Volume 125, Issue 3, 5 May 2006, Pages 483-495 https://doi.org/10.1016/j.cell.2006.03.027Get rights and content
Product Overview: Methylation of histone H3 at lysine 9 (K9) mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing expression at euchromatic sites. H3(K9) demethylation is regulated by at least two classes of demethylases, JMJD1 (which can remove mono- and di-methylation from H3(K9)) and JMJD2 (which can remove trimethylation from H3(K9)). EpiSeeker Histone H3 (K9) Demethylase Activity Quantification Assay Kit (ab113458) allows the user to measure activity/inhibition of H3(K9) specific histone demethylases using cell/tissue extracts. The kit is ready-to-use and provides all the essential components needed for measuring histone demethylase H3(K9) specific activity/inhibition from a broad range of species including mammalian cells/tissues, plants, and bacteria. Display options Format AbstractPubMedPMID
The hairless (HR) protein contains a Jumonji C (JmjC) domain that is conserved among a family of proteins with histone demethylase (HDM) activity. To test whether HR possesses HDM activity, we performed a series of in vitro demethylation assays, which demonstrated that HR can demethylate monomethylated or dimethylated histone H3 lysine 9 (H3K9me1 or me2). Moreover, ectopic expression of wild-type HR, but not JmjC-mutant HR, led to pronounced demethylation of H3K9 in cultured human HeLa cells. We also show that two missense mutations in HR, which we and others described in patients with atrichia with papular lesions, abolished the demethylase activity of HR, demonstrating the role of HR demethylase activity in human disease. By ChIP-Seq analysis, we identified multiple new HR target genes, many of which play important roles in epidermal development, neural function, and transcriptional regulation, consistent with the predicted biological functions of HR. Our findings demonstrate for the first time that HR is a H3K9 demethylase that regulates epidermal homeostasis via direct control of its target genes. Keywords: ChIP-Seq; epigenetics.
Figure 1. HR is a JmjC demethylase.… Figure 1. HR is a JmjC demethylase. A ) Schematic depiction of major functional domains… Figure 1.HR is a JmjC demethylase. A) Schematic depiction of major functional domains of the human HR protein, including a JmjC domain in its C terminus. Two APL patient mutations (D1012N and V1056M) are also indicated. B) Left panel: homology model of the HR protein JmjC domain structure, produced on the basis of homology to JmjC domains of known structure, revealed that the putative active site C-E-H residues are close in space, likely forming an atypical metal-binding pocket (indicated by asterisk). Right panel: aa 1012 and 1056 are indicated on the close-up view of the JmjC domain. C) In vitro demethylation assay using recombinant partial HR protein containing the JmjC domain and premethylated histone H3 peptides: H3K9me1, H3K9me2, H3K9me3, and H3K4me3. D) In vitro demethylation assay using recombinant partial HR and native histone proteins purified from HeLa cells. E) Representative Western blot analysis results illustrating the demethylase activity of HR and JMJD1A. Iron concentration is 50 μM for JMJD1A and 100 μM for HR. Reaction without enzyme input was included as a negative control. F) Graphic illustration of the relative HDM activities of HR and JMJD1A on H3K9me1 at 3 different amounts (10, 20, and 30 μg) of enzyme input. The y axis depicts the relative level of H3K9me1 (determined by the ratio of the intensity between H3K9me1 and total H3), as compared to the no enzyme input control sample (which was assigned a relative value of 1). Results from 5 repeats for each demethylation experiment were used for statistical analyses by t test to determine the significance of differences between different experimental conditions. *P < 0.05, **P < 0.01.
Figure 2. Demethylation activity of HR in… Figure 2. Demethylation activity of HR in human cells. A ) Detection of demethylase activity… Figure 2.Demethylation activity of HR in human cells. A) Detection of demethylase activity by wild-type HR (pCMV-Flag-HR) or mutant HR (pCMV-Flag-HRmutated, with mutations in the putative metal-binding motif in the JmjC domain) in cultured human HeLa cells by immunofluorescence staining and colocalization of HR (green) and H3K9me1 (red). White arrowheads and circles highlight cells overexpressing wild-type HR with reduced genomic H3K9me1. Yellow arrowheads and circles highlight cells overexpressing mutant HR and normal genomic H3K9me1. B) HR-expressing dermal papilla (DP) cells in human anagen hair follicle (HF) display reduced levels of genomic H3K9me1. a–c) HR expression detected by immunofluorescence staining with DAPI (blue; a), anti-HR (green; b), and merged image (c) in HF. e–g) H3K9me1 methylation detected by immunofluorescence staining with DAPI (blue; e), anti- H3K9me1 (green; f), and merged image (g) in human HF. Dotted white line separates DP cells from surrounding matrix cells. d, h) Graphic illustration of the relative intensity of HR (d) and H3K9me1 (h) staining (green) vs. DAPI staining (blue) from a representative population of DP and matrix cells (indicated by the yellow dotted arrow in a and e) for each antibody. DP cell population is highlighted by a dotted yellow border. C) H3K9me1 demethylation by wild-type HR and patient-specific mutant HR found in patients with APL (D1012N and V1056M). Total histones from HeLa cells were used as the demethylation substrate. *P < 0.05.
Figure 3. Demethylation targets of HR in… Figure 3. Demethylation targets of HR in human cells. A ) Comparison of H3K9me1 and… Figure 3.Demethylation targets of HR in human cells. A) Comparison of H3K9me1 and H3K9me2 levels in the promoter or TREs of Wise, Mxi1, and Caspase14 between HR-expressing and nonexpressing HEK293 cells by ChIP assays. For each gene, 4 different fragments (as schematically depicted on each gene map) were assessed by ChIP PCR. Intensity of each PCR product correlates positively with the level of H3K9 methylation in each fragment assessed by ChIP PCR. B) Effect of ectopic HR expression on its target gene regulation in HEK293 cells and NHKs, respectively. C, empty-vector transfected cells; HR, HR-transfected cells. *P < 0.05, **P < 0.01.
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Histone h3 k9 demethylase activity assay
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