A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes

Objectives Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF. Results We developed a hybrid RNA FISH-IF protocol for use on Drosophila melanogaster polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in Drosophila melanogaster polytene chromosomes. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-023-06482-0.


INTRODUCTION
The relationships between a DNA locus and the proteins that target that locus affect fundamental processes such as DNA replication, transcription, and repair (1). A common technique used to investigate protein-DNA localization is ChIP-seq, which captures positional information of proteins across the genome. However, ChIP-seq involves several caveats: it is expensive, it requires access to sequencing platforms (2), and it is difficult to perform by inexperienced users. As an alternative to ChIP-seq, investigators utilize microscopy to reveal protein localization (3), monitor biochemical interactions between proteins and DNA (4), and quantify binding mechanisms that lead to the formation of protein-DNA complexes (5). To confirm whether a protein of interest is targeting a specific genomic locus, investigators have historically combined DNA fluorescent in situ hybridization (DNA FISH) with protein immunofluorescence (IF) (6,7). However, the protocol for DNA FISH is often incompatible with protein immunohistochemistry; it involves a DNA denaturation step that can reverse chemical crosslinks and denature protein epitopes, thus hindering primary antibody binding (8)(9)(10).
Combining DNA FISH with IF on Drosophila polytene chromosomes is a historically invaluable method for cytological analysis. Polytene chromosomes are formed through repetitive cycles of DNA endoreduplication without nuclear division. These polyploid cells can contain up to 1024 copies of the genome (11). This amplification of the genome gives rise to defined chromosomal banding patterns that represent chromatin regions and allows investigators to analyze relatively high-resolution protein binding patterns (12,13). Although researchers have applied DNA FISH and IF on polytenes in the past (6), the aforementioned incompatibility presents a need for an alternative to investigate protein-DNA interactions. To address this need, we developed a hybrid RNA FISH -IF method that indirectly marks genes on Drosophila polytene chromosomes.
Unlike DNA FISH, RNA FISH identifies the RNA transcripts that surround its parent gene (14), thus providing indirect genomic locus information. As RNA is single-stranded, RNA FISH does not require a denaturation step (15), which renders it more compatible with immunohistochemistry and more accessible for less-experienced trainees than DNA FISH.
We tested the ability of RNA FISH probes to mark genomic loci on polytene chromosomes, determined if single molecule RNA (smRNA) probes hybridize to local RNA or DNA, and optimized our hybrid RNA FISH and IF protocol. We used the D. melanogaster histone gene array as our model system. In the wildtype D. melanogaster genome, the endogenous histone locus consists of five histone genes arranged in a single histone array (HA). Arrays are tandemly repeated ~107 times at a single locus (16). We also used engineered fly lines that carry transgenes with varying numbers of HAs, where even a single HA can attract specific transcription factors (17,18). Notably, this gene array titration allowed us to incrementally optimize the sensitivity of our RNA FISH -IF hybrid assay towards single-gene detection. We verified expected protein-DNA interactions on polytenes marked with histone smRNA FISH probes by immunostaining with Multi-sex combs (Mxc), a protein that only targets HAs (including both endogenous and transgenic HAs) (19)(20)(21). We sought to expand this technique for those working with any Drosophila transgene marked by reporters, specifically by leveraging smRNA probes targeting common markers like mini-white. However, we found that most singlecopy genes did not give strong signal by smRNA FISH, suggesting that transcriptional level in salivary gland tissue contributes to detection limits. Overall, we present a protocol for investigating and visualizing protein binding at specific genomic locations that is inexpensive, quick, and accessible to Drosophila investigators at all levels.

Fly stocks
We maintained all Drosophila melanogaster fly lines on standard cornmeal-molasses food at 18°C. We used third instar larvae for dissections. We obtained fly stocks from Bloomington Drosophila Stock Center (y,w: stock #1495) or stocks as gifts from the Duronio and Marzluff laboratories (17,22).

