1  Introduction

• 1.1 N-terminal acetylation of proteins

• 1.2 N^α-acetyltransferases in S. cerevisiae

• 1.3 NatA – the major N^α-acetyltransferase complex of S. cerevisiae

• 1.4 Chromatin and gene regulation

• 1.5 Chromatin modifying processes

• 1.6 Silencing in S. cerevisiae

• 1.7  Silencing proteins investigated in this thesis

• 1.8 Outline of this thesis

2  Materials and Methods

• 2.1 Materials

  • 2.1.1  E. coli strains

  • 2.1.2 Yeast strains

  • 2.1.3 Growth conditions and media

  • 2.1.4  Plasmid constructions

  • 2.1.5 Oligonucleotides

  • 2.1.6 Buffers

• 2.2 Methods

  • 2.2.1 Yeast strain construction

  • 2.2.2  Molecular cloning techniques

  • 2.2.3 Silencing assays

  • 2.2.4 Two-hybrid assay

  • 2.2.5 Immunofluorescence on yeast cells

  • 2.2.6 Biochemical techniques

3  Results

• 3.1 Nat1 was required for repression of the HM loci, telomeres and the rDNA locus

• 3.2 Orc1 required N^α-acetylation by NatA for its function in telomeric silencing

  • 3.2.1 Tethering of Orc1 or Sir1 to the silencer bypassed the requirement for NatA in silencing

  • 3.2.2 Orc1 was N-terminally acetylated by NatA

  • 3.2.3 Unacetylated orc1 mutants displayed telomeric derepression

  • 3.2.4  HM silencing was not affected by the lack of N-terminal acetylation of Orc1

  • 3.2.5 N^α‑acetylation was not required for the protein stability of Orc1

  • 3.2.6 NatA activity, but not N^α-acetylation of Orc1, was required for replication

  • 3.2.7 Synthetic lethality between nat1∆ and SUM1-1 was suppressed by orc1∆1‑235

• 3.3 N-terminal deletions of Orc1 caused silencing defects distinct from those of nat1Δ

  • 3.3.1  HMR silencing was disrupted in N-terminally truncated orc1 mutants

  • 3.3.2  Alpha‑factor sensitivity was reduced in N-terminally truncated orc1 mutants

  • 3.3.3 N-terminal truncations of Orc1 enhanced the α‑factor resistance of sir1Δ

  • 3.3.4 Telomeric silencing was affected by N-terminal truncations of Orc1

  • 3.3.5  Replication was not disturbed by N-terminal truncations of Orc1

  • 3.3.6 The N-terminal 51 amino acids of Orc1 were required for its two-hybrid interaction with Sir1

• 3.4 Sir3 was a substrate of NatA

  • 3.4.1 Sir3 was N^α-acetylated by NatA

  • 3.4.2 NatA activity was required to localize Sir3 to perinuclear foci

• 3.5 A genetic screen for multicopy suppressors of the nat1Δ silencing defect

  • 3.5.1 Screening for restored silencing of HMR SS ΔI in a nat1Δ strain

  • 3.5.2  Overexpression of SSF2 suppressed the nat1Δ mating defect

  • 3.5.3 Overexpression of ORC1 did not suppress the mating defect caused by nat1Δ

4  Discussion

• 4.1 Relevance of N^{α -acetylation for Orc1}

• 4.2 Function of the N-terminal 100 amino acid domain of Orc1

• 4.3 A model of the role of NatA in silencing

• 4.4 N^α-acetylation as a conserved eukaryotic protein modification

References

Abbreviations

Curriculum vitae

Publications

Acknowledgements

Declaration

Table 1: Characteristics of the three NAT complexes in S. cerevisiae*

Table 2.1: Yeast strains used in this study.

Table 2.1 (continued)

Table 2.1 (continued)

Table 2.2: Plasmids used in this study.

Table 3.1: Positive candidates from a screen for multi-copy suppressors of the nat1Δ mating defect in the MATαHMR SS ΔI strain AEY1273.

