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J Widom

Publications and source records attributed to J Widom.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

Structure, dynamics, and function of chromatin in vitro.

The substrates for the essential biological processes of transcription, replication, recombination, DNA repair, and cell division are not naked DNA; rather, they are protein-DNA complexes known as chromatin, in one or another stage of a hierarchical series of compactions. These are exciting times for students of chromatin. New studies provide incontrovertible evidence linking chromatin structure to function. Exceptional progress has been made in studies of the structure of chromatin subunits. Surprising new dynamic properties have been discovered. And, much progress has been made in dissecting the functional roles of specific chromatin proteins and domains. This review focuses on in vitro studies of chromatin structure, dynamics, and function.

Animals↗
PubMed1997

Coupled-enzymatic assays for the rate and mechanism of DNA site exposure in a nucleosome.

The packaging of DNA in nucleosomes presents obstacles to the action of gene regulatory proteins and polymerases on their natural chromatin substrates. We recently reported that nucleosomes exist in a conformational equilibrium, transiently exposing stretches of their DNA off the histone surface. Such "site exposure" processes potentially provide the needed access of proteins to DNA in chromatin. However, the experiments that reveal site exposure are carried out on timescales of tens of minutes to hours. The actual rates of site exposure are not known. Here we use T7 RNA polymerase and exonuclease III as probes to obtain a more relevant lower bound on the rate of nucleosomal site exposure. We find that the organization of DNA into nucleosomes detectably slows the elongation rate of the polymerase, but that full-length elongation, which requires access to all of the DNA, occurs on the seconds timescale. Independent experiments with exonuclease III, which probes the outermost DNA segments only, similarly show that site exposure in these regions occurs on a timescale of seconds or faster. We conclude that site exposure is sufficiently rapid that it may play a role in the initial binding of regulatory proteins to nucleosomal target sites. These rapid rates argue against a nucleosome sliding model for the mechanism of site exposure. Surprisingly, the measured rates may be too slow to account for the known rates of polymerase elongation in vivo. Mechanisms by which polymerase progression through nucleosomes might be catalyzed are discussed.

DNA↗
PubMed1997

Cloning and characterization of lung-endothelial cell adhesion molecule-1 suggest it is an endothelial chloride channel.

Lung-endothelial cell adhesion molecule-1 (Lu-ECAM-1) is an endothelial cell surface molecule that mediates adhesion of metastatic melanoma cells to lung endothelium. Here we analyze the organization of the Lu-ECAM-1 protein complex, report the sequence of Lu-ECAM-1 cDNAs, and reveal a novel function of the protein. Lu-ECAM-1 immunopurified from bovine aortic endothelial cells (BAEC) consists of tightly associated glycoproteins of 90, 38, and 32 kDa, with minor components of 130 and 120 kDa. We present evidence that all of these protein species are encoded by a single open reading frame whose initial translation product is proteolytically processed to yield the other products. Correct processing in vitro was demonstrated by transfection of the longest cDNA into human embryonic kidney 293 cells; immunoblot analysis showed that the approximately 120-kDa precursor gave rise to 90- and 38-kDa products. RNA blots of BAEC mRNA detected messages in agreement with the sizes of the cDNA clones in addition to several of high molecular weight. DNA blot analysis showed that Lu-ECAM-1 is conserved throughout its length in all mammals tested, usually as a single or low copy gene. In the bovine, Lu-ECAM-1 protein is 88% identical to a calcium-dependent chloride channel described recently in tracheal epithelium, Ca-CC. Probes for Lu-ECAM-1 mRNA and protein confirmed the presence of a homolog in this tissue. We show that messages for both proteins are present in lung while only Ca-CC is present in trachea and only Lu-ECAM-1 is present in BAEC. These results suggest that endothelial cells express a chloride channel that is related to, but distinct from, that expressed in tracheal epithelium. They further suggest that an adhesion molecule can also be a chloride channel.

Amino Acid Sequence↗
PubMed1997

Chromatin: the nucleosome unwrapped.

