Comparison between experimental results and simulation results of the transcription induction of Rig-I and of interferon.

<p>The panels show the amount in individual cells (dots), and an interpolated contour plot of the 2D histogram (solid lines). The two left panels show the experimental results and the two right panels show simulation results. The two top panels and two bottom panels show results obtained at 6 hours and 10 hours, respectively. Significant similarity can be seen between the experimental results and the simulation results. A small population of early responders can be seen in the simulation results at 6 hours, corroborating the early responders hypothesis.</p

Consistent behavior of the synchronized Gillespie algorithm.

<p>Histograms of the intervals between consecutive occurrences of processes in two stochastic simulations. Both simulation were performed with two processes whose rates do not change during the simulation. The first simulation used the original Gillespie algorithm (dots and X's), and the second one used the modified Gillespie algorithm (open circles and open squares) without changing the state of the system during synchronization. Both processes display the same exponential distribution with the correct mean.</p

Fitting simulation results to analytical solution.

<p>Results of a deterministic diffusion simulation in which at each time step half the molecules at each grid square are equally distributed to neighboring squares. molecules were placed in the middle of the grid at time . A. The concentration in the middle of the grid as a function of simulation steps (dots) and a fit to the inverse of time (solid line). B. The concentration in a horizontal section through the middle of the grid after 2000 simulation steps (dots), and a fit to a Gaussian function (solid line).</p

DNA polymerase <b>η</b> mutational signatures are found in a variety of different types of cancer

<p>DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in Y<u>C</u>G motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide.</p

Conditional <i>Klf6</i> inactivation in vivo causes selective loss of differentiating oligodendrocytes.

<p><b>(A–F)</b> Confocal and morphometric analysis of oligodendrocyte numbers in developing <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and control <i>Klf6</i>^<i>fl/fl</i> spinal cords. Panel <b>(A)</b> shows samples from E12.5, immunostained for Olig2 and either Mnr (upper panels) or NeuN (lower panels). At E12.5, Olig2⁺ numbers in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and control samples are identical, and no differences are seen in neuronal markers. In controls, Olig2⁺ cell numbers then increase from E16.5 through P14, but no increase is seen in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> samples <b>(B–D)</b>. Areas outlined in panel <b>(B)</b> are shown at higher magnification, inset. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4B Fig</a>. At P1, myelin proteins are expressed in controls, but not in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> spinal cords <b>(C)</b>. <b>(E,F)</b> Analysis of stage-specific markers. Early events in differentiation, such as Nkx2.2 co-expression at E14.5, occur normally in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mice, but differentiating cells are selectively lost at subsequent timepoints <b>(E)</b>. In contrast, numbers of Olig2⁺Sox9⁺ OLP remain identical to controls <b>(F)</b>. There are no changes in Mnr⁺ motor neurons, which share the same origin as ventral OLP in the pMN domain (<b>A</b> upper panels, <b>B, G</b>). <b>(H,I)</b> Selective loss of differentiating cells is associated with increased apoptosis <b>(H)</b>, but OLP proliferation is unaffected <b>(I)</b>. <b>(J–L)</b> Postnatal stage-specific analysis confirms absence of differentiating (Apc⁺) oligodendrocytes from <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mice <b>(J,L)</b>, whereas OLP (Olig2⁺Ng2⁺) numbers are comparable to controls <b>(K,L)</b>. Representative cells are arrowed. <b>(M)</b> Analysis of <i>NG2creER</i>^<i>–</i>:<i>Klf6</i>^<i>fl/fl</i> mice, in which <i>Klf6</i> inactivation is inducibly targeted to OLP. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4C and S4D Fig</a>. Similar to <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> embryos, these mice also display selective loss of differentiating oligodendrocytes. Data are mean ± SEM. Statistics, <b>(D–I,L)</b> ANOVA plus Bonferroni post test, <b>(M)</b> Student’s <i>t</i> test, <i>***p <</i> 0.001. Scale: <b>(A,B)</b> 100 μm, inset 20 μm, <b>(C)</b> 250 μm, <b>(J,K)</b> 10 μm. Data shown are from lumbar sections of two to six mice per genotype per timepoint. Thoracic sections showed compatible findings. Individual values are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p

RNA sequencing identifies Klf6-dependence of gp130-driven transcriptional patterns.

