scholarly journals GAGA Factor and the TFIID Complex Collaborate in Generating an Open Chromatin Structure at the Drosophila melanogaster hsp26 Promoter

2002 ◽  
Vol 22 (17) ◽  
pp. 6148-6157 ◽  
Author(s):  
Boris A. Leibovitch ◽  
Quinn Lu ◽  
Lawrence R. Benjamin ◽  
Yingyun Liu ◽  
David S. Gilmour ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Chromatin Structure ◽  
Random Sequence ◽  
Regulatory Region ◽  
Open Chromatin ◽  
Gaga Factor ◽  
Factor Binding ◽  
Hypersensitive Sites ◽  
Tfiid Complex

ABSTRACT The upstream regulatory region of the Drosophila melanogaster hsp26 gene includes two DNase I-hypersensitive sites (DH sites) that encompass the critical heat shock elements. This chromatin structure is required for heat shock-inducible expression and depends on two (CT) n •(GA) n elements bound by GAGA factor. To determine whether GAGA factor alone is sufficient to drive formation of the DH sites, we have created flies with an hsp26/lacZ transgene wherein the entire DNA segment known to interact with the TFIID complex has been replaced by a random sequence. The replacement results in a loss of heat shock-inducible hsp26 expression and drastically diminishes nuclease accessibility in the chromatin of the regulatory region. Chromatin immunoprecipitation experiments show that the decrease in TFIID binding does not reduce GAGA factor binding. In contrast, the loss of GAGA factor binding resulting from (CT) n mutations decreases TFIID binding. These data suggest that both GAGA factor and TFIID are necessary for formation of the appropriate chromatin structure at the hsp26 promoter and predict a regulatory mechanism in which GAGA factor binding precedes and contributes to the recruitment of TFIID.

scholarly journals Long-Range Nucleosome Ordering Is Associated with Gene Silencing in Drosophila melanogaster Pericentric Heterochromatin

2001 ◽  
Vol 21 (8) ◽  
pp. 2867-2879 ◽  
Author(s):  
Fang-Lin Sun ◽  
Matthew H. Cuaycong ◽  
Sarah C. R. Elgin
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Silencing ◽  
Long Range ◽  
Chromatin Structure ◽  
Regulatory Region ◽  
Pericentric Heterochromatin ◽  
Dnase I ◽  
Micrococcal Nuclease ◽  
Hypersensitive Sites ◽  
Nucleosome Array

ABSTRACT We have used line HS-2 of Drosophila melanogaster, carrying a silenced transgene in the pericentric heterochromatin, to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is ca. 10 bp larger than that observed for bulkDrosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing.

(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene

1993 ◽  
Vol 13 (5) ◽  
pp. 2802-2814
Author(s):  
Q Lu ◽  
L L Wallrath ◽  
H Granok ◽  
S C Elgin
Keyword(s):  
Heat Shock ◽  
Chromatin Structure ◽  
Previous Analysis ◽  
Heat Shock Factor ◽  
Dnase I ◽  
Inducible Expression ◽  
P Element ◽  
Enzyme Treatment ◽  
Lacz Gene ◽  
Gaga Factor

Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L. Glaser, G.H. Thomas, E.S. Siegfried, S.C.R. Elgin, and J.T. Lis, J. Mol. Biol. 211:751-761, 1990). This (CT)n element appears to contribute to formation of the wild-type chromatin structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q. Lu, L.L. Wallrath, B.D. Allan, R.L. Glaser, J.T. Lis, and S.C.R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE. We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the chromatin structure and to heat shock inducibility. hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity. The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin structure and heat-inducible expression of the transgenes. These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro. In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the chromatin structure of the transgenes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.

scholarly journals A heat-shock-activated cDNA encoding GAGA factor rescues some lethal mutations in the Drosophila melanogaster Trithorax-like gene

2001 ◽  
Vol 78 (1) ◽  
pp. 13-21 ◽  
Author(s):  
H. GRANOK ◽  
B. A. LEIBOVITCH ◽  
S. C. R. ELGIN
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Position Effect ◽  
Efficient Production ◽  
Position Effect Variegation ◽  
Chromosomal Protein ◽  
Gaga Factor ◽  
Genetic Rescue ◽  
Regulation Of Synthesis

