Supplementary Figure 9: The FACT complex facilitates transcription-associated pruning of macroH2A2 (related to Fig. 7).

(a) Western blot of isolated chromatin showing the expression level of GFP-tagged macroH2A isoforms in dKO iDFs using antibodies targeting respective macroH2A isoforms. Chromatin of WT iDFs is used to show the level of endogenous macroH2A isoforms. Histone H3 and amido black staining of histones used as loading control. (b) Western blot of whole cell extracts using anti-GFP antibody showing a similar expression level of GFP-tagged histones. GAPDH used as loading control. (c) Agarose gel electrophoresis analysis showing the distribution of DNA isolated from nucleosomes used as input for MNase IP. Bands corresponding to DNA of mono-/di-/tri-nucleosomes are labeled. (d) Western blot validation of MNase IP—qMS showing the interaction between macroH2A1.1 and PARP1. An empty lane between input and IP was removed for clarity. (e) Western blot analysis showing that endogenous macroH2A2 interacts with FACT subunits SPT16 and SSRP1 in iDF soluble nuclear extracts. MacroH2A2 IP with macroH2A dKO iDFs was used as control. (f) Diagram showing domain architecture of macroH2A2 and design of studied macroH2A2 fragments. (g) Western blot analysis showing interactions between FACT subunit SPT16 and different regions of macroH2A2 in iDF soluble nuclear extracts. For SPT16, a shorter (upper) and a longer (lower) exposure are shown. Lanes between the left and right panels were removed for clarity. (h) RT-qPCR analysis of iDFs with SPT16 knockdown. 18 S rRNA is used as internal reference. Error bars represent s.d. from n = 3 independent experiments. P values are calculated from two-tailed t test comparing shSPT16 with shScr (*P < 0.05, ** P < 0.01). (i) Model of the dynamics of de novo macroH2A2 deposition at distinct chromatin regions. The diagram depicts the absolute level (left) and background-normalized relative level (right) of chromatin incorporated macroH2A2 at indicated regions during de novo deposition. Green-shaded areas highlight the time window where transient macroH2A2 peaks are present. (j) Western blot of IP with chromatin-free cell extract in iDFs showing the interaction of macroH2A2 and NAP-1. Different salt concentrations were applied during immunoprecipitation as indicated.