Csm4, in Collaboration with Ndj1, Mediates Telomere-Led Chromosome Dynamics and Recombination during Yeast Meiosis

Chromosome movements are a general feature of mid-prophase of meiosis. In budding yeast, meiotic chromosomes exhibit dynamic movements, led by nuclear envelope (NE)-associated telomeres, throughout the zygotene and pachytene stages. Zygotene motion underlies the global tendency for colocalization of NE-associated chromosome ends in a “bouquet.” In this study, we identify Csm4 as a new molecular participant in these processes and show that, unlike the two previously identified components, Ndj1 and Mps3, Csm4 is not required for meiosis-specific telomere/NE association. Instead, it acts to couple telomere/NE ensembles to a force generation mechanism. Mutants lacking Csm4 and/or Ndj1 display the following closely related phenotypes: (i) elevated crossover (CO) frequencies and decreased CO interference without abrogation of normal pathways; (ii) delayed progression of recombination, and recombination-coupled chromosome morphogenesis, with resulting delays in the MI division; and (iii) nondisjunction of homologs at the MI division for some reason other than absence of (the obligatory) CO(s). The recombination effects are discussed in the context of a model where the underlying defect is chromosome movement, the absence of which results in persistence of inappropriate chromosome relationships that, in turn, results in the observed mutant phenotypes

Exile Vol. XXXIX No. 1

Title Page by Ellen Gurley \u2793 i Epigraph by Ezra Poind ii Table of Contents iii-iv Remaining a Soldier by Kristin Kruse \u2793 1-2 Vietnam War Memorial by Brooke MacKaye 3 We both ride in back by Chris Macaluso \u2793 4 Artwork by Jamie Oliver \u2794 5 Liberal Dirge #1 by Charis Brummitt \u2796 6-7 Artwork (anonymous) 7 Two ex-lovers and a dirty glass door by Chris Macaluso \u2793 8 The Salt of the Air by Kristen Padden \u2793 9-12 Artwork (anonymous) 13 Artwork by Ellen Gurley \u2793 14 Sun-Child by Jen Rudgers \u2796 15 Crazy Horse by Kevin Nix \u2794 16 The Fall of the Western Field by Rich Croft \u2793 17 In the Closet by Beth Widmaier \u2795 18 Winter Strawberries by Katy Rudder \u2793 19 Still Life (anonymous) 19 For This and Much Beyond This Poem by Matt Wanat \u2795 20-21 Artwork by Peggy Ryan \u2793 22 The Cycle Repeats: Apathy by Ishak Kang \u2793 23 The Judge by Ellen Gurley \u2793 24 Pear Colored by Erin Dempsey \u2793 25-26 4-Square by Trey Dunham \u2794 27 Artwork by Jamie Oliver \u2794 28 Ink & Heroine by Rich Croft \u2793 29 Figments by Craig Bowers \u2793 30-31 Malfi Coast (anonymous) 31 Suzanne (anonymous) 32 Hey Stella by Carey Chistie \u2795 33 Turning Leaves by Erin Lott \u2796 34-35 Reclining Nude (anonymous) 35 Blazon by Matt Wanat \u2795 36-37 Artwork by Holly Aikens \u2793 38 Awake by A. Fair \u2796 39 Dell the Barber by Kevin Nix \u2795 40 Artwork by Holly Aikens \u2793 40 Tree House by Katy Rudder \u2793 41-46 Jailbait by Ellison J. Stind \u2795 47 Mother by Charis Brummitt \u2796 48-49 Artwork by Bess Hammer \u2795 49 Private Origami by Trey Dunham \u2794 50 Among the Tendrils of Sleep by J. Trevett Allen \u2795 51 Poet of the Unforgiven by Carey Christie \u2795 52 Stuntman Steve by Andrew Zobay \u2793 53 sculpture by Lily Streett \u2794 53 Wonderings of an Adopted Son by Andy Heckert \u2793 54-55 Artwork by Holly Aikens \u2793 55 Odd Binge by C. N. Polumbus \u2793 56-57 Artwork by Holly Aikens \u2793 57 Artwork by Peggy Ryan \u2793; untitled by Jennifer Wendell \u2794 (superimposed) 58 Shadows of Pearl by Travis Brady \u2793 59-60 October/Rt. 161 by Annette Gallagher 61 Artwork by Jamie Oliver \u2794 61 The Influx by Craig Bowers \u2793 62 Artwork by Michael Norpell \u2794 63 editorial board 64 Editorial decision is shared equally among the Editorial Board. -64 Cover: Jamie Oliver -64 NOTE: With the exeption of Malfi Coast , all artwork listed as anonymous in the published table of contents appears to be signed by Ellen Gurley. 37th Yea