<p><b>(A)</b> Overview of approach used to define key Klf6 effectors. Initial RNA-seq analysis of primary mouse cultures identifies 212 unique Klf6-regulated transcripts in OLP and 91 in iOL, of which 40 are shared. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s009" target="_blank">S1</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s011" target="_blank">S3</a> Tables and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s007" target="_blank">S5A and S5B Fig</a>. <b>(B,C)</b> Results of functional inference <b>(B)</b> and GO analysis <b>(C)</b> of RNA-seq data, from primary OLP (red), iOL (blue), or both (purple). In <b>(B)</b>, numbers of Klf6-regulated genes are indicated for each function. Implicated signaling pathways in OLP are presented as a smaller Venn diagram, inset. <b>(D)</b> Examples of qPCR validation of RNA-seq data for select OLP, iOL, and shared genes. A larger cohort of validation data is presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s007" target="_blank">S5C Fig</a>. <b>(E)</b> Gp130 sensitivity of select validated differentially expressed transcripts. Results are shown from Oli-neu cells treated with Cntf (100 ng/ml) for up to 24 h. Colored areas indicate the time period before peak response of Klf6 to Cntf. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s007" target="_blank">S5D Fig</a>. <b>(F)</b> Klf6-dependence of Cntf-induced responses. qPCR analysis of <i>Klf6-</i>silenced and control Oli-neu cells treated with Cntf for up to 24 h. Cntf sensitivity of differentially expressed targets is blunted in <i>Klf6</i>-silenced samples. Note also that some genes are Cntf-independent but Klf6-dependent during differentiation. Data are mean ± SEM. Statistics, <b>(D)</b> Student’s <i>t</i> test, <b>(F)</b> Two-way ANOVA plus Bonferroni post-test, <i>*p <</i> 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Data are representative of two to three independent studies. RNA-seq data are presented in full in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s009" target="_blank">S1</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s011" target="_blank">S3</a> Tables and are available on the GEO website (<a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">http://www.ncbi.nlm.nih.gov/geo/</a>) (Accession number GSE79245). Individual values for all other quantifications are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p

Klf6 is induced by gp130-Stat3 signals and is required for CNS myelination.

