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

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

10.1101/329284 ◽

2018 ◽

Cited By ~ 1

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.

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

PLoS Genetics ◽

10.1371/journal.pgen.1007442 ◽

2018 ◽

Vol 14 (8) ◽

pp. e1007442 ◽

Cited By ~ 5

Author(s):

Olga Kyrchanova ◽

Amina Kurbidaeva ◽

Marat Sabirov ◽

Nikolay Postika ◽

Daniel Wolle ◽

...

Keyword(s):

Bithorax Complex ◽

Response Element ◽

Polycomb Response Element ◽

Chromatin Boundary

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The Enhancer-Blocking Activity of the Fab-7 Boundary From the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites

Genetics ◽

10.1534/genetics.104.029561 ◽

2004 ◽

Vol 168 (3) ◽

pp. 1371-1384 ◽

Cited By ~ 77

Author(s):

Susan Schweinsberg ◽

Kirsten Hagstrom ◽

Daryl Gohl ◽

Paul Schedl ◽

Ram P. Kumar ◽

...

Keyword(s):

Binding Sites ◽

Bithorax Complex ◽

Gaga Factor ◽

Factor Binding ◽

Blocking Activity ◽

Enhancer Blocking

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The Mcp Element from the bithorax Complex Contains an Insulator That Is Capable of Pairwise Interactions and Can Facilitate Enhancer-Promoter Communication

Molecular and Cellular Biology ◽

10.1128/mcb.25.9.3682-3689.2005 ◽

2005 ◽

Vol 25 (9) ◽

pp. 3682-3689 ◽

Cited By ~ 62

Author(s):

Natalia Gruzdeva ◽

Olga Kyrchanova ◽

Alexander Parshikov ◽

Andrey Kullyev ◽

Pavel Georgiev

Keyword(s):

Gene Expression ◽

Regulatory Region ◽

Boundary Elements ◽

Bithorax Complex ◽

Response Element ◽

Polycomb Response Element ◽

Pairwise Interactions ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene

ABSTRACT Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3′ cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.

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