The Variable CTCF Site from Drosophila melanogaster Ubx Gene is Redundant and Has no Insulator Activity

– CTCF is the most thoroughly studied chromatin architectural protein and it is found in both Drosophila and mammals. CTCF preferentially binds to promoters and insulators and is thought to facilitate formation of chromatin loops. In a subset of sites, CTCF binding depends on the epigenetic status of the sur-rounding chromatin. One such variable CTCF site ( vCTCF ) was found in the intron of the Ubx gene, in close proximity to the BRE and abx enhancers. CTCF binds to the variable site in tissues where Ubx gene is active, suggesting that the vCTCF site plays a role in facilitating contacts between the Ubx promoter and its enhancers. Using CRISPR/Cas9 and attP/attB site-specific integration methods, we investigated the functional role of vCTCF and showed that it is not required for normal Drosophila development. Furthermore, a 2161-bp fragment containing vCTCF does not function as an effective insulator when substituted for the Fab-7 boundary in the Bithorax complex. Our results suggest that vCTCF function is redundant in the regulation of Ubx .

Parasegment-specific expression of the Ubx, abd-A, and Abd-B homeotic genes in the Drosophila melanogaster Bithorax complex (BX-C) is controlled by nine autonomous regulatory domains, which are separated by special elements called boundaries or insulators [1]. Boundaries ensure autonomy by blocking contacts between regulatory elements in one domain with regulatory elements in adjacent domains. Boundaries can also prevent enhancers from interacting with promoters [2][3][4]. In addition to insulator activity, some boundaries have an ability to specifically interact with their target gene in BX-C, enabling enhancers in distant regulatory domains to stimulate their target promoter [5]. These properties of the boundaries ensure correct parasegment-specific expression of the BX-C genes during Drosophila development. Consistent with this idea, Fab-6, Fab-7, and Fab-8 were shown to specifically interact with the promoter upstream region of Abd-B gene [6]. It is likely that this interaction determines the correct topological positioning of the corresponding regulatory domains (iab5 -iab7) with Abd-B promoter in parasegments 10-12.
Most of the BX-C boundaries contain binding sites for Drosophila CTCF (dCTCF), and these sites are important for the insulator activity of these boundaries ( Fig. 1) [7]. In the intron of the Ubx gene 30 kb downstream from the promoter, a variable dCTCF binding site (vCTCF) was identified ( Fig. 1) [8]. dCTCF does not occupy this site in tissues where Ubx is inactive (imaginal discs of the first pair of legs), but binds to it when the Ubx gene is transcriptionally active (imaginal discs of the third pair of legs). Moreover, dCTCF binding to vCTCF is associated with changes in the topology of the abx/bx regulatory domain: in tissues where Ubx is active an increase in the frequency of vCTCF contacts with the Ubx promoter is observed [8]. A model was proposed according to which binding of dCTCF to vCTCF facilitates tissue-specific interaction of the abx, BRE enhancers with the Ubx promoter [9,10]. The aim of this study was to test this hypothesis.
To study vCTCF function in enhancer-promoter interactions, we used the CRISPR/Cas9 system to delete a 3408-bp DNA fragment (3R:16701239..16704646) that spans the vCTCF site and the bx PRE (polycomb response element) 1 kb downstream, and in its place we introduced an attP site (Δ3.4 attP , Fig. 1). Flies homozygous for Δ3.4 attP deletion show evidence of variable LOF transformations. The deletion transforms the anterior third thoracic segment toward the anterior second thoracic, a phenotype known as bithorax (bx) [11,12]. In mutant flies the anterior third leg resembles the second leg, and in ~10% of flies anterior notal tissue is present on the dorsal surface of the third thoracic segment (Fig. 2). These transformations are caused by a disruption in the interactions of enhancers downstream of vCTCF with Ubx promoter. The Δ3.4 attP deletion overlaps with a previously described 9.5 kb deletion, bx 34e-prv . Like Δ3.4 att , it also has a variable bx phenotype which is caused by a decrease in Ubx   In wt males, A7 segment is absent, A6 sternite is banana-shaped and has no bristles, while A5 sternite is rectangular and covered with bristles. A5 tergite is completely covered with trichomes, while A6 has bristles only along anterior and ventral margins (see dark field). In Fab-7 attP50 males, A6 segment is transformed toward A7 (does not develop) due to the fusion of iab-6 and iab-7 regulatory domains. vCTCF+PRE males also do not develop A6 segment. In order to test vCTCF insulator activity we used Fab-7 attP50 replacement platform (Fig. 1). In this platform, Fab-7 boundary is removed, resulting in the fusion of iab-6 and iab-7 regulatory domains. This leads to ectopic activation of the iab-7 regulatory domain in PS11, which in turn results in the loss of the sixth abdominal segment in adult males [13][14][15]. It was demonstrated previously that PREs are often located in close proximity to insulators and contribute to the formation of a functional boundary [16,17]. Therefore, a fragment containing both bx PRE and vCTCF in reverse orientation, vCTCF+PRE (2161-bp, 3R:16702487..16704647) was tested in Fab-7 attP50 . We found that the 6 th abdominal segment is still missing in males carrying vCTCF+PRE insertion. This finding indicates that the vCTCF+PRE sequence does not have insulator activity.

WT
Altogether, our data do not support a model in which vCTCF is a necessary mediator of enhancerpromoter interactions in abx/bx domain. Moreover, the data suggest that the bx PRE may play that role. However, further research is needed to explore the functions of this element in Ubx regulation. Since the loss of the bx PRE leads only to a variable LOF phenotype, it can be assumed that, in contrast to the Abd-B enhancers, Ubx enhancers are much more autonomous and less dependent on other regulatory elements to form appropriate promoter contacts. ACKNOWLEDGMENTS This work was supported by the Russian Science Foundation projects nos. 20-14-00201 and 19-74-30026. CRISPR/Cas9 mutagenesis and attP/attB site-specific integration were supported by grant 20-14-00201. Phenotype analysis was supported by grant 19-74-30026 from the Russian Science Foundation.

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Conflict of interest. The authors declare that they have no conflicts of interest.
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