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Judith A. Kassis - One of the best experts on this subject based on the ideXlab platform.
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The role of Polycomb-group response elements in regulation of Engrailed transcription in Drosophila
Development (Cambridge England), 2008Co-Authors: Sarah K. Devido, J. Lesley Brown, Deborah Kwon, Judith A. KassisAbstract:Polycomb group proteins are required for long-term repression of many Genes in Drosophila and all metazoans. In Drosophila , DNA fragments called Polycomb-group response elements (PREs) have been identified that mediate the action of Polycomb-group proteins. Previous studies have shown that a 2 kb fragment located from -2.4 kb to -395 bp upstream of the Drosophila Engrailed promoter contains a multipartite PRE that can mediate mini- white silencing and act as a PRE in an Ubx -reporter construct. Here, we study the role of this 2 kb fragment in the regulation of the Engrailed Gene itself. Our results show that within this 2 kb fragment, there are two subfragments that can act as PREs in embryos. In addition to their role in Gene silencing, these two adjacent PRE fragments can facilitate the activation of the Engrailed promoter by distant enhancers. The repressive action of the Engrailed PRE can also act over a distance. A 181 bp subfragment can act as a PRE and also mediate positive effects in an enhancer-detector construct. Finally, a deletion of 530 bp of the 2 kb PRE fragment within the endogenous Engrailed Gene causes a loss-of-function phenotype, showing the importance of the positive regulatory effects of this PRE-containing fragment. Our data are consistent with the model that Engrailed PREs bring chromatin together, allowing both positive and negative regulatory interactions between distantly located DNA fragments.
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An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila Engrailed Gene.
Nucleic acids research, 2005Co-Authors: J. Lesley Brown, Daniel J. Grau, Sarah K. Devido, Judith A. KassisAbstract:Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress Gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila Engrailed Gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.
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an sp1 klf binding site is important for the activity of a polycomb group response element from the drosophila Engrailed Gene
Nucleic Acids Research, 2005Co-Authors: Lesley J Brown, Daniel J. Grau, Sarah K. Devido, Judith A. KassisAbstract:Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress Gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila Engrailed Gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.
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a complex array of dna binding proteins required for pairing sensitive silencing by a polycomb group response element from the drosophila Engrailed Gene
Genetics, 2002Co-Authors: Jeffrey L Americo, Lesley J Brown, Mary Whiteley, James B. Jaynes, Miki Fujioka, Judith A. KassisAbstract:Regulatory DNA from the Drosophila Gene Engrailed causes silencing of a linked reporter Gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transGene and has been called "pairing-sensitive silencing." The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), we tested the activities of each of these Engrailed fragments in a construct designed to detect PRE activity in embryos. Both fragments were found to behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment showed additional silencing capabilities. Using the mini-white reporter Gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggested that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function.
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the drosophila polycomb group Gene pleiohomeotic encodes a dna binding protein with homology to the transcription factor yy1
Molecular Cell, 1998Co-Authors: Lesley J Brown, Diane Mucci, Mary Whiteley, Marieluise Dirksen, Judith A. KassisAbstract:Abstract Genes of the Polycomb group (PcG) of Drosophila encode proteins necessary for the maintenance of transcriptional repression of homeotic Genes. PcG proteins are thought to act by binding as multiprotein complexes to DNA through Polycomb group response elements (PREs); however, specific DNA binding has not been demonstrated for any of the PcG proteins. We have identified a sequence-specific DNA binding protein that interacts with a PRE from the Drosophila Engrailed Gene. This protein (PHO) is a homolog of the ubiquitous mammalian transcription factor Yin Yang-1 and is encoded by pleiohomeotic , a known member of the PcG. We propose that PHO acts to anchor PcG protein complexes to DNA.
Lesley J Brown - One of the best experts on this subject based on the ideXlab platform.
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an sp1 klf binding site is important for the activity of a polycomb group response element from the drosophila Engrailed Gene
Nucleic Acids Research, 2005Co-Authors: Lesley J Brown, Daniel J. Grau, Sarah K. Devido, Judith A. KassisAbstract:Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress Gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila Engrailed Gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.
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a complex array of dna binding proteins required for pairing sensitive silencing by a polycomb group response element from the drosophila Engrailed Gene
Genetics, 2002Co-Authors: Jeffrey L Americo, Lesley J Brown, Mary Whiteley, James B. Jaynes, Miki Fujioka, Judith A. KassisAbstract:Regulatory DNA from the Drosophila Gene Engrailed causes silencing of a linked reporter Gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transGene and has been called "pairing-sensitive silencing." The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), we tested the activities of each of these Engrailed fragments in a construct designed to detect PRE activity in embryos. Both fragments were found to behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment showed additional silencing capabilities. Using the mini-white reporter Gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggested that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function.
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the drosophila polycomb group Gene pleiohomeotic encodes a dna binding protein with homology to the transcription factor yy1
Molecular Cell, 1998Co-Authors: Lesley J Brown, Diane Mucci, Mary Whiteley, Marieluise Dirksen, Judith A. KassisAbstract:Abstract Genes of the Polycomb group (PcG) of Drosophila encode proteins necessary for the maintenance of transcriptional repression of homeotic Genes. PcG proteins are thought to act by binding as multiprotein complexes to DNA through Polycomb group response elements (PREs); however, specific DNA binding has not been demonstrated for any of the PcG proteins. We have identified a sequence-specific DNA binding protein that interacts with a PRE from the Drosophila Engrailed Gene. This protein (PHO) is a homolog of the ubiquitous mammalian transcription factor Yin Yang-1 and is encoded by pleiohomeotic , a known member of the PcG. We propose that PHO acts to anchor PcG protein complexes to DNA.
Sarah K. Devido - One of the best experts on this subject based on the ideXlab platform.
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The role of Polycomb-group response elements in regulation of Engrailed transcription in Drosophila
Development (Cambridge England), 2008Co-Authors: Sarah K. Devido, J. Lesley Brown, Deborah Kwon, Judith A. KassisAbstract:Polycomb group proteins are required for long-term repression of many Genes in Drosophila and all metazoans. In Drosophila , DNA fragments called Polycomb-group response elements (PREs) have been identified that mediate the action of Polycomb-group proteins. Previous studies have shown that a 2 kb fragment located from -2.4 kb to -395 bp upstream of the Drosophila Engrailed promoter contains a multipartite PRE that can mediate mini- white silencing and act as a PRE in an Ubx -reporter construct. Here, we study the role of this 2 kb fragment in the regulation of the Engrailed Gene itself. Our results show that within this 2 kb fragment, there are two subfragments that can act as PREs in embryos. In addition to their role in Gene silencing, these two adjacent PRE fragments can facilitate the activation of the Engrailed promoter by distant enhancers. The repressive action of the Engrailed PRE can also act over a distance. A 181 bp subfragment can act as a PRE and also mediate positive effects in an enhancer-detector construct. Finally, a deletion of 530 bp of the 2 kb PRE fragment within the endogenous Engrailed Gene causes a loss-of-function phenotype, showing the importance of the positive regulatory effects of this PRE-containing fragment. Our data are consistent with the model that Engrailed PREs bring chromatin together, allowing both positive and negative regulatory interactions between distantly located DNA fragments.
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An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila Engrailed Gene.
Nucleic acids research, 2005Co-Authors: J. Lesley Brown, Daniel J. Grau, Sarah K. Devido, Judith A. KassisAbstract:Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress Gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila Engrailed Gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.
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an sp1 klf binding site is important for the activity of a polycomb group response element from the drosophila Engrailed Gene
Nucleic Acids Research, 2005Co-Authors: Lesley J Brown, Daniel J. Grau, Sarah K. Devido, Judith A. KassisAbstract:Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress Gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila Engrailed Gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.
Thomas B. Kornberg - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of the Drosophila Engrailed protein at a site outside its homeodomain enhances DNA binding.
The Journal of biological chemistry, 1995Co-Authors: Hm Bourbon, Enrique Martín-blanco, David B. Rosen, Thomas B. KornbergAbstract:Abstract The Engrailed Gene encodes a homeodomain-containing phosphoprotein that binds DNA. Here, we show that Engrailed protein is posttranslationally modified in embryos and in embryo-derived cultured cells but is essentially unmodified when expressed in Escherichia coli. Engrailed protein produced by bacteria can be phosphorylated in nuclear extracts prepared from Drosophila embryos, and phosphotryptic peptides from this modified protein partly reproduce two-dimensional maps of phosphotryptic fragments obtained from metabolically labeled Engrailed protein. The primary embryonic protein kinase modifying Engrailed protein is casein kinase II (CK-II). Analysis of mutant proteins revealed that the in vitro phosphoacceptors are mainly clustered in a region outside the Engrailed homeodomain and identified serines 394, 397, 401, and 402 as the targets for CK-II phosphorylation. CK-II- dependent phosphorylation of an N-truncated derivative of Engrailed protein purified from bacteria increased its DNA binding 2-4-fold.
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Creating a Drosophila wing de novo, the role of Engrailed, and the compartment border hypothesis
Development (Cambridge England), 1995Co-Authors: Tetsuya Tabata, Elizabeth Gustavson, Zehra Ali, Carol Schwartz, Thomas B. KornbergAbstract:Anterior/posterior compartment borders bisect every Drosophila imaginal disc, and the Engrailed Gene is essential for their function. We analyzed the role of the Engrailed and invected Genes in wing discs by eliminating or increasing their activity. Removing Engrailed/invected from posterior wing cells created two new compartments: an anterior compartment consisting of mutant cells and a posterior compartment that grew from neighboring cells. In some cases, these compartments formed a complete new wing. Increasing Engrailed activity also affected patterning. These findings demonstrate that Engrailed both directs the posterior compartment pathway and creates the compartment border. These findings also establish the compartment border as the pre-eminent organizational feature of disc growth and patterning.
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SELECTION AND CHARACTERIZATION OF SEQUENCES WITH HIGH AFFINITY FOR THE Engrailed PROTEIN OF DROSOPHILA
Mechanisms of development, 1995Co-Authors: Maria Teresa Saenz-robles, Tetsuya Tabata, Florence Maschat, Matthew P. Scott, Thomas B. KornbergAbstract:The Engrailed Gene helps to direct Drosophila melanogaster development by encoding a homeodomain-containing DNA binding protein. To identify Genes whose transcription Engrailed regulates, we developed a method to isolate genomic sequences to which Engrailed protein binds with high affinity. Fragments of genomic DNA were fractionated on an Engrailed protein affinity column, and fragments that were retained in the presence of 0.4-1.0 M KC1 were isolated and cloned. The isolated fragments include regions of the Engrailed and cubitus interruptus Gene promoters, both of which are candidate targets of Engrailed, and most fragments contain regions that Engrailed protein protects from DNaseI digestion. Chromosomal deletions that remove some of the Engrailed binding sites (located either at 64D, 96B or 99D) interact Genetically with Engrailed. Characterization of a transcript encoded in region 64D revealed its dependence on Engrailed protein.
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Allele-specific quantification of Drosophila Engrailed and invected transcripts
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Andrew S. Goldsborough, Thomas B. KornbergAbstract:Abstract Changes in levels of transcription can be difficult to gauge in animals with lethal mutations. For example, mutations in a regulatory region of an essential Gene can have secondary consequences that complicate attempts to quantify the transcripts produced by the mutant Gene. We describe a method that circumvents this problem by revealing the relative amount of transcript produced from each allele in a heterozygote. With this method, recessive lethal mutations can be analyzed in animals that are phenotypically wild type. We used this technique to analyze mutations in the regulatory region of the Drosophila Engrailed Gene and found that truncations reduce transcription to levels that depend both upon the tissue and upon the location of the chromosomal break. We also found that these mutations affect expression of the linked invected Gene, suggesting that Engrailed and invected share a complex set of regulatory elements that operate over at least 85 kb. We suggest that this technique will have General utility for the quantitation of allele-specific transcripts, even when amounts of tissue are limiting.
J. Lesley Brown - One of the best experts on this subject based on the ideXlab platform.
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The role of Polycomb-group response elements in regulation of Engrailed transcription in Drosophila
Development (Cambridge England), 2008Co-Authors: Sarah K. Devido, J. Lesley Brown, Deborah Kwon, Judith A. KassisAbstract:Polycomb group proteins are required for long-term repression of many Genes in Drosophila and all metazoans. In Drosophila , DNA fragments called Polycomb-group response elements (PREs) have been identified that mediate the action of Polycomb-group proteins. Previous studies have shown that a 2 kb fragment located from -2.4 kb to -395 bp upstream of the Drosophila Engrailed promoter contains a multipartite PRE that can mediate mini- white silencing and act as a PRE in an Ubx -reporter construct. Here, we study the role of this 2 kb fragment in the regulation of the Engrailed Gene itself. Our results show that within this 2 kb fragment, there are two subfragments that can act as PREs in embryos. In addition to their role in Gene silencing, these two adjacent PRE fragments can facilitate the activation of the Engrailed promoter by distant enhancers. The repressive action of the Engrailed PRE can also act over a distance. A 181 bp subfragment can act as a PRE and also mediate positive effects in an enhancer-detector construct. Finally, a deletion of 530 bp of the 2 kb PRE fragment within the endogenous Engrailed Gene causes a loss-of-function phenotype, showing the importance of the positive regulatory effects of this PRE-containing fragment. Our data are consistent with the model that Engrailed PREs bring chromatin together, allowing both positive and negative regulatory interactions between distantly located DNA fragments.
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An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila Engrailed Gene.
Nucleic acids research, 2005Co-Authors: J. Lesley Brown, Daniel J. Grau, Sarah K. Devido, Judith A. KassisAbstract:Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress Gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila Engrailed Gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.