Figure 7. UTX and UTY associate in common protein complexes and are capable of H3K27 demethylase independent gene regulation.

(A) Co-transfection of HA-UTX with Flag-UTX or Flag-UTY demonstrates that HA-UTX can immunoprecipitate with both Flag-UTX and Flag-UTY. (B) Immunoprecipitation of Flag-UTX and Flag-UTY reveal interaction with RBBP5, a component of the H3K4 methyl-transferase complex. Flag vector transfection was used as a negative control for immunoprecipitation. (C) Fnbp1, a gene targeted directly by UTX, has intermediate downregulation in X^Utx− Y^Uty+ MEFs (68% of WT, t-test p-value = 0.002), but was further compromised in X^Utx− X^Utx− (42% of WT, t-test p-value relative to X^Utx− Y^Uty+ = 0.001) and X^Utx− Y^Uty− (48% of WT, t-test p-value relative to X^Utx− Y^Uty+ = 0.02, N>4 independent MEF lines per genotype) MEFs. MEFs were generated from the X^UtxGT2Δ and Y^UtyGT alleles. (D) Fnbp1 is similarly mis-expressed in X^UtxGT2fl allelic combinations of E12.5 MEFs. X^Utx− X^Utx− and X^Utx− Y^Uty− MEFs significantly differ from X^Utx− Y^Uty+ MEFs (t-test p-value = 0.05 and 0.02 respectively, N>4 independent MEF lines per genotype). (E) H3K27me3 ChIP was performed on E12.5 X^Utx+ Y^Uty+ control (green) and X^Utx− X^Utx− (red) MEFs. An IgG antibody control is indicated in grey. Quantitative PCR for the ChIP was performed over a negative control region (an intergenic region) as well as a positive control (HoxB1). Fnbp1 failed to accumulate H3K27me3 in X^Utx− X^Utx− MEFs (t-test p-value = 0.5, N = 4 independent MEF lines per genotype). (F) H3K4me3 ChIP was performed on E12.5 X^Utx+ Y^Uty+ control (green) and X^Utx− X^Utx− (red) MEFs. An IgG antibody control is indicated in grey. Quantitative PCR for the ChIP was performed over a negative control region (intergenic region) as well as a positive control (Npm1). The WT Fnbp1 promoter exhibited significant H3K4me3 accumulation, which was reduced in X^Utx− X^Utx− MEFs (t-test p-value = 0.005, N = 3 independent MEF lines per genotype).