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Modulation of lysine methylation in myocyte enhancer factor 2 during skeletal muscle cell differentiation

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dc.contributor.advisor윤홍덕-
dc.contributor.author최진미-
dc.date.accessioned2017-07-14T01:42:01Z-
dc.date.available2017-07-14T01:42:01Z-
dc.date.issued2014-02-
dc.identifier.other000000016735-
dc.identifier.urihttps://hdl.handle.net/10371/122257-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 의과학과, 2014. 2. 윤홍덕.-
dc.description.abstractMyocyte enhancer factor 2 (MEF2) is a family of transcription factors that regulates many processes, including muscle differentiation. Due to its many target genes, MEF2D requires tight regulation of transcription activity over time and by location. Epigenetic modifiers have been suggested to regulate MEF2-dependent transcription via modifications of histones and MEF2. However, the modulation of MEF2 activity by lysine methylation, an important posttranslational modification that alters the activities of transcription factors, has not been studied. We report the reversible lysine methylation of MEF2D by G9a and LSD1 as a regulatory mechanism of MEF2D activity and skeletal muscle differentiation. G9a methylates lysine-267 of MEF2D and represses its transcriptional activity, but LSD1 counteracts it. This residue is highly conserved between MEF2 members in mammals. The methylation status of MEF2D modulates chromatin binding affinity. During myogenic differentiation of C2C12 mouse skeletal muscle cells, the G9a-mediated methylation of MEF2D decreased. Concordantly, MEF2D-dependent myogenic genes were upregulated. The methylation defective mutant of MEF2D readily activated myogenin gene transcription. Thus, we have identified lysine-267 as a methylation/demethylation site and demonstrate that the lysine methylation state of MEF2D regulates its transcriptional activity and skeletal muscle cell differentiation.-
dc.description.tableofcontentsAbstract i
Contents iii
List of figures v
List of tables x
List of Abbereviations xi

I. Introduction 1
1-1. Posttranslational modification (PTM) 2
1-2. Skeletal muscle differentiation 6
1-3. Myocyte enhancer factor 2 (MEF2) 8

II. Material and Methods 12
2-1. Cell culture and transient expression 13
2-2. DNA constructs and site-directed mutagenesis 13
2-3. Antibodies and reagents 14
2-4. Immunoprecipitation and reporter gene assay 15
2-5. Retroviral infection 15
2-6. Lentivirus production 15
2-7. Immunofluorescence 16
2-8. In vitro methylation and demethylation assay 16
2-9. ESI-LC-MS analysis 17
2-10. Quantitative real-time PCR and chromatin immunoprecipitation assay 17
2-11. Statistical analysis 18

III. Results 20
3-1. MEF2D is methylated at K267 21
3-2. MEF2D is methylated by G9a 31
3-3. MEF2D interacts with G9a 33
3-4. MEF2D is demethylated by LSD1 48
3-5. G9a inhibits MEF2D transcriptional activity by regulating its recruitment to chromatin 53
3-6. Knockdown of G9a enhances MEF2D-dependent transcription 64
3-7. Overexpression of G9a attenuates MEF2D transcriptional activity 75

IV. Discussion 84

V. References 90

VI. Abstract in Korean 103
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dc.formatapplication/pdf-
dc.format.extent2936627 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectMEF2-
dc.subjectG9a-
dc.subjectmethylation-
dc.subjectC2C12-
dc.subjectmuscle cell differentiation-
dc.subject.ddc610-
dc.titleModulation of lysine methylation in myocyte enhancer factor 2 during skeletal muscle cell differentiation-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pagesxi, 105-
dc.contributor.affiliation의과대학 의과학과-
dc.date.awarded2014-02-
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