# Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva

Solène Vanderperre, Samir Merabet

► To cite this version:

Solène Vanderperre, Samir Merabet. Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva. Cells, 2024, 13 (7), pp.613. 10.3390/cells13070613. [hal-04931328](https://hal.science/hal-04931328v1)

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# Visualization of dimeric protein complex association on specific enhancers in salivary gland nuclei of the *Drosophila* larva

Solène Vanderperre¹ and Samir Merabet ¹,#

¹Institut de Génomique Fonctionnelle de Lyon (IGFL), UMR5242, Ecole Normale Supérieure de Lyon (ENSL), CNRS, Université de Lyon, Lyon, France.

²email: samir.merabet@ens-lyon.fr

**Abstract:** Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein-protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity *in vivo*.

Over the last years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied for the analysis of different types of PPIs. In particular, BiFC has been applied for analyzing PPIs with hundreds of TFs in the nucleus of live *Drosophila* embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that could be coupled with BiFC. Here, we present a novel experimental strategy that we called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables to precisely quantify the enrichment of specific dimeric protein complexes on target enhancers in *Drosophila* salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more largely in other tissues during *Drosophila* development. Our work set up the experimental basis for these future applications.

**Keywords:** protein-protein interaction; BiFC; ANCHOR; ParB-INT; Hox

## 1. Introduction

Cell fate is determined by specific gene expression programs that are primarily occurring at the level of transcription. This key regulatory process leads to the production of precise doses of mRNA molecules that will subsequently be translated into proteins. Among the different cis-regulatory elements that control mRNA production are the so-called "enhancers", which are DNA elements of relatively small size (less than 1 kilobases) and located at various distances from the transcription start site [1]. Enhancers contain specific DNA sequences that are recognized by transcription factors (TFs). Importantly, the full activity of an enhancer relies on the combinatorial binding and activity of several TFs with their cofactors [2]. Therefore, understanding the molecular rules that govern enhancer activity requires the visualization of protein-protein interactions (PPIs) that are specifically occurring on it in the nucleus.

Several techniques have been developed to visualize the genomic regions of interest (ROIs). These techniques rely on the use of either fluorescent oligonucleotide probes for doing fluorescent in situ hybridization (FISH), or DNA-binding proteins fused to fluorescent proteins (FPs). FISH provides kilobase resolution but requires stringent experimental procedures, such as DNA denaturation, that may affect PPIs ([3], see also results). Alternatively, different DNA-binding proteins have been fused to different FPs to target and label a genomic ROI in the nucleus. These strategies depend on multiplexing for getting
enough FPs bound to the target locus for subsequent analysis by microscopy. For example, fluorescent dCas9 (catalytically dead Cas9) has been used to label repeated sequences in the genome [4–6]. Fluorescent dCas9 can also be used to label non-repetitive cis-regulatory elements by multiplexing guide RNAs (gRNAs) oligonucleotides [7]. However, these Cas9-based strategies have only been established in culture cells and await further development for applications in a multicellular organism. Other strategies exist to label genomic ROI in the nucleus. For example, the Lactose Operon Repressor/Operator (LacI/LacO) and Tetracycline Operon Repressor/Operator (TetR/TetO) systems have been used to visualize chromosome translocations in human live cells [8]. Still, this system requires intensive engineering since it is based on the insertion of a high number of LacO and TetO DNA sequences for exploiting fluorescent signals emitted by the FP-LacI and FP-TetR fusion proteins. Another more promising approach is based on the Partition Protein B/Binding Sequence (ParB-INT or ANCHOR) system. In this system, multimerization of the fluorescent ParB proteins is achieved through PPIs on the *INT* sequence. First applied in yeast [9] and human cell culture [10,11], this system has also been developed to visualize enhancer-promoter interactions [12] or a DNA locus in *Drosophila* [13]. Moreover, a recent analysis showed that the ANCHOR system did not locally perturb transcription in *Drosophila* (in contrast to the LacI/lacO system: [14]).

