleosome remodeling has been shown, in many cases, to involve cis displacement of nucleosomes on the DNA. This process seems similar to the long-recognized random diffusion of nucleosomes along DNA, but the remodeling process is unidirectional and ATP dependent. Several years ago, we developed a model for nucleosome migration, based on the diffusion of "twist-defects" within the nucleosomal DNA. This has been modified into a model that incorporates ATP-dependent defect generation, and can account for many observations concerning remodeling. However, certain experimental studies in recent years have cast doubt on the applicability of the twist-diffusion model for remodeling, and seem to favor instead a "reptation" model. We discuss herein these problems and propose a resolution.

Adenosine Triphosphate↗

PubMed1999

The nucleosome core particle: does it have structural and physiologic relevance?

Although the nucleosomal core particle has been extensively studied as the basic building block of chromatin, the biological significance of a unit carrying exactly 146 bp of DNA remains unclear. Herein, we present data to show that the histone octamer can stably accommodate anywhere from about 100 to 170 bp of DNA. The unfolded structures containing less than 146 bp may well be of greater biological importance than the canonical core particle.

Animals↗

PubMed1999

The site of binding of linker histone to the nucleosome does not depend upon the amino termini of core histones.

Using nucleosomes reconstituted on a defined sequence of DNA, we have investigated the question as to whether the N-terminal tails of core histones play a role in determining the site of binding of a linker histone. Reconstitutes used histone cores of three types: intact, lacking the N-terminal H3 tails, or lacking all tails. In each case the same, single defined position for the histone core was observed, using high-resolution mapping. The affinity for binding of linker histone H1(o) was highest for the intact cores, lowest for the tailless cores. However, the location of the linker histone, as judged by micrococcal nuclease protection, was exactly the same in each case, an asymmetric site of about 17 bp to one side of the core particle DNA.

Binding Sites↗

PubMed1999

Chromatin structure revisited.

Independently of the enormous progress in our understanding of the structure of the core particle, there remain a multitude of structural questions still to be answered. The main points discussed here can be summarized as follows: (1) The meaning of the term 'core particle' should be widened to reflect the fact that the actual length of DNA wrapped around the histone octamer in the context of the chromatin fiber may vary between approximately 100 and approximately 170 bp. (2) In the chromatosome, the linker histone forms a bridge between one terminus of the chromatosomal DNA and a point close to the dyad axis. (3) The particle that contains one molecule of HMG1 may be classified as a bona fide chromatosome. (4) In the extended fiber, the partition of the nucleosomal DNA into core and linker is a dynamic feature, responding to environmental influences; fiber structure-related constraints demand that linker length be beyond a certain minimal value. (5) The compact fiber structure seems to be rather irregular; the precise nature of this structure is still to be determined. Finally, the term 30-nm fiber should be dropped as a designator of the compact or condensed chromatin fiber structure.

Animals↗

PubMed1998

The non-histone chromatin protein HMG1 protects linker DNA on the side opposite to that protected by linker histones.

Linker histones and HMG1/2 constitute the two major proteins that bind to linker DNA in chromatin. While the location of linker histones on the nucleosome has attracted considerable research effort, only a few studies have addressed the location of HMG1 in the particles. In this study, we use a procedure based on micrococcal nuclease digestion of reconstituted nucleosomal particles to which HMG1 has been bound, followed by analysis of the protected DNA by restriction nuclease digestion, to locate the HMG1 binding site. Nucleosomal particles were reconstituted on a 235-base pair DNA fragment, which is known to be a strong nucleosome positioning sequence. The results unequivocally show that HMG1 protects linker DNA on one side of the core particle. Importantly, and possibly of physiological relevance, the linker DNA site protected by HMG1 was located on the side opposite to that already shown to be protected by linker histone binding.

Animals↗

PubMed1998

Differential silver-staining sodium dodecyl sulfate-polyacrylamide gel electrophoresis: a nonisotopic method for characterizing gel-separated histone-DNA complexes.

