McpandFab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex ofDrosophila melanogaster

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.

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A Stage-Specific Factor Confers Fab-7 Boundary Activity during Early Embryogenesis in Drosophila

Molecular and Cellular Biology ◽

10.1128/mcb.01622-07 ◽

2007 ◽

Vol 28 (3) ◽

pp. 1047-1060 ◽

Cited By ~ 18

Author(s):

Tsutomu Aoki ◽

Susan Schweinsberg ◽

Julia Manasson ◽

Paul Schedl

Keyword(s):

Adult Stage ◽

Early Embryogenesis ◽

Specific Factor ◽

Bithorax Complex ◽

Recognition Sequence ◽

Regulatory Domains ◽

Nuclear Extracts ◽

Early Embryos ◽

Blocking Activity ◽

Enhancer Blocking

ABSTRACT The Fab-7 boundary is required to ensure that the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax complex can function autonomously. Though Fab-7 functions as a boundary from early embryogenesis through to the adult stage, this constitutive boundary activity depends on subelements whose activity is developmentally restricted. In the studies reported here, we have identified a factor, called early boundary activity (Elba), that confers Fab-7 boundary activity during early embryogenesis. The Elba factor binds to a recognition sequence within a Fab-7 subelement that has enhancer-blocking activity during early embryogenesis, but not during mid-embryogenesis or in the adult. We found that the Elba factor is present in early embryos but largely disappears during mid-embryogenesis. We show that mutations in the Elba recognition sequence that eliminate Elba binding in nuclear extracts disrupt the early boundary activity of the Fab-7 subelement. Conversely, we find that early boundary activity can be reconstituted by multimerizing the Elba recognition site.

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Redundant enhancers in the iab-5 domain cooperatively activate Abd-B in the A5 and A6 abdominal segments of Drosophila

10.1101/2021.05.22.445252 ◽

2021 ◽

Author(s):

Nikolay Postika ◽

Paul Schedl ◽

Pavel Georgiev ◽

Olga Kyrchanova

Keyword(s):

Regulatory Elements ◽

Regulatory Domain ◽

Bithorax Complex ◽

Tissue Specific ◽

Regulatory Domains ◽

Male Specific ◽

Necessary And Sufficient

The homeotic Abdominal-B (Abd-B) gene belongs to Bithorax complex and is regulated by four regulatory domains named iab-5, iab-6, iab-7 and iab-8, each of which is thought to be responsible for directing the expression of Abd-B in one of the abdominal segments from A5 to A8. It is assumed that male specific features of the adult cuticle in A5 is solely dependent on regulatory elements located in iab-5, while the regulatory elements in the iab-6 are both necessary and sufficient for the proper differentiation of the A6 cuticle. Unexpectedly, we found that this long held assumption is not correct. Instead, redundant tissue-specific enhancers located in the iab-5 domain are required for the proper activation of Abd-B not only in A5 but also in A6. Our study of deletions shows that the iab-5 initiator is essential for the functioning of the iab-5 enhancers in A5, as well as for the correct differentiation of A6. This requirement is circumvented by deletions that remove the initiator and most of the iab-5 regulatory domain sequences. While the remaining iab-5 enhancers are inactive in A5, they are activated in A6 and contribute to the differentiation of this segment. In this case, Abd-B stimulation by the iab-5 enhancers in A6 depends on the initiators in the iab-4 and iab-6 domains.

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

10.1101/423103 ◽

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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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 ◽

10.1242/dev.127.4.779 ◽

2000 ◽

Vol 127 (4) ◽

pp. 779-790 ◽

Cited By ~ 12

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.

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Complete reconstitution of bypass and blocking functions in a minimal artificial Fab-7 insulator from Drosophila bithorax complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1907190116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13462-13467 ◽

Cited By ~ 4

Author(s):

Olga Kyrchanova ◽

Marat Sabirov ◽

Vladic Mogila ◽

Amina Kurbidaeva ◽

Nikolay Postika ◽

...

