15 research outputs found
ARCHITECTURE OF DROSOPHILA DOSAGE COMPENSATION COMPLEX
In heterogametic organisms, male and female cells harbor structurally different sex chromosome pairs. The difference in the transcriptional output of these sex chromosomes is epigenetically balanced, a phenomenon dubbed as dosage compensation. In Drosophila, male cells up-regulate their one X chromosome roughly two times by the help of a molecular machine called MSL complex. This ribonucleoprotein enzymatic complex, specifically formed in male cells, localizes to the X-chromosome, changing its structure mostly by histone H4 Lysine 16 acetylation, thus enabling enhanced transcription. The details of how the complex finds the chromosome and how it regulates transcription are not thoroughly understood. In this thesis, with the help of X-ray crystallography, we derived point mutations on the scaffold Msl1 protein that create partial complexes to study the contribution of each subunit to X chromosome recognition, RNA integration and spreading alone the X chromosome. In the first part of this thesis, we focused on the PEHE region of Msl1 protein that binds Mof and Msl3. We generated point mutants of Msl1 that cannot bind Mof or Msl3. We showed that loss of either Mof or Msl3 prevents spreading of the MSL complex on the body of the X-linked genes whereas Msl1 promoter binding remained unaffected. We observed qualitative differences between high affinity sites (HAS), initial binding platforms of MSL complex, and noticed that promoter located HAS can bind Msl1 independent of Mof and Msl3 whereas other HAS depend on the presence of intact PEHE module for optimal binding. In the second part of the thesis, we examined the interaction of Msl1 and Msl2 and showed that Msl1 forms a homodimer through its coiled coil region and this homodimer creates a platform for Msl2 binding. Msl2 binding happens through helices surrounding the RING finger domain. We showed that Msl2 RING finger can function as a ubiquitin ligase, and Msl1 is an in vitro substrate of Msl2 ubiquitination. By point mutational analysis on Msl1 we showed that Msl1 forms a dimer independent of Msl2 in both male and female cells. Dimerization is required for Msl2 binding, roX2 RNA integration to the complex, X chromosome recognition and spreading along the body of X-linked genes. This clearly showed that functionality of MSL complex entirely depends on its dimeric configuration. We identified roX2 HAS as an elementary HAS where its recognition only happens through Msl1-Msl2 dimer interface. Furthermore we discovered that Msl1 binds to promoters in a dimer/Msl3/Mof/Msl2 independent fashion. This binding occurs also at the autosomes and in both sexes suggesting a general function of Msl1 at promoters of Drosophila. We showed that promoters are also occupied by Msl2 but not by Msl3, indicating that Msl3 can have an important role for distinguishing promoter bound complex and canonical MSL complex. In order to support the in vivo importance of amino acid residues that had been point mutated, we generated transgenic flies that express Msl1 and its mutated forms from the identical genomic location. We showed Msl1 mutants are unable to rescue the Msl1 null male lethality and also cause male specific lethality upon over-expression in wild type background confirming the importance of these mutated residues
Scarless Gene Tagging with One-Step Transformation and Two-Step Selection in Saccharomyces cerevisiae and Schizosaccharomyces pombe
Gene tagging with fluorescent proteins is commonly applied to investigate the localization and dynamics of proteins in their cellular environment. Ideally, a fluorescent tag is genetically inserted at the endogenous locus at the N- or C- terminus of the gene of interest without disrupting regulatory sequences including the 5’ and 3’ untranslated region (UTR) and without introducing any extraneous unwanted “scar” sequences, which may create unpredictable transcriptional or translational effects. We present a reliable, low-cost, and highly efficient method for the construction of such scarless C-terminal and N-terminal fusions with fluorescent proteins in yeast. The method relies on sequential positive and negative selection and uses an integration cassette with long flanking regions, which is assembled by two-step PCR, to increase the homologous recombination frequency. The method also enables scarless tagging of essential genes with no need for a complementing plasmid. To further ease high-throughput strain construction, we have computationally automated design of the primers, applied the primer design code to all open reading frames (ORFs) of the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe), and provide here the computed sequences. To illustrate the scarless N- and C-terminal gene tagging methods in S. cerevisiae, we tagged various genes including the E3 ubiquitin ligase RSP5, the proteasome subunit PRE1, and the eleven Rab GTPases with yeast codon-optimized mNeonGreen or mCherry; several of these represent essential genes. We also implemented the scarless C-terminal gene tagging method in the distantly related organism S. pombe using kanMX6 and HSV1tk as positive and negative selection markers, respectively, as well as ura4. The scarless gene tagging methods presented here are widely applicable to visualize and investigate the functional roles of proteins in living cells.United States. National Institutes of Health (NS087557)American Parkinson Disease Association, Inc
X chromosomal regulation in flies: when less is more
In Drosophila, dosage compensation of the single male X chromosome involves upregulation of expression of X linked genes. Dosage compensation complex or the male specific lethal (MSL) complex is intimately involved in this regulation. The MSL complex members decorate the male X chromosome by binding on hundreds of sites along the X chromosome. Recent genome wide analysis has brought new light into X chromosomal regulation. It is becoming increasingly clear that although the X chromosome achieves male specific regulation via the MSL complex members, a number of general factors also impinge on this regulation. Future studies integrating these aspects promise to shed more light into this epigenetic phenomenon
Cartoon of gene tagging methods in yeast.
