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D Lohr

Publications and source records attributed to D Lohr.

At least 19 recordsLinked to original sources
PubMed2005

A fluorescence resonance energy transfer-based probe to monitor nucleosome structure.

Nucleosomes are the basic units of eukaryotic chromatin structure. By restricting factor access to regulatory DNA sequences, nucleosomes significantly impact genomic processes such as transcription, and various mechanisms to alter nucleosome structure to relieve this repression have evolved. Both nucleosomes and processes that alter them are inherently dynamic in nature. Thus, studies of dynamics will be necessary to truly understand these relief mechanisms. We describe here the characteristics of a novel fluorescence resonance energy transfer-based reporter that can clearly signal the formation of a canonical nucleosome structure and follow conformational and compositional changes in that structure, both at the ensemble-average (bulk) and at the single molecule level. Labeled nucleosomes behave conformationally and thermodynamically like typical nucleosomes; thus they are relevant reporters of nucleosome behavior. Nucleosomes and free DNA are readily distinguishable at the single-molecule level. Thus, these labeled nucleosomes are well suited to studies of dynamic changes in nucleosome structure including single-molecule dynamics.

DNA Probes↗
PubMed2004

A statistical thermodynamic model applied to experimental AFM population and location data is able to quantify DNA-histone binding strength and internucleosomal interaction differences between acetylated and unacetylated nucleosomal arrays.

Imaging of nucleosomal arrays by atomic force microscopy allows a determination of the exact statistical distributions for the numbers of nucleosomes per array and the locations of nucleosomes on the arrays. This precision makes such data an excellent reference for testing models of nucleosome occupation on multisite DNA templates. The approach presented here uses a simple statistical thermodynamic model to calculate theoretical population and positional distributions and compares them to experimental distributions previously determined for 5S rDNA nucleosomal arrays (208-12,172-12). The model considers the possible locations of nucleosomes on the template, and takes as principal parameters an average free energy of interaction between histone octamers and DNA, and an average wrapping length of DNA around the octamers. Analysis of positional statistics shows that it is possible to consider interactions between nucleosomes and positioning effects as perturbations on a random positioning noninteracting model. Analysis of the population statistics is used to determine histone-DNA association constants and to test for differences in the free energies of nucleosome formation with different types of histone octamers, namely acetylated or unacetylated, and different DNA templates, namely 172-12 or 208-12 5S rDNA multisite templates. The results show that the two template DNAs bind histones with similar affinities but histone acetylation weakens the association of histones with both templates. Analysis of locational statistics is used to determine the strength of specific nucleosome positioning tendencies by the DNA templates, and the strength of the interactions between neighboring nucleosomes. The results show only weak positioning tendencies and that unacetylated nucleosomes interact much more strongly with one another than acetylated nucleosomes; in fact acetylation appears to induce a small anticooperative occupation effect between neighboring nucleosomes.

Acetylation↗
PubMed2004

Single-molecule recognition imaging microscopy.

Atomic force microscopy is a powerful and widely used imaging technique that can visualize single molecules and follow processes at the single-molecule level both in air and in solution. For maximum usefulness in biological applications, atomic force microscopy needs to be able to identify specific types of molecules in an image, much as fluorescent tags do for optical microscopy. The results presented here demonstrate that the highly specific antibody-antigen interaction can be used to generate single-molecule maps of specific types of molecules in a compositionally complex sample while simultaneously carrying out high-resolution topographic imaging. Because it can identify specific components, the technique can be used to map composition over an image and to detect compositional changes occurring during a process.

Animals↗
PubMed2004

Using atomic force microscopy to study nucleosome remodeling on individual nucleosomal arrays in situ.

In eukaryotes, genomic processes like transcription, replication, repair, and recombination typically require alterations in nucleosome structure on specific DNA regions to operate. ATP-dependent nucleosome remodeling complexes provide a major mechanism for carrying out such alterations in vivo. To learn more about the action of these important complexes, we have utilized an atomic force microscopy in situ technique that permits comparison of the same individual molecules before and after activation of a particular process, in this case nucleosome remodeling. This direct approach was used to look for changes induced by the action of the human Swi-Snf remodeling complex on individual, single-copy mouse mammary tumor virus promoter nucleosomal arrays. Using this technique, we detect a variety of changes on remodeling. Many of these changes are larger in scale than suggested from previous studies and involve a number of DNA-mediated events, including a preference for the removal of a complete turn (80 basepairs) of nucleosomal DNA. The latter result raises the possibility of an unanticipated mode of human Swi-Snf interaction with the nucleosome, namely via the 11-nm histone surface.

