Document Detail

Small-molecule-based inhibition of histone demethylation in cells assessed by quantitative mass spectrometry.
MedLine Citation:
PMID:  20583823     Owner:  NLM     Status:  MEDLINE    
Post-translational modifications on histones are an important mechanism for the regulation of gene expression and are involved in all aspects of cell growth and differentiation, as well as pathological processes including neurodegeneration, autoimmunity, and cancer. A major challenge within the chromatin field is to develop methods for the quantitative analysis of histone modifications. Here we report a mass spectrometry (MS) approach based on ultraperformance liquid chromatography high/low collision switching (UPLC-MS(E)) to monitor histone modifications in cells. This approach is exemplified by the analysis of trimethylated lysine-9 levels in histone H3, following a simple chemical derivatization procedure with d(6)-acetic anhydride. This method was used to study the inhibition of histone demethylases with pyridine-2,4-dicarboxylic acid (PDCA) derivatives in cells. Our results show that the PDCA-dimethyl ester inhibits JMJD2A catalyzed demethylation of lysine-9 on histone H3 in human HEK 293T cells. Demethylase inhibition, as observed by MS analyses, was supported by immunoblotting with modification-specific antibodies. The results demonstrate that PDCA derived small molecules are cell permeable demethylase inhibitors and reveal that quantitative MS is a useful tool for measuring post-translational histone modifications in cells.
Mukram M Mackeen; Holger B Kramer; Kai-Hsuan Chang; Matthew L Coleman; Richard J Hopkinson; Christopher J Schofield; Benedikt M Kessler
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of proteome research     Volume:  9     ISSN:  1535-3907     ISO Abbreviation:  J. Proteome Res.     Publication Date:  2010 Aug 
Date Detail:
Created Date:  2010-08-06     Completed Date:  2010-12-07     Revised Date:  2014-02-24    
Medline Journal Info:
Nlm Unique ID:  101128775     Medline TA:  J Proteome Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4082-92     Citation Subset:  IM    
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MeSH Terms
Cell Line
Chromatography, Liquid / methods
Gene Expression Regulation / physiology*
Histone Demethylases / antagonists & inhibitors
Histones / metabolism*
Jumonji Domain-Containing Histone Demethylases / metabolism
Lysine / metabolism
Mass Spectrometry / methods*
Protein Processing, Post-Translational / physiology*
Proteomics / methods*
Pyridines / pharmacology
Grant Support
084655//Wellcome Trust; G0501068//Medical Research Council; //Biotechnology and Biological Sciences Research Council; //Wellcome Trust
Reg. No./Substance:
0/Histones; 0/Pyridines; 499-80-9/2,4-pyridinedicarboxylic acid; EC 1.14.11.-/Histone Demethylases; EC 1.14.11.-/Jumonji Domain-Containing Histone Demethylases; EC 1.5.-/KDM4A protein, human; K3Z4F929H6/Lysine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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