Figure 7.

PARylation of NSD2 disrupts its association with chromatin in the cell. A, NTKO cells pretreated with 5 μm Olaparib for 24 h were treated with 0.5 mm hydrogen peroxide for 15 min and harvested to extract nuclear proteins. PARylated proteins were then affinity captured with a poly(ADP-ribose)-binding macrodomain resin and immunoblotted for PARP1 and NSD2 as indicated. B, nuclear fractionation assay of KMS11 cells. Soluble and insoluble nuclear fractions were analyzed by immunoblot for NSD2. HDAC2 was used as soluble loading control and histone H3 was used as insoluble loading control. Relative soluble and insoluble NSD2 was quantified using ImageJ. C, ChIP-qPCR against NSD2 on the NSD2-regulated genes JAM2 and CLS2 promoters and gene bodies using NTKO cells with or without treatment of hydrogen peroxide. IgG was used as negative control. Enrichment was calculated as a percentage of total input DNA. Three independent biological replicates are shown.