Characterizing the effects of pathogenic WAS missense mutations on histone H2A-to-H2A.Z exchange and transcription elongation in vivo. (A-B) Single and sequential MNase-ChIP assays performed on nuclear extracts from T_H1-skewed, WAS^null T_H cells stably expressing the indicated WASp mutants. Other details are as described in the legend to Figures 2C and 3B. Data represent the averages of triplicates, with bars indicating the standard error from at least 3 biological replicates. (C) PCR-based quantitation of the absolute DNaseI hypersensitivity at the indicated gene promoter loci in nonskewed T_H0 or T_H1-skewed, normal, and WAS^null T_H cells stably expressing the indicated WASp mutants, or controls (UT, untransfected control; FL, full-length WASp transfected) is shown. T_H nuclei were treated with DNaseI, and the number of amplicons lost was quantified by RT-qPCR and is displayed as percent of DNaseI-untreated control. Data represent the average of triplicate values from 1 experiment. (D) MNase-ChIP assays performed on nuclear extracts from T_H1-skewed, WAS^null T_H cells stably expressing the indicated WASp mutants. The location of 8 different genomic positions (position 1 is near TSS at 5′UTR; position 8 is at 3′UTR immediately after the last coding exon) within the IFNG locus, where the ChIP-qPCR primer/probes were designed is indicated and numbered (in red). DNaseI HS (DHS) profile for the primary human peripheral T_H1 cells (in gray) publicly available from the ENCODE–University of Washington was aligned alongside our custom track to give context to the location of our ChIP-qPCR primer/probes. “Normal” denotes an HTLV-1–immortalized CD4⁺T cell line generated from a healthy donor. UT, untransfected WAS-null patient-derived T cells line.