Characterization of the trp5-27 allele used to monitor drug-induced mitotic gene conversion in the Saccharomyces cerevisiae tester strain D7

Mitotic gene conversions, among other recombinagenic events, can play an important role in the multistep process of carcinogenesis. The ability of chemicals to induce such gene conversions can easily be monitored in the Saccharomyces cerevisiae tester strain YHE2, a derivative of strain D7. For the detection of drug-induced gene conversions, two mutations in the TRP5 locus are used, trp5-12 and trp5-27. Here we report on the characterization of the stable allele trp5-27. Our analysis revealed two relevant mutations in trp5-27: (a) a transition C to T at position 121 after ATG that results in an amber stop codon and abolishes gene expression and (b) a transversion A to T at position 1555 that creates an ochre stop codon. Simultaneous amber and ochre suppression with the suppressors SUP3 and SUP11, respectively, was capable of relieving the tryptophan-requiring phenotype of strains carrying the trp5-27 allele. These findings have implications on the length of gene conversion tracts in conversion events between trp5-12 and trp5-27: conversion tracts can cover several kilobases, if the site of the mutation in trp5-12 lies outside of the positions mutated in trp5-27. Conversely, the maximal length is limited to 1435 bp, if the mutation in trp5-12 is located between the positions mutated in trp5-2

Control of replication initiation by the Sum1/Rfm1/Hst1 histone deacetylase

<p>Abstract</p> <p>Background</p> <p>Replication initiation at origins of replication in the yeast genome takes place on chromatin as a template, raising the question how histone modifications, for instance histone acetylation, influence origin firing. Initiation requires binding of the replication initiator, the Origin Recognition Complex (ORC), to a consensus sequence within origins. In addition, other proteins bind to recognition sites in the vicinity of ORC and support initiation. In previous work, we identified Sum1 as an origin-binding protein that contributes to efficient replication initiation. Sum1 is part of the Sum1/Rfm1/Hst1 complex that represses meiotic genes during vegetative growth via histone deacetylation by the histone deacetylase (HDAC) Hst1.</p> <p>Results</p> <p>In this study, we investigated how Sum1 affected replication initiation. We found that it functioned in initiation as a component of the Sum1/Rfm1/Hst1 complex, implying a role for histone deacetylation in origin activity. We identified several origins in the yeast genome whose activity depended on both Sum1 and Hst1. Importantly, <it>sum1</it>Δ or <it>hst1</it>Δ caused a significant increase in histone H4 lysine 5 (H4 K5) acetylation levels, but not other H4 acetylation sites, at those origins. Furthermore, mutation of lysines to glutamines in the H4 tail, which imitates the constantly acetylated state, resulted in a reduction of origin activity comparable to that in the absence of Hst1, showing that deacetylation of H4 was important for full initiation capacity of these origins.</p> <p>Conclusion</p> <p>Taken together, our results demonstrate a role for histone deacetylation in origin activity and reveal a novel aspect of origin regulation by chromatin. These results suggest recruitment of the Sum1/Rfm1/Hst1 complex to a number of yeast origins, where Hst1 deacetylated H4 K5.</p

Interactions within the mammalian DNA methyltransferase family

BACKGROUND: In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. RESULTS: The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. CONCLUSIONS: The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase

Functional analysis of archaeal MBF1 by complementation studies in yeast

Contains fulltext : 95916.pdf (publisher's version ) (Open Access)BACKGROUND: Multiprotein-bridging factor 1 (MBF1) is a transcriptional co-activator that bridges a sequence-specific activator (basic-leucine zipper (bZIP) like proteins (e.g. Gcn4 in yeast) or steroid/nuclear-hormone receptor family (e.g. FTZ-F1 in insect)) and the TATA-box binding protein (TBP) in Eukaryotes. MBF1 is absent in Bacteria, but is well- conserved in Eukaryotes and Archaea and harbors a C-terminal Cro-like Helix Turn Helix (HTH) domain, which is the only highly conserved, classical HTH domain that is vertically inherited in all Eukaryotes and Archaea. The main structural difference between archaeal MBF1 (aMBF1) and eukaryotic MBF1 is the presence of a Zn ribbon motif in aMBF1. In addition MBF1 interacting activators are absent in the archaeal domain. To study the function and therefore the evolutionary conservation of MBF1 and its single domains complementation studies in yeast (mbf1Delta) as well as domain swap experiments between aMBF1 and yMbf1 were performed. RESULTS: In contrast to previous reports for eukaryotic MBF1 (i.e. Arabidopsis thaliana, insect and human) the two archaeal MBF1 orthologs, TMBF1 from the hyperthermophile Thermoproteus tenax and MMBF1 from the mesophile Methanosarcina mazei were not functional for complementation of an Saccharomyces cerevisiae mutant lacking Mbf1 (mbf1Delta). Of twelve chimeric proteins representing different combinations of the N-terminal, core domain, and the C-terminal extension from yeast and aMBF1, only the chimeric MBF1 comprising the yeast N-terminal and core domain fused to the archaeal C-terminal part was able to restore full wild-type activity of MBF1.However, as reported previously for Bombyx mori, the C-terminal part of yeast Mbf1 was shown to be not essential for function. In addition phylogenetic analyses revealed a common distribution of MBF1 in all Archaea with available genome sequence, except of two of the three Thaumarchaeota; Cenarchaeum symbiosum A and Nitrosopumilus maritimus SCM1. CONCLUSIONS: The absence of MBF1-interacting activators in the archaeal domain, the presence of a Zn ribbon motif in the divergent N-terminal domain of aMBF1 and the complementation experiments using archaeal- yeast chimeric proteins presented here suggests that archaeal MBF1 is not able to functionally interact with the transcription machinery and/or Gcn4 of S. cerevisiae. Based on modeling and structural prediction it is tempting to speculate that aMBF1 might act as a single regulator or non-essential transcription factor, which directly interacts with DNA via the positive charged linker or the basal transcription machinery via its Zn ribbon motif and the HTH domain. However, also alternative functions in ribosome biosynthesis and/or functionality have been discussed and therefore further experiments are required to unravel the function of MBF1 in Archaea

