scholarly journals In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycomb-response element

Development ◽  
1997 ◽  
Vol 124 (9) ◽  
pp. 1809-1820 ◽  
Author(s):  
J. Mihaly ◽  
I. Hogga ◽  
J. Gausz ◽  
H. Gyurkovics ◽  
F. Karch
Keyword(s):  
Boundary Element ◽  
Domain Boundary ◽  
P Element ◽  
Chromatin Domain ◽  
Homeotic Genes ◽  
Bithorax Complex ◽  
Specific Expression ◽  
Response Element ◽  
Regulatory Domains ◽  
Polycomb Response Element

Parasegmental (PS)-specific expression of the homeotic genes of the bithorax-complex (BX-C) appears to depend upon the subdivision of the complex into a series of functionally independent cis-regulatory domains. Fab-7 is a regulatory element that lies between iab-6 and iab-7 (the PS11- and PS12-specific cis-regulatory domains, respectively). Deletion of Fab-7 causes ectopic expression of iab-7 in PS11 (where normally only iab-6 is active). Two models have been proposed to account for the dominant Fab-7 phenotype. The first considers that Fab-7 functions as a boundary element that insulates iab-6 and iab-7. The second model envisages that Fab-7 contains a silencer element that keeps iab-7 repressed in parasegments anterior to PS12. Using a P-element inserted in the middle of the Fab-7 region (the bit transposon), we have generated an extensive collection of new Fab-7 mutations that allow us to subdivide Fab-7 into a boundary element and a Polycomb-respond element (PRE). The boundary lies within 1 kb of DNA on the proximal side of the bit transposon (towards iab-6). Deletions removing this element alone cause a complex gain- and loss-of-function phenotype in PS11; in some groups of cells, both iab-6 and iab-7 are active, while in others both iab-6 and iab-7 are inactive. Thus, deletion of the boundary allows activating as well as repressing activities to travel between iab-6 and iab-7. We also provide evidences that the boundary region contains an enhancer blocker element. The Polycomb-response element lies within 0.5 kb of DNA immediately distal to the boundary (towards iab-7). Deletions removing the PRE alone do not typically cause any visible phenotype as homozygotes. Interestingly, weak ectopic activation of iab-7 is observed in hemizygous PRE deletions, suggesting that the mechanisms that keep iab-7 repressed in the absence of this element may depend upon chromosome pairing. These results help to reconcile the previously contradictory models on Fab-7 function and to shed light on how a chromatin domain boundary and a nearby PRE concur in the setting up of the appropriate PS-specific expression of the Abd-B gene of the BX-C.

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.

scholarly journals A Polycomb and GAGA Dependent Silencer Adjoins the Fab-7 Boundary in the Drosophila Bithorax Complex

Genetics ◽  
1997 ◽  
Vol 146 (4) ◽  
pp. 1365-1380 ◽  
Author(s):  
Kirsten Hagstrom ◽  
Martin Muller ◽  
Paul Schedl
Keyword(s):  
Domain Boundary ◽  
Expression Patterns ◽  
Regulatory Region ◽  
Polycomb Group ◽  
Homeotic Genes ◽  
Bithorax Complex ◽  
Gaga Factor ◽  
Independent Manner ◽  
Specific Expression ◽  
Restricted Pattern

The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the Frontabdominal-7 cis-regulatory region (Fab-71) dominantly transforms parasegment 11 into parasegment 12. Previous studies suggested that removal of a domain boundary element on the proximal side of Fab-71 is responsible for this gain-of-function phenotype. In this article we demonstrate that the Fab-71 deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment and plays a different role in regulating parasegment-specific expression patterns of the Abd-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of a BXD, Ubx/lacZ reporter transgene. Both silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste1 allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans a nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor.

