Full-sib family size distribution in four broods of <i>Littorina saxatilis</i>.

<p>Observed number of offspring per male is estimated in COLONY. Expected number of sires is approximated by a truncated Poisson distribution.</p

The number of sires in four half-sib families of <i>Littorina saxatilis</i>.

<p>Four females and their offspring were genotyped at five microsatellite DNA loci. The most likely number was estimated using the likelihood-based software COLONY and the minimum number was calculated using MINSIRES.</p

The effect of sample size on the estimated number of sires.

<p>Most likely number of sires is estimated using COLONY in random subsamples of offspring from a single brood of <i>Littorina saxatilis</i>; subsample size varies from 10 to 77 (i.e. the whole brood).</p

Structural features determining OTUB2’s broader cleavage specificity as compared to OTUB1.

<p>The structural models of (<b>A</b>) OTUB1 (adapted from <i>14</i>) and (<b>B</b>) OTUB2 (this study) are shown in blue, the proximal ubiquitin in purple and the distal ubiquitin in red (note that the proximal ubiquitin (purple) in (<b>B</b>) is not part of the structure). The N-terminal α-helix of OTUB1 (dark blue cylinder) that is absent in OTUB2 makes direct contact with the proximal ubiquitin and hence restricts its binding to an orientation presenting Lys48 towards the catalytic site (red arrows). This restriction is not present in OTUB2, thereby allowing a more permissive ubiquitin recognition mode.</p

Comparison of OTUB2-Ub with other OTU-Ub complexes.

<p>Superposition of OTUB2-Ub (blue and red) with yeast OTU1-Ub (grey) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115344#pone.0115344.ref020" target="_blank">20</a>] (<b>A</b>), vOTU-Ub (grey) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115344#pone.0115344.ref023" target="_blank">23</a>] (<b>B</b>) and OTUB1-Ubal-UBC13-Ub (grey / yellow) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115344#pone.0115344.ref013" target="_blank">13</a>] (<b>C</b>) complexes. The free donor Ub is shown in yellow and the UBC13 is omitted in (<b>C</b>) for clarity.</p

Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells

<div><p>The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development.</p></div

OTUB2 has a broader cleavage profile than OTUB1.

<p>(<b>A</b>) Ubiquitin (Ub), Nedd8, ISG15 and SUMO1 were conjugated to the biotinylated peptide VKAKIQD (Ub_26–32) as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115344#pone.0115344.ref019" target="_blank">19</a>] and subjected to cleavage by recombinant OTUB2, UCH-L3 or crude cell lysate (- represents untreated control), followed by SDS-PAGE separation and analysis by streptavidin-HRP immunoblotting. (<b>B</b>) di-SUMO2/3 was incubated with DMSO, OTUB2, OTUB2delta, OTUB1 or crude lysate for the indicated times, followed by SDS-PAGE separation and analysis by immunoblotting. (<b>C</b>) Linear di-Ubiquitin (di-Ub) was incubated with OTUB2, UCH-L3 or DMSO for the indicated times, followed by SDS-PAGE separation and analysis by immunoblotting. (<b>D</b>) di-Ub substrates with the linkages Lys6, 11, 27, 29, 33, 48 or 63 were incubated with either wild type (Wt) or catalytically inactive C51S mutant (M) OTUB2 for four hours, followed by SDS-PAGE and immunoblotting analysis.</p

OTUB N-terminal tails modulate DUB activity and Ub chain linkage specificity.

<p>(<b>A</b>) Design of OTUB1-(N-term)-OTUB2 (Otub1–2) and OTUB2-(N-term)-OTUB1 (Otub2–1) chimera constructs and recombinant proteins (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115344#pone.0115344.s002" target="_blank">S2 Fig.</a>). <b>(B)</b> Active site labeling using HA-UbBr2 (Br2) or HA-Ub-VME (VME) revealed that the OTUB1 N-terminal tail affects labeling selectivity towards the VME probe. (<b>C</b>) Ub-Rhodamine activity assay revealing the restricting effect of the N-terminal tail of OTUB1 on both, OTUB1 and OTUB2. (<b>D</b>) Magnification of the assay scale shown in (<b>C</b>) to reveal enzymatic activities of the OTUB1 (Otub1, Otub 2–1) proteins. (<b>E</b>) K-values calculated from the Ub-Rhodamine assays (<b>C-D</b>) demonstrating the restricting effect of the OTUB1 N-terminal tail. (<b>F-G</b>) Lys48 and Lys63 tetra-Ubiquitin cleavage activities are affected by OTUB1/2 N-terminal tails. For the quantitation of the relative Ub-cleavage shown in (<b>F</b>), the sum of the intensities of the bands corresponding to cleaved tetra-ubiquitin (Ub/Ub2/Ub3 observed in (<b>G</b>), upper panel) was normalized to the intensity of the band corresponding to the enzyme used (observed in the anti-his immunoblot, (<b>G</b>), bottom panel). All values in (F) are shown relative to the values observed for OTUB2 (mean of OTUB2 is set to 1), and the error bars are S.E. (n = 4).</p

Overall structure of the OTUB2-Ub complex.

<p>(<b>A</b>) A ribbon diagram showing OTUB2-Ub complex with OTUB2 colored in blue and Ub in red. The active site residues are shown as sticks with carbon atoms colored in cyan; the last two residues of Ub and the covalent linker to Cys51 are drawn as orange sticks. (<b>B</b>) Representative <i>|2Fo-Fc|</i> map contoured at 1σ showing well defined electron density for residues around the active site. (<b>C</b>) Comparison of ligand bound and apo (grey) structures of OTUB2, the large structural changes due to Ub binding are indicated by arrows. (<b>D</b>) Close-up view of the active site of ligand bound and apo OTUB2. (<b>E</b>) Electrostatic surfaces of OTUB2 (bottom panel) and Ub (top panel). Ub is moved upwards and rotated 90° to show the positively charged surface patch that has complementary interactions with OTUB2.</p

Large TF families oscillate and show differential distribution during the cell cycle.

<p>(A-D) θ-value plots for all TFs and TF families with a significantly differing pattern in (A, C) HeLa-Fucci cells and (B, D) U2OS-Fucci cells (FDR ≤0.001; logCPM ≥1; FC ≥1.5). (E) θ-value plot of cell-cycle-synchronized TFs in HeLa-Fucci cells versus U2OS-Fucci cells, with suggested phase-specific expression groups denoted I-VI and the main TFs involved in G1 restriction and onset of S phase indicated in red. (F) Schematic showing how repressor activity may shape transcriptional boundaries during the cell cycle, exemplified by E2F7/8 repression of E2F1/2 expression.</p