Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal reproducibility and low sample success rate as accepted caveats. However, low sample success rates are an obvious disadvantage when polytene chromosomes are used for more high-throughput approaches, such as genetic or antibody screens, or for experiments requiring high-quality chromosome structure preservation. Here we present an exceptionally reproducible squashing and fluorescence staining protocol, which generates high-quality fluorescence images on well-spread chromosomes. This is followed by our novel, semi-automated MATLAB analysis program used to determine correlations between fluorescence signals of interest at a single site on polytene chromosomes, in a pixel-by-pixel manner. In our case, we have used this approach to assess chromatin changes at genomic sites, ectopically targeted by nuclear pore proteins. The use of our analysis program increases the ability to make unbiased conclusions on changes in chromatin structure, or in protein recruitment to chromatin, regardless of sample variation in immunofluorescence staining. As it is simply based upon differences in fluorescence intensity at a defined location, the provided analysis program is not limited to analysis of polytene chromosome, and could be applied to many different contexts where correlation between fluorescent signals at any particular location is of interest.
Keywords: Polytene chromosome squashes, Chromatin structure, Protein recruitment, Pearson correlation coefficients, Immunofluorescence analysis
Polytene chromosomes from Drosophila larval salivary glands have an unusually large size (visible under a typical cell-culture microscope). This is due to altered cell cycle processes that result in ~1,000 copies of each chromosome that do not segregate from each other. The resulting large chromosomes have stereotyped and reproducible alternating bands of condensed and decondensed chromatin, as chromosomes are aligned and locus-specific chromatin structure is visible upon polytenization. The nature of these chromosomes makes the analysis of chromatin structure uniquely visible relative to diploid cell types, enabling analysis of chromatin changes throughout development, transcriptional activation, or upon perturbation. Additionally, genome-wide and site-specific chromatin binding of proteins can be visualized by immunofluorescence staining of polytene chromosomes (Paro, 2008; Cai et al., 2010 ), allowing for investigation of the relationships between candidate proteins and chromatin condensation, transcriptional activity, and histone modifications. In this manner, polytene chromosome staining provides a rapid and inexpensive alternative or supplement to molecular assays such as Chromatin Immunoprecipitation, coupled to sequencing (ChIP-seq).
Our squashing and fluorescence analysis pipeline was specifically designed for analysis of chromatin structure and protein recruitment/changes at single bands of interest, originally for a band of LacI-tethered protein bound to a genomic lacO repeat array ( Kuhn et al., 2019 ). In this study, we targeted specific nuclear pore components, fused to LacI, to lacO arrays, integrated at defined sites in the genome. We then aimed to determine whether such targeting leads to recruitment of candidate interacting partners, and also to changes in target chromatin compaction. This approach involved identification of a single band of interest (harboring the lacO site) within an entire genome of spread chromosomes, which required high-quality chromosome spreading for increased visibility and analysis of chromosome banding patterns. Additionally, screening through multiple antibodies to assay for many candidate interacting partners required an extremely reproducible squashing protocol, with minimal sample failure rate. To achieve these goals, we developed this optimized polytene squashing protocol, which can be utilized to improve image quality and sample success rate. It is also especially useful for chromosome ‘mappability’ (determining genomic location by cytological mapping of polytene banding patterns) and for more high-throughput applications.
In order to reliably analyze site-specific changes in chromatin structure (i.e., changes in chromatin condensation as assessed by DNA stain fluorescence intensity), and in protein recruitment, we developed a semi-automated MATLAB function. This function, PCCcalc, calculates the Pearson Correlation Coefficients (PCC) between multiple fluorescence signals at a defined genomic site on a pixel-by-pixel basis in an unbiased manner. This greatly reduces data analysis time and subjectivity derived from manual analysis and user-based variability. Additionally, this approach circumvents the impact of slide-to-slide variability in fluorescence background or image acquisition, as the analysis focuses on the relationship between fluorescence signals more than absolute values of a given signal. This method can be used on any easily identifiable site on polytene chromosomes to determine binding correlations between multiple protein factors, or to find correlations between protein of interests and associated changes in chromatin compaction. Furthermore, we believe this analysis method can be extended to determine fluorescence correlation beyond the described use in polytene chromosomes, and can be used to analyze co-localization or co-occurrence of fluorescently labeled biological markers in many other contexts and cell types.