The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain

Development ◽  
2000 ◽  
Vol 127 (4) ◽  
pp. 779-790 ◽  
Author(s):  
S. Barges ◽  
J. Mihaly ◽  
M. Galloni ◽  
K. Hagstrom ◽  
M. Muller ◽  
...  
Keyword(s):  
Higher Order ◽  
Specific Pattern ◽  
Bithorax Complex ◽  
Response Element ◽  
Regulatory Domains ◽  
Polycomb Response Element ◽  
Distal Side ◽  
Blocking Activity ◽  
Enhancer Blocking ◽  
Order Domain

The Drosophila bithorax complex Abdominal-B (Abd-B) gene specifies parasegmental identity at the posterior end of the fly. The specific pattern of Abd-B expression in each parasegment (PS) determines its identity and, in PS10-13, Abd-B expression is controlled by four parasegment-specific cis-regulatory domains, iab-5 to iab-8, respectively. In order to properly determine parasegmental identity, these four cis-regulatory domains must function autonomously during both the initiation and maintenance phases of BX-C regulation. The studies reported here demonstrate that the (centromere) distal end of iab-7 domain is delimited by the Fab-8 boundary. Initiators that specify PS12 identity are located on the proximal iab-7 side of Fab-8, while initiators that specify PS13 identity are located on the distal side of Fab-8, in iab-8. We use transgene assays to demonstrate that Fab-8 has enhancer blocking activity and that it can insulate reporter constructs from the regulatory action of the iab-7 and iab-8 initiators. We also show that the Fab-8 boundary defines the realm of action of a nearby iab-8 Polycomb Response Element, preventing this element from ectopically silencing the adjacent domain. Finally, we demonstrate that the insulating activity of the Fab-8 boundary in BX-C is absolutely essential for the proper specification of parasegmental identity by the iab-7 and iab-8 cis-regulatory domains. Fab-8 together with the previously identified Fab-7 boundary delimit the first genetically defined higher order domain in a multicellular eukaryote.

Transcription through the iab-7 cis-regulatory domain of the bithorax complex interferes with maintenance of Polycomb-mediated silencing

Development ◽  
2002 ◽  
Vol 129 (21) ◽  
pp. 4915-4922 ◽  
Author(s):  
Ilham Hogga ◽  
François Karch
Keyword(s):  
Domain Boundary ◽  
Regulatory Region ◽  
Chromatin Domain ◽  
Dependent Manner ◽  
Regulatory Domain ◽  
Bithorax Complex ◽  
Regulatory Domains ◽  
Repression Complex ◽  
Ectopic Activation ◽  
Functional Autonomy

The Fab-7 chromatin domain boundary insures functional autonomy of the iab-6 and iab-7 cis-regulatory domains in the bithorax complex (BX-C). We have previously shown that chromatin insulators such asgypsy or scsmin are potent insulators that cannot substitute for Fab-7 function within the BX-C. During the early stages of these swapping experiments, we initially used a fragment of scs that was slightly larger than a minimal scs element (scsmin). We report that this scs fragment, unlike scsmin, interferes in an orientation-dependent manner with the output of a regulatory region covering 80 kb of DNA (from iab-4 to iab-8). At the core of this orientation-dependent phenotype is a promoter located immediately adjacent to the scs insulator. In one orientation, the promoter traps the activity of theiab-3 through iab-5 cis-regulatory domains, diverting them from the abd-A gene. In the opposite orientation, the promoter is transcribing the iab-7 cis-regulatory domain, resulting in ectopic activation of the latter. Our data suggest that transcription through aPolycomb-Response Element (PRE) interferes with the maintenance of aPolycomb repression complex.

scholarly journals Boundary Element-Associated Factor 32B Connects Chromatin Domains to the Nuclear Matrix

2007 ◽  
Vol 27 (13) ◽  
pp. 4796-4806 ◽  
Author(s):  
Rashmi U. Pathak ◽  
Nandini Rangaraj ◽  
Satish Kallappagoudar ◽  
Krishnaveni Mishra ◽  
Rakesh K. Mishra
Keyword(s):  
Boundary Element ◽  
Dna Sequences ◽  
Nuclear Matrix ◽  
Domain Boundary ◽  
Coiled Coil ◽  
Boundary Elements ◽  
Structural Basis ◽  
Chromatin Domain ◽  
Eukaryotic Genomes

