Fig 2: Analysis of the BHLHE40 gene promoter in HCT116 cells. (A) Chromatin immunoprecipitation assay. Four different regions (see scheme on top) of the BHLHE40 promoter were amplified after immunoprecipitation with indicated antibodies. (B) Activation of the (-2000/+50) BHLHE40 promoter luciferase reporter construct or the parental vector pGL2-Basic upon ETV1 overexpression. Shown are means with standard deviations (n=4). One-way ANOVA with post hoc Tukey’s multiple comparisons test. (C) Similar, joint stimulation of the (-2000/+50) BHLHE40 promoter luciferase activity by ETV1 and either JMJD1A or JMJD2A; “mut” indicates catalytically inactive mutants of JMJD1A (H1120A/D1122G) or JMJD2A (H188A). Shown are means with standard deviations (n=6). Statistical significance was determined by one-way ANOVA with post hoc Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant.

Fig 3: Regulation of BHLHE40 expression by ETV1 and associated JMJD cofactors. (A) Downregulation of ETV1, JMJD1A or JMJD2A with respective shRNAs in HCT116 cells reduced BHLHE40 protein levels. Asterisks mark BHLHE40 or ETV1, while other bands are unspecific in the respective blots. (B) Corresponding quantitative RT-PCR examining BHLHE40 mRNA levels. Shown are means with standard deviations (n=3). One-way ANOVA with post hoc Dunnett’s multiple comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Fig 4: Complex formation between JMJD1A and JMJD2A. (A) Coimmunoprecipitation experiment with 6Myc-JMJD2A and Flag-JMJD1A in 293T cells. The top panel shows immunoprecipitation (IP) with anti-Flag antibodies followed by anti-Myc Western blotting, while the bottom two panels show input levels for 6Myc-JMJD2A and Flag-JMJD1A. (B) Likewise, interaction between 6Myc-JMJD1A and Flag-JMJD2A. (C) Endogenous coimmunoprecipitation of JMJD2A with JMJD1A in HCT116 cells. Either control IgG or anti-JMJD1A antibody was used for immunoprecipitation. (D) Coimmunoprecipitations of indicated Flag-tagged JMJD1A amino acids with 6Myc-JMJD2A. Top shows a sketch of JMJD1A. (E) Coimmunoprecipitation of 6Myc-JMJD1A with indicated Flag-tagged JMJD2A amino acids. Top shows a sketch of JMJD2A.

Fig 5: KDM4A regulates H3K9me3 methylation on the target genes of ETV2iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs were differentiated ± Dox and EBs at day 4 were cross-linked, sonicated, and the fragmented genomic DNAs were subsequently immunoprecipitated with rabbit anti-mouse H3K9me3 antibody or rabbit IgG antibody. The immunoprecipitated DNA fragments were qPCR-amplified with primers corresponding to the promoter regions of the indicated genes. DNA enrichment is shown as a percentage of input DNA. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗p < 0.05.