#Finetunes distribution software

The point mutation in the JmjC domain is indicated with an asterisk. The PHD domain and JmjC domain are indicated. (B) Schematic representation of different Jhd1 mutants. *, conserved Fe(II) binding site #, α-KG binding site $, Tyr315. (A) Sequence alignment of the JmjC domain of Jhd1 with that from several known human histone demethylases.

The JmjC domain and its adjacent sequences are important for Jhd1 enzymatic activity. NS, two nonspecific cross-reactive bands. Overexpression of JHD1 was confirmed by anti-Flag antibodies (top panel), and deletion of JHD1 was confirmed by PCR (data not shown). Relative methylation levels are indicated above each blot.

#Finetunes distribution software

Changes in methylation levels were quantified with NIH ImageJ software and normalized with histone H3. Equal amount of whole-cell lysates from wild-type, JHD1 overexpression, or jhd1Δ strains were analyzed by Western blotting using the various antibodies indicated. (F) Overexpression of Jhd1 results in a subtle decrease of H3K36me2 and me3 methylation levels, but the jhd1 deletion does not. Results presented are averages of three independent experiments with standard deviations.

Demethylase assays were performed under the same conditions as for panel D.

(E) Effects of cofactors Fe(II), α-ketoglutarate (α-keto), and ascorbate on the enzymatic activity of Jhd1. Demethylation activities are presented as averages of three independent experiments with standard deviations. About 2 μg (∼24 pmol) of recombinant GST-Jhd1 protein was incubated with 4 μg (∼37 pmol, based on the molecular weight of histone octamer) of radioactively labeled H3K36me2 substrates (100,000 cpm total) at 37☌ for 1 h and analyzed in formaldehyde release assays. (D) In vitro histone demethylase activities of recombinant GST-Jhd1 toward different radioactive histone substrates. (C) In vitro radioactive formaldehyde release assays using immunoprecipitates analyzed in panel B and substrates that are methylated on H3K4me1, H3K36me2, or H3K79me, using GST-SET7, CBP-Set2-Flag, and GST-hDOT1L, respectively. Input (In), flowthrough (Ft), and immunoprecipitates (IP) were fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting using an anti-Flag antibody. (B) Western blot analysis of immunoprecipitates from five Flag-tagged JmjC domain-containing proteins using whole-cell extracts. (A) Schematic representation of the five JmjC domain-containing proteins in S. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units. Finally, chromatin immunoprecipitation-coupled microarray studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and alpha-ketoglutarate as cofactors. Here we report further characterization of Jhd1. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in Saccharomyces cerevisiae, is an H3K36-specific demethylase. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Steady-state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Histone methylation plays important roles in the regulation of chromatin dynamics and transcription.