Keyword(s):

Cross Talk ◽

Binding Sites ◽

Multiprotein Complex ◽

Bithorax Complex ◽

Artificial Boundary ◽

Long Distance ◽

Specific Expression ◽

Regulatory Domains ◽

Architectural Proteins ◽

Distance Communication

Boundaries in the bithorax complex (BX-C) delimit autonomous regulatory domains that drive parasegment-specific expression of the Hox genes Ubx, abd-A, and Abd-B. The Fab-7 boundary is located between the iab-6 and iab-7 domains and has two key functions: blocking cross-talk between these domains and at the same time promoting communication (boundary bypass) between iab-6 and the Abd-B promoter. Using a replacement strategy, we found that multimerized binding sites for the architectural proteins Pita, Su(Hw), and dCTCF function as conventional insulators and block cross-talk between the iab-6 and iab-7 domains; however, they lack bypass activity, and iab-6 is unable to regulate Abd-B. Here we show that an ∼200-bp sequence of dHS1 from the Fab-7 boundary rescues the bypass defects of these multimerized binding sites. The dHS1 sequence is bound in embryos by a large multiprotein complex, Late Boundary Complex (LBC), that contains the zinc finger proteins CLAMP and GAF. Using deletions and mutations in critical GAGAG motifs, we show that bypass activity correlates with the efficiency of recruitment of LBC components CLAMP and GAF to the artificial boundary. These results indicate that LBC orchestrates long-distance communication between the iab-6 regulatory domain and the Abd-B gene, while the Pita, Su(Hw), and dCTCF proteins function to block local cross-talk between the neighboring regulatory domains iab-6 and iab-7.

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Redundant enhancers in the iab-5 domain cooperatively activate Abd-B in the A5 and A6 abdominal segments of Drosophila

Development ◽

10.1242/dev.199827 ◽

2021 ◽

Author(s):

Nikolay Postika ◽

Paul Schedl ◽

Pavel Georgiev ◽

Olga Kyrchanova

Keyword(s):

Additive Model ◽

Bithorax Complex ◽

Tissue Specific ◽

Regulatory Domains ◽

Necessary And Sufficient

The Abdominal-B (Abd-B) gene belongs to Bithorax complex and its expression is controlled by four regulatory domains, iab-5, iab-6, iab-7 and iab-8, each of which is thought to be responsible for directing the expression of Abd-B in one of the abdominal segments from A5 to A8. A variety of experiments have supported the idea that BX-C regulatory domains are functionally autonomous and that each domain is both necessary and sufficient to orchestrate the development of the segment they specify. Unexpectedly, we discovered that this model does not always hold. Instead, we find that tissue-specific enhancers located in the iab-5 domain are required for the proper activation of Abd-B not only in A5 but also in A6. Our findings indicate that the functioning of the iab-5 and iab-6 domains in development of the adult cuticle A5 and A6 in males fit better with an additive model much like that first envisioned by Ed Lewis.

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

Development ◽

10.1242/dev.124.9.1809 ◽

1997 ◽

Vol 124 (9) ◽

pp. 1809-1820 ◽

Cited By ~ 5

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.

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Mobile Element 297 in the Abd-B gene of Drosophila melanogaster, Not Delta 88, Is Responsible for the tuh-3 Mutation

Genetics ◽

10.1093/genetics/147.2.679 ◽

1997 ◽

Vol 147 (2) ◽

pp. 679-688

Author(s):

Judith A Mack ◽

Ronald D Smith ◽

David T Kuhn

Keyword(s):

Drosophila Melanogaster ◽

Maternal Effects ◽

Molecular Analysis ◽

Maternal Effect ◽

Mobile Element ◽

Bithorax Complex ◽

Genital Tissue

The tumorous-head-3 (tuh-3) mutation has been associated with the insertion of mobile element Delta 88 at +200 on the bithorax complex (BX-C) DNA map, 5′ of all Abdominal-B (Abd-B) transcripts. Different phenotypes of tuh-3 are regulated by the tumorous-head-1 (tuh-1) maternal effect locus. In the presence of the recessive tuh-1h maternal effect, tuh-3 offspring produce homeotic abdominal and genital tissue in the head. In the presence of the dominant tuh-1g maternal effect, tuh-3 offspring have normal heads but now show genital defects. One other mutant, I127B, produces flies with identical defects to that of tuh-3 in the presence of both maternal effects. Molecular analysis of I127B revealed the insertion of mobile element 297 in the Abd-B gene, ∼25 kb downstream of the Delta 88 insertion in tuh-3. No other abnormalities were detected. Reexamination of our tuh-3 strain revealed a 297 insertion in an identical region to that of I127B, in addition to the Delta 88 insertion. Recombinants of tuh-3, carrying 297 only, produced homeotic head defects and genital defects in the presence of the tuh-1h and tuh-1R maternal effects, respectively. Recombinants of tuh-3, carrying Delta 88 only, failed to produce any defects in the presence of either maternal effect. Based upon these results, we propose that it is the 297 insertion in the Abd-B gene, not Delta 88, that is responsible for the tuh-3 mutation.

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

Development ◽

10.1242/dev.129.21.4915 ◽

2002 ◽

Vol 129 (21) ◽

pp. 4915-4922 ◽

Cited By ~ 1

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.

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