Fig. 5

PU.1 depletion fails to block HDACi-induced chromatin accessibility changes. a Schematic of PU.1 shRNA knockdown strategy. K562 cells were transfected with a control vector, a vector containing an shRNA that targets PU.1, or mock transfected. Following 10 days of puromycin selection, stable knockdown cultures were obtained and immediately subjected to SAHA or vehicle control 72-h treatment followed by DNase-seq and RNA-seq profiling. b Western blot showing PU.1 levels before and after SAHA treatment for vector control-, shRNA 1-, or shRNA 2-transfected K562. β-actin was used as loading control (n = 2 replicates). c MA plot of fold-change in chromatin accessibility (DNase-seq) between vector control and PU.1 knockdown (shRNA 1) K562 over average signal found at each site. There are no significant DHS sites at P < 0.10 (n = 2 replicates). d MA plot of fold-change in chromatin accessibility between vector control and PU.1 knockdown (shRNA 1) K562 following 72-h SAHA treatment over average signal found at each site. There are no significant DHS sites at P < 0.10 (n = 2 replicates). e Boxplots of fold-change in chromatin accessibility between vehicle control and SAHA-treated K562 with vector control or shRNA knockdown of PU.1. Three sets of DHS sites are shown for comparison—PU.1-bound (overlaps ChIP-seq peak) sites that did not open in original SAHA treatment, PU.1-bound sites that open with original SAHA treatment, and unbound sites that open with original SAHA treatment. f Representative DHS site located downstream of the SYF2 gene that opens with original 72-h SAHA treatment (DNase-seq) and contains a PU.1 ChIP-seq peak. The DHS site still opens in PU.1 shRNA knockdown cells