Real zero polynomials and Polya-Schur type theorems

BackgroundProtein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and body mass, and has been implicated in endoplasmic reticulum (ER) stress. Herein, we assess the role of PTP1B in ER stress in brown adipocytes, which are key regulators of thermogenesis and metabolic response.Methodology/principal findingsTo determine the role of PTP1B in ER stress, we utilized brown adipose tissue (BAT) from mice with adipose-specific PTP1B deletion, and brown adipocytes deficient in PTP1B and reconstituted with PTP1B wild type (WT) or the substrate-trapping PTP1B D181A (D/A) mutant. PTP1B deficiency led to upregulation of PERK-eIF2α phosphorylation and IRE1α-XBP1 sub-arms of the unfolded protein response. In addition, PTP1B deficiency sensitized differentiated brown adipocytes to chemical-induced ER stress. Moreover, PERK activation and tyrosine phosphorylation were increased in BAT and adipocytes lacking PTP1B. Increased PERK activity resulted in the induction of eIF2α phosphorylation at Ser51 and better translatability of ATF4 mRNA in response to ER stress. At the molecular level, we demonstrate direct interaction between PTP1B and PERK and identify PERK Tyr615 as a mediator of this association.ConclusionsCollectively, the data demonstrate that PTP1B is a physiologically-relevant modulator of ER stress in brown adipocytes and that PTP1B deficiency modulates PERK-eIF2α phosphorylation and protein synthesis

Unique profile of ordered arrangements of repetitive elements in the C57BL/6J mouse genome implicating their functional roles.

The entirety of all protein coding sequences is reported to represent a small fraction (~2%) of the mouse and human genomes; the vast majority of the rest of the genome is presumed to be repetitive elements (REs). In this study, the C57BL/6J mouse reference genome was subjected to an unbiased RE mining to establish a whole-genome profile of RE occurrence and arrangement. The C57BL/6J mouse genome was fragmented into an initial set of 5,321 units of 0.5 Mb, and surveyed for REs using unbiased self-alignment and dot-matrix protocols. The survey revealed that individual chromosomes had unique profiles of RE arrangement structures, named RE arrays. The RE populations in certain genomic regions were arranged into various forms of complexly organized structures using combinations of direct and/or inverse repeats. Some of these RE arrays spanned stretches of over 2 Mb, which may contribute to the structural configuration of the respective genomic regions. There were substantial differences in RE density among the 21 chromosomes, with chromosome Y being the most densely populated. In addition, the RE array population in the mouse chromosomes X and Y was substantially different from those of the reference human chromosomes. Conversion of the dot-matrix data pertaining to a tandem 13-repeat structure within the Ch7.032 genome unit into a line map of known REs revealed a repeat unit of ~11.3 Kb as a mosaic of six different RE types. The data obtained from this study allowed for a comprehensive RE profiling, including the establishment of a library of RE arrays, of the reference mouse genome. Some of these RE arrays may participate in a spectrum of normal and disease biology that are specific for mice

Unique Profile of Ordered Arrangements of Repetitive Elements in the C57BL/6J Mouse Genome Implicating Their Functional Roles

The entirety of all protein coding sequences is reported to represent a small fraction (,2%) of the mouse and human genomes; the vast majority of the rest of the genome is presumed to be repetitive elements (REs). In this study, the C57BL/ 6J mouse reference genome was subjected to an unbiased RE mining to establish a whole-genome profile of RE occurrence and arrangement. The C57BL/6J mouse genome was fragmented into an initial set of 5,321 units of 0.5 Mb, and surveyed for REs using unbiased self-alignment and dot-matrix protocols. The survey revealed that individual chromosomes had unique profiles of RE arrangement structures, named RE arrays. The RE populations in certain genomic regions were arranged into various forms of complexly organized structures using combinations of direct and/or inverse repeats. Some of these RE arrays spanned stretches of over 2 Mb, which may contribute to the structural configuration of the respective genomic regions. There were substantial differences in RE density among the 21 chromosomes, with chromosome Y being the most densely populated. In addition, the RE array population in the mouse chromosomes X and Y was substantially different from those of the reference human chromosomes. Conversion of the dot-matrix data pertaining to a tandem 13repeat structure within the Ch7.032 genome unit into a line map of known REs revealed a repeat unit of,11.3 Kb as a mosaic of six different RE types. The data obtained from this study allowed for a comprehensive RE profiling, including the establishment of a library of RE arrays, of the reference mouse genome. Some of these RE arrays may participate in

PTP1B dephosphorylation of PERK.

<p>(<b>A</b>) PERK was immunoprecipitated from BAT lysates of Cre, fl/fl and adipose-PTP1B KO (KO) male mice fed HFD for 30 weeks, and then immunoblotted with anti-phosphotyrosine antibodies, phospho-specific antibodies for PKR Tyr293 (PERK Y615) and PERK (Thr980). Blots were also probed for PERK to control for loading. Each lane represents brown adipose tissue from a different animal. (<b>B</b>) Differentiated WT, KO and D/A brown adipocytes and PERK−/− and PERK+/+ fibroblasts were treated with thapsigargin (TG) for the indicated times. Immunoprecipitates of PERK were immunoblotted with anti-phosphotyrosine antibodies. Blots were also probed for PERK to control for loading. (<b>C</b>) PTP1B KO preadipose cells were co-transfected with PTP1B WT and substrate-trapping D181A mutant (D/A) and Myc-tagged PERK wild type. In addition, PERK KO fibroblasts were co-transfected with PTP1B D/A and Myc-tagged PERK WT and Y615F mutant. Cell were treated with TG for the indicated times then lysed in NP40 or RIPA (R), with or without pervanadate (V) treatment. Lysates were immunoprecipitated using mouse (m) and human (h) PTP1B antibodies and immunoblotted using anti-c-Myc and anti-(m/h) PTP1B antibodies. (<b>D</b>) PTP1B KO brown preadipocytes were transfected with hPTP1B WT, or hPTP1B D/A (red) and c-Myc-PERK WT or Y615F mutant (green), treated with thapsigargin (TG) for 2 hours and visualized by fluorescence confocal microscopy. Scale bar corresponds to 20 µm.</p

