Enzymatic and Biochemical Characterization of the JARID1C Jumonji Demethylase 公开
Hu, Ruogu (2011)
Abstract
Human diseases result from epigenetic changes in addition to genetic alterations in the cell. A wide range of cancers and neurological disorders have been linked to aberrant patterns of covalent modifications in chromatin. For example, methylations of DNA and histones affect transcription and are controlled by methyltransferases and demethylases, among which Jumonji demethylases play important roles. Jumonji proteins belong to a superfamily of 2-oxoglutarate (2-OG)-Fe (II)-dependent dioxygenases and reverse histone lysine methylation. The evolutionarily conserved JARID1 (Jumonji AT-Rich Interactive Domain 1) family of Jumonji demethylases catalyzes demethylation of histone H3 lysine 4 (H3K4me2/3, a mark for active transcription). Their JmjC domains bear enzymatic activity, and such substrate specificity is potentially affected by the additional chromatin-reader domains (such as Plant Homeodomain, or PHD), representing a common mechanism in regulation of chromatin modifiers. In this study, I biochemically characterized the enzymatic activity of JARID1C on H3K4 methylation with different histone peptides, and explored the binding specificity of the two JARID1C PHD domains. An N-terminal JARID1C recombinant fragment was active on synthetic histone H3K4me2/3 peptides. The end product of demethylation is mono-methylated H3K4. This activity was affected by peptide length, yet independent of the methylation status of H3K9. Additionally, I measured the Michaelis-Menton kinetics of JARID1C demethylation in a formaldehyde release assay and showed that the R and S form of a 2-OG analogue, 2-hydroxyglutarate, could inhibit JARID1C activity with similar IC50 values (0.8 and 0.7 mM, respectively). Using bacterially expressed JARID1C PHD domains in pull-down and isotherm titration calorimetry assays, I showed that PHD1 domain preferentially associates with unmodified H3K4 peptide; but such binding is very weak compared to other known interactions between PHD domains and histone peptides. Similar results were obtained for PHD2. These results revealed the properties of JARID1C catalysis and interaction with histones. JAIRD1C is implicated in X-linked mental retardation and cancers. This work provides a biochemical perspective of the protein which complements current in vivo data in the literature. In the long run, it will help to elucidate mechanisms and roles of histone demethylation in human diseases, potentially leading to development of novel epigenetic targets for therapeutics.
Table of Contents
Table of Contents
Chapter I: Background &
Introduction……………………………………………………………………………………………Page
1
1.1 Epigenetic changes in human diseases and methylation in the
chromatin…………………………...Page
1
1.2 Histone demethylation and modes of transcriptional
control…………………………………………………Page
2
1.3 The JARID1 family of histone demethylases and JARID1C domains
and function…………………Page
6
1.4
Significance……………………………………………………………………………………………………………………………Page
9
Chapter II: Enzymatic Properties of
JARID1C…………………………………………………………………………………Page
10
2.1
Introduction………………………………………………………………………………………………………………………………Page
10
2.2
Results………………………………………………………………………………………………………………………………………Page
10
2.3
Discussion…………………………………………………………………………………………………………………………………Page
22
2.4 Materials &
Methods…………………………………………………………………………………………………………………Page
23
Chapter III: Binding Properties of JARID1C PHD
domains………………………………………………………………Page
34
3.1
Introduction……………………………………………………………………………………………………………………………Page
34
3.2
Results…………………………………………………………………………………………………………………………………Page
38
3.3
Discussion……………………………………………………………………………………………………………………………Page
43
3.4 Materials &
Methods…………………………………………………………………………………………………………………Page
43
Chapter IV: Crystallization Trials and Activity Screens of a
Jumonji Protein
MINA53…………………………Page
47
4.1
Introduction……………………………………………………………………………………………………………………………………Page
47
4.2
Results……………………………………………………………………………………………………………………………………………Page
48
4.3
Discussion………………………………………………………………………………………………………………………………………Page
58
4.4 Materials &
Methods………………………………………………………………………………………………………………………Page
58
5.3 JARID1 demethylase as potential drug targets…………………………………………….............................Page 64 Appendix……………………………………………...................................................................................Page 66 Summary of all JARID1C and other constructs generated…………………………….................................Page 66 References………………………………………………………………………………………………......................................Page 68
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