Here, we present a unique experimental approach coupling the ANCHOR and Biomolecular Fluorescence Complementation (BiFC) systems to visualize and measure the specific enrichment of dimeric protein complexes on a target DNA sequence in the nucleus of *Drosophila* larval salivary gland cells. BiFC relies on the property of monomeric FPs to be reconstituted from two separate sub-fragments upon spatial proximity [15,16]. In particular, BiFC has been extensively used to reveal and analyze PPIs between various classes of TFs in live *Drosophila* embryos [17–19]. As a proof-of-concept, we used the well-characterized enhancer of the salivary gland selector gene *forkhead* (enhancer *fkh250*) as a regulatory model system. This enhancer is specifically regulated by the Hox protein Sex combs reduced (Scr) in association with the Extradenticle (Exd) cofactor *in vivo* [20]. This regulation occurs through the recognition of a unique Scr/Exd DNA-binding site. Importantly, Exd was shown to bind cooperatively with Scr and not with any other Hox protein on this enhancer *in vitro* [20]. The specific regulation by Scr/Exd complexes was confirmed *in vitro* and *in vivo* with mutated enhancers in the Scr or Exd DNA-binding site [20]. Interestingly, the Scr/Exd DNA-binding site was also converted into a more consensus Hox/Exd binding site, allowing the binding of different Hox/Exd complexes *in vitro* and leading to ectopic expression in the embryo [20]. Altogether, the different variants of the *fkh250* enhancer constitute ideal tools for testing the specificity of the ANCHOR and BiFC systems. Our work demonstrates that BiFC and ANCHOR coupling (hereafter called “BiFOR”) is a powerful approach for the sensitive quantification of PPI enrichment on specific target enhancers in *Drosophila* salivary gland nuclei.

## 2. Material and methods 84

### 2.1. Fly lines 85

The salivary gland *sgs3-Gal4* driver was from the Bloomington stock center (line 6870). *ParB1-mCherry* fly lines were given by François Payre (CBI, Toulouse, France).

The *UAS-VC-Ubx*, *UAS-VC-Scr* and *UAS-VN-Exd* fly lines were already established [21].

### 2.2. Generation of the INT1-fkh enhancer fly lines 90

The different enhancers *fkh250*, *fkh250MUT* and *fkh250CONS* were synthesized in ten copies (GenScript) and cloned between the EcoRI and XhoI sites, downstream of the three *INT1* sequences of the *pattB-INT1-hslacZ* vector (provided by F. Payre). All constructs were verified by sequencing before sending for injection (Bestgene Inc. company). *INT1-fkh*
constructs were all inserted in the same landing on the third chromosome through the phiC31 integrase system [22]. 95
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2.3. *Salivary gland preparation for imaging.* 97

Dissected salivary glands were fixed at room temperature for 20 min in phosphate-buffered saline (PBS) containing 3.7% formaldehyde (formaldehyde methanol free, Thermo Fisher Scientific) and washed 2 times for 30 min in 1x PBS. Salivary glands were either mounted in Vectashield with DAPI (Vector labs) between slide and coverslip for directly visualizing BiFC and ParB1-mCherry, or prepared for standard immunostaining before mounting for revealing the VC-Ubx construct (using rabbit anti-GFP (A11122, Molecular Probes, 1:500) recognizing the VC fragment [17], coupled to secondary Alexa Fluor 488 (A11039, Molecular Probes)). 98
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2.4. *FISH probes synthesis for the forkhead gene* 106

The protocol for synthesizing DNA probes is taken from [23]. Briefly, a 12 kb long sequence centered on the *fkh* gene locus was chosen. Twelve pairs of 21 bp oligonucleotides were determined via the Primer3 website to cover the entire region (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Each fragment was then amplified by PCR from genomic DNA (Fig. S6). Each fragment is approximately 1 kb long. PCR products are then digested and labelled with the FISH Tag DNA orange kit (Alexa Fluor 555 dye, Thermofisher), following provider instructions. 107
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2.5. *Acquisition of confocal images for LIGHTNING deconvolution* 114