 Fig. 1.1: The NatA complex is associated with the ribosome. In the current model, the non-catalytic subunit Nat1 mediates the stable contact of NatA with the large ribosomal subunit. Nat1 interacts with the nascent polypeptide chain that emerges from the tunnel exit and guides it to the catalytic subunit Ard1, which transfers an acetyl moiety from acetyl coenzyme A to the N-terminal amino acid of NatA substrates. The putative catalytic subunit Nat5 is also associated with the complex. (adapted from Gautschi et al. 2003)

   Fig. 1.2: The basic structure of chromatin. The 11 nm fiber consists of DNA wrapped in two turns around histone octamers (nucleosomes) at intervals of about 200 bp along the DNA. Further folding creates a spiral structure, the 30nm fiber. Positively charged (deacetylated) histone tails (arrows) facilitate higher-order folding, whereas the acetylation of histone tails (bars) promotes the unfolded state corresponding to active chromatin. The two chromatin states are well-defined in electron micrographic images. (adapted from http://sgi.bls.umkc.edu/waterborg/chromat/chroma09.html)

 Fig. 1.3: Histone tail modifications. The amino termini of core histones contain diverse posttranslational modifications. The diagram indicates known modifications at specific residues of human histones H3 and H4. M = methylation, A = acetylation, P = phosphorylation. (adapted from Lachner et al. (2003))

Abb. 1.4: Mating-type loci and HM silencers. The mating-type loci MAT, HML and HMR are localized on chromosome III of S. cerevisiae. HML and HMR are repressed due to the nearby silencers E and I, which consist of binding sites for ORC, Rap1 and Abf1. The silencers are nucleation sites for silencing complexes, as depicted for HMR-E. The Sir complex interacts with nucleosomes and spreads into the HMR locus thereby creating a silenct chromatin structure.

 Fig. 1.5: Silent chromatin at a yeast telomere. The telomeric (TG_1-3) repeats provide binding sites for Rap1, which recruits the Sir complex. The subtelomeric CoreX element contains a binding site for ORC and acts likewise as a nucleation site for the Sir complex. Due to interactions of the silencing proteins the telomere folds back and forms a loop, which further stabilizes the chromatin structure.

 Fig. 1.6: Schematic structure of the rDNA array in S. cerevisiae. The rDNA locus is an array of tandemly repeating units containing the coding regions for ribosomal RNA seperated by non-transcribed spacer regions NTS1 and NTS2. The latter holds a binding site for ORC. Binding sites for the silencing RENT complex are depicted by arrows. (adapted from Huang and Moazed (2003))

 Abb. 3.1: NatA activity was required for HM, telomeric and rDNA silencing. (A) The deletion of NAT1 resulted in derepression of HML and HMR SS ΔI, as measured by the reduced mating ability of MATa and MATα strains, respectively. Patch-mating assays were performed with MATa strains AEY2 (WT) and AEY80 (nat1Δ), and MATαHMR SS ΔI strains AEY5 (WT) and AEY1273 (nat1Δ). (B) Silencing of MET15 inserted into the rDNA locus was impaired by nat1Δ, as indicated by the brighter colony color of strain AEY 2786 (nat1Δ) compared to AEY160 (WT) on lead indicator medium. (C) Silencing of URA3 inserted near the left telomere of chromosome VII depended on functional NatA. Serial dilutions of strains AEY1017 (WT) and AEY2371 (nat1Δ) were assayed on 5-FOA containing medium counterselecting for URA3-expressing cells.

 Fig. 3.2: The silencing function of NatA was genetically linked to ORC1. (A) The deletion of the binding site for Rap1, but not for ORC or Abf1, from HMR-E disrupted HMR silencing in nat1Δ mutants. HMR silencing was tested by the α-mating ability of wild-type and nat1Δ strains with HMR-E lacking the binding site for ORC (AEY84, AEY2146), Rap1 (AEY81, AEY2144) and Abf1 (AEY71, AEY2148). Results from quantitative mating assays are given relative to a value of 1.0 for AEY2. (B) Tethered silencing by Orc1, but not the other ORC subunits was independent of NAT1 and required SIR1. In MATα strains AEY1275 (WT), AEY1276 (nat1Δ) and AEY 2947 (nat1Δsir1Δ), the ORC binding site of the synthetic HMR-E silencer was replaced by five Gal4-binding sites (HMR SSΔI, 5xGal4-RAP-ABF). The strains carried plasmids encoding the Gal4 DNA binding domain fused N-terminally to Orc1 (5-267aa) (pAE408), Orc2 (pAE108), Orc3 (pAE595), Orc4 (pAE597), Orc5 (pAE109), Orc6 (pAE516) and Sir1 (pAE100) and were tested for HMR silencing in patch-mating assays.

