New Orthogonal Transcriptional Switches Derived from Tet Repressor Homologues for <i>Saccharomyces cerevisiae</i> Regulated by 2,4-Diacetylphloroglucinol and Other Ligands

Here we describe the development of tightly regulated expression switches in yeast, by engineering distant homologues of <i>Escherichia coli</i> TetR, including the transcriptional regulator PhlF from <i>Pseudomonas</i> and others. Previous studies demonstrated that the PhlF protein bound its operator sequence (phlO) in the absence of 2,4-diacetylphloroglucinol (DAPG) but dissociated from phlO in the presence of DAPG. Thus, we developed a DAPG-Off system in which expression of a gene preceded by the phlO-embedded promoter was activated by a fusion of PhlF to a multimerized viral activator protein (VP16) domain in a DAPG-free environment but repressed when DAPG was added to growth medium. In addition, we constructed a DAPG-On system with the opposite behavior of the DAPG-Off system; <i>i.e.</i>, DAPG triggers the expression of a reporter gene. Exposure of DAPG to yeast cells did not cause any serious deleterious effect on yeast physiology in terms of growth. Efforts to engineer additional Tet repressor homologues were partially successful and a known mammalian switch, the <i>p</i>-cumate switch based on CymR from <i>Pseudomonas</i>, was found to function in yeast. Orthogonality between the TetR (doxycycline), CamR (d-camphor), PhlF (DAPG), and CymR (<i>p</i>-cumate)-based Off switches was demonstrated by evaluating all 4 ligands against suitably engineered yeast strains. This study expands the toolbox of “On” and “Off” switches for yeast biotechnology

Multichange Isothermal Mutagenesis: A New Strategy for Multiple Site-Directed Mutations in Plasmid DNA

<u>M</u>ultichange <u>ISO</u>thermal (MISO) mutagenesis is a new technique allowing simultaneous introduction of multiple site-directed mutations into plasmid DNA by leveraging two existing ideas: QuikChange-style primers and one-step isothermal (ISO) assembly. Inversely partnering pairs of QuikChange primers results in robust, exponential amplification of linear fragments of DNA encoding mutagenic yet homologous ends. These products are amenable to ISO assembly, which efficiently assembles them into a circular, mutagenized plasmid. Because the technique relies on ISO assembly, MISO mutagenesis is additionally amenable to other relevant DNA modifications such as insertions and deletions. Here we provide a detailed description of the MISO mutagenesis concept and highlight its versatility by applying it to three experiments currently intractable with standard site-directed mutagenesis approaches. MISO mutagenesis has the potential to become widely used for site-directed mutagenesis

A high throughput mutagenic analysis of yeast sumo structure and function

<div><p>Sumoylation regulates a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using <i>S</i>. <i>cerevisiae</i>, a model organism with a single essential SUMO gene (<i>SMT3</i>), we developed a library of >250 mutant strains with single or multiple amino acid substitutions of surface or core residues in the Smt3 protein. By screening this library using plate-based assays, we have generated a comprehensive structure-function based map of Smt3, revealing essential amino acid residues and residues critical for function under a variety of genotoxic and proteotoxic stress conditions. Functionally important residues mapped to surfaces affecting Smt3 precursor processing and deconjugation from protein substrates, covalent conjugation to protein substrates, and non-covalent interactions with E3 ligases and downstream effector proteins containing SUMO-interacting motifs. Lysine residues potentially involved in formation of polymeric chains were also investigated, revealing critical roles for polymeric chains, but redundancy in specific chain linkages. Collectively, our findings provide important insights into the molecular basis of signaling through sumoylation. Moreover, the library of Smt3 mutants represents a valuable resource for further exploring the functions of sumoylation in cellular stress response and other SUMO-dependent pathways.</p></div

Analysis of <i>smt3</i> SIM binding mutant alleles.

