DOI: 10.6078/d1tq67
title: Data from: HP1a dynamics in the early drosophila embryo

description: The spatial segregation of heterochromatin into distinct, membrane-less
 nuclear compartments involves the binding of the heterochromatin protein 1
 (HP1) to H3K9me2/3-rich genomic regions. While HP1 exhibits liquid-liquid
 phase separation properties in vitro, its mechanistic role in vivo on the
 structure and dynamics of heterochromatin remains largely unresolved.
 Here, using biophysical modeling, we systematically investigate the mutual
 coupling between self-interacting HP1-like molecules and the chromatin
 polymer. We reveal that the specific affinity of HP1 for H3K9me2/3 loci
 facilitates coacervation in nucleo, and promotes the formation of stable
 heterochromatin condensates at HP1 levels far below the concentration
 required in vitro to observe phase separation in purified protein assays.
 These heterotypic HP1-chromatin interactions give rise to a strong
 dependence of the nucleoplasmic HP1 density on the HP1-H3K9me2/3
 stoichiometry, consistent with the thermodynamics of multicomponent phase
 separation. The dynamical crosstalk between HP1 and the visco-elastic
 chromatin scaffold also leads to anomalously-slow equilibration kinetics,
 which strongly depend on the genomic distribution of H3K9me2/3 domains and
 result in the coexistence of multiple long-lived, microphase-separated
 heterochromatin compartments. The morphology of these complex coacervates
 is further found to be governed by the dynamic establishment of the
 underlying H3K9me2/3 landscape, which may drive their increasingly
 abnormal, aspherical shapes during cell development. These findings
 compare favorably to 4D microscopy measurements of HP1 condensates that we
 perform in live Drosophila embryos, and suggest a general quantitative
 model of heterochromatin formation based on the interplay between
 HP1-based phase separation and chromatin mechanics.
description: Flies homozygous for the expression of ectopic GFP-HP1a on the
 second chromosome (+, GFP-HP1a/CyO; +) were allowed to lay embryos on
 apple juice plates supplemented with yeast paste at 25°C. Embryos were
 collected by hand and dechorionated in 50% bleach before being mounted for
 live imaging. Embryos were imaged using a Zeiss 880 Airyscan microscope
 with a 63x/1.4 oil immersion objective. Time-lapse image stacks were
 collected every 30 seconds with a Z-spacing of 0.36μm. Imaging at least
 part of nuclear cycle 13 (NC 13) allowed the start of NC14 (time=0 sec) to
 be clearly defined as the first time point where circular nuclei appear
 following mitosis. All movies were Airyscan-processed using Zen 2.3
 software and the first 72 time points of NC 14 (max36 minutes) were
 analyzed using Arivis software. Briefly, HP1a foci were segmented from the
 overall nuclear signal using an intensity threshold &gt; 75% of the
 fluorescence intensity and a lower size threshold of 0.03
 μm<sup>3</sup> corresponding to approximately 10 voxels (0.085
 μm x 0.085 μm x 0.36 μm). All HP1a segments found to be percolating in
 3-dimensional space were merged into a single segment, and population
 statistics were calculated for each time point. The
 imaging data made accessible here are the 4-D zeiss confocal modies
 converted to .tif stacks in FIJI but unanalyzed and unaltered in any other
 way.  
description: These files can be opened in ImageJ/ FIJI and Arivis. Please
 check the file names for each file to make sure that the time and z axis
 of the data have been properly read by whichever software you would like
 to use. The time and z index is appended to each file name, so a cursory
 read of these will orient the user.