Lu Zhongjian, Poliakov Eugenia, Redmond T Michael
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute/NIH, 7 Memorial Drive, Bethesda, MD 20892, USA.
Curr Eye Res. 2006 May;31(5):457-66. doi: 10.1080/02713680600678059.
We wish to identify transcriptional factors involved in regulation binding to the proximal promoter region of the RPE65 gene that confers RPE-specific expression.
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 mouse Rpe65 gene promoter/reporter constructs. SiRNA interference was used to suppress ZNF492 expression.
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 mouse Rpe65 gene 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 decreased Rpe65 promoter activity.
We have identified ZNF492, a KRAB-zinc finger protein, by its interaction with immobilized RPE65 promoter DNA sequence. This KRAB-zinc finger protein serves as a moderate transcriptional factor for Rpe65 gene upregulation. In ZNF492, absence of KRAB-A might reduce or prevent co-repressor binding to account for the modest upregulation of Rpe65 gene expression.
我们希望鉴定参与调控与赋予视网膜色素上皮（RPE）特异性表达的RPE65基因近端启动子区域结合的转录因子。
我们将人D407 RPE细胞核提取物与基于RPE65基因近端启动子的双链（5'端生物素化的正义链）寡核苷酸一起孵育，该寡核苷酸与链霉亲和素-磁珠结合。洗脱结合的核蛋白，在SDS-PAGE上分离，并通过质谱分析。肽序列用于鉴定cDNA克隆，这些克隆被亚克隆到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。使用小干扰RNA（siRNA）干扰来抑制ZNF492的表达。
我们鉴定出一种与生物素化-DNA/链霉亲和素磁珠结合的D407核蛋白，它是编码名为ZNF492的蛋白质的克隆KIAA1473的产物。ZNF492有一个531个氨基酸的开放阅读框，N端截短，缺少通常的Krüppel相关框-A（KRAB-A），而KRAB-B保持完整，并有12个串联排列的C2H2锌指。在D407细胞中共同表达ZNF492蛋白并未激活TR1，即具有49 bp 5'侧翼序列的小鼠Rpe65基因启动子/报告基因构建体，但确实激活了包含188 bp 5'侧翼序列的构建体TR2，激活倍数为2.5倍，以及包含655 bp 5'侧翼序列的更长构建体TR4和包含1240 bp 5'侧翼序列的TR5，激活倍数约为2倍。在D407细胞中，siRNA介导的ZNF492抑制导致Rpe65启动子活性降低。
我们通过ZNF492与固定化的RPE65启动子DNA序列的相互作用鉴定出了一种KRAB-锌指蛋白ZNF492。这种KRAB-锌指蛋白作为Rpe65基因上调的中度转录因子。在ZNF492中，KRAB-A的缺失可能减少或阻止共抑制因子的结合，从而导致Rpe65基因表达的适度上调。
