RNA Polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes

Greenleaf, Arno L.; Plagens, Ulrich; Jamrich, Milan; Bautz, Ekkehard K. F.

doi:10.1007/bf00329465

Cited by 45 publications

(12 citation statements)

References 19 publications

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“…As a reaction to an environmental stress like heat shock, chromatin undergoes rapid and dynamic remodeling to turn off most of the gene expression and to turn on the genes specifically associated with heat shock responses (reviewed in reference 31). The induction of heat shock loci, which results in the synthesis of heat shock proteins and RNA, is accomplished by the recruitment of RNA pol II, transcription factors, and other chromosomal proteins including topo I to heat shock puffs (15,19,24,46,53,58). Our immunofluorescence data demonstrated that the topo I N-terminal sequence enables rapid mobilization of topo I to the heat shock puffs.…”

Section: Discussionmentioning

confidence: 69%

Targeting to Transcriptionally Active Loci by the Hydrophilic N-Terminal Domain of Drosophila DNA Topoisomerase I

Shaiu

Hsieh

1998

Molecular and Cellular Biology

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DNA topoisomerase I (topo I) from Drosophila melanogaster contains a nonconserved, hydrophilic N-terminal domain of about 430 residues upstream of the conserved core domains. Deletion of this N terminus did not affect the catalytic activity of topo I, while further removal of sequences into the conserved regions inactivated its enzymatic activity. We have investigated the cellular function of the Drosophila topo I N-terminal domain with top1-lacZ transgenes. There was at least one putative nuclear localization signal within the first 315 residues of the N-terminal domain that allows efficient import of the large chimeric proteins into Drosophila nuclei. The top1-lacZ fusion proteins colocalized with RNA polymerase II (pol II) at developmental puffs on the polytene chromosomes. Either topo I or the top1-lacZ fusion protein was colocalized with RNA pol II in some but not all of the nonpuff, interband loci. However, the fusion proteins as well as RNA pol II were recruited to heat shock puffs during heat treatment, and they returned to the developmental puffs after recovery from heat shock. By immunoprecipitation, we showed that two of the largest subunits of RNA pol II coprecipitated with the N-terminal 315-residue fusion protein by using antibodies against ␤-galactosidase. These data suggest that the topo I fusion protein can be localized to the transcriptional complex on chromatin and that the N-terminal 315 residues were sufficient to respond to cellular processes, especially during the reprogramming of gene expression.Type I DNA topoisomerase (topo I) catalyzes a cycle of transesterification reactions, during which it generates transient single-strand DNA breaks; these reversible reactions result in the topological transformation of either single-strand or double-strand DNA molecules (8,20,55). Biochemical and genetic studies have demonstrated that topo I plays important roles in DNA replication, transcription, and recombination and in chromosome condensation and the maintenance of genome stability. The top1 gene is not essential for viability in yeast, and genetic analysis suggests that some of the biological functions of topo I can be performed by topo II (59). However, recent genetic screens of Saccharomyces cerevisiae have identified four complementation groups of mutants with mutations that, in combination with a top1 mutation, result in a lethal phenotype. Some of these synthetic lethal mutants have mutations in genes other than top2 (44). Therefore, it is possible that not all the biological functions of topo I can be substituted by topo II. Furthermore, because top1 is an essential gene in Drosophila melanogaster (30) and in mice (37), the functions of topo I may not be efficiently substituted by topo II in multicellular organisms.One of the functions of topo I is to provide a swivel to facilitate the unwinding of the DNA duplex during the process of transcription or replication (56). Diametric DNA supercoiling can transiently accumulate in the front and in the wake of a transcriptional fork as proposed...

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Section: Discussionmentioning

confidence: 69%

Targeting to Transcriptionally Active Loci by the Hydrophilic N-Terminal Domain of Drosophila DNA Topoisomerase I

Shaiu

Hsieh

1998

Molecular and Cellular Biology

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“…Heat shock in Drosophila results in a disappearance of most mRNAs and corresponding protein synthesis and the concomitant appearance of high levels of a few heat shock mRNAs and their corresponding heat shock protein products. This dramatic change in gene expression is accompanied by a rapid mobilization of Pol II and associated factors to a small number of heat shock loci, which can be visualized by immunofluorescent localization (17). If SRCAP is a general transcription coactivator for Pol II, we would expect to find it similarly concentrated at heat shock puff sites.…”

Section: Resultsmentioning

confidence: 99%

Human SRCAP and Drosophila melanogaster DOM Are Homologs That Function in the Notch Signaling Pathway

