Leão Felipe B, Vaughn Lauren S, Bhatt Dev, Liao Will, Maloney Dillon, Carvalho Brener C, Oliveira Leonardo, Ghosh Sankar, Silva Aristóbolo M
Laboratory of Inflammatory Genes, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, Brazil.
Department of Microbiology & Immunology, Columbia University, College of Physicians and Surgeons, New York, NY 10031, USA.
Int J Biochem Cell Biol. 2020 Oct;127:105840. doi: 10.1016/j.biocel.2020.105840. Epub 2020 Aug 28.
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）结构域家族成员1b由巨噬细胞中表达的一种Toll样受体（TLR）诱导基因编码，但其表达调控的转录机制仍不清楚。在此，我们对Rasgef1b的5'侧翼序列进行了功能表征并分析了其转录激活情况。我们确定炎症反应性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保守，含有一簇五个NF-κB结合位点。荧光素酶报告基因检测表明该启动子对TLR激活以及RelA或cRel转录因子有反应，但对RelB无反应。此外，定点诱变表明κB结合位点是LPS诱导最大启动子激活所必需的。利用测序的转座酶可及染色质分析（ATAC-seq）显示该启动子位于可及染色质区域。更重要的是，染色质免疫沉淀测序（ChIP-seq）表明在LPS刺激骨髓来源的巨噬细胞后，RelA被招募到启动子区域。最后，对Rela缺陷型巨噬细胞的研究或用Bay11-7082进行的药理学抑制表明，NF-κB是TLR激动剂诱导最佳Rasgef1b表达所必需的。我们的数据提供了NF-κB介导的调节机制的证据，该机制促进巨噬细胞中TLR激活后Rasgef1b的表达。
