Uncoupling Transcription from Covalent Histone Modification

<div><p>It is widely accepted that transcriptional regulation of eukaryotic genes is intimately coupled to covalent modifications of the underlying chromatin template, and in certain cases the functional consequences of these modifications have been characterized. Here we present evidence that gene activation in the silent heterochromatin of the yeast <i>Saccharomyces cerevisiae</i> can occur in the context of little, if any, covalent histone modification. Using a SIR-regulated heat shock-inducible transgene, <i>hsp82-2001</i>, and a natural drug-inducible subtelomeric gene, <i>YFR057w</i>, as models we demonstrate that substantial transcriptional induction (>200-fold) can occur in the context of restricted histone loss and negligible levels of H3K4 trimethylation, H3K36 trimethylation and H3K79 dimethylation, modifications commonly linked to transcription initiation and elongation. Heterochromatic gene activation can also occur with minimal H3 and H4 lysine acetylation and without replacement of H2A with the transcription-linked variant H2A.Z. Importantly, absence of histone modification does not stem from reduced transcriptional output, since <i>hsp82-ΔTATA</i>, a euchromatic promoter mutant lacking a TATA box and with threefold lower induced transcription than heterochromatic <i>hsp82-2001</i>, is strongly hyperacetylated in response to heat shock. Consistent with negligible H3K79 dimethylation, <i>dot1Δ</i> cells lacking H3K79 methylase activity show unimpeded occupancy of RNA polymerase II within activated heterochromatic promoter and coding regions. Our results indicate that large increases in transcription can be observed in the virtual absence of histone modifications often thought necessary for gene activation.</p></div

Signcryption Efficiency Comparison.

<p>|<i>G</i>₁|: the length of the elements in <i>G</i>₁; |ID|: the length of ID; |<i>M</i>|: the length of the plaintext <i>M</i>;</p><p><i>m</i>: the number of signers (<i>m</i> = 1 in schemes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063562#pone.0063562-Yu1" target="_blank">[9]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063562#pone.0063562-Elkamchouchi1" target="_blank">[11]</a> and our scheme); <i>n</i>: the number of recipients.</p

Comparison of merits and demerits.

<p><b>Note:</b> (*) refers to schemes prone to the <i>cross-comparison attack</i> and <i>joint conspiracy attack</i>.</p

Notations.

<p>Notations.</p

Symbol Definition.

<p>Symbol Definition.</p

Western blots for TH and DAT in purified N27-A and unpurified N27 cells.

<p><b>(A)</b>: Representative images show TH, DAT and β-actin Western blots from purified and unpurified N27 cells. Strong TH bands were seen in purified N27-A cells, while much reduced TH protein levels were seen in unpurified N27 cells. There were moderate DAT protein levels in purified N27-A cells but only faint DAT bands in unpurified N27 cells. <b>(B):</b> Quantification of TH and DAT Western blots relative to β-actin bands. The TH and DAT levels in unpurified N27 cells were set at 100%. Results show that purified N27-A cells have four-fold higher TH and three-fold higher DAT protein levels compared to unpurified N27 cells. (n = 6, **<i>p</i><0.01)</p

De-signcryption Efficiency Comparison.

<p>|<i>M</i>|: the length of the plaintext <i>M</i>; <i>m</i>: the number of signers (<i>m</i> = 1 in schemes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063562#pone.0063562-Yu1" target="_blank">[9]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063562#pone.0063562-Elkamchouchi1" target="_blank">[11]</a> and our scheme); <i>n</i>: the number of recipients.</p><p>Note: N/A refers to a single-recipient scheme where the message is transmitted using a unicast communication channel, thus it is unnecessary for the recipient to judge whether he/she is authorized.</p

Tunable Assembly of Colloidal Crystal Alloys Using Magnetic Nanoparticle Fluids

We demonstrate a magnetic technique for assembling bidisperse and tridisperse colloidal particle fluids into a variety of complex structures with dimensionality ranging from 0-D (rings) to 1-D (chains) to 2-D (tiles). Compared with prior work on bidisperse particles that are commensurate in size, here we explore the assembly of different sized particles, and we show that due to packing constraints, new particle structures can be realized experimentally. Extending these experiments to a tridisperse system, we demonstrate that at low concentrations the smallest particle does not change the underlying crystal structures of the bidisperse system; however, it can assist in the formation of crystallite structures that were not stable in a bidisperse system. Additionally, we discovered that the smallest particle mimics the role of the ferrofluid, by shifting the locations in phase space where the bidisperse crystal structures can be experimentally obtained. Finally, we demonstrate that 3-particle crystal structures can be tuned by varying the strength of the external field, which is not possible in a 2-particle system

<i>SIR</i>-regulated heat shock transgene system.

<p>(a) Summary of <i>hsp82</i> transgenes used in this study with location, orientation and dosage of integrated <i>HMRE</i> silencers indicated by arrows (see Inset for location of silencer binding proteins ORC, Rap1 and Abf1). Transgenes occupy the native chromosomal <i>HSP82</i> locus (located ∼95 kb from TEL16L) and contain the indicated silencer insertions with no extraneous DNA sequence (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#s4" target="_blank">Materials and Methods</a>). Note that the transcription start site lies 60 bp upstream of the start codon and the 3′ integration site lies ∼50 bp 3′ of the mapped transcription termination site <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004202#pgen.1004202-Farrelly1" target="_blank">[74]</a>. The ORF is indicated as a black rectangle; coordinates are numbered relative to the ATG codon. (b) Transcriptional output of <i>hsp82</i> transgenes under non-inducing (30°C) and inducing conditions (20 min heat shock at 39°C). Depicted are bar graph summaries of Northern analyses of <i>SIR⁺</i> and <i>sir4Δ</i> cells bearing the indicated <i>hsp82</i> transgenes (arrows symbolize integrated silencers as in A); <i>HSP82</i>⁺ was analyzed in the parental strain. Transcript abundance was normalized to <i>ACT1</i> and is presented relative to the non-induced <i>HSP82</i>⁺ level set at 100 (depicted are means ± S.D.; N = 2).</p

Growth properties of purified N27-A and unpurified N27 cells.

<p><b>(A-F):</b> Both cell types were plated at 20,000 cells in each well of 6-well plates. Representative images were taken at Day 1, 3, and 5 for each cell type. Purified N27-A cells (<b>Images A-C</b>) grew more slowly than unpurified N27 cells (<b>Images D-F</b>). <b>(G)</b>: Growth charts of purified N27-A (red line) and unpurified N27 cells (green line) from Day 0 to Day 7. Data present the average cell number from two wells of purified and unpurified cells in three experiments (n = 6, each cell type). Bar, 20 μm for <b>A-F</b>.</p