Biomedical subjects

S Pennings

Publications and source records attributed to S Pennings.

12 recordsLinked to original sources

PubMed1997

CpG methylation remodels chromatin structure in vitro.

One of the mechanisms proposed to explain how CpG methylation effects gene repression invokes a DNA methylation-determined chromatin structure. Previous work implied that this DNA modification does not influence nucleosome formation in vitro, thus current models propose that certain non-histone proteins or a preferential affinity by linker histones for methylated DNA may mediate changes in chromatin structure. We have reinvestigated whether CpG methylation alters the chromatin structure of reconstitutes comprising only core histones and DNA. We find that DNA methylation prevents the histone octamer from interacting with an otherwise high affinity positioning sequence in the promoter region of the chicken adult beta-globin gene. This exclusion is attributed to methylation-determined changes in DNA structure within a triplet of CpG dinucleotides. In the affected nucleosome, this sequence motif is located 1.5 helical turns from the dyad axis and is oriented towards the histone core. These findings establish that DNA methylation does have the capacity to modulate chromatin structure directly, at its most fundamental level. Furthermore, our observations strongly suggest that a very limited number of nucleotides can make a decisive contribution to the translational positioning of nucleosomes.

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PubMed1997

Nucleoprotein gel electrophoresis for the analysis of nucleosomes and their positioning and mobility on DNA.

Nucleoprotein gel electrophoresis, as well as its use in chromatin research, is reviewed from its early application in the characterization of native nucleosome composition to current uses in analyzing transcription factor-nucleosome complexes and in visualizing multiple nucleosome positioning. Despite our incomplete understanding of the principles behind the separation of particles in native polyacrylamide, gels, powerful new applications of the method have emerged in recent years. This offers the potential for novel experimental strategies, such as those that have led to the discovery of nucleosome mobility.

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PubMed1995

Periodicity of strong nucleosome positioning sites around the chicken adult beta-globin gene may encode regularly spaced chromatin.

Positioned nucleosomes contribute to both the structure and the function of the chromatin fiber and can play a decisive role in controlling gene expression. We have mapped, at high resolution, the translational positions adopted by limiting amounts of core histone octamers reconstituted onto 4.4 kb of DNA comprising the entire chicken adult beta-globin gene, its enhancer, and flanking sequences. The octamer displays extensive variation in its affinity for different positioning sites, the range exhibited being about 2 orders of magnitude greater than that of the initial binding of the octamer. Strong positioning sites are located 5' and 3' of the globin gene and in the second intron but are absent from the coding regions. These sites exhibit a periodicity (approximately 200 bp) similar to the average spacing of nucleosomes on the inactive beta-globin gene in vivo, which could indicate their involvement in packaging the gene into higher-order chromatin structure. Overlapping, alternative octamer positioning sites commonly exhibit spacings of 20 and 40 bp, but not of 10 bp. These short-range periodicities could reflect features of the core particle structure contributing to the pronounced sequence-dependent manner in which the core histone octamer interacts with DNA.

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PubMed1995

Deposition of histone H1 onto reconstituted nucleosome arrays inhibits both initiation and elongation of transcripts by T7 RNA polymerase.

The effect of histone H1 on transcription by bacteriophage T7 RNA polymerase was examined using reconstituted chromatin templates. A 3.8 kb linear DNA template consisting of a specific transcription promoter for T7 RNA polymerase placed upstream of 18 tandem repeats of a 207 bp nucleosome positioning sequence derived from the 5S rRNA gene of Lytechinus variegatus was used as a template for chromatin reconstitution. Regularly spaced arrays of nucleosome cores were assembled onto this DNA template from donor histone octamers by salt step dialysis. Histone H1 was incorporated onto free DNA or reconstituted chromatin templates and double label transcription assays were performed. The experiments indicated that histone H1 has a strong inhibitory effect on both transcription initiation and elongation. These effects are especially pronounced on chromatin templates, where both transcription initiation and elongation are virtually halted. The inhibition of transcription elongation appears to result from a dramatic increase in premature termination of transcripts. These experiments indicate that assembly of histone H1 into chromatin can result in structures which are completely repressed with respect to transcription.

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PubMed1994

Linker histones H1 and H5 prevent the mobility of positioned nucleosomes.

We have previously identified a generally occurring short-range mobility of nucleosome cores on DNA in relatively low ionic strength conditions. Here we report that this mobility of histone octamers positioned on constructs of 5S rDNA is suppressed by the binding of histone H1 or H5 to the nucleosome. Histone H5 is the more potent inhibitor of nucleosome mobility, in accordance with its higher affinity for chromatin. We propose that this reversible restraint on chromatin dynamics may play a role in local regulation of processes that require access to the DNA.

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PubMed1992

Effect of glycerol on the separation of nucleosomes and bent DNA in low ionic strength polyacrylamide gel electrophoresis.

We report that glycerol changes the separation characteristics of polyacrylamide nucleoprotein gels in which it is included as a stabilizing agent. Polyacrylamide gel electrophoresis fractionates DNA and nucleosomes according to net negative charge, mass and conformation. With glycerol included, fractionation seems to be largely based on particle mass and charge. The conformation factor in separation is progressively lost with increasing glycerol concentrations. Nucleosome positions on the same DNA fragment are no longer resolved, while the difference in electrophoretic mobility between core particles and nucleosomes carrying longer DNA becomes smaller and is eventually lost. The retardation of bent DNA is also much reduced. Using the differences in separation characteristics between glycerol-containing and regular nucleoprotein gels could be a new means to obtain information on macromolecules in solution.

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PubMed1992

Mobile nucleosomes--a general behavior.

We have previously reported the mobility of positioned nucleosomes on sea urchin 5S rDNA. In this study we demonstrate the temperature dependence and the range of this mobility on 5S rDNA constructs. We find that this dynamic behavior also applies to bulk mononucleosomes and nucleosomes reconstituted onto sequences of the Alu family of ubiquitous repeats. We conclude that short range sliding is potentially a general phenomenon that is dependent on the underlying sequence and its position on the histone octamer. The nucleoprotein gel analysis used also reveals the dramatic effect on gel electrophoretic migration caused by the location of the histone octamer on DNA fragments. The usefulness of this technique for studying nucleosome positioning and its dynamics is demonstrated.

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PubMed1991

Chromatosome positioning on assembled long chromatin. Linker histones affect nucleosome placement on 5 S rDNA.

Long chromatin containing linker histones H1 or H5 was assembled on tandemly repeated 172 or 207 base-pair nucleosome positioning sequences from a sea urchin 5 S RNA gene. The effects of H1 and H5 on spacing and positioning of nucleosomes were assessed. In the absence of linker histones, precise determinations of core particle boundaries showed that, although a large proportion of the histone octamers occupy a unique position, there is a small group of other, less populated sites located around this major site. The dominant position was found 10 to 15 base-pairs upstream from the unique position previously reported for the histone octamer on the monomer 260 base-pair sequence. Linker histones do not override the underlying DNA signals that induce the very regular spacing of nucleosomes in chromatins assembled on these strongly positioning multimer DNA sequences. They were nevertheless found to be decisive in determining the chromatosome positions and their distributions, and as such define the chromatosome as a positioning entity.

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PubMed1991

Mobility of positioned nucleosomes on 5 S rDNA.

We report on a dynamic aspect of nucleosome positioning, in the absence of transcription-related events, on sea urchin 5 S rDNA. On tandem repeats of nucleosome length DNA of this strongly positioning sequence, histone octamers assemble in one dominant position surrounded by minor positions, ten base-pairs apart and therefore with identical rotational setting of the DNA coil. The existence of this cluster of positions, determined using micrococcal nuclease is confirmed by the results from DNase I footprinting and restriction enzyme analysis. The results from these techniques and from two-dimensional nucleoprotein polyacrylamide gel analysis indicate that the cluster of octamer positions is in dynamic equilibrium, in low ionic conditions, suggesting that the minor positions reflect fluctuations around the major nucleosome site. Histone octamer mobility appears to be temperature dependent and is reversibly inhibited by Mg2+.