Adenosine Triphosphate↗
PubMed2003

Development of a fluorescent probe for the study of nucleosome assembly and dynamics.

To develop a probe for use in real-time dynamic studies of nucleosomes, core histones (from Drosophila) were conjugated to a DNA-intercalating dye, thiazole orange, by a reaction targeting Cys 110 of histone H3. In the absence of DNA, the conjugated histones are only very weakly fluorescent. However, upon reconstitution into nucleosomes by standard salt dialysis procedures, the probe fluoresces strongly, reflecting its ability to intercalate into the nucleosomal DNA. The probe is also sensitive to the nature of the DNA-histone interaction. Nucleosomes reconstituted by stepwise salt dialysis give a fluorescence signal quite different from that of the species formed when DNA and histones are simply mixed in low salt. In addition, changing either the DNA length or the type of sequence (nucleosome positioning sequences versus random DNA of the same size) used in the reconstitution alters the resulting fluorescence yield. The results are all consistent with the conclusion that a more rigid, less flexible nucleosome structure results in less fluorescence than a looser structure, presumably due to structural constraints on dye intercalation. This probe should be well suited to analyzing nucleosome dynamics and to following factor-mediated assembly and remodeling of nucleosomes in real time, particularly at the single-molecule level.

Animals↗
PubMed2003

Nucleosomal arrays can be salt-reconstituted on a single-copy MMTV promoter DNA template: their properties differ in several ways from those of comparable 5S concatameric arrays.

Subsaturated nucleosomal arrays were reconstituted on a single-copy MMTV promoter DNA fragment by salt dialysis procedures and studied by atomic force microscopy. Up to an occupation level of approximately eight nucleosomes on this 1900 bp template, salt reconstitution produces nucleosomal arrays which look very similar to comparably loaded 5S rDNA nucleosomal arrays; i.e., nucleosomes are dispersed on the DNA template. Thus, at these occupation levels, the single-copy MMTV template forms arrays suitable for biophysical analyses. A quantitative comparison of the population features of subsaturated MMTV and 5S arrays detects differences between the two: a requirement for higher histone levels to achieve a given level of nucleosome occupation on MMTV templates, indicating that nucleosome loading is thermodynamically less favorable on this template; a preference for pairwise nucleosome occupation of the MMTV (but not the 5S) template at midrange occupation levels; and an enhanced salt stability for nucleosomes on MMTV versus 5S arrays, particularly in the midrange of array occupation. When average occupation levels exceed approximately eight nucleosomes per template, MMTV arrays show a significant level of mainly intramolecular compaction; 5S arrays do not. Taken together, these results show clearly that the nature of the underlying DNA template can affect the physical properties of nucleosomal arrays. DNA sequence-directed differences in the physical properties of chromatin may have important consequences for functional processes such as gene regulation.

DNA↗
PubMed2002

Mapping nucleosome locations on the 208-12 by AFM provides clear evidence for cooperativity in array occupation.

Concatameric sea urchin 5S rDNA templates reconstituted with histones provide very popular chromatin models for many kinds of in vitro studies. We have used AFM to characterize the locational aspects of nucleosome occupation on one such array, the 208-12, by determining the internucleosomal- and end-distance distributions for arrays reconstituted to various subsaturating levels with nonacetylated or hyperacetylated HeLa histones. A simulation analysis of the experimental distributions confirms the qualitative conclusions and provides quantitative parameter values for the identified features. For nonacetylated arrays, the end-distance data demonstrate the nucleosome positioning ability of the 5S sequence and detect an enhanced preference for nucleosomes to bind at DNA termini. The internucleosomal-distance data provide clear evidence for cooperativity in nucleosome location on these templates, detectable even at subsaturated loading levels. Hyperacetylated arrays show no change in the preference of nucleosomes to bind at termini and a slight change in nucleosome positioning behavior but, most strikingly, little or no evidence for cooperativity in nucleosome location. Thus, acetylation of the N-terminal histone tails abolishes the cooperativity.

