Ras Guanine Exchange Factor (RasGEF) domain family member 1b is encoded by a Toll-like receptor (TLR)-inducible gene expressed in macrophages, but transcriptional mechanisms that govern its expression are still unknown. Here, we have functionally characterized the 5′ flanking Rasgef1b sequence and analyzed its transcriptional activation. We have identified that the inflammation-responsive promoter is contained within a short sequence (-183 to +119) surrounding the transcriptional start site. The promoter sequence is evolutionarily conserved and harbors a cluster of five NF-κB binding sites. Luciferase reporter gene assay showed that the promoter is responsive to TLR activation and RelA or cRel, but not RelB, transcription factors. Besides, site-directed mutagenesis showed that the κB binding sites are required for maximal promoter activation induced by LPS. Analysis by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that the promoter is located in an accessible chromatin region. More important, Chromatin Immunoprecipitation sequencing (ChIP-seq) showed that RelA is recruited to the promoter region upon LPS stimulation of bone marrow-derived macrophages. Finally, studies with Rela-deficient macrophages or pharmacological inhibition by Bay11-7082 showed that NF-κB is required for optimal Rasgef1b expression induced by TLR agonists. Our data provide evidence of the regulatory mechanism mediated by NF-κB that facilitates Rasgef1b expression after TLR activation in macrophages.

Ras鸟嘌呤交换因子（RasGEF）域家族成员1b由在巨噬细胞中表达的Toll样受体（TLR）诱导型基因编码，但是控制其表达的转录机制仍然未知。在这里，我们已经对5'侧翼Rasgef1b的功能进行了表征序列并分析其转录激活。我们已经确定，炎症反应启动子包含在转录起始位点周围的短序列（-183至+119）内。该启动子序列在进化上是保守的，并且具有五个NF-κB结合位点的簇。萤光素酶报告基因检测表明，该启动子对TLR激活和RelA或cRel具有响应，但对RelB转录因子无响应。此外，定点诱变表明κB结合位点是LPS诱导的最大启动子激活所必需的。使用测序（ATAC-seq）通过转座酶可及染色质的分析分析显示，启动子位于可及染色质区域。更重要，染色质免疫沉淀测序（ChIP-seq）显示，经LPS刺激源自骨髓的巨噬细胞后，RelA被募集到启动子区域。最后，研究相对缺乏的巨噬细胞或Bay11-7082的药理抑制作用表明，TLR激动剂诱导的最佳Rasgef1b表达需要NF-κB 。我们的数据提供了由巨噬细胞中TLR激活后促进Rasgef1b表达的NF-κB介导的调节机制的证据。