Single-molecule biochemistry coming of age.
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Journal Article↗
Biomedical subjects
J Zlatanova
Publications and source records attributed to J Zlatanova.
The archaeal histone-fold protein HMf organizes DNA into bona fide chromatin fibers.
BACKGROUND: The discovery of histone-like proteins in Archaea urged studies into the possible organization of archaeal genomes in chromatin. Despite recent advances, a variety of structural questions remain unanswered. RESULTS: We have used the atomic force microscope (AFM) with traditional nuclease digestion assays to compare the structure of nucleoprotein complexes reconstituted from tandemly repeated eukaryal nucleosome-positioning sequences and histone octamers, H3/H4 tetramers, and the histone-fold archaeal protein HMf. The data unequivocally show that HMf reconstitutes are indeed organized as chromatin fibers, morphologically indistinguishable from their eukaryal counterparts. The nuclease digestion patterns revealed a clear pattern of protection at regular intervals, again similar to the patterns observed with eukaryal chromatin fibers. In addition, we studied HMf reconstitutes on mononucleosome-sized DNA fragments and observed a great degree of similarity in the internal organization of these particles and those organized by H3/H4 tetramers. A difference in stability was observed at the level of mono-, di-, and triparticles between the HMf particles and canonical octamer-containing nucleosomes. CONCLUSIONS: The in vitro reconstituted HMf-nucleoprotein complexes can be considered as bona fide chromatin structures. The differences in stability at the monoparticle level should be due to structural differences between HMf and core histone H3/H4 tetramers, i.e., to the complete absence in HMf of histone tails beyond the histone fold. We speculate that the existence of core histone tails in eukaryotes may provide a greater stability to nucleosomal particles and also provide the additional ability of chromatin structure to regulate DNA function in eukaryotic cells by posttranslational histone tail modifications.
Animals↗
Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers.
Single chromatin fibers were assembled directly in the flow cell of an optical tweezers setup. A single lambda phage DNA molecule, suspended between two polystyrene beads, was exposed to a Xenopus laevis egg extract, leading to chromatin assembly with concomitant apparent shortening of the DNA molecule. Assembly was force-dependent and could not take place at forces exceeding 10 pN. The assembled single chromatin fiber was subjected to stretching by controlled movement of one of the beads with the force generated in the molecule continuously monitored with the second bead trapped in the optical trap. The force displayed discrete, sudden drops upon fiber stretching, reflecting discrete opening events in fiber structure. These opening events were quantized at increments in fiber length of approximately 65 nm and are attributed to unwrapping of the DNA from around individual histone octamers. Repeated stretching and relaxing of the fiber in the absence of egg extract showed that the loss of histone octamers was irreversible. The forces measured for individual nucleosome disruptions are in the range of 20-40 pN, comparable to forces reported for RNA- and DNA-polymerases.
Animals↗
DNA methylation-dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone.
Dynamic alterations in chromatin structure mediated by postsynthetic histone modifications and DNA methylation constitute a major regulatory mechanism in DNA functioning. DNA methylation has been implicated in transcriptional silencing, in part by inducing chromatin condensation. To understand the methylation-dependent chromatin structure, we performed atomic force microscope (AFM) studies of fibers isolated from cultured cells containing normal or elevated levels of m5C. Chromatin fibers were reconstituted on control or methylated DNA templates in the presence or absence of linker histone. Visual inspection of AFM images, combined with quantitative analysis of fiber structural parameters, suggested that DNA methylation induced fiber compaction only in the presence of linker histones. This conclusion was further substantiated by biochemical results.
3T3 Cells↗
Gel-immobilized microarrays of nucleic acids and proteins. Production and application for macromolecular research.
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Acrylic Resins↗
Single molecule force spectroscopy in biology using the atomic force microscope.
The importance of forces in biology has been recognized for quite a while but only in the past decade have we acquired instrumentation and methodology to directly measure interactive forces at the level of single biological macromolecules and/or their complexes. This review focuses on force measurements performed with the atomic force microscope. A general introduction to the principle of action is followed by review of the types of interactions being studied, describing the main results and discussing the biological implications.
Biophysics↗
Linker histone binding and displacement: versatile mechanism for transcriptional regulation.
In recent years, the connection between chromatin structure and its transcriptional activity has attracted considerable experimental effort. The post-translational modifications to both the core histones and the linker histones are finely tuned through interactions with transcriptional regulators and change chromatin structure in a way to allow transcription to occur. Here we review evidence for the involvement of linker histones in transcriptional regulation and suggest a scenario in which the reversible and controllable binding/displacement of proteins of this class to the nucleosome entry/exit point determine the accessibility of the nucleosomal DNA to the transcriptional machinery.
Acetylation↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
Linker histone binding to superhelical DNA: electrophoretic and filter binding studies.
It has been known for years that linker histones bind preferentially to supercoiled DNA. This preference has been demonstrated by a number of different techniques including deoxyribonucleoprotein electrophoresis, sedimentation, and filter binding under non-competitive conditions. In an attempt to further study this issue, we used one- and two-dimensional electrophoretic gels and filter binding under competitive conditions, with all DNA forms of interest being simultaneously present in the incubation mixture. Comparison between results obtained by the two methods showed that whereas the preference for superhelical molecules was clearly seen in the electrophoretic gels, the filter binding assay failed to reveal this preference. These results reveal limitations to the filter binding technique, which must be borne in mind in studies involving superhelical DNA molecules.
Animals↗