Connecting the histone acetyltransferase complex SAS-I to the centromere in S. cerevisiae

Dissertation

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

im Fach Biologie
eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Dipl. biol. Stefanie Seitz
geb. 18.06.1975, Frankfurt/Main

Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Jürgen Mlynek

Dean:
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Michael Linscheid

Gutachter:
1. Prof. Dr. Ann Ehrenhofer-Murray
2. Prof. Dr. Harald Saumweber
3. Prof. Dr. Francis Stewart

Tag der mündlichen Prüfung: 20.10.2004

Abstract

The essential histone H3 variant Cse4 plays a crucial role at the centromere in S. cerevisiae, where it replaces histone H3 in that it assembles centromere specific (Cse4-H4)2 tetrameres. We found in our study that the histone H3 variant was able to interact over its unique N-Terminus with two subunits of the histone acetyltransferase complex SAS-I: Sas2 and Sas4. Mutations within the acetyl-CoA binding site (HAT domain) or the zink-finger of Sas2 disrupted the binding to Cse4, although an indirect interaction was found with co-immunoprecipitation experiments.

Additionally, the N-terminus of Cse4 interacted with Cac1, the largest subunit of the chromatin assembly factor CAF-I and Asf1 – two histone chaperones that assemble histones H3 and H4 into nucleosomes. Our findings further suggest a role of Cac1 independent of Cac2 and Cac3 as no binding to Cse4 could be detected. A role for Sas2 at the centromere was further confirmed in that a sas2 deletion (sas2 delta) disrupted the binding of Cse4 to Ctf19. Additionally, sas2 delta partially rescued the temperature sensitivity of a cse4-103 mutated strain at elevated temperatures, suggesting a role for Sas2 in improving centromere stability. An important question resulted from our studies: is Sas2 able to acetylate the histone H3 variant Cse4 ? We have circumstantial evidence that Cse4 was indeed acetylated in the cell, but whether Sas2 accounts for the acetylation remains to be determined.

Keywords: epigenetics, centromere, histone acetylation, chromatin assembly, histone code

Zusammenfassung

Die essentielle Histon H3 Variante Cse4 ersetzt am Centromer das Standard Histon H3 und bildet zusammen mit Histon H4 funktionelle Cse4-H4 Tetramere aus. In dieser Studie konnte gezeigt werden, das Cse4 über seinen einzigartigen N-Terminus mit zwei Komponenten des Histon-Acetyltransferase-Komplexes SAS-I interagiert: der enzymatischen Untereinheit Sas2 und Sas4. Mutationen innerhalb des atypischen C2HC Zink-Fingers oder der HAT-Aktivierungsdomäne von Sas2 verhindern eine Bindung an Cse4, obwohl mit Hilfe von Co-Immunopräzipitationsexperimenten eine indirekte Interaktion nachgewiesen werden konnte.

Weiterhin wurde gezeigt, dass Cse4 mit Cac1, der größten Untereinheit des Chromatin-Assemblierungsfaktors CAF-I und Asf1 interagiert – zwei Histon Chaperonen, die Histon H3 und H4 in Chromatin assemblieren. Unsere Ergebnisse lassen weiterhin auf eine separate Rolle von Cac1, unabhängig von den beiden anderen Untereinheiten schließen. Die Interaktion von Cse4 und Ctf19 wird durch eine Deletion von Sas2 verhindert. Ebenfalls kann die Temperatur-Sensitivität eines cse4-103 mutierten Hefestamms durch eine Sas2-Deletion partiell supprimiert. Somit kann man darauf schließen, dass Sas2 eine Funktion bei der Stabilisierung des Centromers aufweist.

Die bisherigen Ergebnisse lassen die Frage aufkommen, ob Cse4 in der Zelle acetyliert ist und ob es möglicherweise als Histon H3 Variante ebenfalls ein Substrat von SAS-I darstellt. Wir konnten zeigen, dass Cse4 tatsächlich in einem acetylierten Status vorliegt, ob SAS-I jedoch für die Acetylierung verantwortlich ist bleibt nachzuweisen.

Eigene Schlagworte: Epigenetik, Centromer, Histon Acetylierung, Chromatin Assemblierung, Histon Code