TERRA and the histone methyltransferase Dot1 cooperate to regulate senescence in budding yeast

<div><p>The events underlying senescence induced by critical telomere shortening are not fully understood. Here we provide evidence that TERRA, a non-coding RNA transcribed from subtelomeres, contributes to senescence in yeast lacking telomerase (<i>tlc1Δ</i>). Levels of TERRA expressed from multiple telomere ends appear elevated at senescence, and expression of an artificial RNA complementary to TERRA (anti-TERRA) binds TERRA <i>in vivo</i> and delays senescence. Anti-TERRA acts independently from several other mechanisms known to delay senescence, including those elicited by deletions of <i>EXO1</i>, <i>TEL1</i>, <i>SAS2</i>, and genes encoding RNase H enzymes. Further, it acts independently of the senescence delay provided by <i>RAD52</i>-dependent recombination. However, anti-TERRA delays senescence in a fashion epistatic to inactivation of the conserved histone methyltransferase Dot1. Dot1 associates with TERRA, and anti-TERRA disrupts this interaction <i>in vitro</i> and <i>in vivo</i>. Surprisingly, the anti-TERRA delay is independent of the C-terminal methyltransferase domain of Dot1 and instead requires only its N-terminus, which was previously found to facilitate release of telomeres from the nuclear periphery. Together, these data suggest that TERRA and Dot1 cooperate to drive senescence.</p></div

The anti-TERRA senescence delay is independent of Exo1 and Rad52 but is dependent on Dot1.

<p>(A) Anti-TERRA delays senescence in <i>tlc1Δ exo1Δ</i> mutants. <i>TLC1/tlc1Δ EXO1/exo1Δ</i> were sporulated and senescence assays of <i>tlc1Δ</i> and <i>tlc1Δ exo1Δ</i> (n = 5 each) were performed with anti-TERRA either induced or uninduced. Anti-TERRA and <i>exo1Δ</i> each significantly delay senescence (<i>tlc1Δ</i> uninduced versus <i>tlc1Δ</i> induced, 10 PD, p < 0.002 and <i>tlc1Δ</i> uninduced versus <i>tlc1Δ exo1Δ</i> uninduced, 9 PD, p = 0.001). Together they delay senescence even further (<i>tlc1Δ exo1Δ</i> uninduced versus <i>tlc1Δ exo1Δ</i> induced, 13 PD, p = 0.0016). (B) Anti-TERRA delays senescence in <i>tlc1Δ rad52Δ</i> mutants but not <i>tlc1Δ rad52Δ dot1Δ</i> mutants. <i>TLC1/tlc1Δ RAD52/rad52Δ DOT1/dot1Δ</i> diploids were sporulated and senescence assays of <i>tlc1Δ rad52Δ</i> (n = 5) and <i>tlc1Δ rad52Δ dot1Δ</i> (n = 6) were performed with anti-TERRA either induced or uninduced. Both anti-TERRA and <i>dot1Δ</i> delay senescence in the absence of Rad52 (<i>tlc1Δ rad52Δ</i> uninduced versus <i>tlc1Δ rad52Δ</i> induced, 10 PD, p < 0.0001 and <i>tlc1Δ rad52Δ</i> uninduced versus <i>tlc1Δ rad52Δ dot1Δ</i> uninduced, 9 PD, p = 0.005), but anti-TERRA does not cause a further delay in the absence of Dot1 (<i>tlc1Δ rad52Δ dot1Δ</i> uninduced versus <i>tlc1Δ rad52Δ dot1Δ</i> induced, p = 0.563). (C) Same as in (A) except that <i>dot1</i> deletion was tested instead of <i>exo1</i> deletion. Anti-TERRA and <i>dot1Δ</i> each delay senescence (<i>tlc1Δ</i> uninduced versus <i>tlc1Δ</i> induced, 8 PD, p = 0.002 and <i>tlc1Δ</i> uninduced versus <i>tlc1Δ dot1Δ</i> uninduced, 9 PD, p = 0.007). But again, anti-TERRA does not cause a further delay in the absence of Dot1 (<i>tlc1Δ dot1Δ</i> uninduced vs. <i>tlc1Δ dot1Δ</i> induced 1.5 PD, p = 0.238). In all panels, each data point represents the mean PD versus the mean and SEM of the cell density.</p

Dot1 associates with TERRA and anti-TERRA disrupts this interaction.

