A role for Sum1 in HML silencing and replication initiationin Saccharomyces cerevisiae

Dissertation

zur Erlangung des akademischen Grades
Doktor rerum naturalium
(Dr. rer. nat.)

im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Diplom-Biologe
Horst Irlbacher
geb. 11.05.1971 in Schwandorf/Bay.

Präsident der Humboldt-Universität zu Berlin

Prof. Dr. Jürgen Mlynek

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Thomas Buckhout, PhD

Gutachter:
1. Prof. Dr. Harald Saumweber
2. Prof. Dr. Ann Ehrenhofer-Murray
3. Prof. Dr. Jörn Walter

Tag der mündlichen Prüfung: 14.07.2005

Zusammenfassung

In der eukaryotischen Ontogenese ist die Etablierung differenzierungsspezifischer Genexpression eng an die Unterteilung des Genoms in funktionell getrennte Domänen gekoppelt. Solche Domänen lassen entweder erhöhte transkriptionelle Aktivität zu oder unterdrücken sie und werden Eu- bzw. Heterochromatin genannt. Heterochromatin enthält spezielle Proteine, die zur Ausbildung dieser repressiven Chromatinstruktur beitragen. Eine der Hauptfragen in der Heterochromatinbiologie ist, wie solche Proteine rekrutiert werden. Dieser Prozess ist entscheidend damit einzelnde Regionen im Genom koordiniert zeit- und ortsabhängig reprimiert werden können. In Saccharomyces cerevisiae entsteht Hetero-chromatin an den silent-mating-type Loci HMR a und HMLα durch die zielgerichtete Rekrutierung des Sir-Komplexes über eine Gruppe von Proteinen, die an sogenannte silencer-DNA Sequenzen binden. In diese Arbeit wird gezeigt, daß das Protein Sum1, bisher bekannt als Repressor meiotischer Gene im vegetativen Zellzyklus, als Heterochromatin-Rekrutierungsfaktor für HMLα fungiert. Sum1 konnte in vitro und in vivo an HMLα über ein funktionelles Element innerhalb des HML-E silencers binden und die Deletion von SUM1 verursachte einen Verlust von Repression an HMLα.

SUM1 beeinflußte außerdem die Fähigkeit von HML-E als Replikationsstartpunkt (origin) zu agieren, was eine Rolle von Sum1 in der Replikation nahelegt. Die Beobachtung, daß orc2-1 und orc5-1 mit sum1Δ synthetisch lethal waren und daß cdc6-1, cdc7-1 oder cdc45-1 mit sum1Δ einen synthetischen Wachstumsdefekt aufwiesen unterstützt die Vermutung, daß SUM1 eine globale Rolle in der Replikationsinitiation besitzt. In einer genomweiten Suche wurden ARS Elemente gefunden, die sowohl Sum1 als auch ORC rekrutieren. Dabei konnte gezeigt werden, daß die Replikationsaktivität dieser ARS Elemente von Sum1 bzw. Sum1 Bindungsstellen abhängig war. Als Repressor von meiosespezifischen Genen interagiert Sum1 oft mit der Histondeacetylase Hst1. In diesem Zusammenhang konnte gezeigt werden, daß SUM1-regulierte origins ebenfalls HST1 zur vollen Aktivität benötigten. Zusammenfassend schlagen wir Sum1 als neuartigen Modulator für die Replikationsinitiation an einer Untergruppe chromosomaler Replikationsstartpunkte vor.

Eigene Schlagworte:

Eigene Schlagworte: Chromatin, Silencing, Replikationsstartpunkt, Replikation, ARS, Sum1, Hst1

Abstract

The division of eukaryotic chromatin into functionally distinct domains is critical to implement gene expression programs that drive the development of multicellular organisms. Regions termed euchromatin exist in the genome that are generally conducive to transcription, whereas heterochromatin contains specialized chromatin binding proteins that repress transcription in these regions. A central question in heterochromatin biology is how the heterochromatin factors are targeted to specific genomic regions, a process that is crucial to ensure that the designated domains, and only they, are repressed in the appropriate spatial and temporal fashion. In Saccharomyces cerevisiae heterochromatinization at the silent mating-type loci HMR a and HMLα is achieved by targeting the Sir complex to these regions via a set of anchor proteins that bind to the silencers. Here, we have identified a novel heterochromatin targeting factor for HMLα, the protein Sum1, a repressor of meiotic genes during vegetative growth. Sum1 bound both in vitro and in vivo to HMLα via a functional element within the HML-E silencer, and deletion of SUM1 caused HMLα derepression. Significantly SUM1 was also required for origin activity of HML-E, suggesting a role of Sum1 in replication initiation. Our observations of a synthetic lethality between orc2-1 or orc5-1 and sum1Δ as well as a synthetic growth defect of cdc6-1, cdc7-1 and cdc45-1 with sum1 support the notion that SUM1 has a global role in replication initiation. In a genome-wide search for Sum1-regulated origins, we identified a set of autonomous replicative sequences (ARS elements) that bound both the origin recognition complex and Sum1. Full initiation activity of these origins required Sum1, and their origin activity was decreased upon removal of the Sum1 binding site. In its role as a repressor of meiosis specific genes, Sum1 often works in concert with the histone deacetylase Hst1. We found that SUM1-regulated origins also required HST1 for full activity. Taken together we propose that Sum1 is a novel replication initiation modulator for a subset of chromosomal origins.