The structure of the nucleosome core particle has been determined by X-ray crystallography at 2.8 A resolution. The structure has several significant surprises, and provides important new insights into the structure and function of chromatin.

Crystallography, X-Ray↗
PubMed1997

Nucleosome packaging and nucleosome positioning of genomic DNA.

The goals of this study were to assess the extent to which bulk genomic DNA sequences contribute to their own packaging in nucleosomes and to reveal the relationship between nucleosome packaging and positioning. Using a competitive nucleosome reconstitution assay, we found that at least 95% of bulk DNA sequences have an affinity for histone octamer in nucleosomes that is similar to that of randomly synthesized DNA; they contribute little to their own packaging at the level of individual nucleosomes. An equation was developed that relates the measured free energy to the fractional occupancy of specific nucleosome positions. Evidently, the bulk of eukaryotic genomic DNA is also not evolved or constrained for significant sequence-directed nucleosome positioning at the level of individual nucleosomes. Implications for gene regulation in vivo are discussed.

Animals↗
PubMed1996

The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism.

We have isolated the predominant cytoplasmic histone acetyltransferase activity from Saccharomyces cerevisiae. This enzyme acetylates the lysine at residue 12 of free histone H4 but does not modify histone H4 when packaged in chromatin. The activity contains two proteins, Hat1p and Hat2p. Hat1p is the catalytic subunit of the histone acetyltransferase and has an intrinsic substrate specificity that modifies lysine in the recognition sequence GXGKXG. The specificity of the enzyme in the yeast cytoplasm is restricted relative to recombinant Hat1p suggesting that it is negatively regulated in vivo. Hat2p, which is required for high affinity binding of the acetyltransferase to histone H4, is highly related to Rbap48, which is a subunit of the chromatin assembly factor, CAF-1, and copurifies with the human histone deacetylase HD1. We propose that the Hat2p/Rbap48 family serve as escorts of histone metabolism enzymes to facilitate their interaction with histone H4.

Acetylation↗
PubMed1996

Short-range order in two eukaryotic genomes: relation to chromosome structure.

Fourier transform techniques have been used to analyze the distributions of all ten independent DNA dinucleotide steps in two eukaryotic genomes and one prokaryotic genome, for periodicities of approximately 2 to 500 bp. The results reveal systematic deviations from random expectation for certain dinucleotide steps over this entire range of periodicities, together with striking peaks at certain spatial periodicities for particular dinucleotide steps. Several dinucleotides yield peaks at a periodicity of approximately 10.2 bp that are unique to the eukaryotic genomes. Certain members of this set of dinucleotide signals were previously identified as involved in nucleosome positioning, while others were previously unrecognized. In real-space, these dinucleotides are uncorrelated or even anticorrelated (relative to random expectation) at distances of 10 and 11 bp, despite having greater than random spectral power at the corresponding periodicity. Real-space correlations of these dinucleotides at distances of 10 and 11 bp are suppressed by another spectral component, a 3 bp periodicity attributed to codons, which has a local minimum probability at approximately 10.5 bp. When the two eukaryotic genomes are encoded for the signal "AA or TT", the peak at approximately 10.2 bp periodicity is strengthened, whereas for the prokaryotic genome such a peak remains absent. For the Caenorhabditis elegans genome, this peak becomes the dominant feature in the transform, surpassing a peak owing to the existence of codons in both height and integrated intensity. These results suggest that the requirements of chromosome structure place significant constraints on eukaryotic genome organization; they reveal additional signals that may be related to nucleosome positioning; and they reveal a wealth of additional new non-random aspects of genome sequence organization.

Animals↗
PubMed1996

A model for the cooperative binding of eukaryotic regulatory proteins to nucleosomal target sites.

