The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination

Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination

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 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