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

1993 ◽  
Vol 13 (12) ◽  
pp. 7522-7530
Author(s):  
A Udvardy ◽  
P Schedl
Keyword(s):  
Heat Shock ◽  
Chromatin Structure ◽  
Topoisomerase Ii ◽  
Chromatin Condensation ◽  
Intergenic Spacer ◽  
Domain Boundaries ◽  
Shock Locus ◽  
Sites Of Action ◽  
Putative Domain

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.

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

1993 ◽  
Vol 13 (12) ◽  
pp. 7522-7530 ◽  
Author(s):  
A Udvardy ◽  
P Schedl
Keyword(s):  
Heat Shock ◽  
Chromatin Structure ◽  
Topoisomerase Ii ◽  
Chromatin Condensation ◽  
Intergenic Spacer ◽  
Domain Boundaries ◽  
Shock Locus ◽  
Sites Of Action ◽  
Putative Domain

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.

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

1991 ◽  
Vol 11 (10) ◽  
pp. 4973-4984
Author(s):  
A Udvardy ◽  
P Schedl
Keyword(s):  
Chromatin Structure ◽  
Topoisomerase Ii ◽  
Double Strand Breaks ◽  
General Mechanism ◽  
Sequence Specificity ◽  
Strand Breaks ◽  
Wide Range ◽  
Primary Determinant ◽  
Sites Of Action

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.

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

1991 ◽  
Vol 11 (10) ◽  
pp. 4973-4984 ◽  
Author(s):  
A Udvardy ◽  
P Schedl
Keyword(s):  
Chromatin Structure ◽  
Topoisomerase Ii ◽  
Double Strand Breaks ◽  
General Mechanism ◽  
Sequence Specificity ◽  
Strand Breaks ◽  
Wide Range ◽  
Primary Determinant ◽  
Sites Of Action

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.

scholarly journals In Vivo HP1 Targeting Causes Large-Scale Chromatin Condensation and Enhanced Histone Lysine Methylation

2005 ◽  
Vol 25 (11) ◽  
pp. 4552-4564 ◽  
Author(s):  
Pernette J. Verschure ◽  
Ineke van der Kraan ◽  
Wim de Leeuw ◽  
Johan van der Vlag ◽  
Anne E. Carpenter ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
Large Scale ◽  
Chromosome Region ◽  
Chromatin Condensation ◽  
Regulation Of Gene Expression ◽  
Lac Repressor ◽  
Homologous Proteins ◽  
Lac Operator

ABSTRACT Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) α (HP1α) and HP1β on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1α and HP1β in vivo targeting. HP1α targeting causes the recruitment of endogenous HP1β to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1α and HP1β targeting is sufficient to induce heterochromatin formation.

scholarly journals Transcriptional supercoiling boosts topoisomerase II-mediated knotting of intracellular DNA

2019 ◽  
Vol 47 (13) ◽  
pp. 6946-6955 ◽  
Author(s):  
Antonio Valdés ◽  
Lucia Coronel ◽  
Belén Martínez-García ◽  
Joana Segura ◽  
Sílvia Dyson ◽  
...  
Keyword(s):  
Dna Replication ◽  
Crucial Role ◽  
Topoisomerase Ii ◽  
Chromatin Condensation ◽  
Chromatin Dynamics ◽  
Eukaryotic Chromatin ◽  
Topo Ii ◽  
Inversion Mechanism

AbstractRecent studies have revealed that the DNA cross-inversion mechanism of topoisomerase II (topo II) not only removes DNA supercoils and DNA replication intertwines, but also produces small amounts of DNA knots within the clusters of nucleosomes that conform to eukaryotic chromatin. Here, we examine how transcriptional supercoiling of intracellular DNA affects the occurrence of these knots. We show that although (−) supercoiling does not change the basal DNA knotting probability, (+) supercoiling of DNA generated in front of the transcribing complexes increases DNA knot formation over 25-fold. The increase of topo II-mediated DNA knotting occurs both upon accumulation of (+) supercoiling in topoisomerase-deficient cells and during normal transcriptional supercoiling of DNA in TOP1 TOP2 cells. We also show that the high knotting probability (Pkn ≥ 0.5) of (+) supercoiled DNA reflects a 5-fold volume compaction of the nucleosomal fibers in vivo. Our findings indicate that topo II-mediated DNA knotting could be inherent to transcriptional supercoiling of DNA and other chromatin condensation processes and establish, therefore, a new crucial role of topoisomerase II in resetting the knotting–unknotting homeostasis of DNA during chromatin dynamics.

scholarly journals P-TEFb kinase recruitment and function at heat shock loci

2000 ◽  
Vol 14 (7) ◽  
pp. 792-803 ◽  
Author(s):  
John T. Lis ◽  
Paul Mason ◽  
J. Peng ◽  
David H. Price ◽  
Janis Werner
Keyword(s):  
Heat Shock ◽  
Rna Polymerase Ii ◽  
Polytene Chromosomes ◽  
Pol Ii ◽  
Shock Locus ◽  
And Function ◽  
Cdk9 Kinase Activity ◽  
Productive Elongation

P-TEFb, a heterodimer of the kinase Cdk9 and cyclin T, was isolated as a factor that stimulates formation of productive transcription elongation complexes in vitro. Here, we show that P-TEFb is located at >200 distinct sites on Drosophila polytene chromosomes. Upon heat shock, P-TEFb, like the regulatory factor HSF, is rapidly recruited to heat shock loci, and this recruitment is blocked in an HSF mutant. Yet, HSF binding to DNA is not sufficient to recruit P-TEFb in vivo, and HSF and P-TEFb immunostainings within a heat shock locus are not coincident. Insight to the function of P-TEFb is offered by experiments showing that the direct recruitment of a Gal4-binding domain P-TEFb hybrid to an hsp70 promoter in Drosophilacells is sufficient to activate transcription in the absence of heat shock. Analyses of point mutants show this P-TEFb stimulation is dependent on Cdk9 kinase activity and on Cdk9's interaction with cyclin T. These results, coupled with the frequent colocalization of P-TEFb and the hypophosphorylated form of RNA polymerase II (Pol II) found at promoter-pause sites, support a model in which P-TEFb acts to stimulate promoter-paused Pol II to enter into productive elongation.

Sign in / Sign up

Export Citation Format

Share Document