Regulation of the potential tumorsuppressor gene mad1 by G-CSF [Elektronische Ressource] / vorgelegt von Kan Jiang

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2006
• Nombre de lectures: 42
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Regulation of the potential tumorsuppressor gene mad1 by G-CSF


Von der Fakultät für Mathematik, Informatik und Naturwissenschafen
der Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften
genehmigte Dissertation


vorgelegt von


Kan Jiang
Master of Science
aus Hubei, VR China

Berichter: Universitätprofessor Dr. Bernhard Lüscher
Privatdozent Dr. Christoph Peterhänsel
Tag der mündlichen Prüfung: 14. 03. 2006
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar Contents I
Contents........................................................................... ...........................................I
Abstract.......................................................................................................................VI
1 Introduction.......................................................................................................... 1
1.1 Myc/Max/Mad network ................................................................................. 1
1.1.1 Myc proteins.......................................................................................... 1
1.1.2 Mad proteins ......................................................................................... 4
1.1.3 Max proteins and others........................................................................ 5
1.2 Structure, expression, and functions of Mad1 protein .................................. 7
1.2.1 Structure of Mad1 protein...................................................................... 7
1.2.2 Expression of Mad1 protein................................................................... 7
1.2.3 Functions of Mad1................................................................................. 8
1.3 Targets and partners of Mad1 .................................................................... 10
1.4 The G-CSF, G-CSFR and signal transduction pathways ........................... 13
1.4.1 G-CSF and G-CSFR ........................................................................... 13
1.4.2 Signaling pathways activated by G-CSF ............................................. 15
1.4.3 Negative regulation of the G-CSF signaling ........................................ 17
1.5 Transcription factors in granulopoiesis....................................................... 18
1.5.1 C/EBPs................................................................................................ 18
1.5.2 Sp proteins.......................................................................................... 21
1.5.3 STATs ................................................................................................. 23
1.5.4 PU.1 .................................................................................................... 25
1.5.5 c-Myb .................................................................................................. 26
1.6 Aim of the study.......................................................................................... 27
2 Results .............................................................................................................. 28
2.1 mad1 expression in different cell lines in response to G-CSF and to TPA 28
2.2 Cis-elements involved in the regulation of the mad1 promoter................... 29
2.2.1 Identification of the mad1 promoter and regulatory regions ................ 31
2.2.2 The activation of mad1 promoter in response to G-CSF is G-CSFR-
dependent ......................................................................................................... 35 Contents II
2.2.3 Role of C/EBP binding sites within the G-CSF-response element ...... 36
2.2.4 Mutation of GC-boxes reduces the mad1 promoter activity but shows
little effect on G-CSF/G-CSFR induction ........................................................... 38
2.2.5 Cooperativity between CCAAT- and GC-boxes .................................. 40
2.3 Transcription factors involved in the regulation of the mad1 gene.............. 42
2.3.1 C/EBP transcription factors activate mad1 reporter genes.................. 42
2.3.2 Involvement of Sp1 protein in the repression of mad1 expression...... 44
2.3.3 Involvement of STAT3 protein in the activation of the mad1 promoter 45
2.4 Transcription factors binding to the endogenous mad1 promoter in response
to G-CSF............................................................................................................... 46
2.4.1 C/EBP transcription factors are recruited to the human mad1 promoter
47
2.4.2 Sp transcription factors interact with the endogenous human mad1
promoter............................................................................................................ 49
2.5 G-CSF/GCSFR signal transduction pathway relevant for mad1 expression50
2.5.1 Structure-function analysis of the G-CSFR: tyrosine residues relevant
for the activity of the mad1 promoter ................................................................. 51
2.5.2 Involvement of the MAPK signal transduction pathways for mad1
promoter activation............................................................................................ 53
2.5.3 STAT3 is activated in response to G-CSF........................................... 54
2.5.4 G-CSF/G-CSFR-activated STAT3 can stimulate gene transcription
through interaction with C/EBPβ........................................................................ 56
3 Discussion ......................................................................................................... 58
3.1 Selection and establishment of a human mad1 expression system ........... 58
3.2 Regulation of the human mad1 promoter at the transcriptional level.......... 60
3.2.1 Identification and characterization of the mad1 promoter.................... 60
3.2.2 Regulation of the activity of the mad1 promoter in response to G-CSF
by C/EBP proteins through functional C/EBP binding sites ............................... 62
3.2.3 Repression of mad1 promoter activity in response to G-CSF by Sp
proteins through the functional SP binding site ................................................. 66 Contents III
3.2.4 Cooperative activation of the mad1 promoter by C/EBP and Sp proteins
through their functional binding sites ................................................................. 68
3.3 G-CSF/G-CSFR signaling transduction pathways relevant for mad1
expression............................................................................................................. 69
3.4 Mechanisms of induction of the mad1 gene ............................................... 72
4 Materials and methods ...................................................................................... 76
4.1 Reagents, chemicals and others ................................................................ 76
4.1.1 Chemicals ........................................................................................... 76
4.1.2 Radiochemicals................................................................................... 76
4.1.3 Cytokines ............................................................................................ 76
4.1.4 Oligonucleotides.................................................................................. 76
4.1.5 Plasmids.............................................................................................. 78
4.1.5.1 Cloning Vectors............................................................................ 78
4.1.5.2 Reporter Gene Plasmids.............................................................. 78
4.1.5.3 Expression Vectors ...................................................................... 81
4.1.6 Antibodies ........................................................................................... 84
4.1.7 Prokaryotic cells and the culture ......................................................... 85
4.1.7.1 E. Coli strains............................................................................... 85
4.1.7.2 Medium ........................................................................................ 85
4.1.8 Eukaryotic cells and culture................................................................. 85
4.1.8.1 Eukaryotic cells ............................................................................ 85
4.1.8.2 Culture conditions ........................................................................ 86
4.1.8.3 Materials for cell culture ............................................................... 86
4.1.8.4 Cryopreservation.......................................................................... 86
4.2 General Molecular Techniques................................................................... 86
4.2.1 Polymerase Chain Reaction (PCR)..................................................... 86
4.2.2 Site Directed Mutagenesis .................................................................. 87
4.2.3 Restriction digestion of plasmid DNA.................................................. 87
4.2.4 DNA agarose gel electrophoresis and extraction ................................ 87 Contents IV
4.2.5 DNA ligation ........................................