Transcriptomics

Dataset Information

26

Identification of Myelin-gene Regulatory Factor as a Critical Transcriptional Regulator Required for CNS Myelination


ABSTRACT: 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. Overall design: 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.

REANALYSED by: GSE119128

INSTRUMENT(S): [Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array

ORGANISM(S): Mus musculus  

SUBMITTER: Ben Emery  

PROVIDER: GSE15303 | GEO | 2009-05-31

SECONDARY ACCESSION(S): PRJNA115835

REPOSITORIES: GEO

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