Histone Deacetylase Inhibitor

A commercial coffee grinder (Gastroback, 42601) was pre-cooled by grinding 30–50g of dry ice twice. The resulting powder of dry ice was discarded. 3g of frozen cells were mixed with ∼90g of dry ice in the coffee mill. Grinding was repeated ten times for 30sec with 30sec breaks to prevent overheating of the coffee mill. Shaking of the coffee mill while grinding prevented the dry ice–cell powder from sticking to the inside wall of the grinding chamber. The fine powder of ground yeast can be stored at −80°C. After evaporation of dry ice, the powder was dissolved in 0.75mL of cold buffer MB200 [20mM Tris–HCl (pH 8), 200mM KCl, 5mM MgAc, 0.5% Triton X-100, 0.1% Tween 20, 1mM DTT] or buffer MB150 [20mM Tris–HCl (pH 8), 150mM KCl, 5mM MgAc, 0.5% Triton X-100, 0.1% Tween 20, 1mM DTT], both supplied with 1× protease and phosphatase inhibitors (Protease and Phosphatase Inhibitor Cocktail 100x, Thermo Fisher Scientific) and 1x histone deacetylase inhibitors (0.5μM Trichostatin A, 25μM Sirtinol), per 1g of ground yeast cells. The respective MB200 or MB150 buffer was then used throughout the complete purification. The cell lysate was cleared from cell debris by centrifugation with 16.000g for 30 min at 4°C. To generate the affinity resin, rabbit IgGs (Sigma) were added to epoxy-activated magnetic beads (BcMag, Bioclone Inc.) in a ratio of 0.17mg IgGs/mg of beads according to a published protocol.³⁴ (link) The IgGs coupled to magnetic beads were equilibrated with buffer MB with 1× protease and phophatase inhibitors (Protease and Phosphatase inhibitor Cocktail 100x, Thermo Fisher Scientific) and 1x histone deacetylase inhibitors (0.5μM Trichostatin A, 25μM Sirtinol) before use. For the purification of the chromatin rings 333μL of magnetic bead slurry with coupled IgGs were added to the cell lysate. The cell lysate-bead suspension was incubated on a rotating wheel for 2h at 4°C. Beads were washed three times with 750μL of cold buffer MB with 1× Protease and Phophatase inhibitors (Protease and Phosphatase Inhibitor Cocktail 100x, Thermo Fisher Scientific) and 1x histone deacetylase inhibitors (0.5μM Trichostatin A, 25μM Sirtinol). Between each washing step, the beads were gently rotated for 5min. Finally, the beads were washed twice with 750μL of cold buffer AC (100mM NH₄Ac pH 7.4 titrated with 2M NH₃, 0.1mM MgCl₂). Chromatin rings were eluted by adding 500μL 0.5M NH₄OH, thorough mixing and incubating at room temperature for 30min. This process was repeated once and both eluates were combined to a final volume of 1mL and frozen at −80°C before submission to mass spectrometry.

Human fibroblasts were seeded, at high (10,000 cells per well) and low (1,000 cells/well) density, into 96-well plates, allowed to adhere, and treated for 24 hours (unless otherwise noted) prior to fixation. The following treatments were used: a histone deacetylase inhibitor, Trichostatin A (TSA, 200 nM), a microtubule depolymerizing agent, Nocodazole (75 nM, 24 h prior to fixation), or an actin depolymerizing agent, Cytochalasin D (CytoD, 10 µM, 60 minutes prior to fixation). A separate human fibroblast line stably modified to express an inducible dominant-negative KASH (DN-KASH) construct was treated with 500 ng/mL Doxycycline for 24 hours prior to fixation to disrupt nucleo-cytoskeletal coupling.