Tomato heat stress transcription factor HsfB1 represents a novel type of general transcription coactivator with a histone-like motif interacting with HAC1-CBP [Elektronische Ressource] / Kapil Bharti

Bhartik

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2005
• Nombre de lectures: 24
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Tomato heat stress transcription factor
HsfB1
represents a novel type of general
transcription coactivator
with a histone-like motif interacting with
HAC1/CBP
Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
Kapil Bharti
vorgelegt
beim Fachbereich Biologie und Informatik
der Johann Wolfgang Goethe-Universität
Frankfurt am Main, September, 2003Acknowledgement
This work has come to completion with the best wishes of my parents
and the expert guidance and invaluable critism of my revered mentor
Prof. Dr. Lutz Nover.
I am also thankful to Dr. Klaus Dieter-Scharf, for his helpful comments
and useful suggestions.
I am indebeted to Angelika for her excellent technical assistance.
I am grateful to Masood and Markus Fauth for their suggestions and
Markus for help with the computer work.
I wish to convey my profound gratitude to members of the big lab for
their constant support, especially Daniela for her neverending help
during my stay in this lab.
The Indian community-Sanjeev, Shravan, Arnab and Sachin deserves
special thanks for providing a warm environment.
No words are enough to thank my wife, Sanita for being a constant
source of encouragement and help throughout.
2Contents
1 Introduction 8
1.1 The heat stress response 8
1.2 Basic structure and classification of Hsfs 9
1.3 Multiplicity and complexity of plant Hsfs 11
1.4 Heat stress element (HSE) containing promoters 14
1.5 Coregulators of Hsf activity 15
1.5.1 TBP-associated factors (TAFs) 16
1.5.2 Histone acetyl transferases (HATs) 16
1.5.3 ATP dependent chromatin remodelling complexes 19
1.6 Enhanceosome: the combinatorial units of gene expression 19
1.7 Aims of this study 21
2 Materials and Methods 22
2.1 General materials and methods 22
2.2 Expression and reporter constructs 22
2.3 Culture and transfection of COS7 cells 23
2.4 Luciferase assays 23
2.5 Purification of recombinant proteins and GST-fused peptides 24
2.6 In vitro pull-down assay 24
2.7 Coimmunoprecipitation 25
2.8 Electrophoretic mobility shift assay 25
3 Results 26
3.1 Synergistic interactions between tomato HsfA1 and HsfB1 26
3.2 Concept of synergism: cooperative binding of HsfB1 with HsfA1 33
3.3 Importance of HSE clusters as independent units for synergistic 36
interactions between Hsfs A1 and B1
3.4 Functional dissection of HSE cluster modules 40
33.5 Structural requirements of Hsfs A1 and B1 for synergistic 45
activation of phsp17*gus reporter
3.6 Importance of a single lysine residue in the CTD of HsfB1 47
for synergistic gene activation
3.7 HsfB1 as a coactivator for cauliflower mosaic virus 35S 49
(CaMV35S) promoter
3.8 HsfB1 as a general coactivator for house-keeping promoters 52
3.9 In vivo role of HsfB1 54
3.10 Synergistic interactions between acidic activators 56
and class B Hsfs from different plants
3.11 Similarity between the –GRGKMMK motif of HsfB1 and N-terminal 60
tails of histones: role of CBP/HAC1 in synergism
3.12 Effect of CBP/HAC1 on reporter gene expression in presence 62
of Hsfs A1 and B1
3.13 Conservation of HsfA1/B1 synergism between plant and 66
animal cells
3.14 In vitro interactions of HAC1/CBP with HsfA1 and HsfB1 68
3.15 Cooperative DNA binding of HsfB1 with HsfA1/NTD and TGA 71
transcription factors
3.16 Presence of a “histone-like motif” in the CTD of HsfB1 73
4 Discussions 76
4.1 Tomato HsfB1: an activator, a repressor or a coactivator? 76
4.1.1 An activator 76
4.1.2 A repressor Hsf 76
4.1.3 A coactivator 78
4.2 HsfB1 as a general coactivator for heat stress inducible, 78
viral and house-keeping gene promoters
4.2.1 Heat stress inducible promoters 78
4.2.2 Viral promoters 81
4.2.3 House-keeping promoters 81
4.3 In vivo functions of HsfB1 82
4.4 Regulation of HsfB1 expression and activity: does HsfB1 recruit 84
4proteasomal subunits to the promoters?
4.4.1 Role of a single lysine residue for both stability and activity 84
of HsfB1
4.4.2 Are the lysine residues in the CTD of HsfB1 responsible for 86
recruitment of proteasome subunits to the promoters?
