The role of Isll/BAF complex during heart development Presented by

The role of Isll/BAF complex during heart
development
Dissertation for the degree for doctor of natural sciences
(Dr.rer.nat)
Presented by
Nitin Tyagi
Submitted to the Faculty of Biology and Chemistry of the
Justus Liebig University, Gieften
Gieften, 2015
1
http://d-nb.info/1069939994
Contents
Abbreviations
7
Summary
10
Zusammenfassung
11
1. Introduction
l.lEmbryonic heart development
12
12
1.1.1 Description of vertebrate cardiogenesis
12
1.1.2 Heart origins from two distinct fields
13
1.2 Cardiac progenitors
1.2.1 The balance between cardiac progenitor proliferation and differentiation
16
17
1.3 Genes Critical for heart formation
18
1.3.1 Gata4
1.3.2 Isll
18
18
1.3.3 Nkx 2.5
19
1.3.4 Tbx proteins
20
1.3.4.1 Tbxl
20
1.3.4.2 Tbx5
20
1.3.5 Hand Proteins ( Handl and Hand2)
21
1.3.6 Mef2s
21
1.4 Epigenetics in cell differentiation
22
1.4.1 Chromatin organization and histone code
23
1.4.2 ATP-dependent chromatin remodeling
23
1.4.3 Chromatin remodeling complexes
24
1.4.3.1 SWI/SNF
24
1.4.3.2 CHD
25
1.4.3.3 ISWI
25
1.4.3.4 IN080
27
1.4.4 The mechanisms of gene regulation by BAF complexes
27
1.4.5 The roles of tissue-specific BAFs
28
1.4.6 Effect of BAF members in heart development
30
Rationale of the Thesis:
36
2 Material and Methods
2.1 Chemicals, equipment and sources of supply
37
37
2.2 Buffers and media
41
2.3 Synthetic oligonucleotides
44
2.4 Vectors and plasmids
45
2.4.1 shRNA knockdown sequences
45
2. Standard molecular biology methods
47
2.6 Standard methods
47
3
2.6.1 RNA extraction using Trizol
48
2.6.2 Real-time PCR
49
2.7. Protein biochemical methods
49
2.7.1 SDS-PAGE/Western blot
49
2.7.2 Stripping
51
2.7.3 Immunoprecipitation
2.7.4 Chromatin-IP
51
52
2.7.5 Isolation of cytoplasmic and nuclear fraction of cells
53
2.8 Cell biology techniques
53
2.8.1 Cell culture
53
2.8.2 Transfection of eukaryotic cells
54
2.8.2.1 PEI-transfection
2.8.2.2 FuGENE®-transfection
54
54
2.8.2.3 Lipofectamine transfection
54
2.8.3 Formation and differentiation of embryoid bodies
55
2.8.4 Establishing of stable ES cell lines
55
2.8.5 Whole mount in situ hybridization in mouse
56
2.8.6 Reporter assay
58
2.8.6.1 Luciferase assay
58
3 Results
59
3.1 Baf60c is a target of Isll
3.1.1 Generation of Isll knockdown stable ES cell line
59
59
3.1.2 Characterization of Isll knockdown stable ES cell line
61
3.1.2.1 Beating
61
3.1.2.2 Isll regulates the expression of cardiomoycytes specific genes
62
3.1.2.3 Isll regulates the expression of smooth muscle specific genes
63
3.1.2.4 Isll does not regulates the expression of endothelial specific genes
3.1.2.5 Isll regulates the expression of cardiac progenitor specific genes
63
64
3.1.2.6 Isll regulates the expression of Baf60c
3.1.3 The expression of Baf60c in mouse embryos
65
66
3.1.3.1 Isll regulates Baf60c transcriptional activity in mice embryo
67
3.1.4 Isll binds at a consensus sequences on Baf60c locus
68
3.1.5 Isll activates Baf60c reporter gene in luciferase assay
70
3.2 Isll interaction with BAF chromatin-remodeling complex
3.2.2 Isll interact with endogenous Brgl in EBs
71
72
3.2.3 Baf60c mediates interaction between Isll and Brgl complex
73
3.2.4 Isll cooperation with Baf60c on Mef2c enhancer
74
3.2.5 Isll cooperation with Brgl on Mef2c enhancer
75
3.3. Role of Baf60c during cardiac differentiation
78
3.3.1 Expression of Baf60c during cardiac differentiation
78
3.3.2 Baf60c regulates the expression of mesodermal germ layer
79
3.3.3 Baf60c regulate the expression of progenitor markers
80
4
3.3.4 Baf60c decide the fate of cardiac progenitor cells
4 Discussion
82
84
4.1 Role of Isll in cardiovascular progenitor cells differentiation
84
4.2 General epigenetic mechanisms in establishing cell fate identity
85
4.3 Isll dependent regulation of Baf60c
86
4.4 Baf60c mediates interaction between Isll and Brgl / BAF chromatin remodeling
complex
87
4.5 Baf60ckd in ES cells affects the proper differentiation
89
References
91
Acknowledgement
104
5