Some nonspecific, DNA-binding proteins, like the linker histones, precipitate DNA upon binding. This is a poorly understood process that limits analysis of such nucleoprotein complexes using standard gel electrophoresis. To circumvent this problem, low concentrations of glutaraldehyde were used to crosslink the linker histones to DNA; then the partially crosslinked complexes were solubilized in SDS2 and separated by SDS-PAGE. Differential detection was accomplished using two different silver staining protocols that preferentially stained either proteins or nucleic acids. A technique was developed which allows the relative proportion of linker histones and DNAs in each detected band to be determined, and is referred to as differential staining SDS-PAGE (DS-SDS-PAGE). DS-SDS-PAGE provides a novel, non-isotopic means for characterizing multiple nucleoprotein bands separated by polyacrylamide gel electrophoresis. In applying this method to a model linker histone-DNA study, we were able to detect both protein-DNA and protein-protein contacts that are important in linker histone assembly onto DNA.

Animals↗

PubMed1998

Self-association of linker histone H5 and of its globular domain: evidence for specific self-contacts.

The ability of avian-specific linker histone H5, and the globular domains of H5 (GH5) and H1(0) (GH1(0), to self-associate either free in solution or when bound to DNA was investigated. All three proteins underwent a salt-dependent increase in turbidity that may be indicative of nonspecific interactions. Dithiobis(succinimidyl propionate) cross-linking was used to measure specific contacts for both H5 and GH5 free in solution and bound to DNA. H5 and GH5 each became cross-linked in solution, with GH5 displaying divalent polymerization interactions, which suggests that two specific surfaces were involved in the assembly process. For GH5-DNA complexes, cross-linking appeared to be largely the consequence of aggregation, but under low concentrations of DSP, cross-linking GH5 was observed to assemble preferentially onto DNA before oligomerizing to form massive aggregates. Both linear and supercoiled DNA facilitated GH5 interactions compared to assembly in solution; differences in the distribution of cross-linked polymer sizes indicates that assembly is dependent on both the presence of DNA and the morphology of the DNA. Finally, on the basis of a technique referred to as quantitative proteolysis, GH5 assembly on DNA appears to involve specific protein-protein contacts involving the C terminus of one partner. Overall, the cumulative results reported here support the premise that linker histones assemble specifically both in solution and on DNA.

Animals↗

PubMed1998

Linker histone protection of chromatosomes reconstituted on 5S rDNA from Xenopus borealis:a reinvestigation.

The location of the linker histone (LH) on the nucleosome has been the subject of recent controversy. Although previous evidence had supported a location over the dyad axis, some recent experiments suggest an asymmetric, off-axis position. In this paper we show that the DNA sequence used to reconstitute chromatosomes in these experiments is prone to artifacts in nuclease digestion: results interpreted as 'protection' by LHs can be obtained with either naked DNA or with reconstituted core nucleosomes, in the absence of LHs. Consequently, we feel that general interpretation or extrapolation of such results must be regarded with the utmost caution. In addition, we show that the protection data on an alternative, previously unreported major core position on this same DNA sequence support a model of asymmetric, off-axis position of the LH, with linker DNA protection on only one side of the core particle.

Animals↗

PubMed1998

Linker histone protects linker DNA on only one side of the core particle and in a sequence-dependent manner.

The protection against micrococcal nuclease digestion afforded to chromatosomal DNA by the presence of a linker histone (H1(o)) has been quantitatively measured in two reconstituted systems. We have used chromatosomes reconstituted at two distinct positions on a DNA fragment containing the 5S rRNA gene from Lytechinus variegatus and at a specific position on a sequence containing Gal4- and USF-binding sites. In all cases, we find asymmetric protection, with approximately 20 bp protected on one side of the core particle and no protection on the other. We demonstrated through crosslinking experiments that the result is not due to any sliding of the histone core caused by either linker histone addition or micrococcal nuclease cleavage. Because the core particle is itself a symmetric object, the preferred asymmetric location of a linker histone must be dictated by unknown elements in the DNA sequence.

