CHD8 is required for progesterone-dependent gene regulation.

<p>(A) Flow diagram depicting the knockdown strategy for CHD8 and the hormone treatment in gene expression and ChIP studies. T47D-MTVL cells were transfected with control siRNA (siCt) or siRNA against CHD8 (siCHD8), subjected to serum deprivation during 48 h and then stimulated with 10 nM R5020 (R5020) or vehicle (EtOH) for 45 min or 6 h, depending on the experiment. (B) Western blot analysis of CHD8 expression upon transfection of T47D-MTVL cells with control siRNA (siCt) or siRNA against CHD8 (siCHD8). (C) Box-and-whisker plots of the change in gene expression of CHD8-dependent genes (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005174#sec016" target="_blank">Materials and Methods</a>) after 6 h of R5020 treatment. siCHD8: cells depleted of CHD8; siCt, control cells. (D) Overlapping between R5020-dependent CHD8-target genes (green) and genes that are differentially regulated in response to R5020 in CHD8-silenced cells with respect to control cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005174#sec016" target="_blank">Materials and Methods</a>) (purple, siCHD8-affected genes). Chip-seq CHD8 peaks were assigned to the closer gene. (E) Effect of CHD8 depletion in progestin-dependent expression of the following genes: <i>HSD11B2</i>, <i>MMTV-Luc</i>, <i>DUSP1</i>, <i>FKBP5</i>, <i>NFE2L3</i> and <i>IL6ST</i>. Level of <i>CHD8</i> expression was determined as control of silencing (upper panel). mRNA levels were determined by RT-qPCR after 45 min or 6 h of stimulation. Data are the mean of at least n = 6 qPCR reactions from three independent experiments. Error bars represent ± SD values. * <i>p</i> < 0.001; ** <i>p</i> < 0.0001 with respect to siCt, using Student’s t-test. (F) ChIP analysis of CHD8 occupancy at the MMTV regulatory region upon stimulation with R5020 during 5 or 45 min. Data are the mean of at least n = 6 qPCR reactions from three independent experiments.