<p>(A) TERRA-like RNA oligonucleotides but not anti-TERRA molecules can pull down native Dot1 from yeast whole cell extracts. Yeast whole cell extracts (WCE) were subject to RNA affinity purification using biotinylated TERRA or control (random sequence) RNA oligonucleotides. Bound materials were eluted with 1M NaCl in lanes 2 and 3 and then the beads were boiled in lanes 4 and 5. Lane 1 is 5% of WCE as input. Proteins were visualized by western blot with anti-Dot1 antibody and anti- -actin antibody as a control. Dot1 protein migrates near 65 kD as a doublet. (B) V5-tagged Dot1 binds TERRA and anti-TERRA prevents this interaction <i>in vitro</i>. Nuclear extracts were subject to RNA pulldown using the indicated RNA templates. Bound materials were eluted with 2X Laemmli buffer by boiling and assayed by western blot with antibodies specific to V5 or Actin. Lanes 5 and 6: TERRA oligonucleotides were annealed to anti-TERRA molecules under G-quadruplex permissive or minimizing conditions (NaCl or LiCl, respectively) and then were then transferred in the standard buffer used for RNA pulldown. The LiCl conditions rule out the possibility that folding of TERRA into G-quadruplexes, rather than forming duplexes with anti-TERRA, explains the loss of Dot1 binding. Lane 1 is 10% of input. Marker size in kD are indicated at left. Both isoforms of Dot1 can be seen around 110 kD. (C) V5-tagged Dot1 binds TERRA and anti-TERRA prevents this interaction <i>in vivo</i>. RNA immunoprecipitation was performed with V5-tagged Dot1 on yeast WCE. TERRA levels are quantified by qRT-PCR and displayed as fold change relative to an untagged Dot1 strain and to input (n = 2).</p

Anti-TERRA RNA binds TERRA <i>in vivo</i> in senescent <i>tlc1Δ</i> mutants.

<p>(A) Transcripts from multiple telomeres increase at senescence. Deletion of the telomerase RNA template, <i>TLC1</i>, causes telomeres to shorten until they undergo senescence. Transcript levels from the indicated telomeres of cells with longer telomeres (non-senescent <i>tlc1Δ</i>) versus cells with shorter telomeres (senescent <i>tlc1Δ</i>) were measured by qRT-PCR and normalized to housekeeping genes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195698#sec002" target="_blank">Materials and Methods</a>). Error bars are the SEM (n = 9). p values ≤ 0.05 are indicated by an asterisk. (B) Anti-TERRA was fused to an MS2 RNA tag and was pulled down from senescent cell extracts using a 6X-His tagged MBP-MS2 coat fusion protein. (C) Anti-TERRA efficiently pulls down native TERRA <i>in vivo</i>. Bar graphs represent the average qPCR-based measurements of the fraction of total cellular anti-TERRA that is pulled down and the fractions of total cellular TERRA from particular telomeres that are pulled down along with anti-TERRA. n = 3 independent experiments. The fraction of anti-TERRA that is pulled down (~55%) is a control that reflects the maximum achievable efficiency of TERRA that could be pulled down along with anti-TERRA if all TERRA molecules are bound by anti-TERRA. The low levels of TERRA recovered in samples from cells in which anti-TERRA is not induced demonstrates the dependence of the TERRA detection on the expression of anti-TERRA.</p

Development of a skin-directed scoring system for Stevens-Johnson syndrome and epidermal necrolysis: a Delphi consensus exercise

Importance Scoring systems for Stevens-Johnson syndrome and epidermal necrolysis (EN) only estimate patient prognosis and are weighted toward comorbidities and systemic features; morphologic terminology for EN lesions is inconsistent.Objectives To establish consensus among expert dermatologists on EN terminology, morphologic progression, and most-affected sites, and to build a framework for developing a skin-directed scoring system for EN.Evidence Review A Delphi consensus using the RAND/UCLA appropriateness criteria was initiated with a core group from the Society of Dermatology Hospitalists to establish agreement on the optimal design for an EN cutaneous scoring instrument, terminology, morphologic traits, and sites of involvement.Findings In round 1, the 54 participating dermatology hospitalists reached consensus on all 49 statements (30 appropriate, 3 inappropriate, 16 uncertain). In round 2, they agreed on another 15 statements (8 appropriate, 7 uncertain). There was consistent agreement on the need for a skin-specific instrument; on the most-often affected skin sites (head and neck, chest, upper back, ocular mucosa, oral mucosa); and that blanching erythema, dusky erythema, targetoid erythema, vesicles/bullae, desquamation, and erosions comprise the morphologic traits of EN and can be consistently differentiated.Conclusions and Relevance This consensus exercise confirmed the need for an EN skin-directed scoring system, nomenclature, and differentiation of specific morphologic traits, and identified the sites most affected. It also established a baseline consensus for a standardized EN instrument with consistent terminology