The mechanism by which gene regulatory proteins gain access to their DNA target sequences in chromatin is not known. We recently showed that nucleosomes are intrinsically dynamic, transiently exposing their DNA to allow sequence-specific protein binding even at buried sites. Here we show that this dynamic behaviour provides a mechanism for cooperativity (synergy) in the binding of two or more proteins to sites on a single nucleosome, even if those proteins do not interact directly with each other in any way. As a consequence of this cooperativity, two proteins binding to the same nucleosome facilitate each other's binding and also control the level of occupancy at each other's sites. This model, with no adjustable parameters, accounts quantitatively for recent reports of cooperative (synergistic) binding to nucleosomes in vitro. We assess the potential importance of this new cooperativity for gene regulation in vivo by comparing its magnitude to free energies of cooperative protein-protein direct contacts having known significance for gene regulation. Possible roles for nucleosome dynamics in eukaryotic gene regulation, and key remaining questions, are discussed.

Chemical Phenomena↗
PubMed1996

Nucleosome transcription studied in a real-time synchronous system: test of the lexosome model and direct measurement of effects due to histone octamer.

We report the development of an alternative approach to studies on nucleosome transcription in vitro. This model system allows us to follow the real-time, synchronous and single passage of RNA polymerase molecules as they progress through DNA packaged in nucleosomes. Results are obtained using phage T7 RNA polymerase with reconstituted nucleosomes prepared from native histones or from histones in which the two H3 Cys 110 thiol groups have been oxidized to form a disulfide bridge. The lengths and concentrations of radiolabeled transcripts produced as a function of time provide direct measurements of the velocities of transcription on naked DNA and on the nucleosomal particles, and allow both relative and absolute efficiencies of initiation, elongation and completion to be determined. These direct measurements of reveal new features of the elongation process. The velocities of elongation on the nucleosomal templates are slightly but reproducibly slower than those on naked DNA. This difference is found to be due to a slight increase in pausing on the nucleosomal templates. Remarkably, the sites of this increased pausing on the nucleosomal templates are also pause sites on the naked DNA. The velocities of elongation on native or oxidized nucleosomal templates are found to be identical to within +/- 10%. We conclude that nucleosomes having covalently bound H3 molecules are substrates for transcription, suggesting that the splitting of the nucleosome postulated in the lexosome model of nucleosome transcription is not a necessary event. Interesting future applications of this methodology are discussed.

Animals↗
PubMed1995

Mechanism of protein access to specific DNA sequences in chromatin: a dynamic equilibrium model for gene regulation.

We present evidence for a mechanism by which regulatory proteins may gain access to their target DNA sequences in chromatin. In this model, nucleosomes are dynamic structures, transiently exposing stretches of their DNA. Regulatory proteins gain access to DNA target sites in the exposed state, and bind with an apparent dissociation constant equal to their dissociation constant for naked DNA divided by a position-dependent equilibrium constant for site exposure within the nucleosome. A sensitive assay, based on the kinetics of restriction digestion of sites within nucleosomes, reveals this dynamic behaviour and quantifies the equilibrium constants for site exposure. Our results have implications for many aspects of chromatin function. They offer new mechanisms for cooperativity (synergy) in regulatory protein binding and for active invasion of nucleosomes.

Base Sequence↗
PubMed1994

Kinetics of compaction during lysozyme refolding studied by continuous-flow quasielastic light scattering.

We recently developed an experiment, termed continuous-flow quasielastic light scattering (QLS), that is capable of monitoring the time evolution of the hydrodynamic diameter of macromolecules or macromolecular assemblies in solution. Here we report the use of this method to directly monitor the kinetics of compaction of the polypeptide chain of hen egg white lysozyme (HEWL) when protein refolding is initiated by 10-fold dilution from 5 M guanidine hydrochloride (GuHCl) at pH 1.5, 23 degrees C. Previously, such information could only be obtained indirectly, by analysis of the kinetics of binding ans release of a fluorescent probe dye. Refolding was also monitored by UV difference absorption spectroscopy to characterize the time scale of the formation of the native environment around the aromatic side chains under the same conditions used in the continuous-flow QLS experiments. We find that HEWL becomes compact within 1 s after the initiation of refolding, the shortest time that is accessible with our first-generation instrument. This time scale is shorter than that for the recovery of the native absorbances in the aromatic region. These results provide direct evidence that the intermediate on the folding pathway of lysozyme is compact. The implications of these results for models of protein folding are discussed.

Animals↗