Functional characterization of histone modifications: survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 102 Bl. Ill., graph. Darst. 30 cm |
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| adam_text | TABLE OF CONTENTS
1. INTRODUCTION 1
1.1 EPIGENETICS AND CHROMATIN 1
1.2 HISTONES AND NUCLEOSOME ASSEMBLY 3
1.3 HISTONE VARIANTS 4
1.4 POSTTRANSLATIONAL MODIFICATIONS OF HISTONES 5
1.4.1 HISTONE METHYLATION 6
1.4.2 HISTONE PHOSPHORYLATION 7
1.5 FUNCTIONS OF HISTONE MODIFICATIONS 8
1.6 NOVEL HISTONE MODIFICATIONS 10
1.6.1 METHYLATION OF HISTONE H4 ASPARTIC ACID 24 11
1.6.2 METHYLATION OF HISTONE H2B PROLINE 1 13
1.6.3 ACID LABILE PHOSPHORYLATION OF HISTONES 15
2. AIMS OF THE STUDY 18
3. RESULTS 19
3.1 METHYLATION OF HISTONE H4
T
AT ASPARTIC ACID 24 (H4D24ME) 19
3.1.1 PRODUCTION AND CHARACTERIZATION OF *-H4D24ME ANTIBODIES 19
3.1.2 H4D24ME IS PRESENT IN SEVERAL CELL LINES AND MOUSE TISSUES 21
3.1.3 PCMT1 METHYLATES H4 AT D24 IN VITRO 22
3.1.4 SIRNA AND SHRNA MEDIATED KNOCKDOWN OF PCMT1 23
3.1.5 H4D24ME IS DEPLETED IN PCMT1 KNOCKOUT MOUSE 24
3.1.6 LOCAL ENRICHMENT OF H4D24ME DETERMINATION BY CHROMATIN
IMMUNOPRECIPITATION 25
3.1.7 GENERATION OF STABLE CELL LINES OVEREXPRESSING PCMT1 27
3.1.8 GENOME-WIDE DISTRIBUTION OF H4D24ME AND PCMT1 BY CHLP-SEQ 28
3.1.9 IDENTIFICATION OF PCMT1 INTERACTION PARTNERS 32
3.1.10 IDENTIFICATION OF H4D24ME READERS 34
3.2 SCREENING OF METHYLTRANSFERASE ACTIVITY TOWARDS HISTONE H2B 36
3.2.1 METTL11A AND METTL9 METHYLATE N-TERMINAL H2B TAIL AND FULL
LENGTH
H2B IN VITRO 37
3.2.2 SIRNA MEDIATED KNOCKDOWN OF METTL9 REDUCES THE LEVELS OF H2BP1 ME2
39
3.3 ACID LABILE PHOSPHORYLATION OF HISTONES 41
3.3.1 PRODUCTION AND CHARACTERIZATION OF SPECIFIC ANTIBODIES 41
3.3.2 ACID LABILITY OF H3R128P AND H4H18P 44
3.3.3 GENOMIC LOCALIZATION OF H3R128P 45
3.3.4 SUBCELLULAR LOCALIZATION OF H3R128P 46
4. DISCUSSION... 48
4.1 METHYLATION OF HISTONE H4 AT ASPARTIC ACID 24 48
4.1.1 H4D24ME SPECIFIC ANTIBODIES 48
4.1.2 PCMT1 METHYLATES H4D24 IN VITRO AND IN VIVO 49
4.1.3 LOCAL AND GENOME-WIDE LOCALIZATION OF H4D24ME/PCMT1 51
4.1.4 POTENTIAL READERS, BINDERS AND INTERACTION PARTNERS 52
4.1.5 PROPOSED MODEL FOR H4D24ME FUNCTION 54
4.2 IDENTIFICATION OF A H2B AT PROLINE 1 METHYLTRANSFERASE 55
4.3 ACID LABILE PHOSPHORYLATIONS OF HISTONES 56
5. SUMMARY 58
6. MATERIALS 60
6.1 PEPTIDES 60
6.2 PRIMERS FOR CLONING AND MUTAGENESIS 60
HTTP://D-NB.INFO/105908029X
6.3 QRT-PCR PRIMERS 61
6.4 CHIP PRIMERS 62
6.5 PLASMIDS 64
6.6 ANTIBODIES 64
6.7 CHEMICALS 65
6.8 BUFFERS 67
6.9 BACTERIAL STRAINS 71
6.10 MAMMALIAN CELL LINES 71
7. METHODS 72
7.1 MOLECULAR BIOLOGY METHODS 72
7.1.1 POLYMERASE CHAIN REACTION 72
7.1.2 PCR MUTAGENESIS 73
7.1.3 AGAROSE GEL ELECTROPHORESIS 73
7.1.4 DIGESTION WITH RESTRICTION ENDONUCLEASES 73
7.1.5 DEPHOSPHORYLATION OF LINEAR DNA 73
7.1.6 LIGATION 74
7.1.7 HEAT SHOCK TRANSFORMATION OF E. COLI 74
7.1.8 PURIFICATION OF PLASMID DNA FROM E.COLI 74
7.1.9 ETHANOL PRECIPITATION OF NUCLEIC ACIDS 74
7.1.10 DNA SEQUENCING 75
7.1.11 QUANTITATIVE PCR 75
7.2 BIOCHEMICAL METHODS 76
7.2.1 AFFINITY PURIFICATION OF RABBIT POLYCLONAL ANTIBODIES 76
7.2.2 WESTERN BLOTTING 76
7.2.3 WESTERN BLOT TRANSFER 77
7.2.4 IMMUNOSTAINING OF WESTERN BLOTS (IMMUNOBLOT) 77
7.2.5 DOT BLOTTING 77