Images were acquired on a confocal Leica SP8 microscope with the 40x or 63x oil objective (HC PL APO 63x/1.40). The Lightning option increases confocal resolution thanks to optimal acquisition parameters for stack thickness, objective used, mounting medium and laser wavelength. These parameters enable maximum details to be extracted from each image voxel. The images acquired are oversampled (a large number of pixels) to enable the processing. The image is scanned by a "LIGHTNING decision mask", and various parameters are measured to determine the image quality of each pixel and adapt the processing according to the result. This differs from traditional methods, which apply reconstruction schemes with global rather than local efficiency. This mask removes much of the background noise and detects signals with a limited number of photons, making the image more resolute (theoretical resolution achieved: 120 nm). Therefore, the essential feature of LIGHTNING results from the preservation of photon number and the sum of all intensities pre- and post-deconvolved images. 115
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Acquisition parameters for each experimental condition were set (2656x2656 pixels; laser scan speed: 574 Hz; x1.5 zoom). A 405 laser is used to image DAPI. A 488 nm laser excites Venus for BiFC signals and a 552 nm laser excites mCherry to localize the *fkh250* enhancer (with Leica HyD detector). See also Fig. S7 for the quantification pipeline of fluorescent immunstaining and BiFC on *fkh* enhancers. 128
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3. Results 133

3.1. *Visualizing Hox/Exd interactions by doing BiFC in salivary gland nuclei* 134

To validate that we could get exploitable BiFC signals in third instar larva (L3) salivary gland nuclei, we used previously published fly lines compatible with the UAS/Gal4 system. More precisely, Hox (Scr and Ultrabithorax, Ubx) and Exd constructs are fused to the C-terminal (UAS-VCScr and UAS-VCUbx) or N-terminal (UAS-VNExd) fragment of Venus [17,21]. Interaction between Hox and Exd fusion proteins should lead to the reconstitution of an excitable Venus fluorescent protein upon complementation between the VN and VC fragments (Fig. 1A-B). The two UAS-VC-Hox constructs are inserted on the same landing site on the second chromosome, allowing having comparable expression levels, as previously verified in the embryo [17,21]. VC-Hox and VN-Exd fusion proteins 135
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were expressed in L3 salivary glands with the *sgs3-Gal4* driver (see materials and methods). The expression of VC-Hox constructs does not seem to affect polytene chromosomes, which look identical to polytene chromosomes of nuclei with no Hox-driven expression (Fig. S1). As expected, immunofluorescent staining of VC-Scr and VC-Ubx with a GFP antibody recognizing the VC fragment (see materials and methods) led to comparable fluorescence intensity in salivary gland nuclei (Fig. S1). However, the pattern was different, with VC-Ubx displaying more distinguishable bands on polytene chromosomes than VC-Scr. VC-Scr displayed a more diffusible and punctual pattern across the nucleus (Fig. S1). It should be noticed that L3 salivary gland nuclei do not contain endogenous Exd given that its nuclear transporter Homothorax (Hth) is repressed by Scr in the embryo (by stage 11, [13]; see also https://flybase.org/reports/FBgn0001235 for RNA-seq data in L3 salivary glands). Thus, this observation suggests that Scr could bind less strongly as a monomere on polytene chromosomes than Ubx.

<figure><img src="image_2.png" /><figcaption>**Figure 1. Visualization of BiFC signals in L3 salivary gland nuclei. A-B.** Principle of the BiFC obtained with the VC-Scr and VN-Exd (A) or VC-Ubx and VN-Exd (B) constructs. A'-B'. Illustrative L3 salivary gland nucleus with BiFC (green) between Scr and Exd (A') or Ubx and Exd (B'), with dapi staining of polytene chromosomes (cyan). Enlargement of a particular zone is shown to highlight both overlapping and non-overlapping staining between BiFC and dapi. Images are confocal acquisition without lightning deconvolution. A"-B". Same illustrative salivary gland nuclei as in A' and B' but with applied lightning acquisition parameters and deconvolution to improve the resolution and signal-to-noise ratio (see also materials and methods). Cyan, green and white arrows highlight regions with dapi only, BiFC only or dapi and BiFC merged signals, respectively. VN: N-terminal fragment of Venus (1-172). VC: C-terminal fragment of Venus (155-235). Scale bars=5µm (upper panels) or 1µm (enlargements).</figcaption></figure>
As expected, the two Hox proteins led to BiFC signals when co-expressed with Exd in salivary gland nuclei (Fig. 1A'-B'). However, the overall quantification of BiFC showed that signals were three times higher on average with Ubx when compared to Scr (see materials and methods and Fig. S2). This differential level of BiFC might be explained by the presence of endogenous Scr (although expressed at very low levels: https://flybase.org/reports/FBgn0003339), which could compete with VC-Scr for complex formation with VN-Exd. Not exclusively, the fusion topology may also affect the interaction between Scr and Exd, therefore BiFC signals. Most likely, the differential BiFC level could also reflect the ability of Ubx/Exd complexes to bind more widely on polytene chromosomes than Scr/Exd complexes. We hypothesize that it could be the case given that the patterns are different: Scr/Exd complexes are visible on a few but intense bands, while many more bands of variable intensities are observed with Ubx/Exd complexes (Fig. 1A'-B'). Such differences in BiFC patterns also suggest that the fusion proteins are not expressed at satuting levels.

