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

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.

中枢神经系统（CNS）髓鞘基因表达的转录调控机制目前尚不清楚。本研究鉴定出基因模型98，将其命名为髓鞘基因调控因子（Myelin-gene Regulatory Factor, MRF），它是中枢神经系统髓鞘形成所必需的转录调控因子。在中枢神经系统中，MRF仅在有丝分裂后少突胶质细胞中特异性表达。MRF是一种核蛋白，含有与酵母转录因子同源的进化保守DNA结合结构域。通过RNA干扰（RNA interference）敲低少突胶质细胞中的MRF，会阻止大多数中枢神经系统髓鞘基因的表达；反之，在培养的少突胶质细胞前体或鸡脊髓中过表达MRF，则会促进髓鞘基因的表达。在少突胶质细胞谱系中缺失MRF的小鼠中，可产生髓鞘前少突胶质细胞，但这些细胞在髓鞘基因表达方面存在严重缺陷，且无法完成髓鞘形成。这些小鼠表现出严重的神经系统异常，并在出生后第三周因癫痫发作死亡。上述研究结果证实，MRF是少突胶质细胞成熟及中枢神经系统髓鞘形成所必需的关键转录调控因子。

我们使用微阵列（microarrays）比较了来自MRF条件性敲除（MRF fl/fl, Olig2 wt/cre）小鼠与同窝对照小鼠的培养少突胶质细胞（体外分化4天），以探究MRF缺乏对髓鞘基因表达的影响。在血小板衍生生长因子（PDGF）存在下体外培养的小鼠少突胶质细胞前体（oligodendrocyte progenitor cells, OPCs）可作为分化前基因表达的基线。总体实验设计：按照此前描述的方法（Cahoy等，2008），从出生后第7天（P7）的小鼠大脑中分离得到MRF条件性敲除（MRF fl/fl, Olig2 wt/cre）小鼠与同窝对照（MRF wt/fl; Olig2 wt/cre）小鼠的少突胶质细胞前体，先用BSL1去除小胶质细胞，再通过PDGFR-α阳性免疫淘选富集目标细胞。按照此前描述的方法（Dugas等，2006）在无血清限定培养基中培养细胞，并添加2% B-27（Invitrogen）。细胞在PDGF-AA（10 ng/ml，PeproTech）存在下增殖数天，随后通过撤除PDGF-AA并添加三碘甲状腺原氨酸（T3，40 ng/ml；Sigma）诱导分化。在诱导分化4天后收集细胞并提取RNA；维持在PDGF-AA中的少突胶质细胞前体可作为少突胶质细胞前体基因表达的基线。使用RNeasy微量试剂盒（Qiagen, Valencia, CA）并结合Qiagen柱上DNase处理去除基因组DNA污染，从细胞中提取总RNA。采用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer, Agilent Technologies）评估RNA完整性，使用NanoDrop ND-1000分光光度计（NanoDrop, Rockland, DE）测定RNA浓度。从1 μg总RNA中制备用于与Affymetrix 3'端阵列杂交的生物素化cRNA，采用Affymetrix双循环靶标标记试剂盒并加入spike-in内参对照（Affymetrix Inc., Santa Clara, CA, 900494）。按照制造商的实验方案，将标记后的cRNA片段化，与小鼠基因组430 2.0阵列（3'-arrays, Affymetrix, 900495）进行杂交。使用Affymetrix GCOS 1.3软件处理原始图像文件，计算单个探针细胞强度数据并生成CEL数据文件。采用GCOS及MAS 5.0算法，对每块芯片的强度数据进行标准化，使目标强度TGT值为500，并计算单个探针集的表达数据及存在/缺失呼叫结果。使用MAS 5.0算法分析的数据对应的基因符号与名称来自Affymetrix Netaffx Mouse430_2注释文件（http://www.affymetrix.com/support/technical/byproduct.affx?product=moe430-20）。质量控制步骤包括：检查原始DAT图像文件是否存在异常，确认每块GeneChip阵列的背景值低于100，监测百分比present呼叫值是否符合该细胞类型的正常范围，以及检查poly(A)内参对照、管家基因及杂交对照，以确认标记与杂交过程的一致性。