The histone deacetylase (HDAC) Rpd3 antagonizes heterochromatin formation at telomeres in Saccharomyces cerevisiae [Elektronische Ressource] / vorgelegt von Stefan Ehrentraut

Ehrentraut Stefan

Sujets

• Biologie

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2008
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

The histone deacetylase (HDAC) Rpd3
antagonizes heterochromatin formation at
telomeres in Saccharomyces cerevisiae





Inaugural-Dissertation
zur
Erlangung des Doktorgrades
Dr. rer. nat.

des Fachbereichs
Biologie und Geografie
an der


Universität Duisburg-Essen




vorgelegt von



Stefan Ehrentraut

geboren in Lauchhammer


Oktober 2008


Die der vorliegenden Arbeit zugrundeliegenden Experimente wurden am Max-Planck-
Institut für Molekulare Genetik in Berlin-Dahlem sowie am Institut für Genetik des
Fachbereichs Biologie und Geografie der Universität Duisburg-Essen durchgeführt.


1. Gutachter: Prof. Dr. Ann E. Ehrenhofer-Murray


2.Gutachter: Prof. Dr. Reinhard Hensel


3.Gutachter:


Vorsitzender des Prüfungsausschusses: Prof. Dr. Daniel Hoffmann


Tag der mündlichen Prüfung: 09. 02. 2009





Table of contents
Table of figures
List of tables
Abbreviations
1 Introduction 9
1.1 DNA packaging in eukaryotes 9
1.2 Organization of chromatin 10
1.3 Histone modifications 11
1.4 Histone acetyltransferases (HATs) 14
1.5 The HAT complex SAS-I 15
1.6 Histone deacetylases (HDACs) 16
1.7 The histone deacetylase (HDAC) Rpd3 17
1.8 Heterochromatin in S. cerevisiae 19
1.8.1 Silencing at the HM loci 19
1.8.2 Telomeric silencing 21
1.8.3 rDNA silencing 23
1.8.4 The Silent Information Regulator (SIR) complex as a key component of
heterochromatin 24
1.9 Restriction of heterochromatin spreading through boundary elements 25
1.10 Replication of chromatin 27
1.11 Outline of this thesis 29
2 Material & Methods 31
2.1 E. coli strains 31
2.2 Media and growth conditions 31
2.3 Saccharomyces cerevisiae strains 31
2.4 Genetic manipulation of S. cerevisiae strains 31
2.4.1 Crossing, sporulation and dissection of asci 31
2.4.2 DNA techniques in S. cerevisiae 32
2.5 Molecular cloning 34
2.6 Chromatin immunoprecipitations 36
2.7 Quantitative reverse transcriptase PCR (qRT-PCR) 38
2.8 Synthetic lethal screen 39
2.9 Yeast genetic assays 39
2.10 Protein-protein interaction assays 39
2.10.1 Yeast-two-hybrid assay 39
























2.10.2 Co-Immunoprecipitation 39
2.11 Yeast protein extracts for SDS-PAGE and Western Blotting 40
2.12 SDS-PAGE and Western Blotting 40
2.13 Computational modelling of the Sir3 structure 40
3 Results 41
3.1 Restriction of heterochromatin spreading by the HDAC Rpd3 41
3.1.1 Deletion of RPD3 was synthetically lethal in the absence of the histone
acetyltransferase complex SAS-I 41
3.1.2 The catalytic activity of the SAS-I complex was required for the survival of rpd3
cells 43
3.1.3 The lethality between rpd3 and sas2 depended on the whole SAS-I complex 44
3.1.4 The lethality between rpd3 and sas2 involved the Rpd3 (L) complex 45
3.1.5 The lethality between rpd3 and sas2 was specific for those two enzymes 46
3.1.6 rpd3 and sas2 changed subtelomeric gene expression 48
3.1.7 The lethality between rpd3 and sas2 was suppressed by sir2 , sir3 and sir4 50
3.1.8 rpd3 caused increased SIR spreading at telomeres 54
3.1.9 rpd3 caused increased silencing in subtelomeric regions by SIR spreading 55
3.1.10 Subtelomeric acetylation was reduced in rpd3 cells 57
3.1.11 Targeted Rpd3 established a boundary at telomeres 59
3.1.12 The boundary function of Rpd3 did not depend on subsequent chromatin modifying
or remodelling activities 62
3.1.13 Enhanced SIR binding through enhanced acetylation in rpd3 mutants? 64
3.1.14 A boundary function for Rpd3 through removal of Sir2 substrates 65
3.1.15 Disruption of the AAA+ domain within Sir3 abrogated SIR spreading 66
3.2 The removal of cytoplasmatic acetylation patterns partially depended on
chromatin assembly factors 71
3.2.1 The INO1 ORF became more acetylated in cac1 and asf1 cells 71
3.2.2 Late-replicating intergenic regions became more acetylated in cac1 and asf1 cells 72
3.2.3 The higher acetylation in cac1 cells was based on an interaction between CAF-I and
Rpd3 75
3.2.4 Rpd3 and Cac1 work epistatic in HM silencing 78
4 Discussion 80
4.1 Rpd3 formed boundaries against heterochromatin 80
4.2 Does boundary formation through Rpd3 require permanent presence of Rpd3 at
the subtelomere? 81
4.3 Is the Rpd3 boundary function related to other chromatin modifying or
remodelling activities? 83