In order to get a better resolution of signals, we applied an adaptative and automated deconvolution method on confocal acquisitions (see materials and methods). This manipulation revealed loci with higher fluorescence intensity and with a pattern following the polytene chromosome organization for Ubx/Exd and to a lesser extent for Scr/Exd complexes (Fig. 1A'' and 1B''). Interestingly, the BiFC profile of Scr/Exd was also different from the binding profile of VC-Scr alone, with less widespread signals and a more defined pattern following the organization of polytene chromosomes. This different binding behavior suggests that Scr/Exd complexes are binding more specific sites with stronger affinity in the genome of salivary gland nuclei than the Scr monomers. This observation is in accordance with the role of Exd in forming a cooperative DNA-binding complex with Scr *in vitro* [20]. Moreover, BiFC signals are not systematically co-localized with DAPI (which depicts AT-rich and more condensed chromatin), underlining that Hox/Exd complexes are bound on different types of chromatin region in salivary gland nuclei (enlargements in Fig. 1A'-A'' and 1B'-B''). Overall, these observations confirm that our fusion constructs and expression system are compatible for doing BiFC in L3 salivary gland nuclei.

## 3.2. *Experimental conditions for DNA-FISH are detrimental for BiFC*

We next wanted to assess whether we could quantify BiFC signals specifically on the *fkh* locus by doing DNA-FISH (Fig. 2A-B). To this end, we designed fluorescent oligonucleotides spanning 12kb upstream and downstream the *fkh* gene (see material and methods) and performed FISH staining on BiFC-positive salivary gland nuclei. Although FISH signals were clearly observable, we could not detect BiFC signals for Scr/Exd complexes (neither in the whole nucleus, nor on the *fkh* locus: Fig. 2A' and Fig. S3). BiFC signals were also extremely weak with Ubx/Exd complexes, making any quantification difficult. A more defined pattern of BiFC signals (Fig. S3), especially after deconvolution (Fig. 2B'), could be observed in a few nuclei. In these rare nuclei, the *fkh* FISH signal was systematically localized in a zone with low or no BiFC (Fig. 2B'), which is in accordance with previous work showing that Ubx/Exd complexes can neither bind the *fkh250* enhancer *in vitro* nor regulate its expression *in vivo* [20]. Altogether, these experiments reveal that experimental conditions for FISH are detrimental for BiFC, underlining the need of using alternative approaches for doing colocalization studies with a genomic ROI.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Experimental conditions for DNA-FISH are detrimental to BiFC. A-B. Principle of the coupling between BiFC resulting from VC-Scr/VN-Exd (A) or VC-Ubx/VN-Exd (B) complex assembly and FISH staining of the *forkhead* (*fkh*) genomic locus. The nucleotide sequence of the DNA-binding site that is specifically recognized by Scr/Exd complexes in the *fkh250* enhancer is indicated. Gray and light-red nucleotides are bound by Exd or the Hox protein, respectively. The two central nucleotides (black) are recognized by the two proteins (based on [20]). The red cross illustrates the inability of Ubx/Exd complexes to bind on the *fkh250* enhancer *in vitro* (based on [20]) A'-B'. Illustrative L3 salivary gland nucleus with BiFC (green) resulting from VC-Scr/VN-Exd (A') or VC-Ubx/VN-Exd (B') complex assembly, FISH of the *fkh* locus (magenta) and dapi staining of polytene chromosomes (cyan). No exploitable BiFC signals is obtained under FISH experimental conditions for VC-Scr/VN-Exd complexes. Very few nuclei showed a weak BiFC pattern with VC-Ubx/VN-Exd. Enlargement in B' highlights the non-overlapping pattern between BiFC and FISH signals. Images are confocal acquisition with lightning deconvolution (see also Fig. S3). Scale bars=5µm (upper panels) or 1µm (enlargements).</figcaption></figure>