Enhanced ATF4 mRNA translation in PTP1B-deficient differentiated brown adipocytes.

<p>Differentiated WT (<b>A, B</b>) and PTP1B KO (<b>C, D</b>) brown adipocytes were left untreated (<b>A, C</b>) or treated (<b>B, D</b>) with thapsigargin (TG, 1 µM) for 2 hr followed by polysome profile analysis using 20–50% sucrose gradients as described in Methods. Gradients were fractionated from top (fraction 1) to bottom (fraction 17). Absorbance of the gradients was measured continuously at 264 nm to provide the polysome profiles. Association of ATF4 mRNA and GAPDH mRNA with the ribosomal interface was detected by RT-PCR. Data present one of two reproducible experiments. <b>E</b>) Protein synthesis in differentiated WT, KO and D/A brown adipocytes before and after TG treatment. Bar chart represents newly incorporated ³⁵S methionine from three independent experiments. (*) indicates significant difference between KO and D/A versus WT at the corresponding treatment time and (^#) indicates significant difference between treated and non-treated cells within each group.</p

Whole-genome view of occurrence and arrangement structure of REs.

<p>The dot-matrix plots of the self-alignment data derived from a total of 5,321 genome units of 0.5 Mb, in addition to 41 subunits of varying sizes, which cover the entire mouse genome, are compiled by chromosome order (1∼19, X, and Y). Each genome unit/subunit is represented by a square and unit identifications are indicated only for the ones on the far left of each row. Genome units without any sequence information (gap) are indicated with a white square. Grey rectangles indicate partial gaps. Ideograms for individual chromosomes, which are adopted from the NCBI mouse genome database, are included as a reference. A set of subunits derived from one genome unit are grouped with a rectangle. A detailed plot view of the RE occurrence and arrangement structure is available through the electronic supplementary figures.</p

Translocation between the tandem RE array of the IgM switch region and the c-<i>Myc</i> gene.

<p>The 3′-end of the immunoglobulin heavy chain (IGh) is presented as a dot-matrix plot with markings for the various switch regions (in blue). The zoomed-in tandem RE array dot-matrix plot represents the IgM switch region (Sμ). The illustration, involving Sμ and c-<i>Myc</i> gene sequences, depicts the putative translocation event between two sections of the IgM switch region on chromosome 12 and a 40-nucleotide stretch within the c-<i>Myc</i> gene on chromosome 15. One unique stretch of 40 nucleotides in the c-<i>Myc</i> sequence, corresponding to a reported translocation breakpoint in the c-<i>Myc</i> gene, had a high homology (80% identity: 5 mismatches and 2 gaps) with two sections of the IgM switch region tandem array. Ch (mouse chromosome), ex (exon), red triangles (putative translocation breakpoints).</p

RE arrays of chromosomes 7, X, and Y.

<p>The RE arrays plotted from the genome units/subunits of chromosomes 7, X, and Y are assembled in order of 5′ to 3′. The same data sets are presented in a smaller scale in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035156#pone-0035156-g002" target="_blank">Fig. 2</a>. The same labeling and identification schedules as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035156#pone-0035156-g002" target="_blank">Fig. 2</a> are used. A set of subunits derived from one genome unit are grouped with a rectangle.</p

Line mapping of REs/genes within the Ch7.032 genome unit.

<p><b>A</b>. The dot-matrix plot is derived from an unbiased self-alignment of the Ch7.032 genome unit. The REs in the area highlighted with a dotted red line are subjected to a line mapping experiment. <b>B</b>. The RE profile within the tandem 13 repeats of the ∼11.3 Kb unit in the highlighted structure are plotted on a scaled line map using color coding to identify RE types. In addition, <i>Obox4</i> gene sequences are marked within each repeat unit. <b>C</b>. The RE profile of the entire Ch7.032 genome unit of 0.5 Mb are plotted on a scaled line map for all RE types with color coding identification. The dotted rectangle represents the location of the first ∼11.3 Kb repeat unit within the Ch7.032 genome unit. High-resolution line maps (for maps on panels B and C) are accessible through <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035156#pone.0035156.s022" target="_blank">Figs. S22</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035156#pone.0035156.s023" target="_blank">S23</a>.</p

Schedule for fragmentation of a genome unit into sequential half-size subunits.

<p>The diagram illustrates how a series of sequential half-size subunits are generated from a genome unit of 0.5 Mb, using the Ch7.134 as an example. During each fragmentation event, the 5′-half is designated as “L" and the 3′-half is designated as “R", and lowercase and uppercase letters are alternated for sequential fragmentation events. The chromosomal ideogram is adopted from the NCBI mouse genome database. *identification of genome unit, **chromosomal location, Ch (chromosome).</p