<p><b>(A)</b> Stage-specific markers in differentiation. Following specification to the Olig2⁺ oligodendrocyte lineage, oligodendrocyte progenitors (OLP) express Ng2, Sox9, and Pdgfrα. Pro-myelinating signals induce differentiation to immature oligodendrocytes (iOL), marked by co-expression of Nkx2.2 and acquisition of Apc and O4. These undergo terminal differentiation to mature OL, which express myelin proteins. <b>(B)</b> Klf/Sp family responses to the pro-myelinating factors Cntf (100ng/ml) and T3 (40ng/ml), as determined by quantitative PCR (qPCR). <b>(C)</b> Immunoblotting for Klf6 in Oli-neu cultures pretreated with 0–1,000 nM Jak Inhibitor I 2 h, then exposed to 100 ng/ml Cntf 1 h. <b>(D)</b> Confocal imaging for Klf6 and the gp130 effector Stat3 in mouse OLP exposed to 100 ng/ml Cntf or vehicle 1 h. Cntf upregulation of Klf6 is associated with translocation to the nucleus, where it colocalizes with Stat3. <b>(E,F)</b> Klf6 expression visualized via confocal imaging in vivo. <b>(E)</b> In the postnatal CNS (spinal cord shown), immunoreactivity is heterogeneous in Olig2⁺ cells and more homogeneous in astrocytes (Gfap⁺) and neurons (NeuN⁺). Boxes and arrows highlight representative cells. <b>(F)</b> Klf6 is highly expressed (arrowheads) in OLP (left panel). In contrast, expression is lower (arrowhead) or undetectable (asterisks) in more mature Apc⁺ iOL (center panel), and Klf6 is not seen in mature Mag⁺ cells (right panel). <b>(G)</b> Still from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s015" target="_blank">S1 Video</a> comparing a P11 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mutant (cko, right) with sex-matched <i>Klf6</i>^<i>fl/fl</i> littermate (ctrl, left). The mutant is ataxic. <b>(H)</b> CNS white matter tracts in P14 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mice display hypomyelination (arrowed), whereas <i>mGfapCre</i>:<i>Klf6</i>^<i>fl/fl</i> mice and <i>Klf6</i>^<i>fl/fl</i> littermate controls show no gross abnormalities. Brains are shown at the same magnification (scale bar, upper right). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s003" target="_blank">S1 Fig</a>. <b>(I,J)</b> Confocal analysis of lumbar spinal cords of P14 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and <i>mGfapCre</i>:<i>Klf6</i>^<i>fl/fl</i> mice and <i>Klf6</i>^<i>fl/fl</i> controls. Myelin proteins are almost absent from <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> spinal cord, whereas the peripheral nervous system (PNS) appears normal (arrowed). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s004" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s005" target="_blank">S3 Figs</a>. <b>(K,L)</b> Electron micrographs of P14 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and <i>Klf6</i>^<i>fl/fl</i> spinal cords and optic nerves. Almost no myelin sheaths are present in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> CNS samples. <b>(M)</b> Gene ontology of BeadArray profiling of P1 CNS from <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> pups and sex-matched <i>Klf6</i>^<i>fl/fl</i> littermates. The five most significant results are shown for physiologic and disease relevance. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4 Fig</a>. Data are mean ± SEM. Statistics: <b>(B,J)</b> ANOVA plus Bonferroni test, *<i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001. Data are representative of two to four mice per genotype (for confocal imaging data) or three mice per genotype (for electron microscopy data). Scale, <b>(A)</b> 5 μm, <b>(E)</b> 20 μm, <b>(F)</b> 10 μm, <b>(H)</b> 3 mm <b>(I)</b> 150 μm, inset 15 μm. Magnifications, <b>(K)</b> x5,000, inset x20,000, <b>(L)</b> x3,000. Individual values are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p

Klf6 is required for differentiation regardless of the initiating stimulus.

<p><b>(A–H)</b> Results of confocal analysis of primary mouse OLP subjected to <i>Klf6</i> silencing as described in Materials and Methods, and treatments indicated. <i>Klf6</i> silencing has no effect on active proliferation, shown by BrdU immunoreactivity in proliferating cultures <b>(A)</b>, nor on cell cycle exit index (Ki67^-BrdU⁺/BrdU⁺) in differentiating OLP cultures <b>(B)</b>. <b>(C–F)</b> Early events in differentiation, such as Nkx2.2 co-expression, occur normally in <i>Klf6</i>-silenced OLP differentiated with either 40 ng/ml T3 or 100 ng/ml Cntf <b>(C)</b>. However, expression of markers of subsequent stages of differentiation, such as O4 and Apc, is delayed in silenced cultures compared with non-targeting (NT) controls, and these cultures fail to mature to Mbp⁺ mOL <b>(D–F)</b>. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4G Fig</a>. These changes are associated with increased apoptosis <b>(G,H)</b>. <b>(I–L)</b> Confocal imaging analysis of primary mouse OLP nucleofected with GFP-labeled <i>Klf6</i>-overexpression construct or GFP vector control, and either left untreated or exposed to 40 ng/ml T3 for 72 h. Klf6 overexpression accelerates differentiation triggered by a pro-myelinating signal (T3), as measured by induction of O4 and Mbp <b>(I–K)</b>. However, Klf6 overexpression alone does not initiate differentiation <b>(L)</b>. <b>(M-O)</b> Confocal imaging analysis of <i>Klf6</i>-silenced and NT control mouse OLP pretreated with 2 μM caspase inhibitor Q-VD-OPh or vehicle for 2 h, then differentiated with T3 for 48 h. Apoptosis is almost absent from cultures exposed to the inhibitor, and Olig2⁺ cell numbers in <i>Klf6</i>-silenced and control conditions are almost identical <b>(M,N)</b>. However, rescue of viability does not restore differentiation, as measured by the percentage of O4⁺ cells <b>(M,O)</b>. Statistics, <b>(A,E,G,J,K,L)</b> Student’s <i>t</i> test, <b>(B,C,D,H,N,O)</b> ANOVA plus Bonferroni post-test, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Data are representative of at least three independent studies in separate cultures. Individual values are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p

