Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of topologically associating domains (TADs). Here, we demonstrate an interaction between proteins that associate with the gypsy insulator and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components prevents stable H2Av phosphorylation in polytene chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av is a component of insulator bodies, and that phosphatase activity is required for insulator body dissolution after recovery from osmotic stress. Together, our results indicate a novel mechanism linking the H2A variant γH2Av to insulator function. 

This dataset consists of fluorescence microscopy imaging of polytene chromosomes and S2 cells. Samples were immunostained with antibodies anti-insulator proteins Su(Hw), CP190 and Mod(mdg4), as well as anti-phosphorylated histone variant H2Av (ϒH2Av) and anti-H2Av. Raw images used to make figures and image analysis in the manuscript are all included in the dataset. 

Image analysis was carried out using ImageJ. We used intensity analysis and linescans to interrogate for intensity levels and colocalization of signals from the different markers on wild-type and mutant chromosomes. Macro codes for ImageJ were developed by Ryan Simmons and are also included in a file in this dataset.

The manuscript also contains genomic analysis of ChIP data from data sets published elsewhere. Reference for all ChIP datasets used can be found in the paper. Numerical data used to calculate correlation coefficients and for scatterplots was included in excel spreadsheets in the Figure 4 folder.

Individual figure folders contain all the data used for each figure. Each figure folder contains a file with the corresponding figure in PDF format.

Raw fluorescence images are TIF files.

All TIF files contain metadata.

TIF files can be visualized using any imaging software including Photoshop and ImageJ.

Image files can be analyzed using macro codes included in the dataset. These codes select ROIs and perform the intensity or colocalization analysis (see Material and Methods in the paper).

Spreadsheets from quantitative analysis used to make graphs presented in the figures are included in the dataset. All spreadsheets were generated using ImageJ and Prism and data was saved in Excel files.  Excel files are included in the dataset for each figure.