<caption>3.3. BiFOR allows to reveal the enrichment of specific dimeric protein complexes on a target enhancer in salivary gland nuclei</caption>

Previous work established ANCHOR as a specific and sensitive method for labelling target ROIs in the genome without affecting transcriptional regulation *in vivo* [13,14].

We therefore decided to couple *INT1* sequences to different versions of the *fkh250* enhancer: wild type, mutant (with mutations abolishing the DNA-binding of Scr and Exd: *fkh250MUT*) and consensus (with mutations transforming the specific Scr/Exd into a generic Hox/Exd binding site and allowing the recognition by different Hox/Exd complexes: *fkh-CONS*). These mutations were based on previous work on the *fkh250* regulation by Hox/Exd complexes [20]. *INT1-fkh250* fusion constructs were all inserted at the same landing site in the genome ([22] and material and methods). ParB1-mCherry fusion proteins were expressed in the salivary glands with the UAS/Gal4 system to label the different *INT1-fkh250* fusion constructs (material and methods). All *INT1-fkh250* fusion constructs were
specifically recognized by ParB1-mCherry, leading to a strong red fluorescent signal in the nucleus. This signal can be of variable size and shape depending on the orientation of the nucleus, and was also usually less intense in the case of the *INT1-fkh250mut* enhancer construct.

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<figure><img src="image_4.png" /><figcaption>**Figure 3. Assessing the specificity of BiFOR with the wild type *fkh250* enhancer in salivary gland nuclei. A-C.** Schematic representation of the different conditions. The ParB1-mCherry (mCh) fusion proteins oligomerize on the target *INT1* cassettes that are inserted closed to the *fkh250* enhancer (see material and methods). Only Scr/Exd complexes were shown to cooperatively bind on *fkh250* *in vitro* [20]. No cooperative binding was observed for Ubx/Exd complexes (but Exd improves monomere binding of Ubx *in vitro* [20]). Extremely weak (illustrated with a dotted-red cross) or no (illustrated with a red cross) binding *in vitro* was observed for Scr and Ubx as a monomere, respectively [20]. **A'-D'**. Illustrative confocal acquisition with lightning deconvolution of salivary gland nuclei expressing the different constructs, as indicated. Protein enrichment (green) was analyzed at the level of the inserted *fkh250* enhancers (red) and compared to the overall maximum green fluorescence intensity in the nucleus (see material and methods). Enlargements are shown at the level of the ParB1-mCherry signal to better highlight the presence or absence of colocalization (white arrowheads) with BiFC (A'-B') or VC-Scr and VC-Ubx (C'-D', revealed with an anti-GFP antibody</figcaption></figure>
recognizing the VC fragment, see materials and methods). A"-D". Statistical quantification of BiFC (A"-B") or immunostaining (C"-D") at the level of the ParB1-mCherry signal. The quantification was obtained from three independent biological replicates. Scale bars=5µm (upper panels) or 1µm (enlargements). See also Fig. S4 and S5.

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Assessing the specificity of BiFOR on two different variants of the *fkh250* enhancer in salivary gland nuclei. A-C. Schematic representation of the *fkh250*<sub>MUT</sub> (A) and *fkh250*<sub>CONS</sub> (B-C) enhancer variants. Mutated residues in the Hox and/or Exd DNA-binding site are highlighted in bold-red. Their negative (red cross) or positive effect on Hox/Exd binding is based on previous *in vitro* and *in vivo* studies [20]. A'-B'. Illustrative confocal acquisition with lightning deconvolution of salivary gland nuclei expressing the different constructs, as indicated. BiFC enrichment (green) was analyzed at the level of the *fkh250*<sub>MUT</sub> (A') or *fkh250*<sub>CONS</sub> (B'-C') enhancers (labelled with ParB1-mCherry, red) and compared to the overall maximum green fluorescent intensity in the nucleus (see material and methods). Enlargements are shown at the level of the ParB1-mCherry signal to better highlight the absence (A') or presence (B'-C') of colocalized green and red signals. A"-C". Statistical quantification of BiFC at the level of the ParB1-mCherry signal. This quantification is obtained from three independent biological replicates. Scale bars=5µm (upper panels) or 1µm (enlargements). See also Fig. S5.</figcaption></figure>