Eissenberg¹,

Wong

Chrivia

2005

Molecular and Cellular Biology

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The putative ATPase chromatin-remodeling machine SRCAP was identified in a yeast two-hybrid protein screen by interaction with the histone acetylase CBP. SRCAP is implicated in the transcriptional coactivation of cyclic AMP-and steroid-dependent promoters, but no natural chromosomal targets for SRCAP regulation have been identified. DOM is the unique SRCAP homolog in Drosophila melanogaster. The goal of this study was to test whether SRCAP is a functional homolog of DOM and to identify potential activities and targets of SRCAP in vivo. We show that human SRCAP complements recessive domino mutant phenotypes. This rescue depends on an intact ATPase homology domain. SRCAP colocalizes extensively with DOM on Drosophila polytene chromosomes and is recruited to sites of active transcription, such as steroid-regulated loci, but not to activated heat shock loci. We show that SRCAP recruits Drosophila CBP to ectopic chromosomal sites, providing the first evidence to suggest that SRCAP and CBP interact directly or indirectly on chromosomes. We show that DOM is a Notch pathway activator in Drosophila and that wild-type SRCAP-but not an ATPase domain mutant-can substitute for DOM in Notch-dependent wing development. We show that SRCAP potentiates Notch-dependent gene activation in HeLa cells. Taken together, these data implicate SRCAP and DOM in developmental gene activation.Most regulated gene expression in eukaryotes is the result of transcriptional regulation. Current models of transcriptional regulation invoke the targeted recruitment of enzymes that make covalent changes in histone and/or DNA structure, in ATPases that remodel histone-DNA interactions, and, in cases of gene activation, in RNA polymerase itself. A major challenge is to identify the diverse roles of chromatin modifiers in transcriptional regulation.SRCAP (SNF2-related CBP-activating protein) shares homology with members of the SNF2/SWI2 class of chromatinremodeling enzymes (20). SRCAP was originally identified in a yeast two-hybrid protein screen for proteins that interact with the histone acetyltransferase CBP (CREB-binding protein). Subsequent studies using transiently transfected reporters have implicated SRCAP as a coactivator for CREB-and nuclear hormone receptor-mediated transcriptional activation (30, 31). The mechanism by which SRCAP activates transcription has not been completely elucidated. Results from a number of studies, including mammalian two-hybrid and coimmunoprecipitation experiments, indicate SRCAP may function in part by recruitment of CBP or other coactivators, such as the arginine methyltransferase CARM-1 and the nuclear hormone receptor coactivator GRIP-1 (20, 31). A role for the conserved ATPase/helicase homology domain in regulation of transcription has not been established, since this activity is dispensable for SRCAP activity in transient transfection assays, nor have the natural chromosomal promoters that utilize SRCAP to regulate transcription been identified.In mammals, the protein most homologous to SRCAP is p400. p400 was ...

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“…When DroiQo.hil flies, embryos, or tissue culture cells are removed from their normal environmental temperature and placed Instead at 370C, a distinct set of DNA sequences (those coding for the heat shock proteins [hsp]) rapidly become transcribed at a greatly Increased rate, while transcription of other genes Is reduced to very low levels (18,19). Immunofluorescence studies using antibodies to RNA polymerase 11 to stain polytene chromosomes have shown that most of the RNA polymerase molecules previously bound to transcribing regions are released by heat shock, and much of this polymerase subsequently binds to the heat shock loci (20,21). Because of the rapid, qualitative alterations in gene expression afforded by heat shock, this has been used to study many aspects of gene expression, particularly the nuclear conformation of genes as monitored by sensitivity to nucleases.…”

Section: Introductionmentioning

confidence: 99%

The association of transcribed genes with the nuclear matrix ofDrosophilacells during heat shock

1985

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ABSIRACTUsing the transcriptional modulation afforded by heat shock, we found that the association of active genes with the nuclear matrix was not dependent on their level of transcription. Heat shock genes were matrix associated both before heat shock (when transcription was relatively low), and during heat shock (when transcription was greatly Increased). Conversely, the cytoplasmic actin gene was matrix associated during normal growth conditions (when transcription was high) and during heat shock (when transcription was greatly decreased). Removal of greater than 99.7% of nascent RNA during preparation of the matrices did not affect these findings. Detailed examination of the cytoplasmic actin gene revealed that Its matrix association was apparently mediated by multiple Interactions near the 5' end of the gene.

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RNA Polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes

Cited by 45 publications

References 19 publications

Targeting to Transcriptionally Active Loci by the Hydrophilic N-Terminal Domain of Drosophila DNA Topoisomerase I

Targeting to Transcriptionally Active Loci by the Hydrophilic N-Terminal Domain of Drosophila DNA Topoisomerase I

Human SRCAP and Drosophila melanogaster DOM Are Homologs That Function in the Notch Signaling Pathway

The association of transcribed genes with the nuclear matrix ofDrosophilacells during heat shock

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