<p>(A) Commonly used methods for tagging a gene of interest (yfg = your favorite gene): C-terminal gene tagging using a marker and 40–50 bp homology (Fig 1A-i) or ≥300 bp homology (Fig 1A–ii), and N-terminal gene tagging using the Cre-loxP system (Fig 1A-iii). Methods i and ii use a selection marker that disrupts the 3’ UTR and cannot be eliminated, which might perturb the function of the fusion. For method iii, the loxP-flanked selection marker can be excised with the Cre recombinase, which leaves behind one flippase recognition target (FRT) site “scar” in the 5’ UTR of the tagged ORF. (B) Tagging methods introduced in this study: scarless C-terminal gene tagging (Fig 1B-i), scarless N-terminal gene tagging (Fig 1B-ii), and scarless internal gene tagging (Fig 1B-iii). The scarless tagging methods require a second round of selection to eliminate the <i>URA3</i> marker, which is surrounded by identical GFP sequences. Note that the resulting “GFP scar” becomes a genuine part of the full-length GFP fusion protein after recombination and that the endogenous UTRs are not altered. The scarless N-terminal tagging method can be used for tagging essential genes in haploid yeast cells because a constitutive promoter situated between the <i>URA3</i> marker and the second GFP fragment drives expression of the ‘partial GFP’-tagged gene prior to excision of <i>URA3</i>. Integration cassettes with either a partial GFP tag (as shown here) or a full-length GFP tag (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.s001" target="_blank">S1 Fig</a>) can be used. (C) Detailed description of the steps for scarless C-terminal tagging of a gene of interest with GFP. The integration cassette is built using two-step PCR synthesis. The homology arms H1 and H2 are amplified in the first round of PCRs and then used as primers together with F1 and R2 in the second round of PCRs. The primer-binding sites of H1 and H2 are unique, which is important for the efficient PCR amplification of the integration cassettes. Excision of the <i>URA3</i> marker is not shown in this cartoon but is identical to the depiction above (Fig 1B-i).</p
DNA gels and Western blot confirm strain construction.
<p>(A) PCR amplification of homology arms H1 and H2. (B) Integration cassettes for C-terminal tagging of <i>PRE1</i> with mNeonGreen (mNG) and the <i>TRP1</i> selection marker using either 50-bp or ≥300-bp homology. (C) Integration cassette for scarless C-terminal tagging of <i>PRE1</i> with mNeonGreen using ≥300-bp homology. (D) Colony PCR confirming correct chromosomal integration of the fusions at the <i>PRE1</i> gene locus. (E) Integration cassette for the scarless N-terminal tagging of <i>YPT1</i> and <i>RSP5</i> with mNeonGreen. (F) Colony PCR confirming correct integration of the mNG<sub>1-177</sub>-<i>URA3</i>-P<sub>EF1α</sub>-mNG<sub>60-236</sub> construct at the <i>YPT1</i> gene locus with primer sets for the upstream (left) and downstream flank of the integration site (middle). Growth in rich medium followed by selection on 5-FOA leads to the identification of cells that have recombined the mNeonGreen fragments and lost the <i>URA3</i> marker, resulting in <i>YPT1</i> tagged N-terminally with mNeonGreen (right). Colony PCR of a strain with an unmodified, wild-type <i>YPT1</i> gene locus is shown as control. (G) Colony PCR confirms correct integration of the mNG<sub>1-177</sub>-<i>URA3</i>-P<sub>EF1α</sub>-mNG<sub>60-236</sub> construct at the <i>RSP5</i> gene locus (left and middle). Recombination results in full-length mNeonGreen-<i>RSP5</i> at the endogenous gene locus (right). (H) Western blot analysis using an anti-Rsp5 antibody (upper) with <i>S</i>. <i>cerevisiae</i> strains expressing untagged Rsp5 (lane #1), mNeonGreen-Rsp5 (lane #2), mNeonGreen<sub>60-236</sub>-Rsp5 (lane #3, before <i>URA3</i> excision), mCherry-Rsp5 (lane #4), Rsp5-mNeonGreen (lane #5), untagged Rsp5 and an ectopic copy of mNeonGreen-Rsp5 (lane #6), and untagged Rsp5 and an ectopic copy of Rsp5-mNeonGreen (lane #7). The mNeonGreen<sub>60-236</sub>-Rsp5 fusion displays extensive self-ubiquitination, likely because of the partial mNeonGreen tag. The C-terminal Rsp5 shows a truncation product that most likely corresponds to un-tagged Rsp5. This truncation product is not observed with the N-terminal Rps5 fusions, which appear fully functional. The molecular weights are 92 kDa for untagged, wild-type Rsp5 and ~119 kDa for the fluorescent protein-tagged Rsp5 fusions. Pgk1 is used as loading control (lower). (I) Spotting assay with the strains that were used for the Western blot of Fig 2H.</p
Scarless C-terminal tagging of a gene of interest in <i>S</i>. <i>pombe</i>.