We first performed BiFC by expressing VC-Scr and VN-Exd in ParB1-mCherry/INT1-fkh250 positive salivary gland nuclei (Fig. 3A). In contrast to the previous observation under DNA-FISH condition, VC-Scr/VN-Exd BiFC signals were not affected. Quantification (see also material and methods) showed that BiFC signals were significantly enriched with ParB1-mCherry, demonstrating a preferential localization on the *fkh250* enhancer when

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compared to other genomic loci in the salivary gland nuclei (Fig. 3A'-A'' and Fig. S4-S5). This observation is in accordance with the specific recognition and regulation of *fkh250* by Scr/Exd *in vitro* and *in vivo* [20]. As a first negative control experiment, we repeated the analysis by expressing VC-Ubx in combination with VN-Exd. As expected, VC-Ubx/VN-Exd signals were globally stronger than the ones obtained with VC-Scr/VN-Exd in salivary gland nuclei. Still, the quantification showed a significant diminution of enrichment on the *fkh250* enhancer (Fig. 3B'-B'' and Fig. S5). We performed additional negative control experiments by expressing VC-Ubx or VC-Scr alone, which do not or only poorly bind on *fkh250* *in vitro*, respectively [20]. Expression of VC-Scr or VC-Ubx led to more albeit less defined signals along the polytene chromosomes than the corresponding BiFC complexes with Exd (Fig. 3C'-D'). Still, the quantification showed the absence of a significant enrichment on *INT1-fkh250* enhancers when compared to Scr/Exd complexes (Fig. 3C''-D'' and Fig. S5). This observation underlines that neither Scr, nor Ubx alone or in complexe with Exd could preferentially bind the *fkh250* enhancer in salivary gland nuclei. This result is in accordance with the previous observations *in vitro* [20]. Together with the specific enrichment of VC-Scr/VN-Exd BiFC, our results indicate that BiFOR can recapitulate the specific recognition of target enhancers by a dimeric protein complex in salivary gland nuclei.

**3.4. BiFOR allows to reveal dimeric complex enrichment on specific target enhancers in salivary gland nuclei**

To further confirm the specificity of BiFOR, we generated two additional variants of the *fkh250* enhancer fused to the *INT1* sequences: *fkh250MUT* and *fkh250CONS*. As previously mentioned, mutations in the *flh250MUT* enhancer abolish Hox/Exd binding *in vitro* [20]. Not surprisingly, the analysis in salivary gland nuclei showed the absence of any significant enrichment of BiFC resulting from Scr/Exd complex assembly at the level of the *fkh250MUT*-containing genomic locus (Fig. 4A-A'' and Fig. S5). In contrast, Scr/Exd and Ubx/Exd BiFC signals were similarly and significantly enriched on *fkh250CONS* (Fig. 4B-B'', 4C-C'' and Fig. S5), confirming the previous *in vitro* observations related to the gained ability of this modified enhancer to be recognized by different Hox/Exd complexes [20].

In conclusion, both the use of different Hox/Exd complexes and variants of the *fkh250* enhancer confirmed the specificity and sensitivity of BiFOR for visualizing and quantifying PPI enrichment on a target enhancer of interest in salivary gland nuclei.

# 4. Discussion

**4.1. Advantages of ANCHOR over other DNA-labelling systems for BiFC coupling**

Several techniques exist to visualize genomic ROIs in the nucleus. Here, we first tried to perform FISH for analyzing BiFC on the genomic locus of *fkh*. We found that the experimental conditions of FISH were detrimental for BiFC, forbidding any quantification measurements and underlining the need of using alternative and softer DNA-labelling approaches.