 Fig. 3.3: Orc1 was N-terminally acetylated by NatA. (A) The isoelectric point (pI) of the Orc1 N-terminus shifted to a more basic pH either by the deletion of NAT1 or by the mutation of the penultimate residue alanine to valine or proline. Whole cell protein extracts of strains AEY2719 (WT), AEY2758 (nat1Δ), AEY3107 (orc1-A2P) and AEY3110 (orc1-A2V) were applied to IEF and SDS gels. TAP-tagged Orc1 (amino acids 1-250) was detected in subsequent immunoblots using the PAP antibody. The faster migrating band in the SDS gel was identified as Orc1 by MALDI-TOF analysis and probably is a proteolytic fragment. (B) Theoretical molecular mass of N-terminal peptides of Orc1 generated by proteolysis with AspN or GluC endopeptidase. The molecular mass as calculated using (http://us.expasy.org/tools/peptide-mass.html) increases by 42 Da due to N^α‑acetylation. (C) MALDI time-of-flight mass spectra of Orc1-TAP derived from a wild-type, but not from a nat1Δ strain, identified the mass of an acetylated N-terminal peptide of Orc1. Orc1-TAP was purified for MALDI-TOF analysis from AEY2719 (WT) and AEY2758 (nat1Δ). Data obtained from the AspN and GluC cleaved samples were consistent for each strain with minimal differences to the theoretical value due to the precision of measurements. (D) The MALDI-TOF spectrum of Orc1-TAP from the nat1Δ strain, but not from wild-type strain, contained the mass of an unacetylated N‑terminal Orc1 peptide. Analysis was performed as in Fig. 3.3C.

Fig. 3.4: The N-terminal peptide of Orc1, whose identity was verified by fragmentation, lacked the initial methionine. (A) A mass corresponding to the Orc1 N‑terminal peptide including the initial methionine was detected neither in the wild-type nor in the nat1Δ derived probe. MALDI-TOF spectra of AspN cleaved Orc1-TAP were obtained as in Fig. 3.3C. The result was confirmed by the data of the GluC cleaved samples (not shown). (B) The sequence of the nat1Δ-derived 560.47 Da peptide corresponded to the N-terminus of Orc1. The peptide was sequenced by fragmentation in post-source decay MALDI analysis. The detected N-terminal sequence ions AK (b2=200), AKT (b3=301), AKTL (b4=414), and C-terminal sequence ions K (y1=147), LK (y2=260), TLK (y3=361) and KTLK (y4=489) added up to the amino acid sequence AKTLK of the Orc1 N‑terminus.

 Fig. 3.5: N^α-acetylation of Orc1 was essential for telomeric silencing. (A) A URA3 gene inserted near the left telomere of chromosome VII was derepressed in unacetylated orc1‑A2P and orc1‑A2V mutants. In these mutants as well as in the nat1Δ mutant, the telomeric effect was not suppressed by the overexpression of SSB1. URA3 expression was tested in serial dilution assays of strains AEY1017 (ORC1), AEY3038 (orc1‑A2V), AEY3105 (orc1‑A2P), and AEY2371 (nat1Δ) on 5-FOA containing medium. For SSB1 overexpression, strains were transformed with pAE964. (B) The loss of N^α‑acetylation of Orc1 did not impair silencing of HML and HMR SS ΔI. Patch-mating assays were performed to test HML silencing using MATa strains AEY2867 (ORC1), AEY3102 (orc1‑A2P), AEY2913 (orc1‑A2V), and AEY2912 (nat1Δ), and to test HMR SS ΔI silencing using MATα strains AEY2866 (ORC1), AEY3103 (orc1-A2P), AEY2903 (orc1-A2V), and AEY2916 (nat1Δ). (C) nat1Δ, but not unacetylated orc1, caused the slight derepression of ADE2 inserted at the HMR locus. Serial dilutions of strains AEY743 (WT), AEY3101 (orc1‑A2P), AEY2721 (orc1‑A2V) and AEY3109 (nat1Δ) were grown on medium lacking adenine.

 Fig. 3.6: Orc1 was present in equal amounts in a wild-type and a nat1Δ strain. Whole cell protein extracts of strains AEY3068 (NAT1) and AEY3070 (nat1Δ) expressing HA-tagged ORC1 were loaded on a SDS gel as samples of 22 μg (lanes 1 and 4), 11μg (lanes 2 and 5), and 5.5μg (lanes 3 and 6) protein. HA-tagged Orc1 was detected in a subsequent Western blot using an α‑HA antibody.