ABSTRACT Chromatin domain boundary elements demarcate independently regulated domains of eukaryotic genomes. While a few such boundary sequences have been studied in detail, only a small number of proteins that interact with them have been identified. One such protein is the boundary element-associated factor (BEAF), which binds to the scs′ boundary element of Drosophila melanogaster. It is not clear, however, how boundary elements function. In this report we show that BEAF is associated with the nuclear matrix and map the domain required for matrix association to the middle region of the protein. This region contains a predicted coiled-coil domain with several potential sites for posttranslational modification. We demonstrate that the DNA sequences that bind to BEAF in vivo are also associated with the nuclear matrix and colocalize with BEAF. These results suggest that boundary elements may function by tethering chromatin to nuclear architectural components and thereby provide a structural basis for compartmentalization of the genome into functionally independent domains.

scholarly journals H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Sarah K Bowman ◽  
Aimee M Deaton ◽  
Heber Domingues ◽  
Peggy I Wang ◽  
Ruslan I Sadreyev ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Protein ◽  
Histone H3 ◽  
Polycomb Group ◽  
Homeotic Genes ◽  
Bithorax Complex ◽  
Body Segment ◽  
Regulatory Domains ◽  
Definition Of ◽  
Identity Expression

The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a ‘stairstep’ pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity.

scholarly journals Chromatin domain boundary element search tool for Drosophila

2012 ◽  
Vol 40 (10) ◽  
pp. 4385-4395 ◽  
Author(s):  
Arumugam Srinivasan ◽  
Rakesh K. Mishra
Keyword(s):  
Boundary Element ◽  
Domain Boundary ◽  
Chromatin Domain ◽  
Search Tool

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

PLoS Genetics ◽  
2018 ◽  
Vol 14 (8) ◽  
pp. e1007442 ◽  
Author(s):  
Olga Kyrchanova ◽  
Amina Kurbidaeva ◽  
Marat Sabirov ◽  
Nikolay Postika ◽  
Daniel Wolle ◽  
...  
Keyword(s):  
Bithorax Complex ◽  
Response Element ◽  
Polycomb Response Element ◽  
Chromatin Boundary

scholarly journals Boundaries support specific long-distance interactions between enhancers and promoters in Drosophila Bithorax complex

2018 ◽  
Author(s):  
Nikolay Postika ◽  
Mario Metzler ◽  
Markus Affolter ◽  
Martin Müller ◽  
Paul Schedl ◽  
...  
Keyword(s):  
Target Genes ◽  
Model Systems ◽  
Homeotic Genes ◽  
Bithorax Complex ◽  
Long Distance ◽  
Gene Target ◽  
Regulatory Domains ◽  
Insulator Elements

AbstractDrosophila bithorax complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously. Paradoxically, seven of the BX-C regulatory domains are separated from their gene target by at least one boundary, and must “jump over” the intervening boundaries. To understand the jumping mechanism, the Mcp boundary was replaced with Fab-7 and Fab-8. Mcp is located between the iab-4 and iab-5 domains, and defines the border between the set of regulatory domains controlling abd-A and Abd-B. When Mcp is replaced by Fab-7 or Fab-8, they direct the iab-4 domain (which regulates abd-A) to inappropriately activate Abd-B in abdominal segment A4. For the Fab-8 replacement, ectopic induction was only observed when it was inserted in the same orientation as the endogenous Fab-8 boundary. A similar orientation dependence for bypass activity was observed when Fab-7 was replaced by Fab-8. Thus, boundaries perform two opposite functions in the context of BX-C – they block crosstalk between neighboring regulatory domains, but at the same time actively facilitate long distance communication between the regulatory domains and their respective target genes.Author SummaryDrosophila bithorax complex (BX-C) is one of a few examples demonstrating in vivo role of boundary/insulator elements in organization of independent chromatin domains. BX-C contains three HOX genes, whose parasegment-specific pattern is controlled by cis-regulatory domains flanked by boundary/insulator elements. Since the boundaries ensure autonomy of adjacent domains, the presence of these elements poses a paradox: how do the domains bypass the intervening boundaries and contact their proper regulatory targets? According to the textbook model, BX-C regulatory domains are able to bypass boundaries because they harbor special promoter targeting sequences. However, contrary to this model, we show here that the boundaries themselves play an active role in directing regulatory domains to their appropriate HOX gene promoter.

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