Key Laboratory of Protein Chemistry and Developmental Biology of Education Ministry of China, College of Life Science, Hunan Normal University, Changsha 410081, China.
Biochem J. 2012 Apr 1;443(1):153-64. doi: 10.1042/BJ20111856.
Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554 bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor-1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions.
Ngb（神经球蛋白）已被确定为一种新型内源性神经保护剂。然而，关于 Ngb 表达的调节机制，尤其是在缺氧条件下，知之甚少。在本研究中，我们将小鼠 Ngb 基因的核心近端启动子定位到一个 554bp 的片段，该片段包含潜在的保守 NF-κB（核因子κB）和 Egr1（早期生长反应因子 1）结合位点。转录因子 p65、p50、Egr1 或 Sp1（特异性蛋白 1）的过表达和敲低分别增加和减少了 Ngb 的表达。通过转染突变型 Ngb 基因启动子构建体、电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）实验评估，证实 NF-κB 家族成员（p65、p50 和 cRel）、Egr1 和 Sp1 在体外和体内与 Ngb 基因近端启动子区结合。此外，还发现了一个κB3 位点，作为负责缺氧诱导的 Ngb 启动子活性的关键顺式元件。NF-κB（p65）和 Sp1 也负责缺氧诱导的 Ngb 表达上调。虽然在小鼠 Ngb 基因的启动子中没有保守的 HREs（缺氧反应元件），但本研究的结果表明，HIF-1α（缺氧诱导因子-1α）也参与了缺氧诱导的 Ngb 上调。总之，我们已经确定 NF-κB、Egr1 和 Sp1 通过与小鼠 Ngb 启动子的特异性相互作用，在调节基础 Ngb 表达中发挥重要作用。NF-κB、Sp1 和 HIF-1α 有助于在缺氧条件下上调小鼠 Ngb 基因的表达。
