Fig 2: 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 3: PASK is expressed in the cytoplasm and the nucleus of muscle satellite cells and associates with H3K4 MLL2 methyltransferase complex. (A) Confocal micrograph of HEK 293T cells immunostained with anti-rabbit PASK antibody (green) and TOPRO3 (blue) to stain the nucleus. Note that PASK is present in both the cytoplasm and nucleus. Note also the speckled appearance in the nucleus (insert). (B) Western blot analysis of the cytoplasmic and nuclear fractions of HEK293T cells with anti-rabbit PASK antibody reveals expression of PASK in both the cytoplasmic and nuclear fractions. GAPDH and H3 were used as markers for cytoplasmic and nuclear fractions. (C) PASK immunoprecipitated with V5 antibody from the nuclear extract of HEK 293T cells transfected with a hPASK expression plasmid with a V5 tag. PASK along with the core members of the H3K4 MLL2 methyltransferase complex, WDR5, ASH2 RBBP5 and the methyltransferase enzyme MLL2 are co-immunoprecipitated in the presence of V5 antibody. Nuclear extract from V5 vector transfected HEK 293T cells was used as control. About 10% of the nuclear extract was used as input. The results shown are a representative of three independent experiments. (D) Direct interaction analysis of PASK with the core members of the H3K4 MLL2 methyltransferase complex was analyzed using recombinant WDR5, ASH2 RBBP5, MLL2 and PASK-V5 proteins. Western blot analysis of V5 agarose beads capture shows PASK directly interacts with WDR5 protein. About 10% of the reaction was used as input. IgG was used as control.

Fig 4: Pask is expressed in the cytoplasm and the nucleus of C2C12 muscle satellite cells and associates with the endogenous mammalian H3K4 Mll2 methyltransferase complex. (A) Confocal micrograph of C2C12 muscle satellite cells immunostained with anti-rabbit Pask antibody (green) and propidium iodide (red) depicting the presence of Pask in both the cytoplasm and nucleus. Insert shows its speckled appearance in the nucleus. (B) Western blot analysis of the cytoplasmic and nuclear fractions of C2C12 cells with anti-rabbit Pask antibody reveals expression of Pask in both the cytoplasmic and nuclear fractions. Gapdh and H3 were used as markers for the cytoplasmic and nuclear fractions. (C) Pask co-immunoprecipitated the core members of the H3K4 Mll2 methyltranferase members Wdr5, Ash2, RbBP5 and Mll2 methyltransferase enzyme from the C2C12 muscle satellite cells when brought down with Pask antibody. Western blots of Pask and Mll1 were also shown for the specificity of the immunoprecipitation. (D) Wdr5 co-immunoprecipitated Pask. Normal rabbit IgG was used as control for immunoprecipitation (C and D). About 10% of the nuclear extract was used as input in both experiments, (C and D). The results shown are a representative of three independent experiments.

Fig 5: Downregulated genes are associated with the senescence phenotype. (A) Commonly downregulated genes in murine KO1 and KO2 cells upon loss of Ash2l were compared to commonly downregulated genes in three primary human cell populations (45) and to downregulated genes in human diploid fibroblasts (44) in response to replicative senescence. The number of genes is indicated that are in common between the different groups: A, KO1/KO2 and the genes in Lackner et al.; B, KO1/KO2 and the genes in Zirkel et al.; M, common genes of all three studies. Comparison KO1/KO2 with Lackner et al. and Zirkel et al.: P-value < 2.2e–16 (Fischer Exact test). (B) The genes in groups A, B and M are listed and the repression of these genes in both KO1 and KO2 cells upon treatment ± HOT for 5 days is indicated. (C) GO pathway analysis of the genes that are in common between the data sets of panel A. Adjusted P-values are indicated; ns, not significant. (D) KO2 cells were treated ± HOT for 5 days. H3K4me3 ChIP-qPCR experiments were performed with primers specific for the promoters of the indicated genes, which belong to the M group (panels A and B). For control, ChIP was carried out using IgG control antibodies. Displayed are mean values ± SD (n = 5) (** < 0.01, *** < 0.001).