GAGA factor is an important chromosomal protein involved in establishing specific nucleosome arrays and in regulating gene transcription in Drosophila melanogaster. We developed a transgenic system for controlled heat-shock-dependent overexpression of the GAGA factor 519 amino acid isoform (GAGA-519) in vivo. Efficient production of stable protein from these transgenes provided genetic rescue of a hypomorphic Trithorax-like (Trl) lethal allele to adulthood. Nevertheless, supplemental GAGA-519 did not suppress position effect variegation (PEV), a phenomenon commonly used to measure dosage effects of chromosomal proteins, nor did it rescue other lethal alleles of Trl. The results suggest requirements for the additional isoforms of GAGA factor, or for more precise regulation of synthesis, to carry out the diverse functions of this protein.

scholarly journals The study of the regulatory region of the Drosophila melanogaster Notch gene by new methods of directed genome editing

2019 ◽  
Vol 23 (2) ◽  
pp. 199-202
Author(s):  
O. V. Andreyenkov ◽  
E. I. Volkova ◽  
N. G. Andreyenkova ◽  
S. A. Demakov
Keyword(s):  
Drosophila Melanogaster ◽  
Genome Editing ◽  
Regulatory Region ◽  
Polytene Chromosomes ◽  
Notch Pathway ◽  
Hereditary Diseases ◽  
Chromatin State ◽  
Open Chromatin ◽  
New Methods ◽  
Notch Gene

The Notch gene plays a key role in the development of organs and tissues of neuroectodermic origin, including the nervous system. In eukaryotic organisms, the Notch pathway is involved in cell fate determination. The Notch gene was first discovered in Drosophila melanogaster. In mammals, the family of Notch receptors includes four homologues. In humans, mutations in the Notch gene cause several hereditary diseases and carcinogenesis. Studies of the regulatory zone of the Notch gene in D. melanogaster have been conducted for several decades. We review their results and methods. The regulatory zone of the Notch gene is in the region of open chromatin state that corresponds to the 3C6/3C7 interband on the cytological map of polytene chromosomes of D. melanogaster salivary glands. The development of new methods for directed genome editing made it possible to create a system for introducing directed changes into the regulatory zone of the gene. Using the CRISPR/Cas9 system, we obtained a directed 4-kilobase deletion including the 5’-regulatory zone, promoter, and the first exon of the Notch gene and introduced the attP site into the first intron of the Notch gene. This approach enabled targeted changes of the sequence of the regulatory and promoter regions of the gene. Thus, it provided a new powerful tool for studies of Notch gene regulation and the organization of the open chromatin state.

scholarly journals (CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene.

1993 ◽  
Vol 13 (5) ◽  
pp. 2802-2814 ◽  
Author(s):  
Q Lu ◽  
L L Wallrath ◽  
H Granok ◽  
S C Elgin
Keyword(s):  
Heat Shock ◽  
Chromatin Structure ◽  
Previous Analysis ◽  
Heat Shock Factor ◽  
Dnase I ◽  
Inducible Expression ◽  
P Element ◽  
Enzyme Treatment ◽  
Lacz Gene ◽  
Gaga Factor

Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L. Glaser, G.H. Thomas, E.S. Siegfried, S.C.R. Elgin, and J.T. Lis, J. Mol. Biol. 211:751-761, 1990). This (CT)n element appears to contribute to formation of the wild-type chromatin structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q. Lu, L.L. Wallrath, B.D. Allan, R.L. Glaser, J.T. Lis, and S.C.R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE. We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the chromatin structure and to heat shock inducibility. hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity. The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin structure and heat-inducible expression of the transgenes. These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro. In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the chromatin structure of the transgenes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.

Chromatin structure of a P-element-transduced hsp-28 gene in Drosophila melanogaster

1986 ◽  
Vol 6 (11) ◽  
pp. 4126-4129
Author(s):  
J C Eissenberg ◽  
S C Elgin
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Chromatin Structure ◽  
Dnase I ◽  
P Element ◽  
Direct Selection ◽  
Wild Type ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
Selection For

The Drosophila hsp-28 gene was heat inducible when transduced to novel chromosomal sites even when no direct selection for transduced gene expression was imposed. The pattern of DNase I-hypersensitive sites 5' to the wild type and transduced copy of hsp-28 was similar. In addition, DNase I-hypersensitive sites occurred within the P-element sequences flanking transduced loci.

scholarly journals Mutations in the Drosophila melanogaster Gene Encoding S-adenosylmethionine Suppress Position-Effect Variegation