<p>(A) Homology arms (H1 and H2) targeting a gene of interest were attached to a DNA cassette comprising a linker (L), NmGFPmut3 (NGFP), the <i>ura4</i> marker, and mGFPmut3 (GFP). The cassette was transformed into <i>S</i>. <i>pombe</i> cells followed by selection on a PMG-ura plate. The surviving colonies were cultured in YES medium for 3 days to allow spontaneous recombination between the GFP fragments and then plated on 5-FOA to select for cells that have <i>yfg</i> tagged scarless with linker-mGFPmut3. (B) The same method described in panel a, except that kanMX6 and HSV1tk were used as positive and negative selection markers, respectively. (C) After selection with G418, <i>tdh1</i> was tagged with NmGFPmut3, which is non-fluorescent and hence no fluorescence was detected by microscopy. (D) After 3 days of culture in YES medium, followed by selection with FUdR, cells with the recombined full-length mGFPmut3 protein attached to <i>yfg</i> showed the expected GFP signal. Scale bar (white) is 5 μm.</p
Pseudocode describing the automated primer design algorithm.
<p>The illustration depicts the primer design for C-terminal gene tagging of a gene of interest (yfg) with a fluorescent protein. N-terminal and internal gene tagging is analogous except for the insertion site. The primers are shown as arrows (red) above and below the respective DNA sequences. GC clamps in the 500-bp upstream and downstream search regions are highlighted in red. The primer sequences for scarless N-terminal, scarless C-terminal, and C-terminal gene tagging with a marker for essentially all <i>S</i>. <i>cerevisiae</i> ORFs are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.s013" target="_blank">S4 Table</a>. The primer sequences for scarless C-terminal tagging of all <i>S</i>. <i>pombe</i> ORFs are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.s014" target="_blank">S5 Table</a>. The code for the automated primer design is contained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.s007" target="_blank">S1 File</a> and available on GitHub (<a href="https://github.com/DXL38/scarless_gene_tagging_in_yeast" target="_blank">https://github.com/DXL38/scarless_gene_tagging_in_yeast</a>). Primers F1 and R1 are used for amplifying the homology arm H1, and primers F2 and R2 are used for amplifying the homology arm H2. The homology arms H1 and H2 are ≥300 bp in size.</p
Fluorescence microscopy of <i>S</i>. <i>cerevisiae</i> cells producing mNeonGreen-tagged fusion proteins.
<p>Yeast strains were constructed by modifying the endogenous gene locus using C-terminal gene tagging with a marker (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.g001" target="_blank">Fig 1A-ii</a>), scarless C-terminal gene tagging (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.g001" target="_blank">Fig 1B-i</a>), or scarless N-terminal gene tagging (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.g001" target="_blank">Fig 1B-ii</a>). <i>PRE1</i> and <i>TDH3</i> were tagged C-terminally with mNeonGreen (mNG) using the <i>TRP1</i> selection marker or scarless. The eleven yeast Rab proteins (i.e. Sec4, Vps21, Ypt1, Ypt6, Ypt7, Ypt10, Ypt11, Ypt31, Ypt32, Ypt52, and Ypt53) are prenylated at their C-termini (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.s003" target="_blank">S3 Fig</a>) and were tagged N-terminally with mNeonGreen using the scarless gene tagging method. The Rab proteins localize to their expected organelles: for example Golgi (Ypt1, Ytp6), trans-Golgi network (Ypt6), vacuole and late endosomes (Ypt7), early endosomes (Vps21), recycling endosomes and post-Golgi exocytic vesicles (Ypt31), and secretory vesicles (Sec4). The ubiquitin ligase <i>RSP5</i> was tagged C-terminally and scarless N-terminally with mNeonGreen. The C-terminal <i>RSP5</i>-mNeonGreen fusion resulted in cell size enlargement (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.s004" target="_blank">S4 Fig</a>) but no growth phenotype was observed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.g002" target="_blank">Fig 2I</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163950#pone.0163950.s005" target="_blank">S5 Fig</a>). The large-cell phenotype was not observed with the scarless N-terminal fusions. The cells producing mCherry-Rsp5 and mCherry-Ypt1 fusions look similar to the cells producing the corresponding N-terminal mNeonGreen fusions, although the mCherry fusions also display some vacuolar localization. <i>PRE1</i>, <i>YPT1</i>, <i>SEC4</i>, and <i>RSP5</i> are essential genes. Scale bar (white) is 5 μm.</p
A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance
Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer “anti-prion drives” that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells