scholarly journals A 1-Megadalton ESC/E(Z) Complex from Drosophila That Contains Polycomblike and RPD3

2003 ◽  
Vol 23 (9) ◽  
pp. 3352-3362 ◽  
Author(s):  
Feng Tie ◽  
Jayashree Prasad-Sinha ◽  
Anna Birve ◽  
Åsa Rasmuson-Lestander ◽  
Peter J. Harte
Keyword(s):  
Glutathione Transferase ◽  
Insertion Site ◽  
Polytene Chromosomes ◽  
Gel Filtration ◽  
Polycomb Group ◽  
Homeotic Genes ◽  
Histone Binding ◽  
Polycomb Response Element ◽  
N Terminus

ABSTRACT Polycomb group (PcG) proteins are required to maintain stable repression of the homeotic genes and others throughout development. The PcG proteins ESC and E(Z) are present in a prominent 600-kDa complex as well as in a number of higher-molecular-mass complexes. Here we identify and characterize a 1-MDa ESC/E(Z) complex that is distinguished from the 600-kDa complex by the presence of the PcG protein Polycomblike (PCL) and the histone deacetylase RPD3. In addition, the 1-MDa complex shares with the 600-kDa complex the histone binding protein p55 and the PcG protein SU(Z)12. Coimmunoprecipitation assays performed on embryo extracts and gel filtration column fractions indicate that, during embryogenesis E(Z), SU(Z)12, and p55 are present in all ESC complexes, while PCL and RPD3 are associated with ESC, E(Z), SU(Z)12, and p55 only in the 1-MDa complex. Glutathione transferase pulldown assays demonstrate that RPD3 binds directly to PCL via the conserved PHD fingers of PCL and the N terminus of RPD3. PCL and E(Z) colocalize virtually completely on polytene chromosomes and are associated with a subset of RPD3 sites. As previously shown for E(Z) and RPD3, PCL and SU(Z)12 are also recruited to the insertion site of a minimal Ubx Polycomb response element transgene in vivo. Consistent with these biochemical and cytological results, Rpd3 mutations enhance the phenotypes of Pcl mutants, further indicating that RPD3 is required for PcG silencing and possibly for PCL function. These results suggest that there may be multiple ESC/E(Z) complexes with distinct functions in vivo.

The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3

Development ◽  
2001 ◽  
Vol 128 (2) ◽  
pp. 275-286 ◽  
Author(s):  
F. Tie ◽  
T. Furuyama ◽  
J. Prasad-Sinha ◽  
E. Jane ◽  
P.J. Harte
Keyword(s):  
Histone Deacetylase ◽  
Binding Protein ◽  
Histone Deacetylation ◽  
Polycomb Group ◽  
Homeotic Genes ◽  
Histone Binding ◽  
Polycomb Response Element ◽  
Chromatin Remodeling Complex

The Drosophila Polycomb Group (PcG) proteins are required for stable long term transcriptional silencing of the homeotic genes. Among PcG genes, esc is unique in being critically required for establishment of PcG-mediated silencing during early embryogenesis, but not for its subsequent maintenance throughout development. We previously showed that ESC is physically associated in vivo with the PcG protein E(Z). We report here that ESC, together with E(Z), is present in a 600 kDa complex that is distinct from complexes containing other PcG proteins. We have purified this ESC complex and show that it also contains the histone deacetylase RPD3 and the histone-binding protein p55, which is also a component of the chromatin remodeling complex NURF and the chromatin assembly complex CAF-1. The association of ESC and E(Z) with p55 and RPD3 is conserved in mammals. We show that RPD3 is required for silencing mediated by a Polycomb response element (PRE) in vivo and that E(Z) and RPD3 are bound to the Ubx PRE in vivo, suggesting that they act directly at the PRE. We propose that histone deacetylation by this complex is a prerequisite for establishment of stable long-term silencing by other continuously required PcG complexes.

Telomeric Associated Sequences of Drosophila Recruit Polycomb-Group Proteins in Vivo and Can Induce Pairing-Sensitive Repression

Genetics ◽  
2003 ◽  
Vol 164 (1) ◽  
pp. 195-208 ◽  
Author(s):  
Antoine Boivin ◽  
Christelle Gally ◽  
Sophie Netter ◽  
Dominique Anxolabéhère ◽  
Stéphane Ronsseray
Keyword(s):  
Position Effect ◽  
Insertion Site ◽  
Polytene Chromosomes ◽  
Polycomb Group ◽  
Dependent Manner ◽  
Trithorax Group ◽  
Telomeric Silencing ◽  
Associated Sequences ◽  
Specific Factors

Abstract In Drosophila, relocation of a euchromatic gene near centromeric or telomeric heterochromatin often leads to its mosaic silencing. Nevertheless, modifiers of centromeric silencing do not affect telomeric silencing, suggesting that each location requires specific factors. Previous studies suggest that a subset of Polycomb-group (PcG) proteins could be responsible for telomeric silencing. Here, we present the effect on telomeric silencing of 50 mutant alleles of the PcG genes and of their counteracting trithorax-group genes. Several combinations of two mutated PcG genes impair telomeric silencing synergistically, revealing that some of these genes are required for telomeric silencing. In situ hybridization and immunostaining experiments on polytene chromosomes revealed a strict correlation between the presence of PcG proteins and that of heterochromatic telomeric associated sequences (TASs), suggesting that TASs and PcG complexes could be associated at telomeres. Furthermore, lines harboring a transgene containing an X-linked TAS subunit and the mini-white reporter gene can exhibit pairing-sensitive repression of the white gene in an orientation-dependent manner. Finally, an additional binding site for PcG proteins was detected at the insertion site of this type of transgene. Taken together, these results demonstrate that PcG proteins bind TASs in vivo and may be major players in Drosophila telomeric position effect (TPE).

scholarly journals The bithorax complex iab-7 Polycomb Response Element has a novel role in the functioning of the Fab-7 chromatin boundary

2018 ◽  
Author(s):  
Olga Kyrchanova ◽  
Amina Kurbidaeva ◽  
Marat Sabirov ◽  
Nikolay Postika ◽  
Daniel Wolle ◽  
...  
Keyword(s):  
Binding Sites ◽  
Boundary Function ◽  
Polycomb Group ◽  
Homeotic Genes ◽  
Bithorax Complex ◽  
Response Element ◽  
Regulatory Domains ◽  
Polycomb Response Element ◽  
Nuclear Extracts

AbstractExpression of the three Bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries, Fab-7. It was initially identified by a deletion, Fab-71, that transformed parasegment (PS) 11 into a duplicate copy of PS12. Fab-71 deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located in between the iab-6 and iab-7 regulatory domains. Transgene and P-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be the iab-7 Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also presented in the replacement construct. Surprisingly, while HS1+HS3 combination has full boundary activity, we discovered that HS1 alone has only minimal function. Moreover, when combined with HS3, only the distal half of HS1, dHS1, is needed. A ∼1,000 kD multiprotein complex containing the GAF protein, called the LBC, binds to the dHS1 sequence and we show that mutations in dHS1 that disrupt LBC binding in nuclear extracts eliminate boundary activity and GAF binding in vivo. HS3 has binding sites for GAF and Pho proteins that are required for PRE silencing. In contrast, HS3 boundary activity only requires the GAF binding sites. LBC binding with HS3 in nuclear extracts, and GAF association in vivo depend upon the HS3 GAF sites, but not the Pho sites. Consistent with a role for the LBC in HS3 boundary activity, the boundary function of the dHS1+HS3mPho combination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for the iab-7 PREs in chromosome architecture.Author SummaryPolycomb group proteins (PcG) are important epigenetic regulators of developmental genes in all higher eukaryotes. In Drosophila, these proteins are bound to specific regulatory DNA elements called Polycomb group Response Elements (PREs). PcG support proper patterns of homeotic gene expression throughout development. Drosophila PREs are made up of binding sites for a complex array of DNA binding proteins, including GAF and Pho. In the regulatory region of the bithorax complex (BX-C), the boundary/insulator elements organize the autonomous regulatory domains, and their active or repressed states are regulated by PREs. Here, we studied the domain organization of the Fab-7 boundary and the neighboring PRE, which separate the iab-6 and iab-7 domains involved in transcription of the Abd-B gene. It was previously thought that PRE recruits PcG proteins that inhibit activation of the iab-7 enhancers in the inappropriate domains. However, here we found that PRE contributes to boundary activity and in combination with a key 242 bp Fab-7 region (dHS1) can form a completely functional boundary. Late Boundary Complex (LBC) binds not only to dHS1 but also to PRE and is required for the boundary activity of both elements. At the same time, mutations of Pho binding sites strongly diminish recruiting of PcG but do not considerably affect boundary function, suggesting that these activities can be separated in PRE.

The domino gene of Drosophila encodes novel members of the SWI2/SNF2 family of DNA-dependent ATPases, which contribute to the silencing of homeotic genes

Development ◽  
2001 ◽  
Vol 128 (8) ◽  
pp. 1429-1441 ◽  
Author(s):  
M.L. Ruhf ◽  
A. Braun ◽  
O. Papoulas ◽  
J.W. Tamkun ◽  
N. Randsholt ◽  
...  
Keyword(s):  
Polytene Chromosomes ◽  
Gene Mutations ◽  
Polycomb Group ◽  
Protein Isoforms ◽  
Homeotic Genes ◽  
Loss Of Function ◽  
Trithorax Group ◽  
A Subunit ◽  
The Common ◽  
Hematopoietic Disorders

The Drosophila domino gene has been isolated in a screen for mutations that cause hematopoietic disorders. Generation and analysis of loss-of-function domino alleles show that the phenotypes are typical for proliferation gene mutations. Clonal analysis demonstrates that domino is necessary for cell viability and proliferation, as well as for oogenesis. domino encodes two protein isoforms of 3202 and 2498 amino acids, which contain a common N-terminal region but divergent C termini. The common region includes a 500 amino acid DNA-dependent ATPase domain of the SWI2/SNF2 family of proteins, which function via interaction with chromatin. We show that, although domino alleles do not exhibit homeotic phenotypes by themselves, domino mutations enhance Polycomb group mutations and counteract Trithorax group effects. The Domino proteins are present in large complexes in embryo extracts, and one isoform binds to a number of discrete sites on larval polytene chromosomes. Altogether, the data lead us to propose that domino acts as a repressor by interfering with chromatin structure. This activity is likely to be performed as a subunit of a chromatin-remodeling complex.

scholarly journals A Drosophila ESC-E(Z) Protein Complex Is Distinct from Other Polycomb Group Complexes and Contains Covalently Modified ESC

2000 ◽  
Vol 20 (9) ◽  
pp. 3069-3078 ◽  
Author(s):  
Joyce Ng ◽  
Craig M. Hart ◽  
Kelly Morgan ◽  
Jeffrey A. Simon
Keyword(s):  
Germ Line ◽  
Gel Filtration ◽  
Developmental Time ◽  
Polycomb Group ◽  
Sex Combs ◽  
Sex Comb ◽  
Z Protein ◽  
Polycomb Group Complexes

ABSTRACT The extra sex combs (ESC) and Enhancer of zeste [E(Z)] proteins, members of the Polycomb group (PcG) of transcriptional repressors, interact directly and are coassociated in fly embryos. We report that these two proteins are components of a 600-kDa complex in embryos. Using gel filtration and affinity chromatography, we show that this complex is biochemically distinct from previously described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, we present evidence that ESC is phosphorylated in vivo and that this modified ESC is preferentially associated in the complex with E(Z). Modified ESC accumulates between 2 and 6 h of embryogenesis, which is the developmental time whenesc function is first required. We find that mutations inE(z) reduce the ratio of modified to unmodified ESC in vivo. We have also generated germ line transformants that express ESC proteins bearing site-directed mutations that disrupt ESC-E(Z) binding in vitro. These mutant ESC proteins fail to provideesc function, show reduced levels of modification in vivo, and are still assembled into complexes. Taken together, these results suggest that ESC phosphorylation normally occurs after assembly into ESC-E(Z) complexes and that it contributes to the function or regulation of these complexes. We discuss how biochemically separable ESC-E(Z) and PC-PH complexes might work together to provide PcG repression.

scholarly journals Structure of a Polycomb Response Element and In Vitro Binding of Polycomb Group Complexes Containing GAGA Factor

2000 ◽  
Vol 20 (9) ◽  
pp. 3187-3197 ◽  
Author(s):  
Béatrice Horard ◽  
Christophe Tatout ◽  
Sylvain Poux ◽  
Vincenzo Pirrotta
Keyword(s):  
Polycomb Group ◽  
Gaga Factor ◽  
Consensus Sequences ◽  
Polycomb Response Element ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Polycomb Response Elements ◽  
Polycomb Group Complexes

ABSTRACT Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.

scholarly journals The iab-7 Polycomb Response Element Maps to a Nucleosome-Free Region of Chromatin and Requires Both GAGA and Pleiohomeotic for Silencing Activity

