DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Georg J. Hoppe :
The Role of Sir2's Enzymatic Activity in the Assembly of Silencing Complexes in Budding Yeast
Die Rolle der enzymatischen Aktivität bei der Bildung von Silencing Komplexen in Hefe

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Abstract

The folding of DNA into high-order chromatin structures appears to play a central role in the regulation of gene transcription. During recent years, various complexes associated with transcriptional activation or repression have been shown to contain chromatin remodeling activity or enzymes that covalently modify histones. The inactivation of large domains of DNA by their compaction into an inaccessible chromatin structure is referred to as chromatin silencing. This type of inactivation is involved in regulation of gene expression and is associated with chromosome structures that are involved in epigenetic inheritance. The silent information regulator 2 (Sir2) protein is a key component of the silencing machinery in yeast. Sir2 is an enzyme that couples protein deacetylation to NAD+ hydrolysis and is required for silencing at the silent mating type loci, the telomeres, and the ribosomal DNA repeats in Saccharomyces cerevisiae. In my thesis, I investigated the role of this enzymatic function in the assembly of silencing complexes at different loci. I have examined the assembly of silencing complexes on DNA in cells carrying the sir2-H364Y and sir2-G262A mutations, which both abolish the enzymatic activity of Sir2. I show that the association of the SIR complex (Sir2/Sir4 and Sir3) with DNA at telomeres and the HML-E silencer, as well as the localization of Sir2-GFP, Sir3-GFP, and GFP-Sir4 to subnuclear telomeric foci, are lost in cells containing an enzymatically inactive Sir2 protein. In contrast, Sir2 associates with rDNA and localizes to the nucleolus with approximately equal efficiency in both SIR2 and sir2-H364Y cells. I find further that histone H4 associated with the rDNA repeats is hypoacetylated. Acetylation of H4 in rDNA and telomeric regions is increased in cells containing either a sir2 deletion_or the sir2-H364Y allele, suggesting that Sir2 directly deacetylates histones in these regions. Consistent with this data, I find that mutation of lysine 16 of histone H4 to glutamine, or deletion of histone H3 N-terminal residues 4-30 result in a dramatic loss of rDNA silencing. Finally, I show that the association of Sir3 with rDNA in a sir4D strain requires the enzymatic activity of Sir2, arguing that Sir3 can localize to chromatin independently of interactions with any of the known silencing proteins. Together, these data suggest that the modification of histones or other chromatin proteins by Sir2 creates a binding site for silencing complexes on chromatin.

Table of Contents

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0 TITLE PAGE AND CONTENTS
1 INTRODUCTION 6
1.1 GENE SILENCING: AN INTRODUCTION 6
1.2 CHARACTERISTICS OF GENE SILENCING 8
1.3 THE GENE SILENCING IN SACCHAROMYCES CEREVISIAE 9
1.4 THE ROLE OF HISTONES IN SILENCING 13
1.5 THE ENZYMATIC ACTIVITY OF SIR2 15
1.6 THESIS GOALS 17
2 MATERIALS AND METHODS 19
2.1 CHEMICALS, BUFFERS, SOLUTIONS, MEDIA 19
2.2 LABORATORY DEVICES 20
2.3 HANDLING OF DNA 21
2.4 ENZYMATIC REACTIONS WITH DNA 22
2.5 CONSTRUCTION OF PLASMIDS 22
2.6 BACTERIA 24
2.7 YEAST 26
2.8 MICROSCOPY 30
2.9 SILENCING ASSAYS 30
2.10 CHROMATIN IMMUNOPRECIPITATION (ChIP) 31
3 RESULTS 35
3.1 THE ROLE OF THE ENZYMATIC ACTIVITY OF SIR2 FOR EFFICIENT ASSOCIATION OF THE SIR COMPLEX WITH DNA 35
3.2 THE ROLE OF SIR2 IN THE RECRUITMENT OF SIR3 TO rDNA 44
3.3 A ROLE FOR SIR3 IN rDNA SILENCING? 48
3.4 THE NAD-DEPENDENT DEACETYLASE ACTIVITY OF SIR2 AND HISTONE H4 DEACETYLATION 49
3.5 THE N-TERMINI OF HISTONES H3 AND H4 IN rDNA SILENCING 51
4 DISCUSSION 54
4.1 THE SIR2-H364Y POINT MUTANT 54
4.2 THE ENZYMATIC ACTIVITY OF SIR2 AND THE ASSEMBLY OF SILENCING COMPLEXES ON CHROMATIN 55
4.3 RECRUITMENT OF SIR3 TO SILENT CHROMATIN 61
4.4 THE ROLE OF HISTONES IN rDNA SILENCING 63
4.5 HISTONES AS IN VIVO SUBSTRATES OF SIR2 51
5 REFERENCES 65
6 APPENDIX 75
6.1 SUMMARY 75
6.2 ZUSAMMENFASSUNG 77
6.3 ABBREVIATIONS 79
6.4 CURRICULUM VITAE 80
6.5 PUBLICATIONS 81

More Information:

Online available: http://www.diss.fu-berlin.de/2002/280/indexe.html
Language of PhDThesis: english
Keywords: Sir2, gene silencing, chromatin
DNB-Sachgruppe: 30 Chemie
Date of disputation: 06-Nov-2002
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Hans Lehrach
Second Referee: Prof. Dr. Danesh Moazed
Contact (Author): gjhoppe@eudoramail.com
Contact (Advisor): danesh@hms.harvard.edu
Date created:07-Dec-2002
Date available:19-Dec-2002

 

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