 Fig. 3.7: nat1Δ affected the replication function of the ORC complex independently of Orc1. (A) Unacetylated orc1 mutants were not temperature-sensitive and thereby differed from nat1Δ. Serial dilutions of strains AEY2866 (ORC1), AEY 3103 (orc1‑A2P), AEY2903 (orc1‑A2V) and AEY2916 (nat1Δ) were grown for two days on complete medium at the indicated temperatures. (B) orc2‑1 nat1Δ double mutants were not viable. orc2‑1 and orc2‑1 nat1Δ segregants from an orc2‑1 nat1Δ double heterozygous cross (AEY24 crossed with AEY1227) were grown for five days on complete medium at 23°C. (C) Viability of the orc2-1 nat1Δ double mutant was rescued by plasmid-borne ORC2. AEY3161 (orc2‑1 nat1Δ pURA3‑ORC2) transformed either with pJR1818 (pHIS3‑ORC2) (Fox et al. 1997) or with pRS313 (vector) was tested for ORC2 dependence by counterselection for pURA3‑ORC2 on 5‑FOA medium. Figures B and C are courtesy of Ann Ehrenhofer-Murray.

 Fig. 3.8: SUM1-1 nat1Δ double mutants were inviable.  SUM1-1 nat1Δ segregants of tetrads dissected from a cross between SUM1‑1 (AEY1224) and nat1Δ (AEY3008) are marked by arrows. Figure is courtesy of A. Ehrenhofer-Murray.

 Fig. 3.9: N-terminal truncations of Orc1 impaired HMR silencing. (A) ADE2 inserted at the HMR locus was derepressed when the N‑terminus of Orc1 was shortened by 51 or 100 amino acids and when the region between 29 and 51 amino acids was deleted. Serial dilutions of strains AEY743 (WT), AEY2587 (Δ1‑10), AEY2589 (Δ1‑28), AEY2333 (Δ1‑51), AEY2335 (Δ1‑100) and AEY2760 (Δ29‑51) were grown on medium lacking adenine to test ADE2 expression. (B) In contrast to HML silencers, synthetic HMR silencer variants were affected by the deletion of the N-terminal 28, 51 or 100 amino acids of Orc1, and the region between amino acids 29 and 51. Patch-mating assays were performed to test silencing at HMR SS ΔI and HMR SS abf1 ^- ΔI using MATα strains AEY2866 and 2864 (WT), AEY2877 and 2883 (Δ1‑10), AEY2907 and 2908 (Δ1‑28), AEY2879 and 2904 (Δ1‑51), AEY2880 and 2905 (Δ1‑100) and AEY2910 (Δ29‑51). HML silencing was tested in patch-mating assays of MATa strains AEY2867 (WT), AEY2887 (Δ1‑10), AEY2937 (Δ1‑28), AEY2888 (Δ1‑51), AEY2889 (Δ1‑100) and AEY2911 (Δ29‑51).

 Fig. 3.10: α‑factor response was abrogated by nat1Δ and diminished in orc1 mutants lacking 51 or 100 amino acids of the N‑terminus or the region of amino acids 29 to 51. 100 cells per strain were analyzed individually after 18 hours of exposure to α‑factor. The ability to respond to α‑factor was measured by the formation of one mating projection per cell (shmoo), whereas α‑factor resistance was indicated by budding and subsequent colony formation. Structures emerging from alternated shmooing and budding are referred to as shmoo clusters. Results of at least three individual experiments per strain are given with respective standard deviations. MATa strains used were depicted in figures 3.5(B) and 3.9(B).

 Fig. 3.11: Deletion of 51 amino acids from the Orc1 N‑terminus enhanced the α‑factor response defect of sir1Δ mutants. The ability to respond to α‑factor was tested as described in Fig. 3.10 using MATa strains AEY2867 (WT), AEY2888 (Δ1‑51), AEY3000 (sir1Δ), AEY3002 (orc1 Δ1‑51 sir1Δ #1) and AEY3003 (orc1 Δ1‑51 sir1Δ #2).

 Fig. 3.12: N‑terminal truncations of 28, 51 and 100 amino acids, as well as removing the region of amino acids 29 to 51 of Orc1, reduced telomeric silencing. Silencing of URA3 inserted near the left telomere of chromosome VII was tested in serial dilution assays of strains AEY1017 (WT), AEY3031 (Δ1‑10), AEY3040 (Δ1‑28), AEY3032 (Δ1‑51), AEY3034 (Δ1‑100) and AEY3036 (Δ29‑51) on 5‑FOA containing medium counterselecting for URA3 expressing cells.

 Fig.3.13: N-terminal deletions of up to 100 amino acids of Orc1 did not affect the temperature sensitivity of the respective mutants. Strains AEY743 (WT), AEY2587 (Δ1‑10), AEY2589 (Δ1‑28), AEY2333 (Δ1‑51), AEY2335 (Δ1‑100) and AEY2760 (Δ29‑51) were grown for two days on complete medium at the indicated temperatures.