<p>(A) The identified lethal and conditional <i>smt3</i> mutants found within the SIM binding surface mapped onto the β2-α1 region of Smt3 (PDB: 1EUV). Residues that gave rise to lethal and conditional phenotypes are boxed in red and magenta, respectively. (B) SIM binding mutants are stress sensitive. The indicated strains were cultured overnight and serially diluted and spotted onto plates with or without hydroxyurea (HU). The plates were incubated at 30°C or 39°C, as indicated. (C) The <i>smt3</i> I35A mutant allele accumulates unusual conjugates. Wild type and <i>smt3</i> I35A strains were grown to mid-log phase, diluted 4 fold then shifted to 39°C for 20 hrs. Samples were collected at 0 and 20 hr time points and analyzed by immunoblot analysis. The stacking portion of the gel was left intact so that ultra-high molecular mass conjugates could be visualized. (D) The <i>smt3</i> I35A mutant allele exhibits cell cycle defects. Wild type and <i>smt3</i> I35A strains were grown to mid-log phase at 30°C and then shifted to 39°C for 20 hrs. Cells were collected at 20 hrs, permeabilized and stained with DAPI. Each bar represents the average of 3 independent experiments in which at least 150 cells were counted per experiment. Vertical bars indicate the standard error. Astericks denote a p-value < 0.05. (E) Phosphorylation of T42 and T43 is not critical for <i>smt3</i> function. The <i>smt3Δ</i> shuffle strain was transformed with plasmids coding for the indicated <i>Smt3</i> alleles. The transformants were grown overnight, serially diluted and spotted onto selective media in the presence or absence of 5-FOA at 30°C and 37°C.</p

Analysis of lethal <i>smt3</i> mutant alleles.

<p>(A) The identified lethal mutations mapped onto the Smt3 crystal structure (PDB: 1EUV). Mutated residues giving rise to lethal phenotypes are highlighted red. Also highlighted are residues predicted or known to be important for conjugation (yellow), deconjugation (blue), conjugation and deconjugation (green) or SIM binding (orange). Lethal mutations in residues important for conjugation, deconjugation or SIM binding are boxed with appropriate corresponding colors. *R71E and G98A mutations were episome remedial. (B) Analysis of the expression and conjugation profiles of lethal <i>smt3</i> mutant alleles. Mutant alleles were expressed in a SUMO1 integrated strain and then analyzed by immunoblot analysis. The asterisk and high molecular mass species seen in the vector only control (also in C and D) represent non-specific, cross-reacting proteins. (C) Analysis of deconjugation in response to ATP depletion. Lethal mutant alleles that form ultra-high molecular mass conjugates were expressed in a SUMO1 integrated strain. Cultures at mid-log phase were grown in normal medium (-) or ATP depletion medium (+) containing sodium azide and 2-deoxyglucose for 10 minutes. Cell lysates were analyzed by immunoblot analysis. (D) Analysis of deconjugation and conjugation following ATP depletion and restoration. The lethal mutants not forming ultra-high molecular mass conjugates were expressed in a SUMO1 integrated strain. Cultures at mid-log phase were grown in normal (-) or ATP depletion media (+) for 10 minutes. Cells were then allowed to recover for 10 minutes in normal medium (+’). Cell lysates were analyzed by immunoblot analysis. (E) Analysis of Smt3 protein localization. The indicated Smt3 proteins were expressed by transforming a SUMO1 expressing strain with the indicated constructs. Transformants were grown to mid-log phase, fixed, spheroplasted and permeabilized. Smt3 localization was determined by immunofluorescence microscopy. DNA was labeled with DAPI. (F) Co-localization of Smt3 foci and Cdc48. A GFP-Cdc48 expressing strain was transformed with empty vector or vectors coding for wild type or F65A mutant Smt3. Transformants were grown to mid-log phase and fixed, spheroplasted and permeabilized. Smt3 and GFP-Cdc48 localization were determined by immunofluorescence microscopy. DNA was labeled with DAPI.</p

Development of a versatile library of yeast SUMO mutants.

<p>(A) Schematic illustration of the <i>SMT3</i> base construct within the pRS413 vector. Positions of the 5’ and 3’ <i>SMT3</i> flanking regions, <i>SMT3</i>, <i>LEU2</i>, <i>ADH1</i> 3’UTR, useful restriction sites and the TAG region, are shown. (B) Illustration of the amino acid substitutions present in the <i>SMT3</i> mutant collection. Individual wild type residues (in white boxes) were substituted with residues shown directly below. (C) The 9 lysine residues in Smt3 mapped onto the Smt3 crystal structure (PDB: 1EUV). (D) Table summarizing the lysine to arginine substitutions included in the <i>SMT3</i> mutant collection. (E) Summary of N- and C-terminal deletions included in the <i>SMT3</i> mutant collection.</p

Analysis of <i>SMT3</i> deletion complementation by human SUMOs.