7.2.6 STRIPPING FOR RE-PROBING WESTERN BLOTS 78
7.2.7 SILVER STAINING 78
7.2.8 IMMUNOFLUORESCENCE 78
7.2.9 PURIFICATION OF NATIVE HISTONES BY ACID EXTRACTION 79
7.2.10 EXPRESSION OF RECOMBINANT PROTEINS 80
7.2.11 PURIFICATION OF UN-TAGGED RECOMBINANT HISTONES 80
7.2..12 PURIFICATION OF HIS
6
-TAGGED HISTONE H4 81
7.2.13 PURIFICATION OF HIS
6
-TAGGED PCMT1 82
7.2.14 PURIFICATION OF GST-FUSION PROTEINS 83
7.2.15 HISTONE METHYLTRANSFERASE (HMT) ASSAY 83
7.2.16 HISTONE PHOSPHORYLATION ASSAY 84
7.2.17 NUCLEAR EXTRACT PREPARATION FOR PEPTIDE PULLDOWNS 84
7.2.18 FLAG-LMMUNOPRECIPITATION OF PCMT1 85
7.2.19 NATIVE CHIP 85
7.2.20 CROSSLINKED CHIP 87
7.2.21 CROSSLINKED CHIP FOR SOLEXA SEQUENCING 88
7.3 CELL BIOLOGY METHODS 88
7.3.1 CELL CULTURE 88
7.3.2 GENERATION OF STABLE CELL LINES 88
7.3.3 SIRNA TRANSFECTION OF MAMMALIAN CELLS 89
7.2.4 RNA ISOLATION AND REVERSE TRANSCRIPTION FROM MAMMALIAN CELLS 89
8. ABBREVIATIONS 90
9. ACKNOWLEDGEMENTS 92
10. REFERENCES 93
|
| any_adam_object | 1 |
| author | Biterge, Burcu |
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| ctrlnum | (OCoLC)896772180 (DE-599)DNB105908029X |
| dewey-full | 572.664 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.664 |
| dewey-search | 572.664 |
| dewey-sort | 3572.664 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
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| spelling | Biterge, Burcu Verfasser (DE-588)1058564390 aut Functional characterization of histone modifications survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase Burcu Biterge 2014 102 Bl. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Freiburg (Breisgau), Univ., Diss., 2014 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027603030&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Biterge, Burcu Functional characterization of histone modifications survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase |
| subject_GND | (DE-588)4113937-9 |
| title | Functional characterization of histone modifications survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase |
| title_auth | Functional characterization of histone modifications survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase |
| title_exact_search | Functional characterization of histone modifications survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase |
| title_full | Functional characterization of histone modifications survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase Burcu Biterge |
| title_fullStr | Functional characterization of histone modifications survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase Burcu Biterge |
| title_full_unstemmed | Functional characterization of histone modifications survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase Burcu Biterge |
| title_short | Functional characterization of histone modifications |
| title_sort | functional characterization of histone modifications survey of novel histone modifications reveals pcmt1 as a new histone methyltransferase |
| title_sub | survey of novel histone modifications reveals PCMT1 as a new histone methyltransferase |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027603030&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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