Noy-Lotan Sharon, Dgany Orly, Lahmi Roxane, Marcoux Nathaly, Krasnov Tanya, Yissachar Nissan, Ginsberg Doron, Motro Benny, Resnitzky Peretz, Yaniv Isaac, Kupfer Gary M, Tamary Hannah
Pediatric Hematology Oncology, Schneider Children's Medical Center of Israel, 14 Kaplan Street, Petah Tiqva 49 202, Israel.
Haematologica. 2009 May;94(5):629-37. doi: 10.3324/haematol.2008.003327. Epub 2009 Mar 31.
Congenital dyserythropoietic anemia type I is an inherited autosomal recessive macrocytic anemia associated with ineffective erythropoiesis and the development of secondary hemochromatosis. Distinct erythroid precursors with internuclear chromatin bridges and spongy heterochromatin are pathognomonic for the disease. The mutated gene (CDAN1) encodes a ubiquitously expressed protein of unknown function, codanin-1. Based on the morphological features of congenital dyserythropoietic anemia type I erythroblasts and data on a role in cell cycle progression of codanin-1 homolog in Drosophila we investigated the cellular localization and possible involvement of codanin-1 during the cell cycle.
Codanin-1 localization was studied by immunofluorescence and immune electron microscopy. Cell cycle expression of codanin-1 was evaluated using synchronized HeLa cells. E2F proteins are the main regulator of G(1)/S transition. An E2F1-inducible cell line (U20S-ER-E2F1) enabled us to study codanin-1 expression following ectopic E2F1 induction. Direct binding of E2F1 to codanin-1 promoter was assessed by chromatin immunoprecipitation. We used a luciferase-reporter plasmid to study activation of CDAN1 transcription by E2F1.
We localized codanin-1 to heterochromatin in interphase cells. During the cell cycle, high levels of codanin-1 were observed in the S phase. At mitosis, codanin-1 underwent phosphorylation, which coincided with its exclusion from condensed chromosomes. The proximal CDAN1 gene promoter region, containing five putative E2F binding sites, was found to be a direct target of E2F1.
Taken together, these data suggest that codanin-1 is a cell cycle-regulated protein active in the S phase. The exact role of codanin-1 during the S phase remains to be determined. Nevertheless this represents the first step towards understanding the function of the proteins involved in congenital dyserythropoietic anemia.
I型先天性红细胞生成异常性贫血是一种常染色体隐性遗传性大细胞性贫血，与无效红细胞生成及继发性血色素沉着症的发生有关。具有核间染色质桥和海绵状异染色质的独特红系前体细胞是该疾病的特征性表现。突变基因（CDAN1）编码一种功能未知、广泛表达的蛋白——科达宁-1。基于I型先天性红细胞生成异常性贫血成红细胞的形态学特征以及果蝇中科达宁-1同源物在细胞周期进程中作用的数据，我们研究了科达宁-1在细胞周期中的细胞定位及可能的作用。
通过免疫荧光和免疫电子显微镜研究科达宁-1的定位。使用同步化的HeLa细胞评估科达宁-1的细胞周期表达。E2F蛋白是G(1)/S期转换的主要调节因子。一种E2F1诱导细胞系（U20S-ER-E2F1）使我们能够研究异位诱导E2F1后科达宁-1的表达。通过染色质免疫沉淀评估E2F1与科达宁-1启动子的直接结合。我们使用荧光素酶报告质粒研究E2F1对CDAN1转录的激活作用。
我们将科达宁-1定位于间期细胞的异染色质中。在细胞周期中，S期观察到高水平的科达宁-1。在有丝分裂时，科达宁-1发生磷酸化，这与其从浓缩染色体中排除同时发生。近端CDAN1基因启动子区域含有五个假定的E2F结合位点，被发现是E2F1的直接靶点。
综上所述，这些数据表明科达宁-1是一种在S期具有活性的细胞周期调节蛋白。科达宁-1在S期的确切作用仍有待确定。然而，这是理解先天性红细胞生成异常性贫血相关蛋白功能的第一步。
