DOI: 10.5061/dryad.95fs76f
title: Data from: ATRX promotes maintenance of herpes simplex virus heterochromatin during chromatin stress

description: The mechanisms by which mammalian cells recognize and epigenetically
 restrict viral DNA are not well defined. We used herpes simplex virus with
 bioorthogonally labeled genomes to detect host factors recruited to viral
 DNA shortly after its nuclear entry and found that the cellular IFI16,
 PML, and ATRX proteins colocalized with viral DNA by 15 min post
 infection. HSV-1 infection of ATRX-depleted fibroblasts resulted in
 elevated viral mRNA and accelerated viral DNA accumulation. Despite the
 early association of ATRX with vDNA, we found that initial viral
 heterochromatin formation is ATRX-independent. However, viral
 heterochromatin stability required ATRX from 4-8 h post infection.
 Inhibition of transcription blocked viral chromatin loss in ATRX-knockout
 cells; thus, ATRX is uniquely required for heterochromatin maintenance
 during chromatin stress. These results argue that the initial formation
 and the subsequent maintenance of viral heterochromatin are separable
 mechanisms, a concept that likely extrapolates to host cell chromatin and
 viral latency.
description: Colocalization
 Distribution Data Analysis ScriptMATLAB script uses
 nuclear masks and foci center x,y coordinate data from each channel
 (results from Foci Detection and Colocalization software) to determine
 distances of nearest neighbor foci in a second channel from foci in the
 reference channel and generate distance frequency data. The script then
 generates the same number of random x,y points within the nuclear mask as
 were detected in the second channel. The distances from the centers of
 reference channel foci to nearest neighbor random points is then
 calculated and reported to within a user defined user defined radius of
 each reference focus. The resulting frequency distributions of
 reference-to-test channel distances and reference-to-random points are
 compared by the non-parametric Kolmogorov-Smirnov test. If the test
 rejects the null hypothesis at 5% significance, the test returns a value
 of 1, and 0 if the null hypothesis is not rejected. The test also returns
 an asymptotic p-value and a list of distances in pixel values which can
 then be plotted and further analyzed using GraphPad Prism (as it was here)
 or other data analysis packages.ColocCode_V02.zipFoci Detection and Colocalization 1052 Software.Foci Detection and Colocalization Software. The Image and Data Analysis Core (IDAC) at Harvard Medical School developed a custom MATLAB based software for nuclear foci and colocalization detection in microscopy images based upon their previously described nuclear foci detection software. In brief, unaltered images are analyzed by the software to detect signal intensity over a set threshold to isolate foci from background. The software then uses DAPI staining to generate a mask that defines nuclear areas in the image. Only foci above the fluorescence threshold and within the nuclear masks are scored. The software determines colocalization based on the distance between the centers of nuclear foci in a reference channel to foci in other channels of the image.ColocMatlabScripts.zip