Domino is expressed ubiquitously during oogenesis and embryogenesis

expression of Dom in early Drosophila development and especially the nervous system.

For this I utilized a fly line expressing a GFP-Dom fusion protein at the dom gene locus and under the endogenous dom promoter, thus reflecting the endogenous expression and localization of Dom (Buszczak et al., 2007). The GFP open reading frame lacks start and stop codons and is inserted in an intronic region upstream to the dom start codon. By sequence analysis I identified an alternative start codon (CTG) two codons upstream in frame with the GFP open reading frame which potentially initiates the expression of the GFP-Dom fusion protein. The resulting GFP-Dom fusion protein would contain two additional N-terminal amino acids prior to the GFP. The splice donor of the inserted GFP sequence enables splicing to the adjacent intron in frame with the start codon of the dom open reading frame. The start codon is located in the beginning of the adjacent intron, resulting in the expression of a GFP-Dom protein with few additional amino acids linking the two open reading frames.

To confirm the expression of a fusion protein and exclude the expression of untagged GFP I performed Western blot analysis. I used ovary lysate, which appears to have high amounts of GFP-Dom protein as determined in preceding experiments (Figure 7 A). The dom gene locus encodes four different protein isoforms DomA, DomD, DomE and DomG, which have molecular weights of 352, 350, 275 and 357 kDA respectively (Figure 5). The isoforms DomA and DomE are both expressed during oogenesis (Börner and Becker, 2016). Thus, I expected to identify at least two GFP bands running higher than the biggest marker band of 170 kDA in the Dom lysate. One band representing the smaller GFP-tagged DomE isoform, and a second band containing the GFP-GFP-tagged larger isoform DomA and potentially also DomD and G. The larger isoforms would most probably be indistinguishable due to their similar size. An antibody directed against GFP detects a single band clearly over 170 kDa in GFP-Dom lysate, which is absent in w¹¹¹⁸ lysate and

lysate containing untagged GFP (expressed under the ubiquitous actin promoter). This band most probably represents the GFP-Dom protein. The absence of a second GFP-Dom band might be due to technical difficulties, like entry of the large protein into the polyacrylamide gel, or due to low expression levels.

Figure 7: The GFP-Dom trap line expresses a GFP fusion protein that binds to polytene chromosomes (A): Western blot from ovary lysate of the GFP-Dom gene trap line, a w¹¹¹⁸ control and a ubiquitously (act promoter driven) GFP-expressing line. A GFP antibody detects untagged GFP (26.9 kDA) in the GFP control but not in GFP-Dom or w¹¹¹⁸. The GFP-Dom fusion proteins have an expected size of roughly between 300 and 390 kDa, dependent on the Dom isoform. A single GFP-Dom band is detected clearly over 170 kDa. (B and C): Polytene chromosome preparations of the GFP-Dom line and a ubiquitously GFP-expressing line.

The GFP signal is visible at the DNA only in the GFP-Dom line (B’). The microscope pictures were taken with the same settings.

I could not detect untagged GFP (26.9 kDa) in the GFP-Dom sample, although the GFP antibody detects several bands between 35 and 80 kDa, which are most probably unspecific as they are also detected in both control samples. A single band at 45 kDa is present in the GFP-Dom lysate that I could not detect in the control samples. This band either reflects a degradation product of the GFP-Dom protein or is also unspecific, but runs too high to represent untagged GFP (Figure 7 A).

Homozygous mutation of dom is early larval lethal, hence the viability of the homozygous GFP-Dom fly line indicates that the fusion protein is functional (Ruhf et al., 2001).

Importantly, Dom is a chromatin remodeler protein which binds to DNA (Eissenberg et al., 2005; Ruhf et al., 2001). Thus, to further confirm the functionality of the GFP-Dom fusion protein, I studied its DNA-binding abilities in polytene chromosome preparations (Figure 7

B). GFP staining can be detected at the DNA in the GFP-Dom fusion line but not in a control line expressing GFP (Figure 7 C), indicating that the fusion protein is capable of binding DNA.

To investigate the expression of Dom in early Drosophila development I analyzed the expression of GFP-Dom in ovaries and embryos by confocal microscopy. GFP-Dom can be detected in all nuclei of ovaries and embryos throughout oogenesis and embryogenesis (Figure 8).

To furthermore confirm the expression of Dom in the Drosophila embryo, I immunostained GFP-Dom embryos with an antibody directed against all isoforms of Dom produced in this study (Figure 9). The Dom antibody signal overlaps with the GFP-Dom staining. Although, I could also detect some background staining, visible as spots, which do not colocalize with the GFP-Dom signal (Figure 9 B’).

Both, the GFP-Dom signal and the Dom staining confirm the expression of Dom in embryonic NBs positive for Baz (Figure 9 C) and the overlying epithelium (Figure 9 B). This finding could further be underpinned using the reporter line dom^k08108 (Figure S 1). Dom is nuclear unless the nuclear envelope has broken down during cell division. Dom staining of polytene chromosomes further validates that Dom binds to DNA and mostly localizes to the euchromatic regions, which are not or weakly stained by Hoechst (Figure 9 D).

Figure 8: Domino expression in ovary and embryo

Confocal microscopy pictures of GFP-Dom ovary and embryos. GFP-Dom is expressed in ovary cells (A) and throughout embryogenesis (B – E). The fusion protein localizes to nuclei.

Figure 9: Domino is expressed in embryonic neuroblasts and binds to polytene chromosomes in euchromatic regions

Confocal microscopy pictures of a stage 13 GFP-Dom embryo (A – C). Dom staining (A’, B’, C’’) overlaps with the GFP-Dom signal (A, B, C’) in all analyzed nuclei, although some background staining is visible. Dom is expressed in the epithelium (B) and in underlying NBs (C, arrow), which are positive for Baz. Dom localizes to the nucleus but is also visible in the cytoplasm after nuclear envelope breakdown. pH3 stains mitotic nuclei. (D) shows polytene chromosome preparations stained for Dom. The Dom staining is strong in Hoechst negative euchromatic regions.