PubMed Health⌕ Search

Biomedical subjects

J Widom

Publications and source records attributed to J Widom.

At least 19 recordsLinked to original sources
PubMed2002

Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation.

Intrinsic nucleosome dynamics termed "site exposure" provides spontaneous and cooperative access to buried regions of nucleosomal DNA in vitro. Two different mechanisms for site exposure have been proposed, one based on nucleosome translocation, the other on dynamic nucleosome conformational changes in which a stretch of the nucleosomal DNA is transiently released off the histone surface. Here we report on three experiments that distinguish between these mechanisms. One experiment investigates the effects on the accessibilities of restriction enzyme target sites inside nucleosomes when extra DNA (onto which the nucleosome may move at low energetic cost) is appended onto one end. The other two experiments test directly for nucleosome mobility under the conditions used to probe accessibility to restriction enzymes: one on a selected nonnatural nucleosome positioning sequence, the other on the well-studied 5S rRNA gene nucleosome positioning sequence. We find from all three assays that restriction enzymes gain access to sites throughout the entire length of the nucleosomal DNA without contribution from nucleosome translocation. We conclude that site exposure in nucleosomes in vitro occurs via a nucleosome conformational change that leads to transient release of a stretch of DNA from the histone surface, most likely involving progressive uncoiling from an end. Recapture at a distal site along DNA that has partially uncoiled would result in looped structures which are believed to contribute to RNA polymerase elongation and may contribute to spontaneous or ATP-driven nucleosome mobility. Transient open states may facilitate the initial entry of transcription factors and enzymes in vivo.

Animals↗
PubMed2001

Polymer reptation and nucleosome repositioning.

We consider how beads can diffuse along a chain that wraps them, without becoming displaced from the chain; our proposed mechanism is analogous to the reptation of "stored length" in more familiar situations of polymer dynamics. The problem arises in the case of globular aggregates of proteins (histones) that are wound by DNA in the chromosomes of plants and animals; these beads (nucleosomes) are multiply wrapped and yet are able to reposition themselves over long distances, while remaining bound by the DNA chain.

Animals↗
PubMed2001

Effects of histone acetylation on the equilibrium accessibility of nucleosomal DNA target sites.

Posttranslational acetylation of the conserved core histone N-terminal tail domains is linked to gene activation, but the molecular mechanisms involved are not known. In an earlier study we showed that removing the tail domains altogether by trypsin proteolysis (which leaves nucleosomes nevertheless intact) leads to 1.5 to 14-fold increases in the dynamic equilibrium accessibility of nucleosomal DNA target sites. These observations suggested that, by modestly increasing the equilibrium accessibility of buried DNA target sites, histone acetylation could result in an increased occupancy by regulatory proteins, ultimately increasing the probability of transcription initiation. Here, we extend these observations to a more natural system involving intact but hyperacetylated nucleosomes. We find that histone hyperacetylation leads to 1.1 to 1.8-fold increases in position-dependent equilibrium constants for exposure of nucleosomal DNA target sites, with an average increase of 1.4(+/-0.1)-fold. The mechanistic and biological implications of these results are discussed.

Acetylation↗
PubMed2001

Poly(dA-dT) promoter elements increase the equilibrium accessibility of nucleosomal DNA target sites.

Polypurine tracts are important elements of eukaryotic promoters. They are believed to somehow destabilize chromatin, but the mechanism of their action is not known. We show that incorporating an A(16) element at an end of the nucleosomal DNA and further inward destabilizes histone-DNA interactions by 0.1 +/- 0.03 and 0.35 +/- 0.04 kcal mol(-1), respectively, and is accompanied by 1.5- +/- 0.1-fold and 1.7- +/- 0.1-fold increases in position-averaged equilibrium accessibility of nucleosomal DNA target sites. These effects are comparable in magnitude to effects of A(16) elements that correlate with transcription in vivo, suggesting that our system may capture most of their physiological role. These results point to two distinct but interrelated models for the mechanism of action of polypurine tract promoter elements in vivo. Given a nucleosome positioned over a promoter region, the presence of a polypurine tract in that nucleosome's DNA decreases the stability of the DNA wrapping, increasing the equilibrium accessibility of other DNA target sites buried inside that nucleosome. Alternatively (if nucleosomes are freely mobile), the presence of a polypurine tract provides a free energy bias for the nucleosome to move to alternative locations, thereby changing the equilibrium accessibilities of other nearby DNA target sites.

Binding Sites↗
PubMed2000

Archaeal histone selection of nucleosome positioning sequences and the procaryotic origin of histone-dependent genome evolution.