Animals↗

PubMed1998

Linker histones versus HMG1/2: a struggle for dominance?

The linker histones (H1, H1 zero, H5, etc.) and a group of abundant non-histone chromosomal proteins (HMG1/2) bind to linker DNA in chromatin and exhibit both generalized and specific effects on gene transcription. The two classes of proteins share many features of DNA binding behaviour, although they are structurally unrelated. While the linker histones and HMG1/2 exhibit direct competition in binding to such structures as four-way junction DNA, whether they compete for binding to the nucleosome has not been investigated. The possibility for either opposite or synergistic effects on gene regulation must be considered at this point.

Animals↗

PubMed1998

Chromatin fiber structure: morphology, molecular determinants, structural transitions.

Despite more than 20 years of research, the structure of the chromatin fiber and its molecular determinants remain enigmatic. Recent developments in high-resolution microscopic techniques, as well as the application of mathematical modeling to chromatin fiber structure, have allowed the acquisition of some new insights into the structure and its determinants. Here we present some of the newest data on the structure of the chromatin fiber in both its extended and compacted states, and bring together this new knowledge with older data in an attempt to provide a unified view of how chromatin components interact with each other to form its various conformations. The structural transitions that are believed to take place during transcriptional activation and its cessation are also discussed. It becomes obvious that despite some progress in our understanding of the fiber structure and its dynamics, huge gaps continue to exist. Bridging these gaps will require further improvements in already available techniques and the introduction of completely new approaches.

Acetylation↗

PubMed1998

Contributions of linker histones and histone H3 to chromatin structure: scanning force microscopy studies on trypsinized fibers.

Little is known about the mechanisms that organize linear arrays of nucleosomes into the three-dimensional structures of extended and condensed chromatin fibers. We have earlier defined, from scanning force microscopy (SFM) and mathematical modeling, a set of simple structural determinants of extended fiber morphology, the critical parameters being the entry-exit angle between consecutive linkers and linker length. Here we study the contributions of the structural domains of the linker histones (LHs) and of the N-terminus of histone H3 to extended fiber morphology by SFM imaging of progressively trypsinized chromatin fibers. We find that cleavage of LH tails is associated with a lengthening of the internucleosomal center-to-center distance, and that the somewhat later cleavage of the N-terminus of histone H3 is associated with a flattening of the fiber. The persistence of the "zigzag" fiber morphology, even at the latest stages of trypsin digestion, can be attributed to the retention of the globular domain of LH in the fiber.

Animals↗

PubMed1998

Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers.

The mechanisms responsible for organizing linear arrays of nucleosomes into the three-dimensional structure of chromatin are still largely unknown. In a companion paper (Leuba, S. H., et al. 1998. Biophys. J. 74:2823-2829), we study the contributions of linker histone domains and the N-terminal tail of core histone H3 to extended chromatin fiber structure by scanning force microscopy imaging of mildly trypsinized fibers. Here we complement and extend these studies by scanning force microscopy imaging of selectively reconstituted chromatin fibers, which differ in subtle but distinctive ways in their histone composition. We demonstrate an absolute requirement for the globular domain of the linker histones and a structural redundancy of the tails of linker histones and of histone H3 in determining conformational stability.

Animals↗

PubMed1998

Binding to four-way junction DNA: a common property of architectural proteins?