<p>(A) Sequence alignments between Smt3 and human SUMO paralogs. (B) SUMO1, but not SUMO2 or SUMO3, complement <i>SMT3</i> deletion. A <i>smt3</i>Δ strain harboring a copy of wild-type <i>SMT3</i> on a <i>URA3</i>-based plasmid (pRS315) was transformed with the indicated plasmids (<i>pSUMO</i>) containing a <i>HIS3</i> selectable marker. The cells were serially diluted and spotted onto histidine minus plates in the absence or presence of 5-FOA and cultured at 30°C. (C) SUMO1 expressing strains are temperature sensitive. Strains containing integrated constructs encoding <i>SMT3</i>, <i>SUMO1</i> precursor or <i>SUMO1</i> mature protein (<i>SUMO1</i>^<i>GG</i>) were grown at 30°C and 39°C for 2 days. (D) Immunoblot analysis of SUMO expression and conjugation in wild type yeast transformed with the indicated plasmids (<i>pSUMO</i>) or in a strain containing integrated SUMO1 (<i>SUMO1</i>). (E) SUMO2 is not activated by the <i>S</i>. <i>cerevisiae</i> E1 activating enzyme. Purified, recombinant <i>S</i>. <i>cerevisiae</i> His-Uba2/Aos1 E1 heterodimer was incubated with Smt3, SUMO1 or SUMO2 in the presence of ATP. Reactions were stopped at the indicated time points by addition of SDS sample buffer with or without β-mercaptoethanol (βME). Uba2~SUMO thioester intermediates were detected by immunoblot analysis. (F) Schematic illustrating the precursor processing and E1 activation defects (red arrows) associated with SUMO2 expression in <i>S</i>. <i>cerevisiae</i>.</p

Analysis of <i>smt3</i> K/R mutant alleles.

<p>(A) and (B) Strains expressing the indicated <i>smt3</i> alleles were grown overnight, serially diluted then spotted onto plates in the presence or absence of HU. The plates were incubated at 30°C or 39°C, as indicated.</p

Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins

<div><p>Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as <i>cdc6-4</i>, are suppressed by deletion of either <i>SIR2</i> or <i>SIR3</i>. While these and other observations indicate that <i>SIR2</i>,<i>3</i> can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a <i>SIR2</i> deletion rescued MCM complex loading at ~80% of euchromatic origins in <i>cdc6-4</i> cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a <i>SIR2</i>-dependent manner in wild type (i.e. <i>CDC6</i>) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin–<i>SIR2</i>-dependent H4K16 hypoacetylation, Sir2, and Sir3 –correlated with how strongly a <i>SIR2</i> deletion suppressed the MCM loading defect in <i>cdc6-4</i> cells. Finally, a screen for histone H3 and H4 mutants that could suppress the <i>cdc6-4</i> growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins.</p></div

Acetylated H4K16 is depleted from origin-adjacent nucleosomes.

<p><b>A.</b> Two groups of loci were analyzed, origins, defined as experimentally-confirmed origins with a confirmed or high-confidence ORC site, n = 259, and intergenic non-origin loci with ORC site matches but for which no origin activity has been observed, n = 179 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref043" target="_blank">43</a>]. These two different groups of loci were compared to determine whether chromatin-states at origins were specific for origin function as opposed to being the result of underlying AT-rich sequence elements present in origins. “ORC in vitro” refers to loci that were bound by purified ORC in a genomic electrophoretic mobility assay [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref073" target="_blank">73</a>]; “ORC in vivo” and “MCM in vivo” refer to the fraction of sites in these two groups that were detected by ORC- or MCM-ChIP, respectively. <b>B.</b> The WebLogo consensus for the ORC sites (or matches) in origins and non-origins, respectively, are shown above the diagram of the fragments used in the analyses of adjacent nucleosomes. Each fragment analyzed was oriented with the T-rich strand of the ORC site 5’ to 3’ on the top strand, and the first nucleotide of the ORC site was designated as position “0”. The fragments were 1201 bp such that six proximal nucleosomes, shown as black ovals, three on each side of the ORC site, were assessed. <b>C.</b> Nucleosome occupancy surrounding the origin and non-origin nucleosome depleted regions are shown using the MNase-ChIP-Seq nucleosome occupancy data from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref032" target="_blank">32</a>]. <b>D.</b> Normalized H4K16ac and H3K9ac for each of the six nucleosomes for the two different groups of loci examined. P-values are derived from Student’s T-test comparing the mean of acetylation status between each nucleosome to the mean acetylation status of nucleosomes from the 239 intergenic control loci. <b>E.</b> <i>SIR2</i>-responsiveness was defined as the ratio of the MCM ChIP-Seq signal in <i>cdc6-4 sir2Δ</i> to <i>sir2Δ</i> cells. The origins were ranked based on <i>SIR2</i>-responsiveness and then divided into quintiles, with the high quintile containing the most <i>SIR2</i>-responsive origins. <b>F.</b> H4K16ac status for the three quintiles of <i>SIR2</i>-responsive origins indicated in ‘<b>E</b>’ was determined as in ‘<b>D</b>’.</p