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 887-896 ◽  
Author(s):  
Jan Larsson ◽  
Jingpu Zhang ◽  
Åsa Rasmuson-Lestander
Keyword(s):  
Drosophila Melanogaster ◽  
Chromatin Structure ◽  
Position Effect ◽  
Regulatory Region ◽  
Polycomb Group ◽  
Position Effect Variegation ◽  
Gene Encoding ◽  
Sex Combs ◽  
S Alleles ◽  
Effect Variegation

Abstract In Drosophila melanogaster, the study of trans-acting modifier mutations of position-effect variegation and Polycomb group (Pc-G) genes have been useful tools to investigate genes involved in chromatin structure. We have cloned a modifier gene, Suppesssm of zeste 5 (Su(z)5), which encodes Sadenosylmethionine synthetase, and we present here molecular results and data concerning its expression in mutants and genetic interactions. The mutant alleles Su(z)5, l(2)R23 and l(2)M6 show suppression of wm4 and also of two white mutants induced by roo element insertions in the regulatory region i.e., wis (in combination with z  1) and wsp1. Two of the Su(z)S alleles, as well as a deletion of the gene, also act as enhancers of PoZycomb by increasing the size of sex combs on midleg. The results suggest that Su(z)5 is connected with regulation of chromatin structure. The enzyme Sadenosylmethionine synthetase is involved in the synthesis of Sadenosylmethionine, a methyl group donor and also, after decarboxylation, a propylamino group donor in the bio-synthesis of polyamines. Our results from HPLC analysis show that in ovaries from heterozygous Su(z)5 mutants the content of spermine is significantly reduced. Results presented here suggest that polyamines are an important molecule class in the regulation of chromatin structure.

scholarly journals Chromatin Remodeling Mediated by Drosophila GAGA Factor and ISWI Activates fushi tarazu Gene Transcription In Vitro

1998 ◽  
Vol 18 (5) ◽  
pp. 2455-2461 ◽  
Author(s):  
Masahiro Okada ◽  
Susumu Hirose
Keyword(s):  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Promoter Region ◽  
Gaga Factor ◽  
Fushi Tarazu ◽  
Nucleosome Remodeling ◽  
Factor Binding ◽  
Chromatin Template

ABSTRACT GAGA factor is known to remodel the chromatin structure in concert with nucleosome-remodeling factor NURF in a Drosophilaembryonic S150 extract. The promoter region of the Drosophila fushi tarazu (ftz) gene carries several binding sites for GAGA factor. Both the GAGA factor-binding sites and GAGA factor per se are necessary for the proper expression of ftz in vivo. We observed transcriptional activation of the ftz gene when a preassembled chromatin template was incubated with GAGA factor and the S150 extract. The chromatin structure within the ftzpromoter was specifically disrupted by incubation of the preassembled chromatin with GAGA factor and the S150 extract. Both transcriptional activation and chromatin disruption were blocked by an antiserum raised against ISWI or by base substitutions in the GAGA factor-binding sites in the ftz promoter region. These results demonstrate that GAGA factor- and ISWI-mediated disruption of the chromatin structure within the promoter region of ftz activates transcription on the chromatin template.

scholarly journals High-resolution mapping of DNase I-hypersensitive sites of Drosophila heat shock genes in Drosophila melanogaster and Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (9) ◽  
pp. 1853-1863 ◽  
Author(s):  
N Costlow ◽  
J T Lis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Heat Shock ◽  
High Resolution ◽  
Consensus Sequence ◽  
Dnase I ◽  
Heat Shock Genes ◽  
Base Pairs ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites

High-resolution analysis of the chromatin structure of the promoter regions of five Drosophila heat shock genes showed a similar location for the hypersensitive sequences relative to the start of transcription. For each of the five genes examined--those coding for hsp27, hsp26, hsp23, hsp70, and hsp83--the DNase I-hypersensitive sites in Drosophila melanogaster nuclei mapped to two regions upstream of the coding region. These sites occurred on the average, 115 and 17 base pairs upstream from the start of transcription of the five heat shock genes examined. This latter site corresponded to sequences at or near the TATA consensus sequence. Sites even further upstream of the hsp27, hsp26, and hsp83 genes were also evident. Additionally, for the two genes examined--hsp70 and hsp83--the DNase I-hypersensitive sites were preserved, at least within this level of resolution (+/- 10 base pairs), when the Drosophila genes were integrated into the Saccharomyces cerevisiae genome. This result indicates that the signals responsible for generating these hypersensitive sites are inherent in the DNA sequences and, in this case, are not highly species specific.

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