Identification of a KRAB-zinc finger protein binding to the Rpe65 gene promoter.
Identification of a KRAB-zinc finger protein binding to the Rpe65 gene promoter.
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鉴定与 Rpe65 基因启动子结合的 KRAB-锌指蛋白。
DOI：
10.1080/02713680600678059
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发表时间：
2006
影响因子：
2
通讯作者：
Redmond,TMichael
中科院分区：
文献类型：
--
作者：
Lu,Zhongjian;Poliakov,Eugenia;Redmond,TMichael
目的：我们希望鉴定参与调控与 RPE65 基因近端启动子区域结合的转录因子，从而赋予 RPE 特异性表达。方法：我们将人 D407 RPE 细胞核提取物与基于 RPE65 近端基因启动子的双链（正义 5 引物生物素化）寡核苷酸一起孵育，并与链霉亲和素-Dynabeads 结合。洗脱结合的核蛋白，在 SDS-PAGE 上分离，并通过质谱分析。使用肽序列来鉴定 cDNA 克隆，将其亚克隆到 pCDNA3.1 中进行表达并共转染到 D407 细胞中，以评估小鼠 Rpe65 基因启动子/报告基因构建体的转录激活。使用SiRNA干扰来抑制ZNF492表达。结果：我们鉴定出与生物素化DNA/链霉亲和素珠结合的D407核蛋白是编码名为ZNF492的蛋白质的克隆KIAA1473的产物。 ZNF492 具有 531 个氨基酸的开放阅读框，N 末端被截短，缺少常见的 Krüppel 相关盒-A (KRAB-A)，而 KRAB-B 保持完整，并具有 12 个串联排列的 C2H2 锌指。 ZNF492 蛋白在 D407 细胞中的共表达不会激活 TR1（一种具有 49 bp 5 引物侧翼序列的小鼠 Rpe65 基因启动子/报告基因构建体），但会激活包含 188 bp 5 引物侧翼序列的构建体 TR2 2.5 倍，以及包含 655 bp 5 引物侧翼序列的较长构建体 TR4 和包含 TR5 的较长构建体。 1240-bp 5-prime 侧翼序列，大约是原来的 2 倍。 D407 中 ZNF492 的 SiRNA 介导抑制导致 Rpe65 启动子活性降低。结论：我们通过其与固定化 RPE65 启动子 DNA 序列的相互作用鉴定了 ZNF492，一种 KRAB 锌指蛋白。这种 KRAB-锌指蛋白可作为 Rpe65 基因上调的中度转录因子。在 ZNF492 中，KRAB-A 的缺失可能会减少或阻止共阻遏物结合，从而解释 Rpe65 基因表达的适度上调。
Purpose: We wish to identify transcriptional factors involved in regulation binding to the proximal promoter region of the RPE65 gene that confers RPE-specific expression.Methods: We incubated human D407 RPE cell nuclear extract with double-stranded (sense 5-prime biotinylated) oligonucleotides, based on the RPE65 proximal gene promoter, bound to streptavidin-Dynabeads. Bound nuclear proteins were eluted, separated on SDS-PAGE, and analyzed by mass spectrometry. Peptide sequence was used to identify cDNA clones that were subcloned into pCDNA3.1 for expression and co-transfection into D407 cells to assess transcriptional activation of mouseRpe65gene promoter/reporter constructs. SiRNA interference was used to suppress ZNF492 expression.Results: We identified a D407 nuclear protein binding to biotinylated-DNA/streptavidin beads as the product of clone KIAA1473 encoding a protein named ZNF492. ZNF492 has an open reading frame of 531 amino acids with a truncated N-terminus and lacks the usual Krüppel-associated box-A (KRAB-A) while KRAB-B remains intact and has 12 C2H2 zinc-fingers in tandem arrangement. Co-expression in D407 cells of ZNF492 protein did not activate TR1, a mouseRpe65gene promoter/reporter construct with 49-bp 5-prime flanking sequence, but did activate construct TR2, containing 188-bp 5-prime flanking sequence, by 2.5-fold, and the longer constructs TR4, containing 655-bp 5-prime flanking sequence, and TR5, containing 1240-bp 5-prime flanking sequence, by about 2-fold. SiRNA-mediated suppression of ZNF492 in D407 resulted in decreasedRpe65promoter activity.Conclusions: We have identified ZNF492, a KRAB-zinc finger protein, by its interaction with immobilizedRPE65promoter DNA sequence. This KRAB-zinc finger protein serves as a moderate transcriptional factor forRpe65gene upregulation. In ZNF492, absence of KRAB-A might reduce or prevent co-repressor binding to account for the modest upregulation ofRpe65gene expression.