HSF2 affects chromatin organization at the hsp70 promoter during mitosis. (A, top) Occupation of phosphorylated histone H3 (H3 S10P) and total histone H3 (H3) at the hsp70 promoter in nonstressed (C) and heat-shocked (HS; 30 min at 42°C) mitotic K562 cells was analyzed by ChIP and quantified with qPCR (n = 4). Cells were transfected with the indicated shRNA constructs and synchronized into mitosis by a thymidine-nocodazole block. The qPCR values of the IP were normalized to the input values and related to the Scrambled unsynchronized control that was arbitrarily set to a value of 1. (A, bottom) Representative immunoblot (n = 4) of H3 S10P levels in K562 cells. Immunoblotting was performed with the indicated antibodies. (B) MNase sensitivity analysis of the hsp70 promoter (−377 to −229 bp) and coding region (+1,286 to +1,397 bp) in mitotic K562 cells as measured by qPCR (n = 4). Cells were transfected, synchronized, and treated as in A. The qPCR values of the MNase-treated samples were related to input and to the Scrambled control sample that was arbitrarily set to a value of 1. (C) RNAPII occupation of the hsp70 promoter in nonstressed mitotic cells. K562 cells were transfected, synchronized and analyzed as in A, analyzed by ChIP, and quantified with qPCR (n = 4). All values represent mean + SEM (error bars). n.s., not specific.