staff

Fabienne Cléard-Karch

Research assistant in Drosophila bithorax complex

  • T: +41 22 379 67 56
  • office 3004b (Sciences III)
  • Different Evolutionary Strategies To Conserve Chromatin Boundary Function in the Bithorax Complex. Genetics 2017 Feb;205(2):589-603. genetics.116.195586. 10.1534/genetics.116.195586.

    abstract

    Chromatin boundary elements subdivide chromosomes in multicellular organisms into physically independent domains. In addition to this architectural function, these elements also play a critical role in gene regulation. Here we investigated the evolution of a Drosophila Bithorax complex boundary element called Fab-7, which is required for the proper parasegment specific expression of the homeotic Abd-B gene. Using a "gene" replacement strategy, we show that Fab-7 boundaries from two closely related species, D. erecta and D. yakuba, and a more distant species, D. pseudoobscura, are able to substitute for the melanogaster boundary. Consistent with this functional conservation, the two known Fab-7 boundary factors, Elba and LBC, have recognition sequences in the boundaries from all species. However, the strategies used for maintaining binding and function in the face of sequence divergence is different. The first is conventional, and depends upon conservation of the 8 bp Elba recognition sequence. The second is unconventional, and takes advantage of the unusually large and flexible sequence recognition properties of the LBC boundary factor, and the deployment of multiple LBC recognition elements in each boundary. In the former case, binding is lost when the recognition sequence is altered. In the latter case, sequence divergence is accompanied by changes in the number, relative affinity, and location of the LBC recognition elements.

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  • Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex. PLoS Genet. 2016 Jul;12(7):e1006188. 10.1371/journal.pgen.1006188. PGENETICS-D-15-02937. PMC4948906.

    abstract

    Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

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  • Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex. Mol. Cell. Biol. 2015 Nov;35(21):3739-52. MCB.00456-15. 10.1128/MCB.00456-15. PMC4589599.

    abstract

    Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C.

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  • Following the intracellular localization of the iab-8ncRNA of the bithorax complex using the MS2-MCP-GFP system. Mech. Dev. 2015 Nov;138 Pt 2():133-40. S0925-4773(15)30021-6. 10.1016/j.mod.2015.08.004.

    abstract

    Homeotic genes are aligned on the chromosome in the order of the segments that they specify along the antero-posterior axis of the fly. In general the genes affecting the more posterior segments repress the more anterior genes, a phenomenon known as "posterior dominance". There is however a noticeable exception to this rule in the central nervous system of Drosophila melanogaster where the posterior Abd-B gene does not repress the immediately more anterior abd-A gene. Instead, abd-A repression is accomplished by a 92 kb-long ncRNA (the iab-8ncRNA) that is transcribed from the large inter-genic region between abd-A and Abd-B. This iab-8ncRNA encodes a microRNA to repress abd-A and also a second redundant repression mechanism acting in cis and thought to be transcriptional interference with the abd-A promoter. Using in situ hybridization, a previous work suggested that the iab8ncRNA transcript forms discrete foci restricted to the nuclear periphery and that this localization may be important for its function. In order to better characterize the intra-cellular localization of the iab-8ncRNA we used the MS2-MCP system, which allows fluorescent labeling of RNA in cells and relies on the interaction between GFP-tagged MS2 coat protein (MCP-GFP) and MS2 RNA stem loops. Our results indicate that the large foci seen in previous studies correspond to the site of iab8ncRNA transcription and that the foci seen may simply be an indication of the level of transcription at the locus. We find no evidence to suggest that this localization is important for its function on abd-A repression. We discuss the idea that the iab-8ncRNA may be a relic of a more general ancient mechanism of posterior dominance during the emergence of the hox clusters that was mediated by transcriptional interference.

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  • DamID as an approach to studying long-distance chromatin interactions. Methods Mol. Biol. 2014 ;1196():279-89. 10.1007/978-1-4939-1242-1_17.

    abstract

    How transcription is controlled by distally located cis-regulatory elements is an active area of research in biology. As such, there have been many techniques developed to probe these long-distance chromatin interactions. Here, we focus on one such method, called DamID (van Steensel and Henikoff, Nat Biotechnol 18(4):424-428, 2000). While other methods like 3C (Dekker et al., Science 295(5558):1306-1311, 2002), 4C (Simonis et al., Nat Genet 38(11):1348-1354, 2006; Zhao et al., Nat Genet 38(11):1341-1347, 2006), and 5C (Dostie et al., Genome Res 16(10):1299-1309, 2006) are undoubtedly powerful, the DamID method can offer some advantages over these methods if the genetic locus can be easily modified. The lack of tissue fixation, the low amounts of starting material required to perform the experiment, and the relatively modest hardware requirements make DamID experiments an interesting alternative to consider when examining long-distance chromatin interactions.

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  • Probing long-distance regulatory interactions in the Drosophila melanogaster bithorax complex using Dam identification. Nat. Genet. 2006 Aug;38(8):931-5. ng1833. 10.1038/ng1833.

    abstract

    A cis-regulatory region of nearly 300 kb controls the expression of the three bithorax complex (BX-C) homeotic genes: Ubx, abd-A and Abd-B. Interspersed between the numerous enhancers and silencers within the complex are elements called domain boundaries. Recently, many pieces of evidence have suggested that boundaries function to create autonomous domains by interacting among themselves and forming chromatin loops. In order to test this hypothesis, we used Dam identification to probe for interactions between the Fab-7 boundary and other regions in the BX-C. We were surprised to find that the targeting of Dam methyltransferase (Dam) to the Fab-7 boundary results in a strong methylation signal at the Abd-Bm promoter, approximately 35 kb away. Moreover, this methylation pattern is found primarily in the tissues where Abd-B is not expressed and requires an intact Fab-7 boundary. Overall, our work provides the first documented example of a dynamic, long-distance physical interaction between distal regulatory elements within a living, multicellular organism.

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