Extended Data Fig. 5: Acetylation site mutant of MED1 alters gene expression in steady-state and stress conditions in ER+ breast cancer cells.
From: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment
a, Workflow depicting the methods employed to replace endogenous MED1 with ectopic MED1 in MCF7 or T47D cells. Lentiviruses expressing gRNAs targeting MED1 were transfected into pools of cells expressing ectopic HA-tagged MED1 (WT or 6KR/Q mutant) with CRISPR-mutated target sequences. b, c, Immunoblotting confirming efficient depletion of MED1, expression of HA-tagged WT and 6KR/Q MED1 (after CRISPR KO) at levels comparable to that of endogenous MED1 in MCF7 (b) and T47D (c) cells. d–f, Poly(A) RNA-seq for 6KR/KO and WT/KO MCF7 cells in steady state. Three biological replicates were detected for each cell type. d, Volcano plot showing Fold-change versus Adjusted P value (two-sided) for each detected gene, with the cut-off for defining DEGs (FC > 2, P adj < 0.05) indicated. e, GSEA comparing the 6KR/KO versus WT/KO gene rank list with KEGG (left) and GOBP (right) gene lists. Adjusted P values are shown in addition to NES reported by bars. f, GSEA revealing that the up-regulated (upper) and down-regulated (lower) genes in MED1 KO versus control cells (strong or mild) are enriched, respectively, in the sets of down-regulated and up-regulated genes of 6KR/KO versus WT/KO cells. NES values are shown. g, Detection by qRT-PCR showing stronger induction of representative hypoxia-induced genes in 6KR/KO versus WT/KO T47D cells treated with 200 μM DFOM. For each of WT/KO or 6KR/KO cells, expression of the gene at each condition was normalized by its expression at control condition in this cell type. N = 3 (biological replicates) for each group. Bars report mean ± SEM. If < 0.05, the P values of two-sided unpaired t-test comparing cell types under specific conditions are shown. For panels b and c, immunoblots are representative of three independent experiments.