Several alternative methods have been developed to label genomic ROIs, the main challenge being to have enough and specific fluorescent signals for proper quantification in the nucleus. Among them are Crispr/Cas9-based approaches, which are long and labor intensive for the cloning and expression of multiple copies of the specific gRNAs. These issues might explain that CrispR/Cas9-based DNA-labeling approaches have not yet been applied in a multicellular organism [4-7]. The LacO/LacI system has been used in *Drosophila*, but it also requires hundred copies of the LacO cassette to exploit fluorescent signals emitted by the DNA-bound LacI-FP fusion protein [8]. In addition, the binding of LacI has been shown to interfere with gene transcription and is therefore not best appropriate for quantifying DNA-bound TFs in the genomic vicinity [14]. In contrast, the ANCHOR system is more neutral on gene transcription [14]. Moreover, the property of ParB proteins to oligomerize on ParS binding sites through protein-protein interactions allows
seeing the ParB-FP without multiplying the number of ParS cassettes (usually three copies are used, [9]). The existence of different ParB proteins recognizing different ParS sequences (ParB1/ParS1 and ParB2/ParS2 for instance) also allows visualizing two different loci simultaneously when each ParB protein is fused to a different FP [14]. Thus, ANCHOR appears as a quite straightforward and neutral DNA-labelling system for visualizing genomic ROIs in the nucleus. Here, we demonstrated that BiFC could be coupled with ANCHOR to visualize PPIs on a specific target enhancer in whole salivary gland nuclei. Moreover, since BiFC is stabilizing the dimeric complex upon complementation (with the formation of covalent bonds between the two sub-fragments of the FP), it allows analyzing not only strong and stable but also weak and transient PPIs on a ROI.

## 4.2 Using BiFC to visualize dimeric complexes in whole-mounted salivary gland nuclei

Given the weak affinity between the complementary split fragments, BiFC could potentially reveal artificial interactions when the two fusion proteins are too strongly expressed. It is therefore critical to control the expression level and have negative controls, when possible. Best negative controls consist in using mutated versions that are known to affect the interaction. Alternative controls like expressing one of the candidate partner as a cold competitor against BiFC could also be performed.

Here, we used the *Sgs3-Gal4* driver, which is specifically expressed in salivary glands during late third instar larval stage [24]. This driver is more specific and less strong than other salivary gland drivers that are expressed at earlier developmental stages and in other tissues (like *fkh-Gal4* [25]). It is therefore the best appropriate for doing BiFC in L3 salivary glands. In addition, the Hox fusion proteins were inserted at the same genomic landing site, allowing having comparable expression levels. Negative controls with Hox and Exd fusion proteins that could not interact together under Hox-like strong expression levels in the embryo have already been published elsewhere [21]. In addition, the distinct binding patterns on polytene chromosomes with VC-Scr and VC-Ubx, either alone or in combination with VN-Exd, underlines that our expression system was not at saturation in salivary gland nuclei. This conclusion is further reinforced by the observation of specific enrichments on the different versions of the *fkh250* enhancer despite the size of the target locus (see below). Along the same line, BiFC has previously been used for visualizing genome-wide binding behavior of a specific dimeric complex on polytene chromosome spreads [26]. BiFOR could potentially have been performed on squashed polytene chromosomes, making the confocal acquisitions of better resolution and quality. However, the purpose of our work was to develop an approach that allows visualizing dimeric complexes on a specific genomic ROI at high resolution when considering the three dimensional space of the whole nucleus, which is not neutral on transcription regulation (especially in the context of chromosome-chromosome contacts for example).

Finally, our results were obtained on fixed salivary glands. Here, the fixation was dictated by the long acquisition time required for the LIGHTNING deconvolution (between 20 and 30 minutes long for a two colors acquisition). Without such a high resolution need, BiFOR could be used on live salivary glands by doing conventional confocal microscopy (with few minutes long acquisition time). Existing post-acquisition deconvolution algorithms could further been applied to improve the resolution (although to a lesser extent than LIGHTNING or other deconvolution methods that require specific acquisition parameters).