Animals↗
PubMed2002

Glutaraldehyde modified mica: a new surface for atomic force microscopy of chromatin.

We have found that mica surfaces functionalized with aminopropyltriethoxysilane and aldehydes bind chromatin strongly enough to permit stable and reliable solution imaging by atomic force microscopy. The method is highly reproducible, uses very small amounts of material, and is successful even with very light degrees of surface modification. This surface is far superior to the widely used aminopropyltriethoxysilane-derivatized mica surface and permits resolution of structure on the nanometer-scale in an aqueous environment, conditions that are particularly important for chromatin studies. For example, bound nucleosomal arrays demonstrate major structural changes in response to changes in solution conditions, despite their prior fixation (to maintain nucleosome loading) and tethering to the surface with glutaraldehyde. By following individual molecules through a salt titration in a flow-through cell, one can observe significant changes in apparent nucleosome size at lower [salt] and complete loss of DNA from the polynucleosomal array at high salt. The latter result demonstrates that the DNA component in these arrays is not constrained by the tethering. The former result is consistent with the salt-induced loss of histones observed in bulk solution studies of chromatin and demonstrates that even histone components of the nucleosome are somewhat labile in these fixed and tethered arrays. We foresee many important applications for this surface in future atomic force microscopy studies of chromatin.

Aluminum Silicates↗
PubMed2001

Population analysis of subsaturated 172-12 nucleosomal arrays by atomic force microscopy detects nonrandom behavior that is favored by histone acetylation and short repeat length.

Concatameric 5 S rDNA templates reconstituted in vitro into nucleosomal arrays provide very popular chromatin models for many kinds of studies. Here, atomic force microscopy is used to determine the population distributions for one such nucleosomal array, the 172-12, reconstituted to various subsaturated levels with nonacetylated or hyperacetylated HeLa histones. This array is a model for short linker length genomes and transcriptionally active and newly replicated chromatins. The analysis shows that as input histone levels increase, template occupation increases progressively as discrete population distributions. The distributions are random at low (n(av) < 4) and high (n(av) > 8) loadings but display specific nonrandom features, such as a deficit of molecules with one nucleosome more or less than the peak species in the distribution and enhanced distribution breadths, in the mid-range (n(av) = 4-8). Thus, the mid-range of occupation on polynucleosomal arrays may be a special range for chromatin structure and/or assembly. The mid-range nonrandom features are enhanced in distributions from short repeat (172-12) arrays, particularly for unacetylated chromatin, and in distributions from hyperacetylated chromatin, particularly for long repeat (208-12) arrays. Thus, short repeat length and acetylation can affect basic chromatin properties, like population tendencies, in very similar ways and therefore may cause similar changes in chromatin structure. Some possible effects are suggested. The data also indicate that it is thermodynamically more difficult for hyperacetylated nucleosomes to assemble onto the 172-12 templates, a result having implications for in vivo chromatin assembly.

Acetylation↗
PubMed2001

Intrinsically bent DNA in the promoter regions of the yeast GAAL1-10 and GAL80 genes.

Circular permutation analysis has detected fairly strong sites of intrinsic DNA bending on the promoter regions of the yeast GAL1-10 and GAL80 genes. These bends lie in functionally suggestive locations. On the promoter of the GAL1-10 structural genes, strong bends bracket nucleosome B, which lies between the UAS(G) and the GAL1 TATA. These intrinsic bends could help position nucleosome B. Nucleosome B plus two other promoter nucleosomes protect the TATA and start site elements in the inactive state of expression but are completely disrupted (removed) when GAL1-10 expression is induced. The strongest intrinsic bend ( approximately 70 degrees ) lies at the downstream edge of nucleosome B; this places it approximately 30 base pairs upstream of the GAL1 TATA, a position that could allow it to be involved in GAL1 activation in several ways, including the recruitment of a yeast HMG protein that is required for the normally robust level of GAL1 expression in the induced state (Paull, T., Carey, M., and Johnson, R. (1996) Genes Dev. 10, 2769-2781). On the regulatory gene GAL80, the single bend lies in the non-nucleosomal hypersensitive region, between a GAL80-specific far upstream promoter element and the more gene-proximal promoter elements. GAL80 promoter region nucleosomes contain no intrinsically bent DNA.