Table of contents

• 1.  Introduction
  • 1.1. Organisation of chromatin
    • 1.1.1. Chromatin assembly
    • 1.1.2. Post-translational modification of histones
    • 1.1.3. Silencing in S. cerevisiae
  • 1.2.  The centromere-kinetochore complex
    • 1.2.1. The centromere in S. cerevisiae
    • 1.2.2. The histone H3 variant Cse4
• 2.  Materials and Methods
  • 2.1. Material
    • 2.1.1. Bacterial strains
    • 2.1.2. Yeast strains
    • 2.1.3. Plasmids
    • 2.1.4. Media
    • 2.1.5. Buffers and Solutions
    • 2.1.6. Antibodies
    • 2.1.7.  Peptides
    • 2.1.8. Primer
  • 2.2. Methods
    • 2.2.1. Molecular methods
      • 2.2.1.1. Cell cultivation
      • 2.2.1.2. Transformation of E. coli and S. cerevisiae
      • 2.2.1.3. DNA isolation
      • 2.2.1.4.  Plasmid constructions
      • 2.2.1.5.  S. cerevisiae strain construction
      • 2.2.1.6. Polymerase chain reaction
      • 2.2.1.7. DNA sequencing
      • 2.2.1.8. Two-hybrid system
        • 2.2.1.8.1. β-galactosidase filter assay
        • 2.2.1.8.2.  HIS3 reporter assay
      • 2.2.1.9. FACS – fluorescent activating cell sorting
    • 2.2.2.  Biochemical methods
      • 2.2.2.1. Protein extract preparation
      • 2.2.2.2.  SDS-PAGE and immunoblotting
      • 2.2.2.3. Detection methods for proteins
      • 2.2.2.4. Concentration of protein solutions
      • 2.2.2.5. Solo- and Co-immunoprecipitation
      • 2.2.2.6. Bacterial expression of Cse4
      • 2.2.2.7. Acetylation assay
• 3.  Results
  • 3.1. Interactions between Cse4, SAS-I and chromatin assembly factors
  • 3.2.  Effect of mutations in SAS-I, CAF-I and Asf1 on centromere function
  • 3.3.  A SAS2-deletion abrogated the interaction between Cse4 and Ctf19
  • 3.4.  Does Sas2 acetylate the histone H3 variant Cse4 ?
• 4.  Discussion
  • 4.1. Cse4 interacts with the SAS-I complex and the chromatin assembly factors Cac1 and Asf1
  • 4.2. The histone acetyltransferase Sas2 has a function at the centromere
  • 4.3. Cse4 exists in an acetylated state in the cell
  • 4.4. A model for chromatin-assembly at the centromere
• Literature
• Curriculum Vitae
• Danksagungen
• Erklärung

Tables

• Table 1 : Two-hybrid interactions between the histone H3 variant Cse4, the SAS-I complex and chromatin assembly factors. The reporter strain L40c was cotransformed with a plasmid expressing Cse4 and a plasmid containing components of the SAS-I complex or chromatin assembly factors. The genes were fused either to a Gal4 activation domain (GAD) or to a DNA binding domain (LexA). Interactions between proteins were measured as expression of lacZ and HIS3. Positive interactions (+) became blue in a β-galactosidase filter assay within 2 h and were able to grow on minimal media lacking histidine, negative interactions (-) did not become blue and were unable to grow on media lacking histidine; n.d. = not determined.