RNase decontamination
We made RNase-free reagents from DEPC-treated water and RNase-free 1X PBS (23). We used RNase AWAY (Spectrum Chemical MFG Corp #970-66898) to clean all surfaces and tools and used filtered pipette tips. We cleaned slide holders by washing them in a mixture of DEPCtreated water and RNase AWAY.

Sample preparation
We dissected salivary glands from third instar larvae and fixed the glands containing polytene chromosomes with three separate fixatives (Fix 1: 4% paraformaldehyde, 1% Triton X-100 in . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 12, 2023. ; https://doi.org/10.1101/2023.06.12.544616 doi: bioRxiv preprint RNase-free 1X PBS) (Fix 2: 4% paraformaldehyde, 50% acetic acid in DEPC-treated water) (Fix 3: 1:2:3 lactic acid:DEPC-treated water:glacial acetic acid) (18) using DEPC-treated reagents and RNase-free materials. We transferred salivary glands to the 1:2:3 fixative (~20 uL) on a siliconized (RainEX) 22mm square coverslip surface. We placed a glass slide on the coverslip containing the salivary glands and third fix and quickly flipped over. We froze slides in liquid nitrogen and immediately removed their coverslips. We briefly stored slides in RNase-free 1X PBS and immediately proceeded with immunohistochemistry.

Immunohistochemistry
We washed slides in 1% Triton X-100 in RNase-free 1X PBS and rocked gently for 10 minutes.
We washed slides twice for 5 minutes in RNase-free 1X PBS and marked the sample perimeter of each slide with an ImmEdge pen (Vector Laboratories #H-4000) in between both washes. We added 250 uL of blocking solution (0.5% UltraPure BSA (Invitrogen #AM2616) in RNase-free 1X PBS) to the sample area of each slide and incubated them at room temperature in a dark humid chamber for 1 hour, shaking gently. Using coverslips, we then incubated slides with 40 uL of diluted primary antibody in blocking solution and incubated them overnight in a dark, sealed, humid chamber at 4°C. We washed slides 3 times for 5 minutes in RNase-free 1X PBS. We applied 40ul of diluted secondary antibody in blocking solution to each slide (including coverslips) and incubated them for 2 hours in a dark humid chamber at room temperature. We washed slides 3 times for 5 minutes in RNase-free 1X PBS. We added 250 uL of post-fixative (4% paraformaldehyde in RNase-free 1X PBS) to each sample area for 3 minutes at room temperature. We then washed each slide 3 times for 5 minutes in RNase-free 1X PBS.

RNA FISH
We washed slides for 5 minutes in ~30 mL Wash Buffer (1:10:1 20x SSC:DEPC-treated water:Deionized Formamide) at room temperature. We added 125 uL of Hybridization Buffer . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 12, 2023. ; https://doi.org/10.1101/2023.06.12.544616 doi: bioRxiv preprint (100 mg/mL dextran sulfate and 10% formamide in 2x SSC and DEPC-treated water) and diluted RNA FISH probe (5 uL of 25uM probe:120 uL Hybridization Buffer) to slides (including coverslips) which were incubated in a dark, humid, sealed chamber at 37°C overnight (~16 hours). We washed slides twice for 10 minutes in prewarmed (37°C) Wash Buffer in the dark.
We then added 250 uL Wash Buffer and diluted DAPI (25 ng/mL DAPI) onto the sample area and incubated slides in a dark, humid chamber at 37°C for 30 minutes. We mounted slides with ~15uL of VECTASHIELD Antifade Mounting Medium (VWR #101098-042) and coverslips. We sealed the samples with nail polish. We immediately proceeded to imaging.

Microscopy
We used a widefield Zeiss AXIO Scope A1 microscope with X-Cite 120 LED Boost fluorescent light source and a 40x Plan-neofluar 0.75 NA objective paired with ZEN 3.6 (blue edition). We visualized .czi files with ImageJ Version 1.53t.

