Regulation of MeCP2 induced heterochromatin remodeling
نویسندگان
چکیده
4.1 Molecular biology methods 28 4.1.1 Construction of expression plasmids 28 4.2 Cell biology methods 29 4.2.1 Cell culture and transfection 29 4.2.2 ImmunoFISH 30 4.2.3 Microscopy, image analysis and statistical evaluation 31 4.3 Biochemical methods 32 4.3.1 In vivo protein interaction assays 32 4.3.2 In vitro protein interaction assays 33 4.3.3 Western blot analysis 34 4.3.4 In vitro poly(ADP-ribosyl)ation assay 34 4.3.5 In vitro poly(ADP-ribose) binding analysis 34 5 RESULTS 36 5.1 MeCP2 RTT mutations affect chromatin organization and inhibition of MeCP2 poly(ADP-ribosyl)ation rescues this defect 36 5.1.1 RTT mutations affect MeCP2 binding and clustering of pericentric heterochromatin 36 5.1.2 Heterochromatin binding and clustering properties map to distinct structures of MeCP2 MBD 40 5.1.3 MeCP2 directly interacts with the nuclear enzyme PARP-1 43 5.1.4 MeCP2 is poly(ADP-ribosyl)ated in vivo 44 5.1.5 Poly(ADP-ribosyl)ation of MeCP2 reduces clustering of pericentric heterochromatin 47 5.1.6 Decrease of poly(ADP-ribosyl)ation rescues chromocenter clustering of RTT mutant MeCP2 50 5.2 Direct interactions of MeCP2 and MBD2 involve poly(ADP-ribosyl)ated domains, that also recognize poly(ADP-ribose) 51 5.2.1 MeCP2 and MBD2 – but not MBD1, MBD3 and MBD4-get poly(ADP-ribosyl)ated in vivo 51 CONTENT 2 5.2.2 MeCP2 and MBD2 poly(ADP-ribosyl)ated domains also recognize poly(ADP-ribose) in a noncovalent manner 53 5.2.3 Direct homo-and hetero-interactions between MeCP2 and MBD2 are partially mediated through their poly(ADP-ribosyl)ated and poly(ADP-ribose) recognizing domains 54 6 DISCUSSION 57 6.1 Regulation of MeCP2 induced heterochromatin remodeling 57 6.1.1 Poly(ADP-ribosyl)ation, poly(ADP-ribose) recognition and interactions among MBDs 59 6.1.2 Interaction of MeCP2 with HP1 and heterochromatin association 61 6.1.3 Poly(ADP-ribosyl)ation of MeCP2 has a regulatory effect on MeCP2 mediated large-scale heterochromatin reorganization 61 6.2 MeCP2 poly(ADP-ribosyl)ation as a therapeutic target 64 6.3 Outlook 66
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