The Drosophila homolog of UTX is an H3 lysine 27 demethylase. (A) Schematic representation of the domain organization of Drosophila UTX (CG5640) and its human homologs. Members of the UTX family have a conserved JmjC domain at the C terminus and 5 to 6 tetratricopeptide repeats (TPR) at the N terminus. Human UTX and UTY are X and Y chromosome-linked homologs, respectively, from a pseudoautosomal portion of the sex chromosomes and are therefore highly similar. Since the Y-linked copy of such X-Y pairs generally mutates at a higher rate (13), the X-linked copy, UTX, can be considered more orthologous to the Drosophila homolog. dUTX shares higher overall sequence identity with UTX than with UTY or JMJD3. (B) Expression of recombinant dUTX in a baculovirus system. N-terminally Flag-tagged dUTX was purified from SF21 cells. Asterisks indicate nonspecific polypeptides. (C) Different amounts of recombinant dUTX (1 and 2 μg) were used in histone demethylation assays using bulk histones as substrates, and reaction mixtures were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting with antibodies to various sites of histone methylation. 1m, monomethylated; 2m, dimethylated; 3m, trimethylated. (D) Expression of a mutant version of dUTX (dUTXm) that bears double mutations (H883A and E885A) in the predicted iron-binding sites of the JmjC domain. Recombinant dUTXm was purified as described for panel B. (E) Comparison of H3K27 demethylase activities with 2 μg each of dUTX or dUTXm. Only the wild-type version of dUTX can demethylate H3K27me3, indicating that the observed enzymatic activity is intrinsic to the dUTX polypeptide.

Colocalization of dUTX with the elongating form of RNA polymerase II (A through D), contrasted with the engaged but paused form (E through H). (B) Costaining for the Ser2-phosphorylated CTD of the elongating form of RNA polymerase. (C and G) Immunolocalization of dUTX on polytene chromosomes. (D) Merge of panels B and C, showing colocalization of Ser2-phosphorylated Pol II and dUTX. (F) Costaining for the engaged but paused Ser5-phosphorylated CTD of Pol II. (H) Merge of panels F and G, showing only partial colocalization. (A and E) Corresponding phase-contrast images.

Demethylase activity in vivo. (A) Western blot demonstrating that anti-dUTX antibodies (α-dUTX) recognize full-length dUTX protein. SF21 cells were infected with a dUTX baculovirus and probed with an affinity-purified rabbit polyclonal antibody recognizing dUTX. Two different amounts of total-cell extract from uninfected (−) and dUTX virus-infected (+) cells were loaded onto the gel. (B to G) Antibodies to dUTX were used to demonstrate reductions in dUTX protein levels after RNAi in Drosophila 3rd-instar larvae. Salivary glands from control (B to D) or RNAi (E to G) larvae were immunostained with anti-dUTX antibodies (D and G). HP1 levels for control and dUTX knockdown larvae were comparable (C and F). (B and E) Corresponding phase-contrast images. (H) H3K27 methylation levels are elevated in dUTX knockdown flies. Western blots of increasing amounts of total histone from lid RNAi knockdown (RNAi ON) or control (RNAi OFF) flies were probed with antibodies to H3K27 methylation and total histone H3.

dUTX is recruited to heat shock genes. (A) Chromatin immunoprecipitation from Schneider 2 (Dmal-2) cells before heat shock (no heat shock [NHS]) and after 5 min of heat shock (HS) with dUTX or RNA polymerase (monoclonal antibody 8WG16). Primers 1 kb into the heat shock gene were used to measure dUTX and Pol II recruitment to the heat shock gene. Rp49 levels are shown for comparison. The percentage of the input DNA recovered in the immunoprecipitate is shown along the y axis. Error bars represent ranges from two experiments. (B to E) Colocalization of dUTX on heat shock puffs of Drosophila salivary glands. (B) Phase-contrast image shows puffing at major heat shock loci, indicated by arrows. (C to E) dUTX (C) and the elongating, Ser2-phosphorylated form of Pol II (D) are colocalized (E) on the three heat shock loci indicated in panel B. (F) Two different antibodies to dUTX or a preimmune serum (Pre-Im) was used in an immunoprecipitation assay with a Drosophila nuclear extract. Interaction of Pol II with dUTX was detected with monoclonal antibody 8WG16.