RNase treatment
After dissecting salivary glands, we treated the glands with 0.1% Triton X-100 for 2 minutes prior to adding 100 ug/mL RNase A (NEB #T3018L) and performed a 1 hour incubation at RT (24) before beginning fixation.
. CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 12, 2023. ;

RESULTS
. CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 12, 2023. ; https://doi.org/10.1101/2023.06.12.544616 doi: bioRxiv preprint A high concentration of transcripts surrounds the parent gene locus in many cells. We therefore hypothesized that RNA FISH would mark genetic loci on Drosophila polytene chromosomes.
We performed RNA FISH on wild-type polytene chromosomes using smRNA FISH probe sets against either histone2b or histone3 transcripts because they are the longest histone genes.
The endogenous histone locus, which carries ~100 tandem histone gene arrays (16), is located on chromosome 2L near the centromere. Our histone2b and histone3 RNA FISH probe sets effectively targeted this region (Fig. 1A). Since most genes do not exist in multiple copies, we next performed the same RNA FISH assay on transgenic lines carrying HA transgenes, either with 12 tandem copies of the histone gene array or a single array. Our histone2b and histone3 probe sets effectively detected these transgenes ( Fig. 1B-C).
. CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 12, 2023. ; https://doi.org/10.1101/2023.06.12.544616 doi: bioRxiv preprint To confirm that our RNA FISH probe sets are detecting histone loci, and to confirm that our RNA FISH protocol is compatible with immunofluorescence (IF), we combined our RNA FISH assay with antibody detection of the histone-locus specific factor Multi-sex combs (Mxc) (22,25).
Mxc also localizes to transgenes carrying histone gene arrays on polytene chromosomes (17,18,22). We observed that Mxc signal colocalizes with histone2b and histone3 RNA FISH, confirming these locations as the endogenous histone locus ( Fig. 2A) and the transgenic loci ( Fig. 2B-C). While developing this protocol, we intentionally conducted IF first because certain RNA FISH regents can alter protein epitopes. We included a postfixative step in between IF and RNA FISH to preserve the IF signal (23), included larger-volume washes, and increased the concentration of probe to 3 uL of 25 uM probe per 100 uL. All of these steps contributed to boosting RNA FISH signals when combined with IF.
Given that we verified Mxc colocalization with histone genes, we wanted to further test the ability of both assays to mark different single-copy genomic loci on polytenes. We tested our . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 12, 2023. ; https://doi.org/10.1101/2023.06.12.544616 doi: bioRxiv preprint procedure on mini-white, a common transgene marker that we use to mark HA transgenes on chromosome 3R, as well as roX2, an X-linked long non-coding RNA that is only expressed in males. RoX2 participates in Drosophila dosage compensation and coats the male Xchromosome (26,27). We did not detect a signal for the mini-white gene (Fig. 3A). While we clearly detected roX2 on male X-chromosomes (Fig. 3B), as previously documented, we saw no RNA FISH signals for the roX2 locus in female polytenes (Fig. 3C). To verify that our RNA FISH probes sets were binding to mRNA transcripts and not binding directly to DNA, we introduced RNase A into the procedure. We observed a large reduction in signal (Fig. 3D-E), suggesting that our RNA FISH probes are binding directly to local mRNA.

DISCUSSION
Our goal in developing a hybrid RNA FISH and IF protocol for polytene chromosomes was to create an alternative method to visualize protein-DNA interactions in Drosophila. Here, we successfully combined RNA FISH and IF to visualize Mxc (a protein that only targets histone genes) colocalizing with histone gene loci. This proof of principle suggests that our hybrid assay could be applied to other proteins and genomic loci of interest. Unfortunately, we found that mini-white, a common transgene marker in Drosophila, was not visible by RNA FISH on polytene chromosomes. Considering that the wild-type white gene has very low expression levels in salivary glands (28), we are not surprised by the absence of mini-white signal. These observations suggest that our RNA FISH technique is more applicable for visualizing loci of genes that are highly expressed in larval salivary glands. Due to the relatively low cost of the protocol and reagents in addition to the 3-day turnaround time, we believe this hybrid RNA FISH . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 12, 2023. ; https://doi.org/10.1101/2023.06.12.544616 doi: bioRxiv preprint