鑑別骨髓衍生巨噬細胞中的c-Maf標的基因

Abstract

c-Maf(v-maf同源肌腱膜纖維肉瘤致癌基因)是Maf家族蛋白的一員，Maf家族蛋白能夠結合Maf辨識元件(MAREs)並活化下游目標基因的轉錄。c-Maf在調控輔助T細胞IL-4、IL-21和IL-22的製造中扮演重要角色。但是在巨噬細胞中的角色仍不明朗。在本研究中，我的研究目標是尋找在巨噬細胞中可能的c-Maf標的基因。利用微陣列法來分析從WT與c-Maf KO小鼠分化的古典活化(M1)與替代性活化(M2)骨髓衍生巨噬細胞(BMDM)。有218個基因在不同組別間表現相差兩倍以上而被選為候選基因。在小鼠與人類中，這些基因的啟動子序列都利用MatInspector (Genometix)和ClustalW (GenomeNet) 分析。基於MARE序列相似度與小鼠/人類的序列比序結果，有36個基因擁有保守的類MARE序列。在這些基因中，針對Fabp4基因進一步研究。Fabp4的表現在WT M2中比M1巨噬細胞高了八倍，並在c-Maf KO巨噬細胞中表現下降。微陣列的結果也經過定量PCR (qPCR) 的確認。另外，染色質免疫共沉澱(ChIP)的結果指出c-Maf能結合Fabp4的啟動子序列。而且針對Fabp4啟動子的螢光素酶法結果也顯示c-Maf能活化Fabp4基因的轉錄。總結，這些研究顯示在巨噬細胞中c-Maf能藉著結合Fabp4啟動子上的MARE位置來活化Fabp4基因的轉錄。

c-Maf (v-maf musculoaponeurotic fibrosarcoma oncogene homolog), a member of the Maf family, is known to transactivate downstream target genes by binding to the Maf-recognition elements (MAREs). It is well known that c-Maf plays an important role in regulating the production of IL-4, IL-21 and IL-22 in TH cells. However, the role of c-Maf in the biology of macrophages remains unclear. In this study, I aimed to search for the possible c-Maf target genes in macrophages. Microarray data were analyzed for gene expression of classically activated (M1) and alternatively activated (M2) bone marrow-derived macrophages from WT and c-Maf KO mice. Two hundred and eighteen genes were selected as candidates based on differential expression with 2-fold change as cutoff. The promoter sequences of these genes in mice and human were analyzed with the MatInspector (Genometix) program and aligned with the ClustalW (GenomeNet) program. The sequences were compared with the sequences of all the known MARE sites. Based on their similarity and alignment analysis of the sequences in mice and human, there were 36 genes with conservative MARE-like sequences. Among them, Fabp4 was chosen for further study, because mRNA microarray analysis showed that the expression of Fabp4 was 8-fold higher in WT M2 than in M1 macrophages and the expression was reduced in c-Maf KO macrophages. Quantitative PCR (qPCR) assay confirmed the microarray results. Chromatin immunoprecipitation (ChIP) assay further revealed that c-Maf binds to Fabp4 promoter. Moreover, Fabp4 promoter reporter assay also showed that c-Maf transactivates Fabp4 gene. Taken together, these findings demonstrated that c-Maf binds to MARE site of Fabp4 promoter and transactivates Fabp4 gene expression in macrophages.