Mouse Rankl Expression Is Regulated in T Cells by c-Fos through a Cluster of Distal Regulatory Enhancers Designated the T Cell Control Region
Mouse Rankl Expression Is Regulated in T Cells by c-Fos through a Cluster of Distal Regulatory Enhancers Designated the T Cell Control Region
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DOI：
10.1074/jbc.m111.231548
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发表时间：
2011-06-10
影响因子：
4.8
通讯作者：
Pike, J. Wesley
中科院分区：
文献类型：
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作者：
Bishop, Kathleen A.;Coy, Heidi M.;Pike, J. Wesley
NF-κ B配体受体激活因子（Rankl）是一种TNF样因子，其诱导负责骨吸收的破骨细胞的形成。虽然T细胞活化上调该基因，但其转录控制的分子机制仍然未知。我们在小鼠原代T细胞和T细胞杂交瘤中使用ChIP芯片分析来定义负责这种上调的调节增强子并表征其特性。升高的H3/H4乙酰化和增加的RNA聚合酶II密度在mRL-D5（一种位于TSS上游76 kb的已知增强子）以及位于甚至更上游约123至约156 kb之间的一簇调控位点（称为T细胞控制区（TCCR））处是明显的。基于钙信号传导和MAPK抑制剂阻断Rankl表达的能力，我们对转录介质c-Fos、NF-κ B和Nfat进行了进一步的ChIP芯片分析。T细胞活化诱导c-Fos在mRL-D5增强子处和TCCR内结合。在转录起始位点和mRL-D5处观察到NF-κ B的相互作用。mRL-D5和TCCR片段在报告基因测定中均表现出强大的转录活性，c-Fos和Nfat元件的位点特异性诱变废除了报告基因活性，表明这两种因子在控制增强子介导的Rankl转录中的作用。最后，染色体构象捕获分析证实，mRL-D5和TCCR的区段位于Rankl基因启动子附近，因此能够潜在地直接影响Rankl基因启动子活性。我们得出结论，mRL-D5和TCCR都代表在T细胞中的Rankl表达中发挥不可或缺的作用的控制区段。
Receptor activator of NF-kappa B ligand (Rankl) is a TNF-like factor that induces the formation of osteoclasts responsible for bone resorption. Although T cell activation up-regulates this gene, the molecular mechanism of its transcriptional control remains unknown. We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this up-regulation and to characterize their properties. Elevated H3/H4 acetylation and increased RNA polymerase II density were evident at mRL-D5, a known enhancer located 76 kb upstream of the TSS, as well as at a cluster of regulatory sites located even further upstream between -123 to -156 kb, termed the T cell control region (TCCR). Based upon the ability of calcium signaling and MAPK inhibitors to block Rankl expression, we conducted further ChIP-chip analysis of the transcriptional mediators c-Fos, NF-kappa B, and Nfat. T cell activation induced c-Fos binding at the mRL-D5 enhancer and within the TCCR. The interaction of NF-kappa B was observed at the transcriptional start site and at mRL-D5. Both mRL-D5 and segments of the TCCR exhibited robust transcriptional activity in reporter assays, and site-specific mutagenesis of c-Fos and Nfat elements abrogated reporter activity, suggesting a role for both factors in the control of enhancer-mediated Rankl transcription. Finally, chromosome conformation capture analysis confirmed that mRL-D5 and segments of the TCCR were located in proximity to the Rankl gene promoter and thus potentially able to influence directly Rankl gene promoter activity. We conclude that both mRL-D5 and the TCCR represent control segments that play an integral role in Rankl expression in T cells.