Andor Udvardy

The 87A7 chromomere: Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains☆

The chromatin fiber of eukaryotic chromosomes is thought to be organized into a series of discrete domains or loops. To learn more about these large-scale structures, we have examined the sequence and chromatin organization of the DNA segments surrounding the two hsp 70 genes at the Drosophila melanogaster cytogenetic locus 87A7. These studies indicate that this heat shock locus is flanked on both the proximal and distal sides by novel chromatin structures, which we have called, respectively, scs and scs (specialized chromatin structures). Each structure is defined by two sets of closely spaced nuclease-hypersensitive sites arranged around a central nuclease-resistant segment. Our findings suggest that these two structures define the proximal and distal boundaries of the 87A7 chromomere and, hence, may be one of the first examples of anchor points for the organization of eukaryotic chromosomes into a series of discrete higher order domains. Moreover, these structures may provide focal points both for the decondensation of the chromomere when the hsp 70 genes are induced by heat shock and for the subsequent rewinding and condensation of the chromomere during recovery from heat shock.

Novel partitioning of DNA cleavage sites for Drosophila topoisomerase II

We have examined the long-range distribution of double-stranded DNA cleavage sites for Drosophila melanogaster topoisomerase II. These studies reveal a novel partitioning of preferred topoisomerase II cleavage sites. In the eukaryotic DNAs examined, major cleavage sites were typically found in nontranscribed spacer segments and close to the 5 and 3 boundaries of genes. In contrast, there were few if any prominent cleavage sites within genes. In addition, most of the major topoisomerase II cleavage sites closely corresponded to naked DNA hypersensitive sites for the prokaryotic enzyme, micrococcal nuclease.

Chromatin structure, not DNA sequence specificity, is the primary determinant of topoisomerase II sites of action in vivo.

In the studies reported here we have used topoisomerase II as a model system for analyzing the factors that determine the sites of action for DNA-binding proteins in vivo. To localize topoisomerase II sites in vivo we used an inhibitor of the purified enzyme, the antitumor drug VM-26. This drug stabilizes an intermediate in the catalytic cycle, the cleavable complex, and substantially stimulates DNA cleavage by topoisomerase II. We show that lysis of VM-26 treated tissue culture cells with sodium dodecyl sulfate induces highly specific double-strand breaks in genomic DNA, and we present evidence indicating that these double-strand breaks are generated by topoisomerase II. Using indirect end labeling to map the cleavage products, we have examined the in vivo sites of action of topoisomerase II in the 87A7 heat shock locus, the histone repeat, and a tRNA gene cluster at 90BC. Our analysis reveals that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase II site selection in vivo. We suggest that chromatin organization may provide a general mechanism for generating specificity in a wide range of DNA-protein interactions in vivo.

Novobiocin blocks the Drosophila heat shock response

In the studies reported here we show that the antibiotic novobiocin, an in vitro inhibitor of topoisomerase II, blocks the Drosophila heat shock response. If novobiocin is added prior to induction, there is no detectable expression of the Drosophila heat shock genes. Moreover, analysis of the chromatin organization of the 87A7 heat shock locus indicates that the antibiotic prevents the structural alterations which normally accompany heat induction. When novobiocin is added after induction, transcription appears to be rapidly turned off, and the chromatin organization of the 87A7 locus is fixed in an active configuration. Novobiocin also prevents the re-establishment of the pre-induced 87A7 chromatin organization which occurs during recovery from heat shock. We have also presented data suggesting that this antibiotic blocks transcription at 25 degrees C. These findings raise the possibility that topoisomerase II may be required in eukaryotes for both gene activation and deactivation.

Chromatin organization of the 87A7 heat shock locus of Drosophila melanogaster.

We have examined the chromatin structure of the hsp 70 gene complex at the 87A7 heat shock locus of Drosophila melanogaster. Our results indicate that this locus has a complex chromatin organization. Heat induction causes highly specific alterations in the chromatin throughout the locus. There are major changes within the heat shock gene transcription units, and in both the upstream and downstream flanking spacers.

Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.

The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery.

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.

Transcriptionally active chromatin is sensitive to Neurospora crassa and S1 nucleases

We have examined the distribution of Neurospora crassa and S1 nuclease cleavage products in the chromatin of the 87A7 heat shock locus of Drosophila melanogaster. Both of these nucleases generate single and double-strand breaks in chromatin at specific sites in the 87A7 locus. Before heat induction, we find that the 5 ends of the two 87A7 hsp 70 genes contain N. crassa and S1 nuclease hypersensitive sites, while there are only a few cleavage products from elsewhere in the locus. With N. crassa nuclease, we observe one major 5 fragment, and this is derived from cleavage in a DNA segment mapping about 90 to 115 base-pairs from the beginning of the transcription unit. With S1 nuclease, we find two 5 cleavage products. The first maps about 120 to 130 base-pairs from the beginning of the gene. Interestingly, this site is also sensitive to S1 nuclease in supercoiled but not linear naked DNA. The other fragment maps very close to the transcription start site (approximately 0 to -15 base-pairs). After heat induction, there is a transition in the chromatin architecture of 87A7. First, there is a marked reduction in the yield of the prominent 5 N. crassa and S1 nuclease fragments. Second, the entire hsp 70 gene, as well as the spacer DNA just downstream from the 3 end of the gene, becomes highly sensitive to both of these nucleases.

Ribosomal RNA genes of Drosophila melanogaster have a novel chromatin structure

We have examined the chromatin organization of the Drosophila melanogaster ribosomal RNA genes using both micrococcal nuclease and DNase I. Several findings are of interest. First, the transcribed DNA segments of the rRNA repeat unit appear to be packaged into an unstable or multiphasic nucleosome structure. Second, the 5 end of the transcription unit is preferentially exposed to nuclease attack. Third, the non-transcribed spacer immediately upstream from the transcription start site has a novel chromatin organization with micrococcal nuclease and DNase I cleavage sites spaced at intervals of about 240 base-pairs. This unusual fragment distribution appears to reflect the underlying sequence organization of the spacer DNA segment, which consists of a series of tandemly repeated 239 base-pair sequence blocks. We have also examined the chromatin structure of the rRNA repeat unit after extraction of nuclei with different concentrations of salt. Our results suggest that the higher order structures may be of importance in determining the novel chromatin organization of the rRNA repeat unit.

Structural polymorphism in DNA

We have used the enzyme micrococcal nuclease and the methylating reagent dimethyl sulfate to examine the structural properties of eukaryotic DNAs. Our studies demonstrate extensive structural polymorphism in the DNA double helix. Moreover, we find that the distribution of helical variants is in some instances correlated with the functional organization of the DNA. These observations raise the possibility that eukaryotic DNAs may be organized into discrete functional units having characteristic structural properties. In addition, we find that boundaries between different functional units are typically marked by DNA segments having unusual conformational properties. Such structural perturbations could serve as signals in the utilization of genetic information in eukaryotes, and may be important in a variety of different protein-DNA interactions.