Fig 2: MUC1-C is necessary for WDR5 and RBBP5 expression.a, b BT-549/tet-MUC1shRNA (a) and MDA-MB-468/tet-MUC1shRNA (b) cells treated with vehicle or DOX for 7 days were analyzed for WDR5 and RBBP5 mRNA levels by qRT-PCR. The results (mean ± SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1) (left). Lysates were immunoblotted with antibodies against the indicated proteins (right). c BT-549/CshRNA and BT-549/MUC1shRNA#2 cells were analyzed for WDR5 and RBBP5 mRNA levels by qRT-PCR. The results (mean ± SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained for CshRNA cells (assigned a value of 1) (left). Lysates were immunoblotted with antibodies against the indicated proteins (right). d BT-549/MUC1shRNA#2 cells transfected with a tet-Flag-MUC1-CD vector were treated with vehicle or DOX for 7 days. Lysates were immunoblotted with antibodies against the indicated proteins. e BT-549 cells treated with vehicle or 5 μM GO-203 for 48 h were analyzed for WDR5 and RBBP5 mRNA levels by qRT-PCR. The results (mean ± SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1) (left). Lysates were immunoblotted with antibodies against the indicated proteins (right). f BT-549 cells grown as monolayers (2D) and as mammospheres (3D) were analyzed for expression of the indicated mRNA levels by qRT-PCR. The results (mean ± SD of 3 or more determinations) are expressed as relative mRNA levels compared to that obtained for 2D cells (assigned as value of 1)(left). Lysates were immunoblotted against the indicated proteins (right). g BT-549/tet-MUC1shRNA 3D mammosphere cells treated with vehicle or DOX for 7 days were analyzed for the indicated mRNA levels by qRT-PCR. The results (mean ± SD of 3 or more determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1) (left). Lysates were immunoblotted against the indicated proteins (right).

Fig 3: The MLL3/4 complexes associate with the mitotic deacetylase complex (MiDAC).Western blot of FLAG-DNTTIP IP from HEK293 cells confirming interaction of DNTTIP1 with the MiDAC components ELMSAN1, HDAC1, HDAC2, and members of the MLL3/4 complexes including UTX, the H3K4 methyltransferases MLL3 and MLL4, and the two core components RBBP5 and ASH2L. KAT2A was used as a negative control. HEK293 cells with a FLAG-tag expressing plasmid were used as an IP control. Nuclear extracts were used as input. Actin was used as a loading control for the inputs.

Fig 4: The interaction interface between UTX and ELMSAN1 forms the nexus between the MLL3/4 complexes and MiDAC.(A) CRISPR strategy to restore MLL3 expression and knockout MLL4 in HCT116 cells. A single base pair deletion in the MLL3 locus was repaired by using a single-stranded oligo donor that contained a blocking modification (G>A mutation at the highlighted position) to create MLL3+/+ MLL4−/− clone 2F11. The newly added G to restore the MLL3 sequence back to WT in HCT116 cells is highlighted in red. The MLL4 promoter was targeted with gRNAs SM124.KMT2D.g4 and SM125.KMT2D.g3 resulting in the deletion of a 9,522-bp sequence within the MLL4 promoter to create MLL3−/− MLL4−/− clone 2G5. The bases at the deletion junction are marked by green and purple, respectively. In addition, a G to A substitution in allele 1 and 2 (highlighted in red) was created 33 bp from the deletion junction. (B) Western blot for members of the MLL3/4 complexes including UTX, the H3K4 methyltransferases MLL3 and MLL4 and the core components RBBP5 and ASH2L from nuclear extracts of MLL3+/+ MLL4+/+ (2F11), MLL3−/− MLL4+/+ (control), and MLL3−/− MLL4−/− (2G5) HCT116 cells. Actin was used as a loading control. (C) Western blot of FLAG-UTX IP from MLL3+/+ MLL4+/+ (2F11), MLL3−/− MLL4+/+ (control), and MLL3−/− MLL4−/− (2G5) HCT116 cells after transfection with FLAG-UTX. UTX is able to associate with MiDAC (DNTTIP1, ELMSAN1, and HDAC1) in the absence of MLL3 and MLL4 suggesting that UTX mediates the interaction between the MLL3/4 complexes and MiDAC. HCT116 cells of the same genotypes transfected with a FLAG-tag expressing plasmid were used as IP controls. Nuclear extracts were used as input. Actin was used as a loading control for the inputs.

Fig 5: MiDAC stability does not depend on the MLL3/4 complexes.Western blot for various subunits of the MLL3/4 complexes and MiDAC from nuclear extracts of WT, Mll3/4 DCD, and Mll3/4 DKO mESCs. As a result of MLL3/4 DKO, UTX protein levels are strongly decreased, whereas protein levels of RBBP5 and MiDAC subunits (DNTTIP1, ELMSAN1, HDAC1, and HDAC2) are unaffected in the absence of MLL3/4. Actin was used as loading control.