Keywords:

Keywords: Chromatin, Silencing, Origin, Replication, ARS, Sum1, Hst1

Inhaltsverzeichnis

• 1  Introduction
  • 1.1  Epigenetics and regulation of gene expression
  • 1.2 Chromatin and gene expression
  • 1.3 Chromatin composition
  • 1.4 Mechanisms that alter chromatin properties
    • 1.4.1  Chromatin remodeling
    • 1.4.2 Chromatin modifications
  • 1.5 Silencing in Saccharomyces cerevisiae
    • 1.5.1  Silencing at the HM loci
    • 1.5.2 Silencing at the telomeres and the rDNA locus
  • 1.6 Replication initiation
    • 1.6.1  Origins of replication
    • 1.6.2 Events during replication initiation
  • 1.7 Regulation of replication initiation
  • 1.8 Proteins investigated in this work
    • 1.8.1  Sum1
    • 1.8.2 Hst1 and Rfm1
  • 1.9  Outline of this thesis
• 2 Materials and Methods
  • 2.1  E.coli strains
  • 2.2 Growth conditions and media
  • 2.3 Yeast strain construction
  • 2.4 Yeast strains
  • 2.5 Plasmid construction
  • 2.6 Silencing assays
  • 2.7 Plasmid loss assay
  • 2.8 Yeast extracts for Western blotting
  • 2.9 SDS PAGE and Immunoblotting
  • 2.10 Co-Immunoprecipitation
  • 2.11 Electrophoretic mobility shift assays (EMSA)
  • 2.12 Chromatin immunoprecipitations
  • 2.13  In vivo replication-origin assay
• 3 Results
  • 3.1  Definition of a core region within the D element
  • 3.2 Genetic interaction of SUM1 and the D element
    • 3.2.1  sum1 caused HMLα derepression
    • 3.2.2  sum1Δwas epistatic to the D element
    • 3.2.3  SUM1 dependent HML silencing was independent of HST1 and RFM1
  • 3.3 Sum1 bound specifically to the D element within HML-E
    • 3.3.1  In vitro binding of Sum1 to HML-E
    • 3.3.2  In vivo localization of Sum1 at HML-E
  • 3.4  sum1Δ decreased origin function of HML-E
  • 3.5  sum1 interacted genetically with orc mutations, cdc6-1, cdc7-1 and cdc45-1
  • 3.6 Sum1 was a replication initiation factor for several origins of replication
    • 3.6.1  Identification of genomic sites of combined Sum1 and ORC binding
    • 3.6.2 Sum1 influenced origin function of ARS1013
    • 3.6.3 Sum1 binding sites controlled origin function of ARS1013
    • 3.6.4 Sum1 affected the chromosomal origin activity of ARS1013
    • 3.6.5 Sum1 influenced origin function of selected origins
    • 3.6.6 Sum1 affected origin function at chromosomes
  • 3.7 Hst1 affected Sum1-modulated replication origins
• 4 Discussion
  • 4.1  Sum1 in silencing
  • 4.2 Sum1 in replication initiation
  • 4.3 Sum1 as a cell programm-dependent replication initiator?
• 5 Appendix  The D-element silencer screen in the “silencing cassette”- plasmid
• References
• Abbreviations
• Curriculum Vitae
• Publications
• Acknowledgements
• Declaration

Tabellen

• Table 2.1: S.cerevisiae strains used in this study
• Table 2.2: Plasmids used in this study
• Table 3.1: Genomic loci that are bound by Sum1 at p<0.01 and ORC at p<0.05
• Table 3.2 : Genomic loci of ORC and Sum1 consensus motif co-occurrence.
• Table 3.3 : Sum1 affected chromosomal origin activity of ARS606 at low temperature.

Bilder

• Figure 1.1: Schematic representation of various DNA compaction levels. Details are described in the text. Image adapted from Sinauer Associates, 2001.
• Figure 1.2: Schematic representation of mating-type loci and silencers
• Figure 1.3: Comparison of ARS elements in S.cerevisiae
• Figure 1.4: Different phases of replication initiation. Details are described in the text.
• Abbildung 1.5: Sum1-1 can establish silencing at HMRaindependently of the Sir-proteins
• Figure 3.1: Silencing properties of the D-element at the HML-E silencer.
• Figure 3.2: Identification of a D-element core region within the HML-E silencer.
• Figure 3.3: SUM1 was required for HMLα silencing and was epistatic to the D element.
• Figure 3.4: Binding of Sum1 to HML-E in vitro
• Figure 3.5: D-specific binding of Sum1 in vitro
• Figure 3.6: In vivo localization of Sum1 at HML-E
• Figure 3.7: sum1Δ#SYMBOL#reduced the ARS activity of HML-E.
• Figure 3.8: Genetic interactions between SUM1 and replication initiation components.
• Figure 3.9: Test for physical interaction of Sum1 and Orc2.
• Figure 3.10: Search for intergenic regions that bind both Sum1 and ORC.
• Figure 3.11: Sum1 influenced origin function of ARS1013
• Figure 3.12: Addition of Sum1 binding sites improved the ARS function of ARS1013-2.
• Figure 3.13: Sum1 was required for chromosomal origin activity of ARS1013
• Figure 3.14: Sum1 affected ARS activity of selected origins of replication.
• Figure 3.15: Chromosomal origin activity of selected ARS sites in wild-type and sum1 strains.
• Figure 3.16: Hst1 affected ARS activity of Sum1 modulated origins of replication.
• Figure 4.1: The different facets of Sum1
• Figure 5.1: Schematic representation of the silencing cassette.
• Figure 5.2: HML-E properties in the silencing cassette