Experimental design to measure genome-wide dissociation rates of newly synthesized H3.3-HA in mouse embryonic stem cells. (A) Schematic of TET-repressible HA-H3.3 expression system. Green and red circles represent endogenous and newly synthesized H3.3, respectively. Ectopically expressed HA-H3.3 incorporates into chromatin over time and reaches an equilibrium level before addition of doxycycline inhibits HA-H3.3 expression. Upon inhibition of its expression, HA-H3.3 dissociates from chromatin over time. (B) Average enrichment of HA-H3.3 ChIP-Seq reads over time. ESCs were cultured for multiple passages with 2 ug/mL DOX before DOX was removed and HA-H3.3 expression induced over a time course of 6 days. ESCs routinely cultured without DOX were exposed to 2 μg/mL DOX to inhibit HA-H3.3 expression. Steady-state levels of HA-H3.3 enrichment (tpnt. 0 h) decline over a time course of 48 h. Horizontal axis depicts time in days during induction of HA-H3.3 expression (- DOX condition) and time in hours during repression (+ DOX condition) of HA-H3.3 expression. (C) Time course western blots showing expression of HA-H3.3 during induction and inhibition of HA-H3.3 expression. ESCs were treated with or without 2 μg/mL DOX. Densitometry was calculated using the Image J software package. (D) Western blot showing comparison between endogenous H3.3 and ectopic HA-H3.3 expression. The asterisk marks transgenic HA-H3.3; the arrow marks endogenous H3.3. (E) Density blot showing the correlation of HA-H3.3 dissociation rates measured in 10 bp windows between two biological replicate experiments. The color represents number of 10 bp windows with highest points (red) and lowest points (dark blue). Pearson’s correlation coefficient (PCC) between two H3.3 dissociation rates is 0.71, P value = 0, df = 6836487. (F) H3.3 dissociation rates are highly correlated with its equilibrium levels. Density blot showing correlation between H3.3 dissociation rates and H3.3 equilibrium levels. PCC = 0.61, df = 6836487, P value = 0.