4.5 Presence of a novel “histone-like motif” in the CTD of HsfB1 86
4.6 Synergism among different types of domains: concept 89
of enhanceosome
4.7 Role of CBP/HAC1 as a scaffold for HsfA1 and HsfB1 90
4.8 Order of recruitment of other coactivators and setting 93
up of a histone code
5 Summary 96
Zusammenfassung 98
6 References 100
7 Appendix 114
8 Curriculum vitae 124
9 Own publications 125
5Abbreviation index
aa amino acid residue
AD activation domain
AHA aromatic, hydrophobic and acidic amino acid residues containing motif
At Arabidopsis thaliana
ATP adenosine triphosphate
bp base pair
CaMV cauliflower mosiac virus
CBP CREB binding protein
cDNA complementary DNA
CLIP cross-linking immunoprecipitation of DNA-protein complexes
Co-IP coimmunoprecipitation
CTAD C-terminal activation domain
CTD C-terminal domain
CMV cytomegalo virus
DBD DNA binding domain
DNA deoxyribonucleic acid
EMSA electrophoretic mobility shift assay
EST expressed sequence tag
Gm Glycine max
GST Glutathione-S-transferase
GUS b-Glucuronidase
HAC1 Homologous to acetyltransferase CBP
HAT Histone acetyl transferase
HTH helix turn helix
HR-A/B heptad repeat-A/B
hs heat stress
HSE heat stress element
Hsf Heat stress transcription factor
HSG heat stress granule
Hsp Heat stress protein
kDa kilo Dalton
Le Lycopersicon esculentum
6Lp Lycopersicon peruvianum
luc Luciferase
NES nuclear export signal
NLS nuclear import signal
Nt Nicotiana tabacum
ntd nucleotide
N-terminus amino terminus of a protein
ORF open reading frame
Os Oryza sativa
PCAT p300/CBP acetyltransferase related protein
PCR polymerase chain reaction
PIC pre-initiation complex
SRC-1 Steroid receptor coactivator-1
rfu relative fluorescence units
RNA ribonucleic acid
RT-PCR reverse transcriptase-polymerase chain reaction
TAFs TATA associated factors
TBP TATA binding protein
One letter code for amino acid residues:
A Alanine M Methionine
C Cysteine N Aspargine
D Aspartic acid P Proline
E Glutamic acid Q Glutamine
F Phenylalanine R Arginine
G Glycine S Serine
H Histidine T Threonine
I Isoleucine V Valine
K Lysine W Tryptophan
L Leucine Y Tyrosine
71 Introduction
1.1 The heat stress response
The pioneering work of the Italian developmental biologist F. Ritossa (Ritossa 1962) led
to one of the most seminative discoveries in the field of molecular cell biology. After a
mistaken increase in the temperature of chamber with Drosophila cultures, a
serendipitous discovery showed extraordinary changes in the gene activity pattern of the
polytene chromosomes in larval salivary glands. It took another 10-15 years before this
unusual gene activity was related to protein synthesis of heat stress proteins (Hsps,
Tissieres et al. 1974) and the corresponding mRNAs were identified (McKenzie and
Meselson 1977). It soon became clear that Ritossa infact had discovered the most
central part of heat stress (hs) response, the unusually strong inducibility which is
inherent to induction of heat stress proteins. Further on it was shown that the principles
of heat stress inducibility of Hsp genes and the Hsp protein families are conserved from
prokaryotes to eukaryotes (for earlier ref. see Ashburner and Bonner 1979; Nover et. al.
1989; Nover 1991). Originally the names of different Hsp families were derived from
their apparent molecular sizes (Nover and Scharf 1997; Forreiter and Nover 1998). A
gene based nomenclature derived from the compilation of related open reading frames
(ORFs) encoding members of Arabidopsis Hsp families can be found in a special issue
of Cell Stress and Chaperones (Nover and Miernyk 2001).
Hsps act as cellular guard under sub-optimal conditions (e.g. temperature, heavy
metals, toxins, oxidants, viral and bacterial infections), both to prevent and repair the
damage caused in the cellular homeostasis. In addition these proteins play important
role in the house-keeping functions by acting as molecular chaperones, participating in
protein folding, topogenesis, translocation and degradation, mostly as multichaperone
machines (Vierling 1991; Parsell and Lindquist 1993; Morimoto et al. 1994; Hartl 1996;
Rutherford and Lindquist 1998; Bharti and Nover 2001; Queitsch et al. 2002). Heat
stress results in a decreased pool of free chaperones, due to their increased demand in
maintaining protein homeostasis during a cellular insult. This decreased pool is
replenished by the new synthesis of Hsp´s, which is attributed to a conserved regulatory
protein, the heat stress transcription factor (Hsf).
81.2 Basic structure and classification of Hsfs
Hsfs, the terminal components of heat stress signalling cascade