Jin W D, Boutillier A L, Glucksman M J, Salton S R, Loeffler J P, Roberts J L
Dr. Arthur M. Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029.
Mol Endocrinol. 1994 Oct;8(10):1377-88. doi: 10.1210/mend.8.10.7854355.
A corticotropin-releasing hormone (CRH) and cAMP-responsive region (-236/-133) in the rat POMC gene promoter previously reported to confer CRH/cAMP responsiveness to heterologous reporter constructs has been characterized. DNAse footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cells. When these individual DNA elements were separately tested in heterologous reporter constructs for CRH induction, only one element, designated PCRH-RE (POMC CRH responsive element, -171/-160) was found to give strong CRH stimulation (5- to 7-fold). This element appears novel as to the possible binding factors, although it has homology to the mouse metallothionein metal regulatory element. Gel shift analyses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stimulation of retarded nucleoproteins following CRH stimulation, suggesting that the possible binding factor(s) may mediate transcriptional regulation at this site. The activity of PCRH-RE binding protein was inhibited by divalent cations, with Cu2+ and Cd2+ being most effective; Zn2+ had no effect, indicating that this binding factor(s) is functionally distinct from the metallothionein metal regulatory element binding protein. A 2.6 kilobase cDNA clone encoding a protein (PCRH-REB-1) binding to this element was isolated by Southwestern screening of an AtT20 expression library with radiolabeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the sequence corresponding to the entire coding region of the protein (PCRH-REB), which proved to be identical to a recently described DNA binding protein of the replication factor C complex, mRFC140/Mouse Southwestern. Primer extension and Northern blot analysis revealed that the size of the full length mRNA is about 4.9 kilobases. PCRH-REB mRNA expression is not restricted to corticotrophs but is present in a broad tissue distribution as evaluated by reverse transcription polymerase chain reaction analysis. A bacterially expressed beta-galactosidase-PCRH-REB-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive element was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCHR-REB protein sequence presents several interesting motifs: one p-Loop motif (ATP binding site), nine protein kinase A phosphorylation sites (implying a possible role in responding to the CRH-induced cAMP signal), and regions of homology to proteins involved in DNA replication and repair. PCRH-REB is, therefore, a potential transacting factor binding to a major CRH-responsive element in the POMC promoter.
先前报道的大鼠阿黑皮素原（POMC）基因启动子中的促肾上腺皮质激素释放激素（CRH）和cAMP反应区域（-236 / -133）已被鉴定，该区域可赋予异源报告基因构建体CRH / cAMP反应性。DNA酶足迹分析显示，该区域的多个元件被表达POMC的AtT20细胞的核蛋白结合。当在异源报告基因构建体中分别测试这些单个DNA元件以进行CRH诱导时，仅发现一个元件，命名为PCRH-RE（POMC CRH反应元件，-171 / -160）可产生强烈的CRH刺激（5至7倍）。尽管该元件与小鼠金属硫蛋白金属调节元件具有同源性，但就可能的结合因子而言，它似乎是新的。用AtT20细胞核提取物对PCRH-RE进行凝胶迁移分析显示，CRH刺激后滞后核蛋白有明显刺激，表明可能的结合因子可能介导该位点的转录调控。PCRH-RE结合蛋白的活性受到二价阳离子的抑制，其中Cu2 +和Cd2 +最有效；Zn2 +没有作用，表明该结合因子在功能上与金属硫蛋白金属调节元件结合蛋白不同。通过用放射性标记的PCRH-RE寡核苷酸对AtT20表达文库进行Southwestern筛选，分离出一个编码与该元件结合的蛋白质（PCRH-REB-1）的2.6千碱基cDNA克隆。该克隆用于分离其他几个cDNA克隆，以确定与该蛋白质（PCRH-REB）的整个编码区相对应的序列，事实证明该序列与最近描述的复制因子C复合物的DNA结合蛋白mRFC140 /小鼠Southwestern相同。引物延伸和Northern印迹分析显示全长mRNA的大小约为4.9千碱基。通过逆转录聚合酶链反应分析评估，PCRH-REB mRNA表达不仅限于促肾上腺皮质激素细胞，而是广泛分布于组织中。细菌表达的β-半乳糖苷酶-PCRH-REB-1融合蛋白显示可有效结合PCRH-RE。此外，PCRH-REB-1融合蛋白与POMC CRH反应元件的结合受到二价阳离子的抑制，其敏感性与使用AtT20细胞核提取物观察到的相似。预测的PCHR-REB蛋白序列呈现出几个有趣的基序：一个p-环基序（ATP结合位点），九个蛋白激酶A磷酸化位点（暗示在响应CRH诱导的cAMP信号中可能起作用），以及与参与DNA复制和修复的蛋白质的同源区域。因此，PCRH-REB是一种潜在的反式作用因子，可结合POMC启动子中的主要CRH反应元件。
