Molekulare Funktion und Regulation des negativen Cofaktors 2, NC2 [Elektronische Ressource] / Elisa Piaia

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
Molekulare Funktion und Regulation
des negativen Cofaktors 2, NC2
Elisa Piaia
aus
Montebelluna, Italien
2005Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von PD Dr. Michael Meisterernst betreut.
Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.
München, 23.11.2004
Elisa Piaia
Dissertation eingereicht am: 23.11.2004
1. Gutachter: PD Dr. Michael Meisterernst
2. Gutachter: Prof. Dr. Patrick Cramer
Mündliche Prüfung am: 15.03.2005Molecular function and regulation
of the negative cofactor 2, NC2 a LinoTABLE OF CONTENTS
SUMMARY ..........................................................................................................1
I. INTRODUCTION ..............................................................................................2
1. Eukaryotic gene expression: from gene sequence to active protein ................................................... 2
2. Promoter structure in eukaryotic class II genes ................................................................................... 3
2.1. The core promoter elements .............................................................................................................. 3
2.1.1. TATA-box .................................................................................................................................. 3
2.1.2. Initiator Elements ...................................................................................................................... 4
2.1.3. DPE ........................................................................................................................................... 4
2.2. Regulatory sequences ........................................................................................................................ 4
2.2.1. Enhancer and Silencer elements ................................................................................................ 4
2.2.2. Insulator ..................................................................................................................................... 5
3. The preinitiation complex: the general transcription factors and the RNA Polymerase II ............. 5
3.1. The general transcription factors (GTFs) .......................................................................................... 5
3.1.1. TBP ............................................................................................................................................ 6
3.1.1.1. TBP paralogues .................................................................................................................. 6
3.1.2. TFIIB ......................................................................................................................................... 7
3.1.3. TFIIF 7
3.1.4. TFIIE 7
3.1.5. TFIIH 8
3.2. RNA Polymerase II ........................................................................................................................... 8
3.2.1. CTD ........................................................................................................................................... 9
3.2.2. The transcription cycle .............................................................................................................. 9
3.2.3. The holoenzyme ...................................................................................................................... 10
4. Transcriptional activators and repressors .......................................................................................... 11
5. Transcriptional cofactors/coregulators ............................................................................................... 11
5.1. TAFs: TBP associated factors ......................................................................................................... 12
5.1.1. TAF paralogues ....................................................................................................................... 12
5.2. TFIIA ............................................................................................................................................... 13
5.3. The Mediator ................................................................................................................................... 13
5.4. USA factors (upstream stimulatory activity) .................................................................................. 14
5.5. BTAF1 ............................................................................................................................................. 15
6. Chromatin structure ............................................................................................................................. 15
6.1. Histone modifications and histone modifying complexes .............................................................. 166.1.1. Acetylation and HATs/HDACs ................................................................................................ 17
6.1.2. Phosphorylation ....................................................................................................................... 17
6.1.3. Methylation and histone methyltransferases (HMTases) ........................................................ 17
6.1.4. Additional histones modifications ........................................................................................... 18
6.2. Non covalent chromatin modification: ATP-dependent Chromatin remodeling complexes ........... 18
7. The negative cofactor 2, NC2 ............................................................................................................... 19
7.1. NC2 is a general transcriptional repressor ...................................................................................... 22
7.2. Interplay between negative and positive effectors .......................................................................... 22
7.3. Positive role of NC2 in transcriptional regulation 23
8. Post-translational protein modifications: the role of phosphorylation in the regulation of
transcription factors ................................................................................................................................. 24
9. Silencing of transcription during mitosis ............................................................................................ 27
10. Protein transport to the nucleus ........................................................................................................ 31
10.1. The nuclear pore complex (NPC) ................................................................................................. 31
10.2. The nuclear localization signal (NLS) .......................................................................................... 32
II. RESULTS ......................................................................................................34
1. Localization studies of NC2.................................................................................................................. 34
1.1. Subcellular distribution of NC2 34
1.1.1. Localization of NC2 α ............................................................................................................. 34
1.1.2. The nucleolar localization of the endogenous NC2 α is restricted to the G1 phase ................ 36
1.1.3. Localization of NC2 β .............................................................................................................. 37
1.2. Import and dimerisation of NC2 ..................................................................................................... 37
1.2.1. Both NC2 α and NC2 β contain an NLS .................................................................................. 37
1.2.2. Is NC2 transported into the nucleus as a dimer or as single subunits? ................................... 40
1.3. Characterization of the NLS ........................................................................................................... 41
1.3.1. NC2 α has a bipartite NLS ....................................................................................................... 41
1.3.2. Mutation of threonine 23 in aspartate abolishes nulceolar localization .................................. 43
1.3.3. NC2 β has a single motif NLS ................................................................................................. 43
1.3.4. Only the first motif of the NC2 α-NLS is important for import of the dimer .......................... 45
1.3.5. Mutation of the NC2 β-NLS affect import of the dimer .......................................................... 45
2. Post-translational modification of NC2: NC2 α is specifically hyperphosphorylated during mitosis 47
2.1. NC2 is neither acetylated nor methylated ....................................................................................... 47
2.2. Monitoring of NC2 level throughout the cell cycle: a new isoform of NC2 α appears during mitosis
(M-NC2 α) .............................................................................................................................................. 49
2.3. The new M-NC2 α form is specific of the mitotic stage ..............................