 Fig. 3.14: Deletion of the N-terminal 51 amino acids abrogated the ability of Orc1 to interact with Sir1 in a two‑hybrid assay. (A) The reporter genes ADE2 and HIS3 were induced in two-hybrid strain AH109 by simultaneous expression of Gal4_BD‑Sir1(346‑678) and Gal4_AD‑Orc1(1‑235), but not Gal4_AD‑Orc1(52‑235). The bait-vector pAE952 was co-transformed with a prey-vector containing either no insert (pAE953), full-length Orc1 (pAE951) or Orc1(52‑235) (pAE966). Two-hybrid interaction was tested by monitoring the expression of HIS3 and ADE2 in serial dilution assays on media lacking histidine or adenine, respectively. (B) The prey protein of Orc1(52‑235) was as abundant as that of Orc1(1‑235) in the two-hybrid strains AEY3028 (Orc1(1‑235)) and AEY3099 (Orc1(52‑235)). A SDS gel of whole cell extracts was analyzed by Western blotting with antibodies against the HA epitope that was part of the prey vector.

Fig. 3.15: Sir3 was acetylated by NatA. (A) The isoelectric point of the Sir3 N-terminus became more basic upon the deletion of NAT1. Whole cell extracts of strains AEY3171 (WT) and AEY3173 (nat1Δ) expressing TAP-tagged Sir3 peptides (amino acids 1‑235) were analyzed as described in Fig. 3.3A. (B) The silencing defect at the synthetic HMR silencer caused by the mutated penultimate amino acid of Sir3 was not enhanced by missing N^α‑acetylation of Orc1. HML silencing was assayed in patch mating assays of MATa strains AEY3144 (sir3‑A2T), AEY3147 (orc1‑A2P sir3‑A2T), AEY3148 (orc1‑A2V sir3‑A2T) and AEY2912 (nat1Δ). Likewise, synthetic HMR silencing was tested in MATα strains AEY3145 (sir3‑A2T), AEY3149 (orc1‑A2P sir3‑A2T), AEY3151 (orc1‑A2V sir3‑A2T), and AEY2916 (nat1Δ).

Fig. 3.16: The association of GFP-tagged Sir3 with telomeric foci was abrogated in nat1Δ cells. Strains AEY160 (WT) and AEY2786 (nat1Δ) transformed with pAE580 were examined by fluorescent microscopy using a FITC filter. Bar, 2 μm.

 Fig. 3.17: The mating defect of nat1Δ was not rescued by overexpressed ORC1. (A) ORC1 expressed under control of the GPD-promoter on a 2μ-based plasmid rescued the silencing defect of orc1Δ1-51 at HMR::ADE2. Strain AEY2333 (orc1Δ1-51) was transformed with pAE866 (p2μGPDp-ORC1) and grown in serial dilutions on medium lacking adenine. Strain AEY743 (WT) was tested in parallel for comparison. (B) In strain AEY1273 (-2μGPDp-ORC1), more Orc1 protein was abundantupon transformation with the overexpressing construct pAE866 (+ 2μGPDp-ORC1). Equal amounts of protein from whole cell extracts were applied to a SDS gel and subsequently to Western blot analysis using antibodies against Orc1 and β tubuline (as loading control). (C) Overexpression of ORC1 did not increase silencing of the synthetic HMR silencer in a nat1Δ background. HMR silencing was determined by the mating ability of MATα-strain AEY1273 (HMR SSΔI nat1Δ) transformed with pRS316 (vector), pAE303 (NAT1) or pAE866 (2μGPDp-ORC1).

 Fig. 4.1: The crystal structure of the N-terminal domain of Orc1. (A) Ribbon presentation. (B) Topology diagram showing the fold of the structure and deletion sites of the orc1 mutants investigated in this study. The BAH core structure is colored blue, the H-domain is shown in magenta, N- and C-terminal helices are shown in red. (adapted from Zhang et al. 2002)

 Fig. 4.2: Model of protein interactions at the N-terminus of Orc1. In addition to Sir1, the Orc1 N-terminus interacts with another, yet unknown protein. This protein requires the N^α‑acetylation of Orc1 for binding and is specifically recruited in the context of telomeric silent chromatin, when Sir1 is absent. In contrast, Sir1 is recruited via the H‑domain of Orc1 specifically to the HM silencers. This binding does not require the N^α‑acetylation of Orc1, but it depends on the integrity of the N‑terminal region of 100 amino acids.