## 4.3. Futures perspectives for BiFOR

Our system was based on transgenic constructs, with ten copies of wild type or modified *fkh* enhancers to ensure the visualization of BiFC signal enrichment in salivary gland nuclei. Salivary gland nuclei are characterized by multi-replicated polytene chromosomes, which also favors the visualization of TF binding events. Our analysis revealed clear and strong signals for both ParB1-mCherry and BiFC, which are approximately covering between 40 and 50 kb. Still, the system was sensitive enough to reveal significant
differences between the different protein and enhancer combinations, and to recapitulate previous observations with purified proteins on single DNA-binding sites *in vitro* [20]. 388 389

Compared to large-scale approaches such as ChIP-Seq or RNA-Seq, BiFOR offers the complementary information for validating the specific regulation and binding on the candidate target enhancer. For example, *in silico* analyses of cis-regulatory sequences of a TF target gene often provides a list of associated candidate TFs that could also bind and participate to the transcriptional regulation. BiFOR opens the possibility to validate whether a candidate TF could interact and form enriched dimeric complexes with the regulatory TF on the candidate target enhancer identified from ChIP-Seq and RNA-Seq analyses. Hundreds of TFs are available for doing BiFC analyses with the UAS/Gal4 expression system [19]. The *INT1*-enhancer construct could be inserted in the genome as shown in this work. Alternatively, given the intensity of ParB1-mCherry and BiFC signals observed with the *fkh250* enhancers, we anticipate that one copy of *INT1* will be enough for visualizing the ROI. In this context, BiFOR could also be deployed by inserting one copy of *INT1* in close proximity of the endogenous candidate enhancer when performing the analysis in salivary gland nuclei. 390 391 392 393 394 395 396 397 398 399 400 401 402 403

The intensity of fluorescent signals also suggests that BiFOR could be exploited in diploid tissues, although it remains to be demonstrated. Along this line, ANCHOR has previously been shown to efficiently label a genomic ROI in *Drosophila* imaginal discs [14]. The exploitation of BiFC might depend on the number of DNA-binding sites in the target enhancer. For example, *fkh250* contains only one Hox/Exd binding site, and a minimum number of copies is probably required for getting exploitable BiFC signals in diploid nuclei under conventional confocal microscope resolution. An alternative strategy to further increase the intensity and/or resolution of BiFC signals in this context could be the use of a BiFC-specific nanobody [27]. Indeed, the reconstitution of the FP upon complementation provides only 20% of the brightness of the original FP used for BiFC. This issue could be circumvented by using a BiFC-specific nanobody fused to a bright FP (such as mNeonGreen, [28]) or other fluorescent-emitting proteins (such as HaloTag, [29]). These imaging tools are compatible for STED microscopy and could therefore also increase the resolution scale of PPI analysis on the target enhancer. The same rational could apply for the ANCHOR labelling system. Here we used ParB1-mCherry fusions proteins, but other ParB proteins fused to brighter fluorescent proteins or systems are available [14]. 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419

## 5. Conclusion

Our work established the coupling of BiFC and ANCHOR for visualizing dimeric protein complexes on a genomic ROI in salivary gland nuclei. This work sets the experimental foundation for future technological developments aiming at visualizing dimeric protein complex association on specific endogenous loci in diploid tissues. These future developments will open new perspectives for understanding transcriptional specificity *in vivo*. The versatility of BiFC and ANCHOR tools makes the BiFOR approach suitable in *Drosophila* and other model organisms. 420 421 422 423 424 425 426 427

**Author Contributions:** S.M. and S.V. designed the study. S.V. performed the experiments and acquired the data. S.M. and S.V. were responsible for data analysis. S.M. wrote the paper, and S.V. amended it. 428 429 430 431 432

**Funding:** This research was funded by FRM (grant 160896), CNRS and ENSL (recurrent funding). 433

**Data Availability Statement:** Dataset available on request from the authors. 434

**Acknowledgments:** We thank Christophe Place for advices on the quantification analysis, Marilyne Duffraisse for the discussions, and Marilyne Duffraisse and Christelle Forcet for comments on the manuscript. We thank the Bloomington stock center, Philippe Valenti and F. Payre for the fly lines, the IGFL imaging facility and the Arthrotools platform of the UAR3444/US8. This work was supported by Fondation pour la Recherche Médicale (Team FRM 160896 and 4th year PhD fellow to S.V.), CNRS and ENS-Lyon. 435 436 437 438 439 440
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