Fig. 3

Bru-seq analysis of transcriptional changes in response to GRHL2 loss in luminal breast cancer MCF7 cells. a Bru-seq sample preparation. Bromouridine (Bru) labeling of nascent RNA was carried out for 30 min at the indicated time points after doxycycline (dox)-induced GRHL2 deletion. b Western blot analysis of GRHL2 expression levels at the indicated time points in sgCTR and sgGRHL2 transduced MCF7 cells. Cas9 induction is monitored and GAPDH serves as loading control. c Bru-seq data analysis approach. Each circle represents a gene set with differential transcription relative to the condition where no doxycycline was added. d Heatmap for genes whose transcription was altered in response to GRHL2 depletion. e Graphs depicting clusters of genes with distinct patterns of transcriptional changes in response to GRHL2 depletion. Graphs represent log₂ AFC of transcription in sgGRHL2(1) and sgGRHL2(2) cells. “Dynamic”: genes with AFC > 2; p < 0.05 at some and AFC < 0.5; p < 0.05 at other time points. “Sustained induction”: genes with AFC > 2; p < 0.05 at all time points. “Sustained repression”: genes with AFC < 0.5; p < 0.05 at all time points. “Induction reset”: genes with AFC > 2; p < 0.05 at early time points followed by a return to 1 < AFC < 2 at day 16. “Repression reset”: genes with AFC < 0.5; p < 0.05 at early time points followed by a return to 0.5 < AFC < 1 at day 16