Transcriptional regulation and functional characterization of the tumor suppressor genes Hugl-1 and Hugl-2 [Elektronische Ressource] / Anubha Kashyap

Anubha Kashyap

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2008
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Transcriptional Regulation and Functional Characterization
of the Tumor Suppressor Genes
Hugl-1 and Hugl-2





Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaften


Am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz




Anubha Kashyap
geb. am 02. März 1976 in Indien

Mainz, 2008
























Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung: 26.08.2008





Table of Contents
Table of Contents
1 Introduction .......................................................................................... 1
1.1 Cell polarity ......................................................................................................... 1
1.1.1 Polarity complex proteins ........................................................................... 2
1.1.1.1 Par complex .................................................................................... 3
1.1.1.2 Crumbs complex............................................................................. 4
1.1.1.3 Scrib complex ................................................................................. 5
1.2 Lethal giant larvae (lgl)........................................................................................ 6
1.2.1 Homologues of lgl ...................................................................................... 7
1.2.1.1 Hugl-1 and Hugl-2.......................................................................... 7
1.2.2 Structural and functional conservation of the lgl........................................ 9
1.2.3 Functions of lgl ......................................................................................... 11
1.2.3.1 Lgl in cell polarity......................................................................... 11
1.2.3.2 Lgl as tumor suppressor ................................................................ 14
1.2.3.3 Lgl as regulator of exocytosis ....................................................... 15
1.2.4 Regulation of lgl function 15
1.3 Epithelial to Mesenchymal Transition (EMT)................................................... 18
1.3.1 EMT in cancer progression....................................................................... 19
1.3.2 Regulators of EMT ................................................................................... 20
1.3.3 EMT and cell polarity............................................................................... 22
1.4 Aim of the study ................................................................................................ 25
2 Materials and Methods ...................................................................... 26
2.1 Instruments and equipments .............................................................................. 26
2.2 Chemicals........................................................................................................... 27
2.3 Antibodies.......................................................................................................... 29
2.4 Software............................................................................................................. 30
2.5 Molecular biological methods ........................................................................... 31
2.5.1 Cloning of target gene............................................................................... 31
2.5.2 Agarose gel electrophoresis...................................................................... 33
2.5.3 Subcloning of DNA fragments ................................................................. 34
2.5.4 Transformation.......................................................................................... 35
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Table of Contents
2.5.5 RT-PCR .................................................................................................... 36
2.5.6 Site directed mutagenesis.......................................................................... 38
2.6 Ecdysone mammalian expression system.......................................................... 38
2.7 Cell biological methods ..................................................................................... 39
2.7.1 Cell culture................................................................................................ 39
2.7.2 Preservation of cells.................................................................................. 41
2.7.3 Transfection .............................................................................................. 42
2.7.4 Migration assay......................................................................................... 42
2.7.5 Luciferase reporter assay .......................................................................... 44
2.7.6 3D Matrigel culture................................................................................... 45
2.7.7 Activation assay........................................................................................ 46
2.7.8 Immunofluorescence staining................................................................... 47
2.8 Biochemical methods......................................................................................... 48
2.8.1 Western blot.............................................................................................. 48
2.8.2 Electrophoretic mobility shift assay (EMSA)........................................... 52
2.8.3 Chromatin immuno precipitation (ChIP).................................................. 58
2.9 Animal experiments........................................................................................... 62
2.9.1 Animal maintenance ................................................................................. 62
2.9.2 Transgenic mice........................................................................................ 63
2.9.2.1 Breeding animals .......................................................................... 64
2.9.2.2 Mice genotyping ........................................................................... 64
2.9.2.3 FACS ............................................................................................ 65
2.9.3 Mice xenograft studies.............................................................................. 67
3 Results.................................................................................................. 69
3.1 Characterization of Hugl-1 and Hugl-2 promoter regions................................. 69
3.1.1 Identification of the Hugl-1 core promoter region.................................... 69
3.1.1.1 Cloning of 5083bp fragment of Hugl-1 gene................................ 70
3.1.1.2 Activity of 5083bp fragment of Hugl-1 putative
promoter region ...................................................................... 72
3.1.1.3 Cloning of 4590bp fragment of Hugl-1 gene................................ 73
3.1.1.4 Activity of 4590bp fragment of Hugl-1 promoter region............. 75
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3.1.1.5 Cloning of 1800bp fragment of Hugl-1 gene................................ 76
3.1.1.6 Activity of 1800bp fragment of Hugl-1 promoter region............. 77
3.1.1.7 Cloning of Hugl-1 promoter region lacking exon-I...................... 78
3.1.1.8 Analysis of Hugl-1 promoter activity ........................................... 79
3.1.1.9 Sequence alignment of Hugl-1 promoter...................................... 80
3.1.2 Identification of Hugl-2 core promoter region ......................................... 82
3.1.2.1 Cloning of Hugl-2 promoter region .............................................. 82
3.1.2.2 Hugl-2 promoter activity .............................................................. 86
3.2 Influence of Snail on Hugl-1 and Hugl-2 promoter activity.............................. 87
3.2.1 Analysis of potential Snail binding sites in Hugl-1 and
Hugl-2 promoter ................................................................................... 88
3.2.2 Influence of Snail on Hugl-2 .................................................................... 89
3.2.2.1 Influence of Snail on Hugl-2 promoter activity............................ 89
3.2.2.2 Influence of Snail on truncated Hugl-2 promoter......................... 91
3.2.2.3 Effect of Snail on mutated Hugl-2 promoter ................................ 92
3.2.2.3.1 Generation of mutated Hugl-2 promoter construct.........92
3.2.2.3.2 Effect of Snail on mutated Hugl-2 promoter activity .....95
3.2.2.4 Confirmation of Snail binding to Hugl-2 promoter by EMSA..... 96
3.2.3 Effect of Snail on Hugl-2 expression........................................................ 98
3.2.3.1 Establishing 293EcR-Snail cell line ............................................. 98
3.2.3.2 Hugl-2 promoter activity in 293EcR-Snail clones........................ 99
3.2.3.3 Expression of Hugl-2 and E-cadherin in 293EcR-Snail
cell line by RT-PCR ............................................................. 100
3.2.3.4 Analysis of Snail binding to Hugl-2 promoter by ChIP ............. 101
3.2.3.5 Functional studies using HEK