Images

• Fig. 1 : Molecular assembly of nucleosomes (picture taken from http://148.216.10.83/CELULA/ 4,2_chromosomes_and_chromatin.htm). The DNA (red) is wrapped around the histone octamer (blue) and both form the nucleosome core particle. This structure is locked in mammals by the linker histone H1 (yellow). The chromatin fiber is further folded into a thicker fiber, the so-called solenoid that is 30 nm in diameter.
• Fig. 2 : Possible modifications of amino acids at the example of the N-terminus of histone H3 from S. cerevisiae. A=acetylation, M=methylation, P=phosphorylation
• Fig. 3 : Structure of the silent mating-type locus HMR in S. cerevisiae with silencer binding proteins and proteins involved in the establishment and maintenance of silencing.
• Fig. 4: Structural and functional regions of the centromere. The model shows the two sister chromatids with their chromosome arms, the centric heterochromatin and the centromere. Attached to the centromeric regions are proteins that assemble to form the outer kinetochore, which in turn serves as the binding site for microtubules.
• Fig. 5: The current model of the yeast centromere. The three centromeric regions CDEI, CDEII and CDEIII serve as binding sites for different centromere binding proteins, e.g. Cse4. Cse4 replaces histone H3 and therefore forms a specialized core nucleosome with histone H2A, H2B and H4. The connection between CDEI, CDEII and the essential CDEIII-region is made by a three protein containing complex (Ctf19-Okp1-Mcm21).
• Fig. 6: Amino acid sequence of the histone H3 variant Cse4. Putative acetylation sites are lysine residues (K) and marked in red.
• Fig. 7: Interactions between Cse4 and the SAS-I complex. The antibodies for precipitation are indicated above the panel. (A) Cse4 immunoprecipitated with Sas2, myc-Sas4 and myc-Sas5. (Top) The protease deficient yeast strain AEY1558 was transformed with plasmids containing 3x-HA-CSE4 (pAE977) and SAS2 (pAE90). Precipitates were detected with α-HA-antibody. (Middle) For immunoprecipitation between 3x-HA-Cse4 (pAE977) and 6x-myc-Sas4 (pAE613) AEY2461 was used. Precipitates were analyzed with α-HA-antibody. (Bottom) Sas5 immunoprecipitated with Cse4. AEY1558 was cotransformed with plasmids containing myc-CSE4 (pAE975) and HA-SAS5 (pAE625). Detection of proteins was performed with α-HA-antibody. (B) Cse4 immunoprecipitated with Sas2 variants, carrying single point mutations in the acetyl-CoA-binding site (HAT-, P213A/P214V) or in the zink-finger (Zn-, C106L). For this purpose, 3x-HA-Cse4 (pAE976/pAE977) and Sas2-Zn- (pAE388) or Sas2-HAT- (pAE249), respectively, were introduced into AEY1559. For detection in precipitates α-HA-antibody was used.
• Fig. 8: Interactions between Cse4 and chromatin assembly factors. The antibodies for precipitation are indicated above the panel. (A) Cse4 immunoprecipitated with Cac1, but not with the other two subunits of the CAF-I complex, Cac2 and Cac3. (Top Left). AEY1808 was cotransformed with plasmids containing 3x-HA-CSE4 (pAE976) and 6x-myc-CAC1 (pAE614). Precipitation was carried out with α-HA-antibody. (Top Right) No interaction was detected between 3x-HA-Cse4 (pAE977) and 6x-myc-Cac2 in AEY1558. Detection of precipitates was performed with α-HA-antibody. (Bottom Left) 3x-HA-Cse4 (pAE977) did not interact with 6x-myc-Cac3 in AEY1558 cac1Δ. Precipitates were analyzed with α-HA-antibody. (B) Cse4 interacted with Asf1 in vivo. Therefore AEY2493 with genomic 3xHA-tagged Asf1 was transformed with 6x-myc-CSE4 (pAE901). For detection of precipitated Cse4 α-myc-antibody was used.
• Fig. 9: Consequences of different mutations on the interaction with Cse4. The antibodies for precipitation are indicated above the panel. (A) The interaction between HA-Cse4 (pAE977) and myc-Sas4 (pAE613) and myc-Cac1 (pAE614), respectively, in a sas2Δ strain (AEY1559). Cse4 was precipitated with α-myc-antibody and precipitates were immunoblotted using α-HA-antibody. (B) HA-Cse4 (pAE977) and Sas2 (pAE90) were still able to interact with each other in a sas4Δ strain (AEY2461), whereas the interaction between HA-Cse4 and myc-Cac1 (pAE614) was disrupted. The detection was carried out with α-HA-antibody. (C) The chromatin assembly factor is not necessary for the interaction between HA-Cse4 and Sas2 (pAE90) or myc-Sas4 (pAE613), respectively. The interaction was tested in a cac1Δ strain (AEY1808). HA-Cse4 was precipitated with α-Sas2 or α-myc, immunoblotting was performed with α-HA-antibody.
• Fig. 10: Partial suppression of the cse4-103 temperature sensitivity by sas2Δ. The strains were spotted in serial dilutions onto selective media and grown for 2 days at 34°C. Strains used in this assay were AEY1194 (wildtype, wt), AEY1781 (cse4-103 sas2Δ), AEY1162 (cse4-103), AEY2373 (cse4-103 cac1Δ) and AEY2374 (cse4-103 sas2Δ cac1Δ).
• Fig. 11: Two-Hybrid interaction between Cse4 and Ctf19 in wt (L40c) and sas2Δ (AEY1695) cells. Yeast strains were plated on YM media and grown at 30°C for 1-2 days. The interaction between Cse4 and Ctf19 was tested with the β-galactosidase filter assay, where positive interactions became blue after incubation at 30°C over night. Picture courtesy of Uta Marchfelder.
• Fig. 12: Immunoprecipitation of HA-Cse4 with α-acetyl-lysine-antibody. HA-Cse4 was isolated with α-HA-antibody from whole cell protein extracts from wildtype (AEY2661) and sas2Δ (AEY2666) cells. The concentrated HA-Cse4 was then incubated with α-acetyl-lysine-antibody and detected via immunoblotting with α-HA-antibody.
• Fig. 13: Acetylation assay with the purified and recombinant His-Cse4 and the SAS-I complex. SAS-I and His-Cse4 were purified from bacteria, whereas histone H4 and PCAF were commercially available. In this assay 2 μg substrate (histone H4, Cse4) was added to 200 ng SAS-I and 500 ng PCAF, respectively, and mixed together with 0,5 μg [14C] acetyl-CoA. After 1 h incubation the samples were loaded onto a 15 % SDS-gel, which was analyzed after the run in a phosphoimager. Figure courtesy of Jacqueline Franke.
• Fig. 14: A model of the histone H3 variant Cse4 and its interactions with the SAS-I complex as well as with a component of the chromatin assembly factors CAF-I and Asf1. A direct interaction is proposed with Sas2, Sas4, Cac1 and Asf1, whereas Sas5 does not bind directly to Cse4.