Design of a minimal silencer for the silent mating-type locus HML of Saccharomyces cerevisiae

The silent mating-type loci HML and HMR of Saccharomyces cerevisiae contain mating-type information that is permanently repressed. This silencing is mediated by flanking sequence elements, the E- and I-silencers. They contain combinations of binding sites for the proteins Rap1, Abf1 and Sum1 as well as for the origin recognition complex (ORC). Together, they recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci. However, the HM silencers exhibit considerable functional redundancy, which has hampered the identification of further silencing factors. In this study, we constructed a synthetic HML-E silencer (HML-SS ΔI) that lacked this redundancy. It consisted solely of Rap1 and ORC-binding sites and the D2 element, a Sum1-binding site. All three elements were crucial for minimal HML silencing, and mutations in these elements led to a loss of Sir3 recruitment. The silencer was sensitive to a mutation in RAP1, rap1-12, but less sensitive to orc mutations or sum1Δ. Moreover, deletions of SIR1 and DOT1 lead to complete derepression of the HML-SS ΔI silencer. This fully functional, minimal HML-E silencer will therefore be useful to identify novel factors involved in HML silencing

Genome-wide H4 K16 acetylation by SAS-I is deposited independently of transcription and histone exchange

The MYST HAT Sas2 is part of the SAS-I complex that acetylates histone H4 lysine 16 (H4 K16Ac) and blocks the propagation of heterochromatin at the telomeres of Saccharomyces cerevisiae. In this study, we investigated Sas2-mediated H4 K16Ac on a genome-wide scale. Interestingly, H4 K16Ac loss in sas2Δ cells outside of the telomeric regions showed a distinctive pattern in that there was a pronounced decrease of H4 K16Ac within the majority of open reading frames (ORFs), but little change in intergenic regions. Furthermore, regions of low histone H3 exchange and low H3 K56 acetylation showed the most pronounced loss of H4 K16Ac in sas2Δ, indicating that Sas2 deposited this modification on chromatin independently of histone exchange. In agreement with the effect of Sas2 within ORFs, sas2Δ caused resistance to 6-azauracil, indicating a positive effect on transcription elongation in the absence of H4 K16Ac. In summary, our data suggest that Sas2-dependent H4 K16Ac is deposited into chromatin independently of transcription and histone exchange, and that it has an inhibitory effect on the ability of PolII to travel through the body of the gene

The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data

Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions

The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae

The acetylation state of histones plays a central role in determining gene expression in chromatin. The reestablishment of the acetylation state of nucleosomes after DNA replication and chromatin assembly requires both deacetylation and acetylation of specific lysine residues on newly incorporated histones. In this study, the MYST family acetyltransferase Sas2 was found to interact with Cac1, the largest subunit of Saccharomyces cerevisiae chromatin assembly factor-I (CAF-I), and with the nucleosome assembly factor Asf1. The deletions of CAC1 (cac1Δ), ASF1 (asf1Δ), and SAS2 (sas2Δ) had similar effects on gene silencing and were partially overlapping. Furthermore, Sas2 was found in a nuclear protein complex that included Sas4 and Sas5, a homolog of TAF(II)30. This complex, termed SAS-I, was also found to contribute to rDNA silencing. Furthermore, the observation that a mutation of H4 lysine 16 to arginine displayed the identical silencing phenotypes as sas2Δ suggested that it was the in vivo target of Sas2 acetylation. In summary, our data present a novel model for the reestablishment of acetylation patterns after DNA replication, by which SAS-I is recruited to freshly replicated DNA by its association with chromatin assembly complexes to acetylate lysine 16 of H4

A novel yeast silencer. the 2mu origin of Saccharomyces cerevisiae has HST3-, MIG1- and SIR-dependent silencing activity.

Silencing in Saccharomyces cerevisiae is found at the mating-type loci HMR and HML, in subtelomeric regions, and at the rDNA locus. Repressed chromatin is built up by the recruitment of the Sir proteins via their interaction with DNA-binding proteins that bind to silencers. Here, we have performed a genetic screen for novel sequence elements within the yeast genome that display silencing activity. We isolated as a novel silencer element the origin of replication from the endogenous 2mu plasmid (2mu ARS). 2mu ARS-mediated silencing was dependent upon the Sir proteins, the origin recognition complex (ORC), and Hst3, a Sir2 histone deacetylase homolog, suggesting that it constituted a novel class of silencing in yeast. Moreover, 2mu ARS carried a binding site for Mig1, a transcriptional repressor of glucose-regulated genes. Both the Mig1-binding site and the MIG1 gene were necessary for full silencing activity of 2mu ARS. Furthermore, Hst3 was physically present at 2mu ARS in a silencing context as well as at the endogenous 2mu plasmid. Also, Hst3 regulated the repression of the flipase gene, although this was likely an indirect effect of HST3 on FLP1 expression

A role for the Saccharomyces cerevisiae RENT complex protein Net1 in HMR silencing.

Silencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD(+)-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2micro-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR