Dataset Information

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

ABSTRACT: 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-?5 (Imp?5), 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 Imp?5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination.

SUBMITTER: Laitman BM

PROVIDER: S-EPMC4877075 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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

Laitman Benjamin M BM Asp Linnéa L Mariani John N JN Zhang Jingya J Liu Jia J Sawai Setsu S Chapouly Candice C Horng Sam S Kramer Elisabeth G EG Mitiku Nesanet N Loo Hannah H Burlant Natalie N Pedre Xiomara X Hara Yuko Y Nudelman German G Zaslavsky Elena E Lee Young-Min YM Braun David A DA Lu Q Richard QR Narla Goutham G Raine Cedric S CS Friedman Scott L SL Casaccia Patrizia P John Gareth R GR

PLoS biology 20160523 5

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 ...[more]

PMID: 27213272

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Project description:The transcriptional control of CNS myelin gene expression is poorly understood. Here we identify gene model 98, which we have named Myelin-gene Regulatory Factor (MRF), as a transcriptional regulator required for CNS myelination. Within the CNS, MRF is specifically expressed by postmitotic oligodendrocytes. MRF is a nuclear protein containing an evolutionarily conserved DNA binding domain homologous to a yeast transcription factor. Knockdown of MRF in oligodendrocytes by RNA interference prevents expression of most CNS myelin genes; conversely, overexpression of MRF within cultured oligodendrocyte progenitors or the chick spinal cord promotes expression of myelin genes. In mice lacking MRF within the oligodendrocyte lineage, pre-myelinating oligodendrocytes are generated but display severe deficits in myelin gene expression and fail to myelinate. These mice display severe neurological abnormalities, and die due to seizures during the third postnatal week. These findings establish MRF as a critical transcriptional regulator essential for oligodendrocyte maturation and CNS myelination. We used microarrays to compare cultured oligodendrocytes (differentiated in vitro for 4 days) from MRF conditional knockouts and control litteramates to look at the effects of MRF deficiency on myelin gene expression. Mouse OPCs grown in vitro in the presence of PDGF serve as a baseline for gene expression prior to differentiation. Mouse OPCs from MRF conditional knockout (MRF fl/fl, Olig2 wt/cre) mice and control littermates (MRF wt/fl; Olig2 wt/cre) were isolated from enzymatically dissociated P7 mouse brains as previously described (Cahoy et al., 2008), positively immunopanning for PDGFR-alpha following a depletion of microglia with BSL1. Cells were grown in defined serum-free media as previously described (Dugas et al., 2006), but with the addition of 2% B-27 (Invitrogen). Cells were proliferated for several days in the presence of PDGF-AA (10 ng/ml, PeproTech) and then differentiation induced by withdrawal of PDGF-AA and addition of triiodothyronine (T3) (40 ng/ml; Sigma). RNA was isolated from cells 4 days after induction of differentiation; OPCs maintained in PDGF-AA serve as a baseline of OPC gene expression. Total RNA was isolated from cells with the RNeasy micro kit (Qiagen, Valencia, CA) using Qiagen on-column DNase treatment to remove any contaminating genomic DNA. The integrity of RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) and RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Rockland, DE). Biotinylated cRNAs for hybridization to Affymetrix 3'-arrays were prepared from 1ug total RNA using the Affymetrix two-cycle target labeling assay with spike in controls (Affymetrix Inc., Santa Clara, CA, 900494). Labeled-cRNA was fragmented and hybridized to Mouse Genome 430 2.0 Arrays (3'-arrays, Affymetrix, 900495) following the manufacturer's protocols. Raw image files were processed using Affymetrix GCOS 1.3 software to calculate individual probe cell intensity data and generate CEL data files. Using GCOS and the MAS 5.0 algorithm, intensity data was normalized per chip to a target intensity TGT value of 500 and expression data and present/absent calls for individual probe sets calculated. Gene symbols and names for data analyzed with the MAS 5.0 algorithm were from the Affymetrix Netaffx Mouse430_2 annotations file (http://www.affymetrix.com/support/technical/byproduct.affx?product=moe430-20). Quality control was performed by examining raw DAT image files for anomalies, confirming each GeneChip array had a background value less than 100, monitoring that the percencelle present calls was appropriate for the cell type, and inspecting the poly(A) spike in controls, housekeeping genes, and hybridization controls to confirm labeling and hybridization consistency.

Dataset Information

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

Publications

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

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