Functional characterization of the Mediator subunit MED25 [Elektronische Ressource] / Lisa Santolin

Lisa Santolin

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 20 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig–Maximilians–Universität München






Functional characterization of the
Mediator subunit MED25














Lisa Santolin
aus
S.Bonifacio, Italien
2006
Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Meisterernst betreut.






Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.



München, am 31.07.2006








Lisa Santolin












Dissertation eingereicht am 31.07.2006

1. Gutachter Prof. Dr. Meisterernst

2. Gutachter Prof. Dr. Jansen

Mündliche Prüfung am 21.11.06
SUMMARY

In this study a structure–function analysis has been employed to analyze transcriptional
regulation through the Mediator subunit MED25. A relationship could be established
between predicted structural domains and functional characteristics of this protein. Most
critically the region responsible for interaction of MED25 with the Mediator was
identified. Immunoprecipitation experiments demonstrated that the so–called VWA
domain (von–Willebrand A domain, amino acids 1–290) is both sufficient and required
for this contact. Site–directed mutagenesis indicates that this binding reaction involves
the non–conserved loop SR2, which is protruding from this domain. Based on the results
of this analysis a model was proposed, in which the primary contact is established by
ionic forces and is further stabilized by hydrophobic interactions.
The previously identified ACID domain was reported to bind to VP16. Targeted
mutagenesis of four different motifs in this region impaired not only transcriptional
activation through MED25 but also led to reduced binding to VP16. In particluar a
lysine–rich motif is also present in two domains of PTOV1, a close homolog of MED25.
Noteworthy, K518 is not conserved in the PTOV1_B domain, which in contrast to
PTOV1_A and the ACID domain of MED25 does not bind to VP16. This led to the
hypothesis that K518 is critically involved in the binding of VP16 to MED25.
Furthermore it could be demonstrated that MED25 contains an intrinsic transcriptional
activation capacity, which is localized in the region 290–715. This indicates additional
recruitment of other factors to promoters through this region. Together with the Mediator
binding VWA–domain and the VP16–interaction domain this region might facilitate
transcriptional activation.
A genome–wide screen showed downregulation of c–Jun and FosB following
overexpression of MED25. Interestingly, expression of GSK3β, a downstream target of
which is cyclin D1, seems to be stimulated by MED25. Together with the finding that
overexpression of MED25 leads to activation of a p21 reporter, this raises the possibility
that MED25 is involved in cell cycle control.
An overlap has been discovered by comparison of MED25 target genes and genes
identified previously as target for the viral activator EBNA2. The close homology
between the activation domains of EBNA2 and VP16 implies a common mechanism of
transcriptional activation by these two viral proteins through MED25. The involvement of
MED25 in gene activation by viral activators might indicate a role for this Mediator
subunit in viral transcription.
This work was contributing to the following publications:

Mittler G., Stühler T., Santolin L., Uhlmann T., Kremmer E., Lottspeich F., Berti L., and
Meisterernst M.
“A novel docking site on Mediator is critical for activation by VP16 in mammalian cells”
The EMBO Journal, 22 (24), 6494-6504, 2003.

Leal A., Huehne K., Bauer F., Sticht H., Berger P., Suter U., Morera B., Saifi M., Lupski
J. R., Ekici A., Pasutto F., Santolin L., Meisterernst M., Reis A., Rautenstrauss B.
“MED25 transcription activator carries an Abelson family SH3 recognition motif altered in
autosomal recessive Charcot–Marie–Tooth disease 2B2” (submitted)






TABLE OF CONTENTS
1. INTRODUCTION..........................................................................................................1
1.1 EUKARYOTIC GENE EXPRESSION: FROM GENES TO PROTEINS........................................1
1.2 EUKARYOTIC TRANSCRIPTION.....................................................................................3
1.2.1 Promoter structure ...........................................................................................3
1.2.2 Chromatin ........................................................................................................6
1.2.2.1 Chromatin modification and chromatin remodelling...................................7
1.2.3 RNA Polymerase II.........................................................................................10
1.2.3.1 The carboxyl–terminal domain (CTD)......................................................11
1.2.4 General transcription factors ..........................................................................12
1.2.5 The Mediator complex....................................................................................16
1.2.5.1 Mammalian Mediator...............................................................................17
1.2.5.2 Mediator–activator interaction..................................................................20
1.2.5.3 Mediator–Pol II interaction.......................................................................22
1.2.6 Activators .......................................................................................................23
1.2.6.1 The viral activator VP16 ..........................................................................24
1.2.6.2 The Epstein–Barr virus nuclear antigen 2 (EBNA2).................................26
1.3 PTOV1..................................................................................................................28
1.4 THE CHARCOT–MARIE–TOOTH (CMT) DISEASE ........................................................29
1.5 ENGINEERING OF THE MOUSE...................................................................................30
1.5.1 The advent of ES cells ...................................................................................30
1.5.2 Traditional gene–targeting .............................................................................31
1.5.3 Site–specific recombinase (SSR) technology ................................................31
1.6 OBJECTIVES ...........................................................................................................35
2 MATERIALS AND METHODS....................................................................................36
2.1 MATERIALS.............................................................................................................36
2.1.1 Chemicals and biochemicals..........................................................................36
2.1.2 Additional material..........................................................................................38
2.1.3 Instruments ....................................................................................................39
2.1.4 General buffers ..............................................................................................40
2.1.5 Enzymes ........................................................................................................43
2.1.6 Antibodies ......................................................................................................44
2.1.7 List of plasmids ..............................................................................................44
2.1.8 List of oligonucleotides...................................................................................47
2.2 MOLECULAR BIOLOGY..............................................................................................51
2.2.1 Cloning...........................................................................................................51
2.2.2 Site directed mutagenesis..............................................................................55
2.2.3 RT–PCR.........................................................................................................56
2.2.4 Microarrays ....................................................................................................57
2.2.5 Dot blot...........................................................................................................59
2.2.6 Genomic DNA extraction................................................................................61
2.2.7 Southern blot..................................................................................................62
2.3 CELL BIOLOGY ........................................................................................................64
2.3.1 Cell lines ........................................................................................................64