Proteins that can be shown to strongly bind in vitro to the four-way (Holliday) junction DNA include not only the obvious candidates such as enzymes involved in recombination, but also a remarkably diverse group of seemingly unrelated proteins. These include the HMG1 box proteins, members of the HMGI-Y family, winged helix proteins (including linker histones), the SWI/SNF complex, and some totally unrelated prokaryotic proteins. What these proteins seem to share is a propensity to bind to bent DNA, to bend DNA upon binding, and/or to preferentially interact with DNA crossings. Thus, they appear to be, in the main, architectural proteins, although some (like the SWI/SNF complex) have very specific functional roles as well. Perhaps because they bind to or promote the formation of particular DNA structures, the four-way junction binding proteins are frequently interchangeable in cellular function. Furthermore, since a given kind of structure can be recognized by many different protein motifs, it is not surprising that apparently unrelated proteins can fall into such a single functional class.

Chromatin↗

PubMed1997

The major chromatin protein histone H1 binds preferentially to cis-platinum-damaged DNA.

Both cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) and trans-diamminedichloroplatinum(II) form covalent adducts with DNA. However, only the cis isomer is a potent anticancer agent. It has been postulated that the selective action of cis-DDP occurs through specific binding of nuclear proteins to cis-DDP-damaged DNA sites and that binding blocks DNA repair. We find that a very abundant nuclear protein, the linker histone H1, binds much more strongly to cis-platinated DNA than to trans-platinated or unmodified DNA. In competition experiments, H1 is shown to bind much more strongly than HMG1, which had been previously considered a major candidate for such binding in vivo.

Animals↗

PubMed1997

Histone H1 preferentially binds to superhelical DNA molecules of higher compaction.

In chromatin, the physiological amount of H1 is one molecule per nucleosome or, roughly, one molecule per 200 bp of DNA. We observed that at such a stoichiometry, H1 selectively binds to supercoiled DNA with magnitude of sigma > or = 0.012 (both negative and positive), leaving relaxed, linear, or nicked DNA molecules unbound. When negative and positive DNA topoisomers of varying superhelicity are simultaneously present in the binding mixture, H1 selectively binds to the molecules with highest superhelicity; less supercoiled forms are gradually involved in binding upon increasing the amount of input protein. We explain this topological preference of H1 as the consequence of an increased probability for more than one H1-DNA contact provided by the supercoiling. The existence of simultaneous contacts of H1 with both intertwined DNA strands in the supercoiled DNA molecules is also inferred by topoisomerase relaxation of H1-DNA complexes that had been prefixed with glutaraldehyde.

Animals↗

PubMed1996

H1 binding unwinds DNA. Evidence from topological assays.

The preference of the linker histones to bind to superhelical DNA in comparison with linear or relaxed molecules suggests that these proteins might, in turn, change the twist and/or writhe of DNA molecules upon binding. In order to explore such a possibility, we looked for changes in the linking number of plasmid pBR322 caused by H1 binding, using assays that involve nicking and resealing of DNA strands. Two types of enzymes were used, eukaryotic topoisomerase I and prokaryotic DNA ligase. The results revealed that H1 binding causes unwinding of the DNA, with the unwinding angle being approximately 10 degrees . The globular domain of histone H1 is also capable of unwinding DNA, but to a lesser degree.

Animals↗

PubMed1996

What determines the folding of the chromatin fiber?

In this review, we attempt to summarize, in a critical manner, what is currently known about the processes of condensation and decondensation of chromatin fibers. We begin with a critical analysis of the possible mechanisms for condensation, considering both old and new evidence as to whether the linker DNA between nucleosomes bends or remains straight in the condensed structure. Concluding that the preponderance of evidence is for straight linkers, we ask what other fundamental process might allow condensation, and argue that there is evidence for linker histone-induced contraction of the internucleosome angle, as salt concentration is raised toward physiological levels. We also ask how certain specific regions of chromatin can become decondensed, even at physiological salt concentration, to allow transcription. We consider linker histone depletion and acetylation of the core histone tails, as possible mechanisms. On the basis of recent evidence, we suggest a unified model linking targeted acetylation of specific genomic regions to linker histone depletion, with unfolding of the condensed fiber as a consequence.

Acetylation↗