PMC

Similarity of MLL3 and ASC-2 at the genetic level. (A) Genotypes of 87 P14. (B) Representative picture of a dying E13.5 MLL3^Δ/Δ embryo (arrow). (C) Representative 2-week-old MLL3^Δ/Δ mouse, retarded in eye opening (arrow), and its wild-type littermate. (D) Representative 4-week-old MLL3^Δ/Δ mouse, with stunted growth, shown with its wild-type littermate. (E) Body weights of MLL3^Δ/Δ (n = 3) mice and their wild-type littermates (n = 4) over a 60-day period. (F) Ten thousand wild-type and MLL3^Δ/Δ MEFs were seeded in 9-cm² wells, and the cells were harvested and counted each day for 6 days. A representative result is shown.

RAR-β2 H3-K4 trimethylation requires ASC-2 and MLL3 or MLL4. (A) E9.5 wild-type (wt), MLL3^Δ/Δ, and ASC-2^−/− MEFs were subjected to ChIP assays for H3-K4 trimethylation and H3/H4 acetylation during treatment with vehicle or 0.1 μM 9-cis-RA (9RA) for 2 h. (B) HepG2 cells transfected with control (con) siRNA or siRNAs for both MLL4s and MLL3 were treated with vehicle or 0.1 μM 9-cis-RA for 2 h, 2 days posttransfection, and they were then subjected to ChIPs for H3-K4 trimethylation. (C) HepG2 cells transfected with control siRNA or siRNA for WDR5 or RbBP5 were treated with vehicle or 0.1 μM 9-cis-RA for 2 h, 2 days posttransfection, and they were then subjected to ChIPs for H3-K4 trimethylation of RAR-β2. These siRNAs had no effect on H3-K4 trimethylation of GAPDH (data not shown). (D) E9.5 wild-type and ASC-2^−/− MEFs, as well as immortalized cell lines derived from wild-type and menin^−/− MEFs, were treated with vehicle or 0.1 μM 9-cis-RA for 12 h, and 9-cis-RA induction of RAR-β2 mRNAs was measured by RT-PCR.

ASC-2 is a key adaptor for RAR-dependent recruitment of MLL3 and MLL4s. (A) HEK293 cells were transfected with siRNA against MLL3 or MLL4s, along with CMV-GFP. These cells were immunostained for MLL3, MLL4s, and ASC-2, 3 days posttransfection. GFP-positive cells (i.e., transfected cells; arrows) were monitored for the expression of MLL3, MLL4s, and ASC-2. (B) HEK293 cells were transfected with control siRNA, siRNA against MLL3 or MLL4s, or siRNAs for both MLL3 and MLL4s. Two days posttransfection, cells were treated with vehicle or 0.1 μM 9-cis-RA (9-RA) for 12 h, and they were then tested for RAR-β2 transcript levels by Q-PCR. (C) Nuclear extracts of HeLa cells were subjected to immunoprecipitations (IP) with IgG and ASC-2 antibody followed by immunoblotting with ASC-2 and WDR5 antibodies. (D) HEK293 cells were transfected with control (con) siRNA or siRNA against WDR5 or RbBP5. Two days posttransfection, cells were treated with vehicle or 0.1 μM 9-cis-RA for 12 h and tested for RAR-β2 and GAPDH (control; data not shown) transcripts by Q-PCR. (E) Immortalized cell lines from wild-type (wt) and ASC-2^−/− MEFs were treated with 0.1 μM 9-cis-RA for 0–45 min and subjected to ChIPs with antibodies against MLL3 and MLL4s.

MLL3^Δ/Δ MEFs support RAR transactivation. (A) Nuclear extracts of E12.5 MEFs from wild-type and MLL3^Δ/Δ mice were immunoblotted (IB) with MLL3, ASC-2, and β-actin antibodies as well as MLL3 antibody preincubated with recombinant MLL3 protein or BSA (data not shown). (B) In Q-PCR, the transcript levels of ASC-2, MLL3, and MLL4s were measured in E12.5 wild-type and MLL3^Δ/Δ MEFs. (C) Immunoprecipitation (IP) of nuclear extracts of E12.5 wild-type and MLL3^Δ/Δ MEFs by IgG and ASC-2 antibody was followed by immunoblotting with MLL3 antibody. (D) E9.5 wild-type (wt), MLL3^Δ/Δ, and ASC-2^−/− MEFs were treated with vehicle or 0.1 μM 9-cis-retinoic acid (RA) for 12 h, and RAR-β2 transcript levels were determined by Q-PCR. (E) E9.5 wild-type and ASC-2^−/− MEFs were transfected with β2-RARE-Luc reporter and an increasing amount of ASC-2 expression vector; then they were treated with vehicle or 0.1 μM 9-cis-RA for 12 h and tested for luciferase activity. (F) Nuclear extracts of HeLa cells were subjected to immunoprecipitations with IgG and antibodies against MLL4s and MLL3 followed by immunoblotting with the indicated antibodies ().

Generation of MLL3^Δ/Δ mice. (A) Wild-type (wt) and targeted MLL3 loci are shown schematically with loxP sites (filled triangles) and the neomycin resistance gene flanked by two Frt sites. Exons 25 and 26 encode 61 aa that include the RYINHS motif, which contributes to the catalytic core region of the SET domain. The genomic positions of the Southern blotting probes and PCR primers are indicated. (B) Southern blot analyses of recombined MLL3 locus. A representative clone (clone 12) generates targeted locus-specific bands of 5.5 and 4.4 kb from BglII and NcoI restriction digestions, respectively. (C) In genomic PCR, primers a/b generate a 650-bp band for the targeted MLL3 locus and a 2,100-bp band for the wild-type MLL3 locus. Primers c/d generate a 350-bp band only from the wild-type MLL3 locus. (D) In RT-PCR, primers e/f generate a 265-bp band for both wild-type and targeted MLL3 mRNAs, whereas primers g/h generate a 356-bp band for wild-type MLL3 mRNAs and a 172-bp band for targeted MLL3 mRNAs. The asterisk indicates primers.