Fig 2: ASH2L associates with MLL1/WDR5 to enhance ERα‐induced transactivation. A, Co‐IP was performed to detect the interactions between endogenous ERα and ASH2L/MLL1/WDR5 in shCtrl and shASH2L Ishikawa cells. B, Interaction domains of ASH2L with MLL1/WDR5, or ERα in Ishikawa cells was tested by Co‐IP. C, Luciferase assay shows the effects induced by deletion of ASH2L on the ERα‐mediated transactivation, which is upregulated by WDR5 or MLL1 in Ishikawa cells. Expression of WDR5 and MLL1 was detected using western blotting. *Asterisks placed to indicate the position of expression of FLAG‐ASH2L truncated mutants. Data are shown as mean ± SD (n = 3), *P < .05, **P < .01, ***P < .001

Fig 3: BAP18 facilitates the recruitment of COMPASS-like core proteins to the cis-regulatory elements of ERα target genes. (A) The Co-IP experiment was performed with anti-ERα and IgG in T47D cells carrying control shRNA or shBAP18 as indicated. Precipitated proteins were determined by western blotting with antibodies against COMPASS-like core proteins orBAP18/ERα as indicated. The location of the star indicates where the protein is expressed. (B) ChIP assays exhibited BAP18/ERα enrichments and modification of H3K4me3 at three independent EREs on upstream of the TSS region of the MYC gene. The location of EREs was indicated in the diagram above (red rectangle). (C) ChIP assays were performed with the antibodies as indicated by demonstrating the recruitment of proteins and the influence of BAP18 on protein recruitment and the histone modification levels as indicated on MYC-ERE3. T47D cells were transfected with control siRNA (siCtrl) or siRNA against BAP18 (siBAP18) with or without estrogen treatment (100 nM). (D, E) The recruitment of ERα (D) or BAP18 (E) together with each component of COMPASS-like core proteins on MYC-ERE3 by ChIP re-IP assays in T47D cells. (F) The additive effect of BAP18 on the co-activation function of COMPASS-like core proteins on the ERα-mediated transactivation. T47D cells were co-transfected with ERα together with BAP18, WDR5, ASH2L or DPY30 expression plasmids as indicated for luciferase analysis. FLAG-tagged PcDNA3 plasmids were used for the control group. (G) The depletion of each component of COMPASS-like core proteins does not affect the interaction between BAP18 and ERα by co-IP experiments in T47D cells. (H) The effect of COMPASS-like core proteins on the recruitment of BAP18 or ERα to the MYC-ERE region using ChIP assays. All data of ChIP assays, ChIP re-IP assays, and luciferase assays were representative of three independent experiments results. Student t-tests were used and error bars represent mean±SD. *P< 0.05, **P<0.01, ***P< 1e−3, ****P< 1e−4, and ns stands for no significance.

Fig 4: COX-2 gene activation leads to histone modifications and requires expression of WDR5. (A) ChIP showing changes in H3K4me3 or H4Ac modifications at the COX-2 promoter (site 8: −1540/−1464). Cells were treated with either mismatched control (MM), RNA12, or RNA12nc at 25 nM. RNAs were transfected at 25 nM. n = 3. See also Supplementary Figures S6A–D. (B, C) Western showing effect of siRNA-mediated silencing of WDR5 on induction of COX-2 protein (B) and mRNA (C) expression by RNA12 and RNA12nc. Cells were first transfected (TRF-1) with either mismatched control (MM) or anti-WDR5 siRNA then transfected with MM, RNA12 or RNA12nc. RNAs were transfected at 25 nM. (C) qPCR showing siRNA-mediated depletion of WDR5 mRNA. (D) ChIP showing changes in H3K4me3 modification at the COX-2 promoter (site 8: −1540/−1464). Cells were first transfected (TRF-1) with either mismatched control (MM) or WDR5 siRNA (WDR5si) then transfected with MM, RNA12 or RNA12nc. RNAs were transfected at 25 nM. n = 3. (E) ChIP showing recruitment of WDR5 at the COX-2 promoter upon addition of RNA12 or RNA12nc (25 nM). (site 1: −81/+56). MM = mismatch control. Error bars are SD. *P < 0.05, **P < 0.01 and ***P < 0.001 (t-test) relative to mismatch control. All experiments were done in A549 cells.

Fig 5: NuRD associates with the MLL methyltransferase complex regulatory subunit Wdr5. A, BN-PAGE migration profile of Wdr5 showing correlation to the NuRD reference profiles of Mta2 and Hdac1. B, Linear regression plot of normalized iBAQ relative to Mta2 for Wdr5, illustrating the comigration trend. C, Western blotting for NuRD subunits Mta2 (FLAG), Hdac2, Chd4, and Rbbp4 following Wdr5 IP. D, An ESC whole cell lysate was subject to size exclusion chromatography. Twenty-eight fractions were collected (3B5–3D8) and analyzed by Western blotting for Mta2-FLAG, Hdac2, Sall4, and Wdr5.