Department of Biology, Emory University, Atlanta, GA, USA.
Piedmont Virginia Community College, Charlottesville, VA, USA.
BMC Res Notes. 2023 Sep 7;16(1):197. doi: 10.1186/s13104-023-06482-0.
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.
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.
研究蛋白质-DNA 相互作用对于理解许多系统中的细胞生长、分化和细胞发育等基本概念至关重要。ChIP-seq 等测序技术可以提供转录因子的全基因组 DNA 结合谱；然而，这种检测方法昂贵、耗时，对于基因组的重复区域可能没有信息，并且严重依赖于抗体的适用性。将 DNA 荧光原位杂交（FISH）与免疫荧光（IF）相结合是一种更快、更经济的方法，历史上常用于研究单个核中的蛋白质-DNA 相互作用。然而，由于 DNA FISH 中需要变性步骤，这可能会改变蛋白质表位，阻碍主要抗体的结合，这些检测方法有时可能不兼容。此外，对于经验不足的受训者来说，将 DNA FISH 与 IF 结合可能具有挑战性。我们的目标是开发一种替代技术，通过将 RNA FISH 与 IF 相结合来研究蛋白质-DNA 相互作用。
我们开发了一种混合的 RNA FISH-IF 方案，用于黑腹果蝇多线染色体铺片中，以可视化蛋白质和 DNA 基因座的共定位。我们证明，该检测方法足够灵敏，可以确定我们感兴趣的蛋白质 Multi-sex combs（Mxc）是否定位于携带组蛋白基因的单拷贝靶转基因。总的来说，本研究为在黑腹果蝇多线染色体上研究蛋白质-DNA 相互作用提供了一种替代的、易于获取的方法，可用于单个基因水平。