2001 ◽  
Vol 21 (4) ◽  
pp. 1311-1318 ◽  
Author(s):  
Rakesh K. Mishra ◽  
Jozsef Mihaly ◽  
Stéphane Barges ◽  
Annick Spierer ◽  
François Karch ◽  
...  
Keyword(s):  
Binding Sites ◽  
Polycomb Group ◽  
Polycomb Group Protein ◽  
Gaga Factor ◽  
Response Element ◽  
Polycomb Response Element ◽  
Free Region ◽  
Group Protein

ABSTRACT In the work reported here we have undertaken a functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila melanogasterbithorax complex (BX-C). Previous studies mapped the iab-7PRE to an 860-bp fragment located just distal to the Fab-7boundary. Located within this fragment is an ∼230-bp chromatin-specific nuclease-hypersensitive region called HS3. We have shown that HS3 is capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of amini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. We show that GAGA and Pho interact with these sequences in vitro and that the consensus binding sites for the two proteins are critical for the silencing activity of theiab-7 PRE in vivo.

polyhomeotic appears to be a target of engrailed regulation in Drosophila

Development ◽  
1995 ◽  
Vol 121 (6) ◽  
pp. 1691-1703 ◽  
Author(s):  
N. Serrano ◽  
H.W. Brock ◽  
C. Demeret ◽  
J.M. Dura ◽  
N.B. Randsholt ◽  
...  
Keyword(s):  
Binding Sites ◽  
Normal Development ◽  
Regulatory Protein ◽  
Polytene Chromosomes ◽  
Polycomb Group ◽  
Germ Band ◽  
High Affinity ◽  
Embryonic Segmentation

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and in normal development of posterior compartments. One of its regulatory targets appears to be polyhomeotic (ph), a Polycomb group gene. We observed, by immunostaining, that Engrailed protein binds to the site of the polyhomeotic locus in region 2D of polytene chromosomes. The same analysis carried out on a transgenic line containing one copy of a P(ph-lacZ) construct shows an additional Engrailed-binding site at the location of the insert. In vivo, polyhomeotic depends on engrailed function in germ-band-elongated embryos, when engrailed and polyhomeotic genes are expressed in similar patterns. By in vitro immunoprecipitations and gel shift assays, we identified two classes of high affinity Engrailed-binding sites upstream of each of the two polyhomeotic transcription units. DNA fragments containing these sites were also immunoprecipitated from embryonic UV crosslinked chromatin in presence of anti-Engrailed antibody. These results suggest that polyhomeotic activation in germ-band-elongated embryos could be mediated by Engrailed-binding to these sites.

The DNA-binding polycomb group protein pleiohomeotic mediates silencing of a Drosophila homeotic gene

Development ◽  
1999 ◽  
Vol 126 (17) ◽  
pp. 3905-3913 ◽  
Author(s):  
C. Fritsch ◽  
J.L. Brown ◽  
J.A. Kassis ◽  
J. Muller
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Point Mutations ◽  
Polycomb Group ◽  
Homeotic Gene ◽  
Homeotic Genes ◽  
Polycomb Group Protein ◽  
Cis Acting ◽  
Group Protein

Polycomb group (PcG) proteins repress homeotic genes in cells where these genes must remain inactive during development. This repression requires cis-acting silencers, also called PcG response elements. Currently, these silencers are ill-defined sequences and it is not known how PcG proteins associate with DNA. Here, we show that the Drosophila PcG protein Pleiohomeotic binds to specific sites in a silencer of the homeotic gene Ultrabithorax. In an Ultrabithorax reporter gene, point mutations in these Pleiohomeotic binding sites abolish PcG repression in vivo. Hence, DNA-bound Pleiohomeotic protein may function in the recruitment of other non-DNA-binding PcG proteins to homeotic gene silencers.

scholarly journals batman Interacts with Polycomb and trithorax Group Genes and Encodes a BTB/POZ Protein That Is Included in a Complex Containing GAGA Factor

2003 ◽  
Vol 23 (4) ◽  
pp. 1181-1195 ◽  
Author(s):  
M. Faucheux ◽  
J.-Y. Roignant ◽  
S. Netter ◽  
J. Charollais ◽  
C. Antoniewski ◽  
...  
Keyword(s):  
Polytene Chromosomes ◽  
Homeotic Genes ◽  
Gaga Factor ◽  
Interaction Domain ◽  
Sex Combs Reduced ◽  
Trithorax Group ◽  
Protein Protein Interaction ◽  
Polycomb Response Element ◽  
Activated State ◽  
Large Protein Family

ABSTRACT Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.

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