DNA, Fungal↗
PubMed2001

Yeast chromatin structure and regulation of GAL gene expression.

Yeast genomic DNA is covered by nucleosome cores spaced by short, discrete length linkers. The short linkers, reinforced by novel histone properties, create a number of unique and dynamic nucleosome structural features in vivo: permanent unpeeling of DNA from the ends of the core, an inability to bind even full 147 bp core DNA lengths, and facility to undergo a conformational transition that resembles the changes found in active chromatin. These features probably explain how yeast can maintain most of its genome in a transcribable state and avoid large-scale packaging away of inactive genes. The GAL genes provide a closely regulated system in which to study gene-specific chromatin structure. GAL structural genes are inactive without galactose but are highly transcribed in its presence; the expression patterns of the regulatory genes can account for many of the features of GAL structural gene control. In the inactive state, GAL genes demonstrate a characteristic promoter chromosomal organization; the major upstream activation sequence (UASG) elements lie in open, hypersensitive regions, whereas the TATA and transcription start sites are in nucleosomes. This organization helps implement gene regulation in this state and may benefit the organism. Induction of GAL expression triggers Gal4p-dependent upstream nucleosome disruption. Disruption is transient and can readily be reversed by a Gal80p-dependent nucleosome deposition process. Both are sensitive to the metabolic state of the cell. Induction triggers different kinds of nucleosome changes on the coding sequences, perhaps reflecting the differing roles of nucleosomes on coding versus promoter regions. GAL gene activation is a complex process involving multiple Gal4p activities, numerous positive and negative cofactors, and the histone tails. DNA bending and chromosomal architecture of the promoter regions may also play a role in GAL regulation. Regulator-mediated competition between nucleosomes and the TATA binding protein complex for the TATA region is probably a central aspect of GAL regulation and a focal point for the numerous factors and processes that contribute to it.

Chromatin↗
PubMed2000

Conformational transition in DNA on a cold surface.

The contour length of DNA fragments, deposited and imaged on mica under buffer, was measured as a function of deposition temperature. Extended DNA molecules (on Ni- and silane-treated surfaces) contract rapidly with falling temperature, approaching the contour length of A-DNA at 2 degrees C. The contraction is not unique to a specific sequence and does not occur in solution at 2 degrees C or on a surface at 25 degrees C, indicating that it arises from a combination of low temperature and surface contact. It is probably a consequence of reduced water activity at a cold surface.

Aluminum Silicates↗
PubMed1999

Evidence for nonrandom behavior in 208-12 subsaturated nucleosomal array populations analyzed by AFM.

Atomic force microscopy was used to determine the population distributions in reconstituted, subsaturated 208-12 nucleosomal arrays. The features found in these distributions vary with the average nucleosome loading per template molecule (n(av)): at n(av) < 4, the distributions show a single peak whose breadth is equal to that expected for a random loading process; at n(av) = 4-8, the distributions are broader than random distributions and are complex; i.e., they contain multiple peaks and/or shoulders. Moreover, the peaks/shoulders typically occur at two nucleosome intervals, i.e., 2, 4, 6 or 3, 5, 7 nucleosomes. This two-nucleosome periodicity is statistically significant. The precise cause for such discrete features within the distributions is unknown, but at least these features would seem to indicate some pairwise preference in nucleosome occupation at these loading levels. In these intermediate-level (n(av) = 4-8) distributions, the major peak contains a larger fraction of the total templates than a random nucleosome loading process would produce. This feature indicates that at these intermediate population levels there is some tendency for correlated nucleosome loading among the templates. Hyperacetylated nucleosomal arrays show only subtle differences in their population distributions compared to nonacetylated arrays and demonstrate the above features. AFM allows one to study unfixed chromatin arrays; we find that nucleosomes on the 208-12 template demonstrate significant lability when they are not glutaraldehyde-fixed.

Acetylation↗
PubMed1995

GAL4/GAL80-dependent nucleosome disruption/deposition on the upstream regions of the yeast GAL1-10 and GAL80 genes.

