A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes

• Type of Material: Article
• Authors: Hannah E Gilbonio, Emory University; Gwyn L Puckett, Piedmont Virginia Community College, Charlottesville; Erica Nguyen, University of Pennsylvania; Leila E Rieder, Emory University
• Language: English
• Date: 2023-06-12
• Publisher: NIH
• Publication Version: Preprint (Prior to Peer Review)
• Copyright Statement: The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
• License: Creative Commons Attribution-NonCommercial 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1101/2023.06.12.544616
• Title of Journal or Parent Work: bioRxiv
• Grant/Funding Information: Financial support was provided by R35GM142724 to LER and R35GM142724-01S1 supplement to HEG.
• Keywords: Drosophila; IF; transcription factor; RNA FISH; polytene chromosomes; Mxc; histone genes
• Research Categories: Biology, Cell; Biology, Molecular; Biology, Genetics

Abstract

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 and 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.