Archaeal histones and the eucaryal (eucaryotic) nucleosome core histones have almost identical histone folds. Here, we show that DNA molecules selectively incorporated by rHMfB (recombinant archaeal histone B from Methanothermus fervidus) into archaeal nucleosomes from a mixture of approximately 10(14) random sequence molecules contain sequence motifs shown previously to direct eucaryal nucleosome positioning. The dinucleotides GC, AA (=TT) and TA are repeated at approximately 10 bp intervals, with the GC harmonic displaced approximately 5 bp from the AA and TA harmonics [(GCN(3)AA or TA)(n)]. AT and CG were not strongly selected, indicating that TA not equalAT and GC not equalCG in terms of facilitating archaeal nucleosome assembly. The selected molecules have affinities for rHMfB ranging from approximately 9 to 18-fold higher than the level of affinity of the starting population, and direct the positioned assembly of archaeal nucleosomes. Fourier-transform analyses have revealed that AA dinucleotides are much enriched at approximately 10. 1 bp intervals, the helical repeat of DNA wrapped around a nucleosome, in the genomes of Eucarya and the histone-containing Euryarchaeota, but not in the genomes of Bacteria and Crenarchaeota, procaryotes that do not have histones. Facilitating histone packaging of genomic DNA has apparently therefore imposed constraints on genome sequence evolution, and since archaeal histones have no structure in addition to the histone fold, these constraints must result predominantly from histone fold-DNA contacts. Based on the three-domain universal phylogeny, histones and histone-dependent genome sequence evolution most likely evolved after the bacterial-archaeal divergence but before the archaeal-eucaryal divergence, and were subsequently lost in the Crenarchaeota. However, with lateral gene transfer, the first histone fold could alternatively have evolved after the archaeal-eucaryal divergence, early in either the euryarchaeal or eucaryal lineages.

Archaea↗
PubMed2000

Effects of core histone tail domains on the equilibrium constants for dynamic DNA site accessibility in nucleosomes.

The N and C-terminal tail domains of the core histones play important roles in gene regulation, but the mechanisms through which they act are not known. These tail domains are highly positively charged and are the sites of numerous post-translational modifications, including many sites for lysine acetylation. Nucleosomes in which these tail domains have been removed by trypsin remain otherwise intact, and are used by many laboratories as a model system for highly acetylated nucleosomes. Here, we test the hypothesis that one role of the tail domains is to directly regulate the accessibility of nucleosomal DNA to other DNA-binding proteins. Three assays are used: equilibrium binding by a site-specific, DNA-binding protein, and dynamic accessibility to restriction enzymes or to a non-specific exonuclease. The effects of removal of the tail domains as monitored by each of these assays can be understood within the framework of the site exposure model for the dynamic equilibrium accessibility of target sites located within the nucleosomal DNA. Removal of the tail domains leads to a 1.5 to 14-fold increase in position-dependent equilibrium constants for site exposure. The smallness of the effect weighs against models for gene activation in which histone acetylation is a mandatory initial event, required to facilitate subsequent access of regulatory proteins to nucleosomal DNA target sites. Alternative roles for histone acetylation in gene regulation are discussed.

Acetylation↗
PubMed2000

The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis.

Mutations in palmitoyl-protein thioesterase 1 (PPT1), a lysosomal enzyme that removes fatty acyl groups from cysteine residues in modified proteins, cause the fatal inherited neurodegenerative disorder infantile neuronal ceroid lipofuscinosis. The accumulation of undigested substrates leads to the formation of neuronal storage bodies that are associated with the clinical symptoms. Less severe forms of PPT1 deficiency have been found recently that are caused by a distinct set of PPT1 mutations, some of which retain a small amount of thioesterase activity. We have determined the crystal structure of PPT1 with and without bound palmitate by using multiwavelength anomalous diffraction phasing. The structure reveals an alpha/beta-hydrolase fold with a catalytic triad composed of Ser115-His289-Asp233 and provides insights into the structural basis for the phenotypes associated with PPT1 mutations.

Amino Acid Sequence↗
PubMed2000

Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites.

We have previously shown that nucleosomes are conformationally dynamic: DNA sequences that in the time-average are buried inside nucleosomes are nevertheless transiently accessible, even to large proteins (or any other macromolecule). We refer to this dynamic behavior as "site exposure". Here we show that: (i) the equilibrium constants describing this dynamic site exposure decrease progressively from either end of the nucleosomal DNA in toward the middle; and (ii) these position-dependent equilibrium constants are strongly dependent on the nucleosomal DNA sequence. The progressive decrease in equilibrium constant with distance inside the nucleosome supports the hypothesis that access to sites internal to a nucleosome is provided by progressive (transient) release of DNA from the octamer surface, starting from one end of the nucleosomal DNA. The dependence on genomic DNA sequence implies that a specific genomic DNA sequence could be a major determinant of target site occupancies achieved by regulatory proteins in vivo, by either governing the time-averaged accessibility for a given nucleosome position, or biasing the time-averaged positioning (of mobile nucleosomes), which in turn is a major determinant of site accessibility.

DNA↗
PubMed2000

Rhizobium etli CE3 carries vir gene homologs on a self-transmissible plasmid.

RosR is a transcriptional regulator important for determining cell-surface characteristics and nodulation competitiveness in Rhizobium etli CE3. We identified a 15-kb region that contains genes with similarity to members of the virB, virC, virG, and virE operons of Agrobacterium tumefaciens and demonstrated that RosR directly regulates one operon in this region. These genes were located on plasmid pa of R. etli CE3, which is self-transmissible between R. etli and A. tumefaciens.