Metabolic reactivation (incubating spheroplasts with galactose and casamino acids) causes disruption of nucleosomes from the upstream regions of the yeast GAL1, GAL10, and GAL80 genes. The disruption is specific. It depends on the transcription activator Gal4; it only occurs in galactose-reactivated chromatin from galactose-grown cells; it only affects upstream region, gene-proximal nucleosomes. Due to this specificity and because some of the same regions have shown induction-dependent changes by in vivo analyses, we suggest that the nucleosome-disrupted structure produced by reactivation is the authentic chromatin structure for these regions under conditions of galactose-induced GAL1-10 and GAL80 expression. It is necessary to carry out a spheroplast reactivation treatment in order to observe this disrupted structure in nuclear chromatin because nucleosomes are redeposited onto these regions during the preliminary steps of nuclear isolation (cell harvest/spheroplast preparation) probably in response to the nonphysiological conditions associated with these steps. However, during the same isolation procedures in cells lacking Gal80 protein, there is no nucleosome deposition on these regions, and the in vivo disrupted structure remains present in the nuclear chromatin. Therefore, the nucleosome deposition process that operates in wild-type cells is dependent on Gal80 protein, defining another activity of this negative regulator.

Chromatin↗
PubMed1995

Transcriptional regulation in the yeast GAL gene family: a complex genetic network.

Regulation of the GAL structural genes in the yeast Saccharomyces cerevisiae is implemented by the products of GAL-specific (GAL4, GAL80, GAL3) and general (GAL11, SWI1, 2, 3, SNF5, 6, numerous glucose repression) genes. Recent work has 1) yielded significant new insights on the DNA binding and transcription activation/Gal80 protein binding functions of the Gal4 activator protein, 2) described the characterization of purified Gal4 protein-Gal80 protein complexes, 3) deconvoluted the multiple and complex glucose repression pathways acting on GAL genes, 4) suggested a new mechanism for the Gal3 protein-mediated induction of GAL structural gene expression, 5) introduced Gal1 protein, a structural gene product, into the regulation scheme, and 6) extended our already substantial understanding of GAL regulatory gene control. The mechanisms which control structural and regulatory gene expression in the GAL family are compared and GAL structural/regulatory gene chromatin structure is discussed.

Chromatin↗
PubMed1993

Chromatin structure and regulation of the eukaryotic regulatory gene GAL80.

The chromatin structure around the 5' end of the yeast regulatory gene GAL80 has been determined. The chromatin organization is very similar to that on the 5' regions of the GAL1-10 structural genes: a constitutive hypersensitive region containing the upstream activating sequence (UAS) element, and nucleosomes around this hypersensitive region. The downstream nucleosome, which is a positioned nucleosome, covers the TATA and transcription start sites. The nucleosome upstream of the hypersensitive region undergoes significant change when cells are grown in galactose, where GAL80 gene expression is induced to maximal levels. The change may be related to the induction process. GAL4 protein binds strongly to the GAL80 UAS in galactose-grown cells, less strongly in glycerol-grown cells, and not at all in glucose-grown cells. These data and published gene expression data are used to develop a model for the regulation of the GAL80 regulatory gene.

Chromatin↗
PubMed1993

Assembly and structural properties of subsaturated chromatin arrays.

Nucleosomes have been reconstituted onto dodecameric repeats of a 208-base pair nucleosome positioning sequence at various subsaturating molar ratios of histone octamer to DNA repeat (r). These reconstitutes have been characterized with respect to lattice site occupancy distributions, nucleosome positioning, chromatin folding, and histone octamer dissociation. Reconstitution at 0.3 < or = r < or = 0.9 produces subsaturated chromatin templates with distinct compositional limits, n +/- 2 nucleosomes, where n is the average number of nucleosomes present on the template at that r value. Nucleosomes on subsaturated arrays occupy the same set of specific positions within the repeat unit as they do on saturated templates, with only very slight differences in the relative frequency of occupation of the various positions. The nucleosomes on very subsaturated templates, i.e. r < or = 0.5, are generally spread out. However, some nucleosome clustering is observed on these very subsaturated templates. These results indicate that assembly of nucleosomes onto this DNA lattice is not a strongly cooperative process. In 200 mM NaCl, subsaturated reconstitutes exhibit reduced levels of folding and histone octamer dissociation compared with saturated arrays. These results provide a framework for understanding the structural features of regions of chromatin partially depleted of nucleosomes.

Animals↗