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How substrate specificity is imposed on a histone demethylase--lessons from KDM2A.

ABSTRACT: Histone lysine methylation and demethylation regulate histone methylation dynamics, which impacts chromatin structure and function. To read and erase the methylated histone residues, lysine demethylases must specifically recognize the histone sequences and methylated sites and discriminate the degree of these methylations. In this issue of Genes & Development, Cheng and colleagues (pp. 1758-1771) determine a crystal structure of histone lysine demethylase KDM2A that specifically targets lower degrees of H3K36 methylation. The results reveal the structural basis for H3K36 substrate specificity and suggest mechanisms of Lys36 demethylation. This KDM2A-H3K36 complex structure, coupled with functional studies, provides needed insight into the process and regulation of histone demethylation.

SUBMITTER: Tsai CL

PROVIDER: S-EPMC4197959 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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How substrate specificity is imposed on a histone demethylase--lessons from KDM2A.

Tsai Chi-Lin CL Shi Yang Y Tainer John A JA

Genes & development 20140801 16

Histone lysine methylation and demethylation regulate histone methylation dynamics, which impacts chromatin structure and function. To read and erase the methylated histone residues, lysine demethylases must specifically recognize the histone sequences and methylated sites and discriminate the degree of these methylations. In this issue of Genes & Development, Cheng and colleagues (pp. 1758-1771) determine a crystal structure of histone lysine demethylase KDM2A that specifically targets lower de ...[more]

PMID: 25128493

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Project description:BackgroundHistone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active 'probe' molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells.ResultsA panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition.ConclusionsHigh-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.

Project description:The methylation status of histones plays a crucial role in many cellular processes, including follicular and oocyte development. Lysine-specific demethylase 2a (KDM2a) has been reported to be closely associated with gametogenesis and reproductive performance, but the specific function and regulatory mechanism have been poorly characterized in vivo. We found KDM2a to be highly expressed in growing follicles and oocytes of mice in this study. To elucidate the physiological role of Kdm2a, the zona pellucida 3-Cre (Zp3-Cre)/LoxP system was used to generate an oocyte Kdm2a conditional knockout (Zp3-Cre; Kdm2aflox/flox, termed Kdm2a cKO) model. Our results showed that the number of pups was reduced by approximately 50% in adult Kdm2a cKO female mice mating with wildtype males than that of the control (Kdm2aflox/flox) group. To analyze the potential causes, the ovaries of Kdm2a cKO mice were subjected to histological examination, and results indicated an obvious difference in follicular development between Kdm2a cKO and control female mice and partial arrest at the primary antral follicle stage. The GVBD and matured rates of oocytes were also compromised after conditional knockout Kdm2a, and the morphological abnormal oocytes increased. Furthermore, the level of 17β-estradiol of Kdm2a cKO mice was only 60% of that in the counterparts, and hormone sensitivity decreased as the total number of ovulated and matured oocytes decreased after superovulation. After deletion of Kdm2a, the patterns of H3K36me2/3 in GVBD-stage oocytes were remarkedly changed. Transcriptome sequencing showed that the mRNA expression profiles in Kdm2a cKO oocytes were significantly different, and numerous differentially expressed genes were involved in pathways regulating follicular and oocyte development. Taken together, these results indicated that the oocyte-specific knockout Kdm2a gene led to female subfertility, suggesting the crucial role of Kdm2a in epigenetic modification and follicular and oocyte development.

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How substrate specificity is imposed on a histone demethylase--lessons from KDM2A.

Publications

How substrate specificity is imposed on a histone demethylase--lessons from KDM2A.

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