Base Sequence↗
PubMed2000

Effects of histone tail domains on the rate of transcriptional elongation through a nucleosome.

The N-terminal tail domains of the core histones play important roles in gene regulation, but the exact mechanisms through which they act are not known. Recent studies suggest that the tail domains may influence the ability of RNA polymerase to elongate through the nucleosomal DNA and, thus, that posttranslational modification of the tail domains may provide a control point for gene regulation through effects on the elongation rate. We take advantage of an experimental system that uses bacteriophage T7 RNA polymerase as a probe for aspects of nucleosome transcription that are dominated by the properties of nucleosomes themselves. With this system, experiments can analyze the synchronous, real-time, single-passage transcription on the nucleosomal template. Here, we use this system to directly test the hypothesis that the tail domains may influence the "elongatability" of nucleosomal DNA and to identify which of the tail domains may contribute to this. The results show that the tail domains strongly influence the rate of elongation and suggest that the effect is dominated by the N-terminal domains of the (H3-H4)(2) tetramer. They further imply that tail-mediated octamer transfer is not essential for elongation through the nucleosome. Acetylation of the tail domains leads to effects on elongation that are similar to those arising from complete removal of the tail domains.

Amino Acid Sequence↗
PubMed1999

Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences.

Our laboratories recently completed SELEX experiments to isolate DNA sequences that most-strongly favor or disfavor nucleosome formation and positioning, from the entire mouse genome or from even more diverse pools of chemically synthetic random sequence DNA. Here we directly compare these selected natural and non-natural sequences. We find that the strongest natural positioning sequences have affinities for histone binding and nucleosome formation that are sixfold or more lower than those possessed by many of the selected non-natural sequences. We conclude that even the highest-affinity sequence regions of eukaryotic genomes are not evolved for the highest affinity or nucleosome positioning power. Fourier transform calculations on the selected natural sequences reveal a special significance for nucleosome positioning of a motif consisting of approximately 10 bp periodic placement of TA dinucleotide steps. Contributions to histone binding and nucleosome formation from periodic TA steps are more significant than those from other periodic steps such as AA (=TT), CC (=GG) and more important than those from the other YR steps (CA (=TG) and CG), which are reported to have greater conformational flexibility in protein-DNA complexes even than TA. We report the development of improved procedures for measuring the free energies of even stronger positioning sequences that may be isolated in the future, and show that when the favorable free energy of histone-DNA interactions becomes sufficiently large, measurements based on the widely used exchange method become unreliable.

Animals↗
PubMed1998

Structure of human methionine aminopeptidase-2 complexed with fumagillin.

The fungal metabolite fumagillin suppresses the formation of new blood vessels, and a fumagillin analog is currently in clinical trials as an anticancer agent. The molecular target of fumagillin is methionine aminopeptidase-2 (MetAP-2). A 1.8 A resolution crystal structure of free and inhibited human MetAP-2 shows a covalent bond formed between a reactive epoxide of fumagillin and histidine-231 in the active site of MetAP-2. Extensive hydrophobic and water-mediated polar interactions with other parts of fumagillin provide additional affinity. Fumagillin-based drugs inhibit MetAP-2 but not MetAP-1, and the three-dimensional structure also indicates the likely determinants of this specificity. The structural basis for fumagillin's potency and specificity forms the starting point for structure-based drug design.

Amino Acid Sequence↗
PubMed1998

Chromatin structure: linking structure to function with histone H1.

A recent study has determined the position and orientation in the nucleosome of the H1 variant 'linker histone' H5; the results focus attention on the unknown function of this highly abundant nuclear protein, and highlight the question of whether H1 is primarily an architectural or a gene-regulatory protein.

Animals↗
PubMed1998

New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.

DNA sequences that position nucleosomes are of increasing interest because of their relationship to gene regulation in vivo and because of their utility in studies of nucleosome structure and function in vitro. However, at present our understanding of the rules for DNA sequence-directed nucleosome positioning is fragmentary, and existing positioning sequences have many limitations. We carried out a SELEX experiment starting with a large pool of chemically synthetic random. DNA molecules to identify those individuals having the highest affinity for histone octamer. A set of highest-affinity molecules were selected, cloned, and sequenced, their affinities (free energies) for histone octamer in nucleosome reconstitution measured, and their ability to position nucleosomes in vitro assessed by native gel electrophoresis. The selected sequences have higher affinity than previously known natural or non-natural sequences, and have a correspondingly strong nucleosome positioning ability. A variety of analyses including Fourier transform, real-space correlation, and direct counting computations were carried out to assess non-random features in the selected sequences. The results reveal sequence rules that were already identified in earlier studies of natural nucleosomal DNA, together with a large set of new rules having even stronger statistical significance. Possible physical origins of the selected molecules' high affinities are discussed. The sequences isolated in this study should prove valuable for studies of chromatin structure and function in vitro and, potentially, for studies in vivo.

Base Composition↗