Development of biochemical tools to characterise human H3K27 histone demethylase JmjD3
<p>Covalent modifications of histone tails play essential roles in mediating chromatin structure and epigenetic regulation. JmjD3 is a JumonjiC domain containing histone demethylase, belongs to the KDM6 subfamily, and catalyses the removal of methyl groups on methylated lysine 27 on histone 3...
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| Format: | Thesis |
| Language: | English |
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2013
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| _version_ | 1826312495431680000 |
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| author | Che, KH |
| author2 | Oppermann, U |
| author_facet | Oppermann, U Che, KH |
| author_sort | Che, KH |
| collection | OXFORD |
| description | <p>Covalent modifications of histone tails play essential roles in mediating chromatin structure and epigenetic regulation. JmjD3 is a JumonjiC domain containing histone demethylase, belongs to the KDM6 subfamily, and catalyses the removal of methyl groups on methylated lysine 27 on histone 3 (H3K27), a critical mark to promote polycomb mediated repression and gene silencing. The importance of JmjD3 has been implicated in development, cancer biology and immunology.</p> <p>In this thesis, I report the recombinant production of active human JmjD3, development of two in vitro screening assays, a cell-based assay, and structural determination of JmjD3 in complex with the inhibitor 8-hydroxy-5-carboxyquinoline (8HQ). A highly selective and potent small molecule inhibitor GSK-J1 was subsequently identified. The inhibitor is active in HeLa cells and promotes a dose-dependent increase of global H3K27 methylation.</p> <p>The inhibitor GSK-J1 was used in two different cell assay systems related to inflammation and differentiation, to understand how H3K27 demethylation controls cellular functions.</p> <p>By inhibiting H3K27me3 demethylation, it is demonstrated that tumor necrosis factor (TNF) and other pro-inflammatory cytokines are regulated by H3K27 demethylase inhibition in M1- type macrophages derived from healthy volunteers and rheumatoid arthritis patients. It is also shown that inhibition of H3K27me3 demethylation abrogates cellular fusion of M2- type macrophages.</p> <p>During RANKL induced osteoclast differentiation, JmjD3 is up-regulated and promotes the expression of the key transcription factor NFATc1. By inhibiting JmjD3, NFATc1 expression is reduced and osteoclastogenesis is inhibited. This mechanism demonstrates a novel anti-resorptive principle of potential utility in conditions of excess bone resorption such as osteoporosis, bone erosion in inflammatory arthritis or cancer of the bone.</p> <p>These experiments further resolve the ambiguity between scaffold and catalytic function associ- ated with the H3K27 demethylase in these biological systems, and demonstrate that its enzymatic activity is crucial for epigenetic regulation of macrophage and osteoclast function.</p> |
| first_indexed | 2024-03-07T05:06:58Z |
| format | Thesis |
| id | oxford-uuid:da42028f-fe7c-4b9e-b3af-d103ae8b9668 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-04-09T03:55:25Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | oxford-uuid:da42028f-fe7c-4b9e-b3af-d103ae8b96682024-03-11T12:55:52ZDevelopment of biochemical tools to characterise human H3K27 histone demethylase JmjD3Thesishttp://purl.org/coar/resource_type/c_db06uuid:da42028f-fe7c-4b9e-b3af-d103ae8b9668Biology (medical sciences)PharmacologyBiochemistryImmunologyEnzymesHigh-Throughput ScreeningCrystallographyOrthopaedicsRheumotologyGenetics (life sciences)Protein chemistryCell Biology (see also Plant sciences)Chemical biologyStructural genomicsEnglishOxford University Research Archive - Valet2013Che, KHOppermann, UYue, W<p>Covalent modifications of histone tails play essential roles in mediating chromatin structure and epigenetic regulation. JmjD3 is a JumonjiC domain containing histone demethylase, belongs to the KDM6 subfamily, and catalyses the removal of methyl groups on methylated lysine 27 on histone 3 (H3K27), a critical mark to promote polycomb mediated repression and gene silencing. The importance of JmjD3 has been implicated in development, cancer biology and immunology.</p> <p>In this thesis, I report the recombinant production of active human JmjD3, development of two in vitro screening assays, a cell-based assay, and structural determination of JmjD3 in complex with the inhibitor 8-hydroxy-5-carboxyquinoline (8HQ). A highly selective and potent small molecule inhibitor GSK-J1 was subsequently identified. The inhibitor is active in HeLa cells and promotes a dose-dependent increase of global H3K27 methylation.</p> <p>The inhibitor GSK-J1 was used in two different cell assay systems related to inflammation and differentiation, to understand how H3K27 demethylation controls cellular functions.</p> <p>By inhibiting H3K27me3 demethylation, it is demonstrated that tumor necrosis factor (TNF) and other pro-inflammatory cytokines are regulated by H3K27 demethylase inhibition in M1- type macrophages derived from healthy volunteers and rheumatoid arthritis patients. It is also shown that inhibition of H3K27me3 demethylation abrogates cellular fusion of M2- type macrophages.</p> <p>During RANKL induced osteoclast differentiation, JmjD3 is up-regulated and promotes the expression of the key transcription factor NFATc1. By inhibiting JmjD3, NFATc1 expression is reduced and osteoclastogenesis is inhibited. This mechanism demonstrates a novel anti-resorptive principle of potential utility in conditions of excess bone resorption such as osteoporosis, bone erosion in inflammatory arthritis or cancer of the bone.</p> <p>These experiments further resolve the ambiguity between scaffold and catalytic function associ- ated with the H3K27 demethylase in these biological systems, and demonstrate that its enzymatic activity is crucial for epigenetic regulation of macrophage and osteoclast function.</p> |
| spellingShingle | Biology (medical sciences) Pharmacology Biochemistry Immunology Enzymes High-Throughput Screening Crystallography Orthopaedics Rheumotology Genetics (life sciences) Protein chemistry Cell Biology (see also Plant sciences) Chemical biology Structural genomics Che, KH Development of biochemical tools to characterise human H3K27 histone demethylase JmjD3 |
| title | Development of biochemical tools to characterise human H3K27 histone demethylase JmjD3 |
| title_full | Development of biochemical tools to characterise human H3K27 histone demethylase JmjD3 |
| title_fullStr | Development of biochemical tools to characterise human H3K27 histone demethylase JmjD3 |
| title_full_unstemmed | Development of biochemical tools to characterise human H3K27 histone demethylase JmjD3 |
| title_short | Development of biochemical tools to characterise human H3K27 histone demethylase JmjD3 |
| title_sort | development of biochemical tools to characterise human h3k27 histone demethylase jmjd3 |
| topic | Biology (medical sciences) Pharmacology Biochemistry Immunology Enzymes High-Throughput Screening Crystallography Orthopaedics Rheumotology Genetics (life sciences) Protein chemistry Cell Biology (see also Plant sciences) Chemical biology Structural genomics |
| work_keys_str_mv | AT chekh developmentofbiochemicaltoolstocharacterisehumanh3k27histonedemethylasejmjd3 |