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PubMed1989

Formation, stability and core histone positioning of nucleosomes reassembled on bent and other nucleosome-derived DNA.

DNA originating from chicken erythrocyte mononucleosomes was cloned and sequenced. The properties of nucleosome reconstruction were compared for two cloned inserts, selected on account of their interesting sequence organization, length and difference in DNA bending. Cloned fragment 223 (182 base-pairs) carries alternatively (A)3-4 and (T)4-5 runs approximately every ten base-pairs and is bent; cloned fragment 213 (182 base-pairs) contains a repeated C4-5ATAAGG consensus sequence and is apparently not bent. Our experiments indicate the preference of the bent DNA fragment 223 over fragment 213 to associate in vitro with an octamer of histones under stringent conditions. We provide evidence that the in vitro nucleosome formation is hampered in the case of fragment 213, whereas the reconstituted nucleosomes were equally stable once formed. For the correct determination of the positioning of the histone octamer with regard to the two nucleosome-derived cloned DNA sequences, the complementary use of micrococcal nuclease, exonuclease III and DNase I is a prerequisite. No unique, but rotationally related, positions of the histone octamer were found on these nucleosome-derived DNA fragments. The sequence-dependent anisotropic flexibility, as well as intrinsic bending of the DNA, resulting in a rotational setting of the DNA fragments on the histone core, seems to be a strong determinant for the allowed octamer positions, Exonuclease III digestion indicates a different histone-DNA association when oligo(d(C.G)n) stretches are involved. The apparent stagger near oligo(d(A.T)n) stretches generated by DNase I digestion on reconstituted nucleosome 223 was found to be inverted from the normal two-base 3' overhang to a two-base 5' overhang. Two possibilities of the oligo(d(A.T)n) minor groove location relative to the histone core are envisaged to explain this anomaly in stagger.

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PubMed1986

Limitations of the poly(glutamic acid) reconstitution method in the reassembly of mono- and dinucleosomes.

Reconstitution of mononucleosomes and dinucleosomes at physiological ionic strength by means of poly(glutamic acid) is not efficient at physiological histone octamer:DNA ratios, unlike that with the salt dialysis method. The shorter the DNA is, the less transfer of octamers from poly(glutamic acid) to DNA occurs. By increasing the octamer:DNA ratio it is possible to involve all the DNA in the assembly, but for DNA longer than core particle length, nucleoprotein particles containing extra histones are concomitantly generated. Except for core particle and chromatosome lengths of DNA reassembled at 0.6:1 or 1:1 octamer:DNA ratio (and thus with low yield), reconstituted nucleoprotein particles proved to be different from native nucleosomes by their insolubility upon isolation. In the aggregates, DNA ends seemed to be sufficiently loose to allow exonuclease III digestion up to a certain limit. This resulted in patterns that for some cloned DNA fragments could give the impression, without knowledge of the above, of resulting from a unique octamer position. In view of the small range of length of DNA and the low yield of faithful reconstitution, the assembly method using poly(glutamic acid) is only of limited use in mono- or dinucleosome reconstitution experiments, at least in our hands.

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PubMedDate not supplied

Comparative filter binding study of H5 to nucleosome core particles, H1, H5 depleted chromatosomes and DNA fragments.

The filter-binding technique with PEI treated glass fiber is used to study the interaction of histone H5 to core particles, chromatosomes and DNA derived from it. By working at very low concentrations of interacting particles we are able to study the effective binding process independent of interfering insoluble complexes. The interactions are characterized by a very high affinity. An intrinsically higher affinity of H5 for cores and chromatosomes versus chromatosome derived DNA is demonstrated. Both chromatosomes and DNA derived from these bind about twice the amount as compared to core particles, which saturate at about one H5 per core particle.

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