Klf6 promotes differentiation via transactivation of importin-α5.

<p><b>(A–C)</b> ChIP-seq <b>(A)</b>, qPCR <b>(B)</b>, and confocal imaging data <b>(C)</b> identifying Klf6 regulation of the downstream importin effector Impα5. Data are also compatible with RNA-seq results in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s009" target="_blank">S1</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s011" target="_blank">S3</a> Tables. <b>(A)</b> ChIP-seq in primary mouse OLP and iOL demonstrates direct Klf6 binding to the promoter region of <i>Impα5</i>, which RNA-seq also identifies as Klf6-regulated (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s011" target="_blank">S3 Table</a>). <b>(B)</b> QPCR for importin family members in <i>Klf6-</i>silenced versus NT control primary mouse OLP, and O4⁺Apc⁺Mbp^- iOL differentiated with T3 for 18 h. Note that Klf6 control of Impα5 expression is selective. <b>(C)</b> Confocal imaging of lumbar spinal cords of E16.5 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and <i>Klf6</i>^<i>fl/fl</i> control embryos (upper panels), and <i>Olig1Cre</i>:<i>Stat3</i>^<i>fl/fl</i> and Stat3^<i>fl/fl</i> control embryos (lower panels), showing reduced Impα5 expression in oligodendrocyte lineage cells in mice with conditional <i>Klf6</i> or <i>Stat3</i> inactivation. Arrowheads in white matter areas (outlined) mark representative cells. Collectively, findings in <b>(A–C)</b> are compatible with the hypothesis that gp130-driven Klf6 uses selective control of Impα5 to regulate oligodendrocyte development <b>(D)</b>. <b>(E–G)</b> qPCR, confocal imaging, and immunoblotting data from primary mouse OLP silenced for <i>Impα5</i>, then differentiated with T3 for 24–72 h. In <b>(E)</b>, data for the myelin marker Cnp are from 72 h; other data are from 24 h. Note that differentiation markers are strongly reduced in <i>Impα5-</i>silenced cultures at both the RNA <b>(E)</b> and protein levels <b>(F,G)</b>. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s008" target="_blank">S6F Fig</a>. <b>(H)</b> OLP cultures from <i>Klf6</i>^<i>fl/fl</i><i>Rosa26</i>^<i>fl/fl</i> mice were exposed to <i>Ad5CMVCre</i> or <i>Ad5CMV</i> control, then nucleofected with either an Impα5 expression construct or empty vector. Cultures were treated with 40 ng/ml T3 and harvested at 72 h. Notably, recombinant Impα5 significantly increased the proportion of cells expressing the differentiation marker O4. Recombinant Impα5 also partially rescued differentiation in Klf6-deficient cultures. Numbers in panels <b>(G,H)</b> refer to proportion of cells positive for the maturation marker per field at 20x magnification, for at least four fields per condition. Data are mean ± SEM. Statistics, <b>(B,E,G)</b> Student’s <i>t</i> test, <b>(H)</b> ANOVA plus Bonferroni post-test, <i>*p <</i> 0.05, ** <i>p</i> <0.01, *** <i>p</i> <0.001. Scalebars, (<b>C,G,H)</b> 20 μm. Data are representative of at least three independent studies. ChIP-seq data are available on the GEO website (<a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">http://www.ncbi.nlm.nih.gov/geo/</a>) (Accession number GSE79245) and in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s012" target="_blank">S4 Table</a>. Individual values for all other quantifications are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p