4.4 Relationship between Rpd3 and other boundary factors 84
4.5 Is Rpd3 required for subtelomeric gene activation? 84
4.6 Boundary formation by HDACs 85
4.7 Which histone residues are important for the Rpd3 boundary function? 87
4.8 A role for the Sir2 metabolite OAADPR in heterochromatin spreading 88
4.9 Is OAADPR transported within the cell? 90
4.10 A mechanism for Rpd3 as a boundary element 91
4.11 Summary and outlook 93
5 Abstract 95
6 Zusammenfassung 96
7 Literature Cited 97
Danksagung 109

Table of figures
Figure 1 DNA in eukaryotic cells is packaged into nucleosomes. .....................................10
Figure 2 Posttranslational modifications within the histone tails.......................................12
+Figure 3 The NAD -dependent acetyl-lysine deacetylation reaction. ................................17
Figure 4 The HM loci of Saccharomyces cerevisiae. ........................................................20
Figure 5 Telomeres in Saccharomyces cerevisiae.............................................................22
Figure 6 The rDNA locus of Saccharomyces cerevisiae. ..................................................23
Figure 7 The deletion of RPD3 was lethal in the absence of Sas2. ....................................43
Figure 8 The lethality between sas2 and rpd3 depended on the catalytic activity of
Sas2. ..................................................................................................................44
Figure 9 Synthetic lethality between rpd3 and sas4 or sas5 . ......................................44
Figure 10 Synthetic lethality between the Rpd3 (L) and the SAS-I complex.......................45
Figure 11 Subtelomeric genes showed similar expression profiles in sas2 and rpd3
cells....................................................................................................................49
Figure 12 The lethality between sas2 and rpd3 was suppressed by sir2 , sir3 and
sir4 . .................................................................................................................51
Figure 13 Suppression of the lethality between sas2 and rpd3 by histone mutants. ........52
Figure 14 Sir2 was mislocalized to subtelomeric regions in the absence of Rpd3................54
Figure 15 The expression of Sir proteins was not increased in the absence of Rpd3............55



Figure 16 Subtelomeric genes were repressed in rpd3 cells in a SIR-dependent
fashion. ..............................................................................................................56
Figure 17 Subtelomeric acetylation is reduced in rpd3 cells.............................................58
Figure 18 Subtelomeric acetylation was influenced by SIR. ...............................................58
Figure 19 Tethered Rpd3 created a boundary at the telomere..............................................59
Figure 20 The targeted boundary function of Rpd3 required the HDAC activity of
Rpd3. .................................................................................................................60
Figure 21 Tethered Rpd3 disrupted silencing at HML and had insulating activity at
HMR.61
Figure 22 Histone mutants lead to telomeric derepression. .................................................65
Figure 23 Targeted boundary function of yeast HDACs depended on their substrate
specificity...........................................................................................................66
Figure 24 Computational modelling of Sir3 revealed a possible OAADPR binding
pocket. ...............................................................................................................67
Figure 25 The putative OAADPR binding domain of Sir3 was necessary for its
function in silencing...........................................................................................68
Figure 26 Disruption of the OAADPR binding motif abrogated the function of Sir3. .........70
Figure 27 The absence of chromatin assembly factors led to higher H4-K12
acetylation in the body of the INO1 gene............................................................72
Figure 28 Late replicating intergenic regions became more acetylated at H4-K12 in the
absence of chromatin assembly factors...............................................................73
Figure 29 The higher H4-K12 acetylation of intergenic regi